G2Cdb::Gene report

Gene id
G00002531
Gene symbol
SNAP25 (HGNC)
Species
Homo sapiens
Description
synaptosomal-associated protein, 25kDa
Orthologue
G00001282 (Mus musculus)

Databases (9)

Curated Gene
OTTHUMG00000031863 (Vega human gene)
Gene
ENSG00000132639 (Ensembl human gene)
6616 (Entrez Gene)
99 (G2Cdb plasticity & disease)
SNAP25 (GeneCards)
Literature
600322 (OMIM)
Marker Symbol
HGNC:11132 (HGNC)
Protein Expression
360 (human protein atlas)
Protein Sequence
P60880 (UniProt)

Synonyms (6)

  • RIC-4
  • RIC4
  • SEC9
  • SNAP-25
  • bA416N4.2
  • dJ1068F16.2

Diseases (9)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000166: Schizophrenia Y Y (11287790) Repeat polymorphism (RP) Y
D00000291: Alagille syndrome N Y (9205123) Deletion (D) ?
D00000179: Attention deficit hyperactivity disorder Y Y (17325713) Single nucleotide polymorphism (SNP) ?
D00000179: Attention deficit hyperactivity disorder Y Y (16088329) Polymorphism (P) ?
D00000166: Schizophrenia Y Y (15823421) Polymorphism (P) Y
D00000153: Obesity N Y (15823421) Polymorphism (P) N
D00000179: Attention deficit hyperactivity disorder Y Y (15007392) Single nucleotide polymorphism (SNP) Y
D00000179: Attention deficit hyperactivity disorder Y Y (15007392) Microsatellite polymorphism (MSP) Y
D00000248: Netherton syndrome N Y (16670861) Deletion (D) Y
D00000140: Diabetes mellitus Type 1 N Y (16519819) Single nucleotide polymorphism (SNP) N
D00000248: Netherton syndrome N Y (16225619) Microinsertion (MI) Y
D00000248: Netherton syndrome N Y (16120162) Single nucleotide polymorphism (SNP) Y
D00000247: Atopic dermatitis N Y (15949016) Polymorphism (P) N
D00000248: Netherton syndrome N Y (15942217) Insertion (I) Y
D00000247: Atopic dermatitis N Y (15753919) Polymorphism (P) Y
D00000315: Food allergy N Y (15753919) Polymorphism (P) Y
D00000240: Asthma N Y (15753894) Single nucleotide polymorphism (SNP) N
D00000247: Atopic dermatitis N Y (15753894) Single nucleotide polymorphism (SNP) N
D00000248: Netherton syndrome N Y (15656819) Single nucleotide polymorphism (SNP) Y

References

  • Support for the MnlI polymorphism of SNAP25; a Korean ADHD case-control study.

    Choi TK, Lee HS, Kim JW, Park TW, Song DH, Yook KW, Lee SH, Kim JI and Suh SY

    Molecular psychiatry 2007;12;3;224-6

  • Netherton syndrome: report of identical twins presenting with severe atopic dermatitis.

    Kilic G, Guler N, Ones U, Tamay Z and Guzel P

    Department of Pediatrics, Division of Pediatric Allergy and Chest Diseases, Istanbul University, Istanbul Medical School, Istanbul, Turkey. kilicgurkan@yahoo.com

    We report the cases of 4-year-old identical twin sisters who presented with severe atopic dermatitis with intractable skin manifestations and multiple food allergies. Netherton syndrome (NS) (OMIM 256500) was suspected due to very high serum IgE levels, growth retardation, severe food allergies and typical hair finding (trichorrhexis invaginata). A definite diagnosis was made by genetic analysis. Our cases are unique in being the first identical twins with NS diagnosed by a novel mutation in the SPINK5 gene. NS should be considered in differential diagnosis in children who have generalized erythema with intractable eczematous lesions and elevated levels of IgE.

    European journal of pediatrics 2006;165;9;594-7

  • Analysis of polymorphisms in 16 genes in type 1 diabetes that have been associated with other immune-mediated diseases.

    Smyth DJ, Howson JM, Payne F, Maier LM, Bailey R, Holland K, Lowe CE, Cooper JD, Hulme JS, Vella A, Dahlman I, Lam AC, Nutland S, Walker NM, Twells RC and Todd JA

    Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Rd, Cambridge, CB2 2XY, UK. debbie.smyth@cimr.cam.ac.uk

    Background: The identification of the HLA class II, insulin (INS), CTLA-4 and PTPN22 genes as determinants of type 1 diabetes (T1D) susceptibility indicates that fine tuning of the immune system is centrally involved in disease development. Some genes have been shown to affect several immune-mediated diseases. Therefore, we tested the hypothesis that alleles of susceptibility genes previously associated with other immune-mediated diseases might perturb immune homeostasis, and hence also associate with predisposition to T1D.

    Methods: We resequenced and genotyped tag single nucleotide polymorphisms (SNPs) from two genes, CRP and FCER1B, and genotyped 27 disease-associated polymorphisms from thirteen gene regions, namely FCRL3, CFH, SLC9A3R1, PADI4, RUNX1, SPINK5, IL1RN, IL1RA, CARD15, IBD5-locus (including SLC22A4), LAG3, ADAM33 and NFKB1. These genes have been associated previously with susceptibility to a range of immune-mediated diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Graves' disease (GD), psoriasis, psoriatic arthritis (PA), atopy, asthma, Crohn disease and multiple sclerosis (MS). Our T1D collections are divided into three sample subsets, consisting of set 1 families (up to 754 families), set 2 families (up to 743 families), and a case-control collection (ranging from 1,500 to 4,400 cases and 1,500 to 4,600 controls). Each SNP was genotyped in one or more of these subsets. Our study typically had approximately 80% statistical power for a minor allele frequency (MAF) >5% and odds ratios (OR) of 1.5 with the type 1 error rate, alpha = 0.05.

    Results: We found no evidence of association with T1D at most of the loci studied 0.02 <P < 1.0. Only a SNP in ADAM33, rs2787094, was any evidence of association obtained, P = 0.0004 in set 1 families (relative risk (RR) = 0.78), but further support was not observed in the 4,326 cases and 4,610 controls, P = 0.57 (OR = 1.02).

    Conclusion: Polymorphisms in a variety of genes previously associated with immune-mediated disease susceptibility and/or having effects on gene function and the immune system, are unlikely to be affecting T1D susceptibility in a major way, even though some of the genes tested encode proteins of immune pathways that are believed to be central to the development of T1D. We cannot, however, rule out effect sizes smaller than OR 1.5.

    Funded by: Medical Research Council: G0000934; Wellcome Trust: 068545/Z/02

    BMC medical genetics 2006;7;20

  • The SNAP25 gene as a susceptibility gene contributing to attention-deficit hyperactivity disorder.

    Feng Y, Crosbie J, Wigg K, Pathare T, Ickowicz A, Schachar R, Tannock R, Roberts W, Malone M, Swanson J, Kennedy JL and Barr CL

    Department of Psychiatry, Cellular and Molecular Division, The Toronto Western Research Institute, University Health Network, Toronto, ON, Canada.

    The synaptosomal-associated protein of 25 kDa gene (SNAP25) has been suggested as a genetic susceptibility factor in attention-deficit hyperactivity disorder (ADHD) based on the mouse strain coloboma. This strain is hemizygous for the SNAP25 gene and displays hyperactivity that responds to dextroamphetamine, but not to methylphenidate. Previously, we reported association of SNAP25 and ADHD using two polymorphisms. To further investigate this gene, we screened the exons for DNA variation and genotyped ten additional polymorphisms in an expanded sample of families from Toronto and a second sample of families collected in Irvine, CA. Significant results were observed in the Toronto sample for four markers, although not in the Irvine sample. The paper discusses the possible influence of the selection criteria on these differential results. The Irvine sample selected subjects that met the DSM-IV combined subtype diagnosis, whereas the Toronto sample included all subtypes. Analysis of the DSM-IV subtypes in the Toronto sample indicated that the differential results were not attributable to ADHD subtype. Differences in ethnicity, differential medication response, and other clinical characteristics of the samples cannot be ruled out at this time. Quantitative analysis of the dimensions of hyperactivity/impulsivity and inattention in the Toronto sample found that both behavioral traits were associated with SNAP25. Our findings continue to support SNAP25 in the susceptibility to ADHD.

    Funded by: NICHD NIH HHS: HD99-004; NIMH NIH HHS: U01:MH50440

    Molecular psychiatry 2005;10;11;998-1005, 973

  • Netherton syndrome in two Japanese siblings with a novel mutation in the SPINK5 gene: immunohistochemical studies of LEKTI and other epidermal molecules.

    Shimomura Y, Sato N, Kariya N, Takatsuka S and Ito M

    Department of Dermatology, Niigata University School of Medicine, Asahimachi-dori, Niigata 951-8510, Japan. yshimo@med.niigata-u.ac.jp

    Background: Netherton syndrome (NS) is a severe autosomal recessive disorder characterized by ichthyosiform erythroderma, bamboo hair and atopy. The disease is caused by mutations in the SPINK5 gene, which encodes a putative serine protease inhibitor, LEKTI (lymphoepithelial Kazal-type-related inhibitor). Previous studies have clearly shown a crucial role for LEKTI in skin barrier formation.

    Objectives: To identify pathogenic mutations in two Japanese siblings with NS, and further to investigate the consequences of the mutations at the protein level.

    Methods: To screen for mutations in the SPINK5 gene, all of its exons and splice junctions were amplified by polymerase chain reaction and directly sequenced. In addition, immunohistochemical staining of LEKTI, desmoglein (Dsg) 1 and elafin was performed with their specific antibodies.

    Results: Mutation analysis resulted in the identification of compound heterozygous mutations, Q713X and R790X, in the SPINK5 gene of both patients. The former one is a novel mutation. Immunohistochemical studies in one patient demonstrated a complete absence of LEKTI and a strong expression of elafin in the patient's skin. Dsg1 was normally expressed in our patient.

    Conclusions: In this report, we describe compound heterozygous mutations in the SPINK5 gene in two Japanese siblings with NS. The result of immunohistochemistry shows LEKTI deficiency and upregulation of elafin in the skin of one patient. Furthermore, our data indicate that degradation of Dsg1 does not always occur in NS.

    The British journal of dermatology 2005;153;5;1026-30

  • A Japanese infant with localized ichthyosis linearis circumflexa on the palms and soles harbouring a compound heterozygous mutation in the SPINK5 gene.

    Mizuno Y, Suga Y, Muramatsu S, Hasegawa T, Shimizu T and Ogawa H

    Department of Dermatology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, Japan.

    We report a 6-month-old Japanese boy showing ichthyosis linearis circumflexa localized on the palms and soles. He showed bamboo hairs and aminoaciduria, and was positive for cow's milk and egg IgE antibodies by radioallergosorbent tests. Trypsin-like hydrolytic activity in the patient's lesional stratum corneum showed an activity seven times higher than that in age-matched controls. DNA analysis showed that the patient harboured the compound heterozygous mutations R790X and 1220+1 G-->C in the SPINK5 gene, compatible with the diagnosis of Netherton syndrome (NS). As the genotype/phenotype correlations in NS have not yet been fully clarified, the position of the premature termination codon in the SPINK5 gene may contribute to explain such a mild form of NS in our patient.

    The British journal of dermatology 2005;153;3;661-3

  • Lack of association of SPINK5 polymorphisms with nonsyndromic atopic dermatitis in the population of Northern Germany.

    Fölster-Holst R, Stoll M, Koch WA, Hampe J, Christophers E and Schreiber S

    The British journal of dermatology 2005;152;6;1365-7

  • The SNAP-25 gene may be associated with clinical response and weight gain in antipsychotic treatment of schizophrenia.

    Müller DJ, Klempan TA, De Luca V, Sicard T, Volavka J, Czobor P, Sheitman BB, Lindenmayer JP, Citrome L, McEvoy JP, Lieberman JA, Honer WG and Kennedy JL

    Neurogenetics Section, Centre for Addiction and Mental Health, Department of Psychiatry, University of Toronto, 250 College Street R30, Toronto, Ont. M5T 1R8, Canada. daniel.mueller@charite.de

    The synaptosomal-associated protein of 25 kDa (SNAP-25) is an essential component of the core complex that mediates presynaptic vesicle trafficking. Thus, SNAP-25 is directly involved in the release of neurotransmitters. Quantitative alterations of SNAP-25 expression have been reported in brain regions and cerebrospinal fluid (CSF) of schizophrenics and in haloperidol treated rats. This observed altered expression may be influenced by genetic variants of SNAP-25. We hypothesized that polymorphisms of the SNAP-25 gene (sites DdeI, MnlI and TaiI in the 3'UTR) are associated with antipsychotic drug response and induced weight gain. A sample of 59 patients with prior suboptimal response to antipsychotic treatment and diagnosed with DSM-IV schizophrenia or schizoaffective disorder was examined. Patients were administered clozapine, haloperidol, olanzapine or risperidone for up to 14 weeks. Clinical response was defined as the difference between the baseline and the endpoint total scores on the Positive and Negative Syndrome Scale (PANSS). Weight was assessed at baseline and at study endpoint. ANOVA revealed that the MnlI and TaiI polymorphisms were associated with response (F[2,53] = 4.57, p = 0.01 and F[2,52] = 3.53, p = 0.03) and with weight gain (F[2,52] = 4.28, p = 0.01 and F[2,51] = 3.38, p = 0.04). When covariates were included, the MnlI polymorphism remained significantly associated with changes of PANSS scores, but not with weight gain. The DdeI polymorphism was not associated with response or weight gain. These findings suggest that SNAP-25 gene variants affect clinical response in patients with prior poor response to antipsychotics. Weight changes do not seem to be associated with polymorphism of the SNAP-25 gene, however, replication in independent samples is warranted.

    Funded by: NIMH NIH HHS: MH MH33127, R10 MH53550

    Neuroscience letters 2005;379;2;81-9

  • Polymorphisms in SPINK5 are not associated with asthma in a Dutch population.

    Jongepier H, Koppelman GH, Nolte IM, Bruinenberg M, Bleecker ER, Meyers DA, te Meerman GJ and Postma DS

    Department of Pulmonary Rehabilitation, Beatrixoord, The Netherlands.

    Background: Asthma and allergic phenotypes are complex genetic diseases with known linkage to chromosome 5q. This region has many candidate genes, including serine protease inhibitor Kazal type 5 (SPINK5), which has been associated with asthma and atopic dermatitis in family-based studies of children with atopic dermatitis.

    Objective: We sought to investigate whether single nucleotide polymorphisms in SPINK5 are associated with asthma, atopic phenotypes, and atopic dermatitis.

    Methods: We investigated whether single nucleotide polymorphisms in SPINK5 (ie, -785 A/G, Asn368Ser, and Lys420Glu) are associated with asthma, atopic phenotypes, and atopic dermatitis in 200 families ascertained by a proband with asthma (nonaffected spouses served as a matched control population) and an independent set of 252 trios with asthma.

    Results: We found no association with asthma, atopic phenotypes, and atopic dermatitis after correction for multiple testing.

    Conclusion: The negative results in this study suggest that SPINK5 is not associated with asthma or atopic phenotypes in individuals ascertained by a proband with asthma. This is consistent with the finding that SPINK5 is not expressed in the lung. Because our patients were ascertained for asthma, a role of SPINK5 in atopic dermatitis cannot be excluded.

    The Journal of allergy and clinical immunology 2005;115;3;486-92

  • SPINK5 polymorphism is associated with disease severity and food allergy in children with atopic dermatitis.

    Kusunoki T, Okafuji I, Yoshioka T, Saito M, Nishikomori R, Heike T, Sugai M, Shimizu A and Nakahata T

    The Journal of allergy and clinical immunology 2005;115;3;636-8

  • Netherton syndrome: report of two Taiwanese siblings with staphylococcal scalded skin syndrome and mutation of SPINK5.

    Chao SC, Richard G and Lee JY

    Department of Dermatology, School of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, Taiwan.

    Netherton syndrome (NS) is a severe autosomal recessive ichthyosis. It is characterized by congenital ichthyosiform erythroderma, trichorrhexis invaginata, ichthyosis linearis circumflexa, atopic diathesis and frequent bacterial infections. Pathogenic mutations in SPINK5 have recently been identified in NS. SPINK5 encodes lymphoepithelial Kazal-type-related inhibitor (LEKTI), a new type of serine protease inhibitor involved in the regulation of skin barrier formation and immunity. We report two Taiwanese brothers with NS. The patients had typical manifestations of NS with an atopic diathesis and recurrent staphylococcal infections, including staphylococcal scalded skin syndrome (SSSS) since birth. Horny layers were obtained by skin surface biopsy for electron microscopy from lesional skin of both patients and from normal controls. All 33 exons and flanking intron boundaries of SPINK5 were amplified for direct sequencing. The ultrastructure of the stratum corneum (SC) was characterized by premature degradation of corneodesmosomes (CDs) with separation of corneocytes. A homozygous 2260A --> T (K754X) mutation of SPINK5 was found in both patients. Staphylococcal exfoliative toxin A (ETA) is a serine protease capable of cleaving desmoglein 1, an important adhesive molecule of CDs, and can cause separation of the SC, resulting in SSSS. The premature degradation of CDs found in our patients may be attributable to insufficient LEKTI, and possibly also to colonization/infection of ETA-producing Staphylococcus aureus. Mechanisms involved in the pathogenesis of the skin barrier defect in NS are proposed. Further study is needed to prove this hypothesis.

    The British journal of dermatology 2005;152;1;159-65

  • Netherton syndrome with extensive skin peeling and failure to thrive due to a homozygous frameshift mutation in SPINK5.

    Geyer AS, Ratajczak P, Pol-Rodriguez M, Millar WS, Garzon M and Richard G

    Department of Dermatology, Columbia University Medical Center, Columbia University, New York, NY, USA.

    Background: Netherton syndrome (NTS) is a rare autosomal recessive multisystem disorder characterized by congenital erythroderma and ichthyosis, hair shaft abnormalities and immune dysregulation. The disorder is caused by deleterious mutations in the SPINK5 gene, encoding the serine protease inhibitor LEKTI.

    Objective: Our objective was to investigate if the erythrodermic variant of peeling skin syndrome is also caused by SPINK5 mutations and to study the consequences of the disease on infantile brain development.

    Methods: In an infant with extensive erythroderma, peeling skin and failure to thrive, we analyzed the SPINK5 gene for pathogenic mutations by direct DNA sequencing and performed repeated brain MRI studies with diffusion-weighted imaging.

    Results: We identified a homozygous 4-base-pair insertion in exon 5 of SPINK5, which introduces a premature termination codon and appears to be a common mutation among West Indies islanders. MRI analyses revealed a persistent diffuse volume loss.

    Conclusion: Our results confirm that early truncation mutations of the coding sequence of SPINK5 produce a severe phenotype and that generalized peeling skin is one of the manifestations of NTS. We further demonstrate for the first time that NTS may be associated with MRI abnormalities indicative of a permanent tissue injury of the brain.

    Funded by: NIAMS NIH HHS: AR02141, AR38923, AR47157

    Dermatology (Basel, Switzerland) 2005;210;4;308-14

  • Haplotype analysis of SNAP-25 suggests a role in the aetiology of ADHD.

    Mill J, Richards S, Knight J, Curran S, Taylor E and Asherson P

    Social, Genetic, and Developmental Psychiatry Research Centre, Institute of Psychiatry, Kings College, London, UK. spjgjsm@iop.kcl.ac.uk

    Several lines of evidence suggest a role for SNAP-25 (synaptosomal-associated protein of 25 kDa) in the genetic aetiology of ADHD. Most notable is the coloboma mouse mutant, which displays spontaneous hyperactivity and is hemizygous for a deletion spanning this gene. We have screened the SNAP-25 gene using denaturing high-performance liquid chromatography and sequencing, and genotyped six polymorphic single-nucleotide polymorphisms and two microsatellites in a clinically ascertained sample of 188 probands. Several markers were found to show association with ADHD, both individually and in combination with other markers to form multimarker haplotypes. Analyses of transmission by parental sex suggested that the association of SNAP-25 with ADHD is largely due to transmission of alleles from paternal chromosomes to affected probands, suggesting that this locus may be subject to genomic imprinting. Overall our data provide some evidence for a role of this gene in ADHD, although the precise causal functional variant is yet to be ascertained.

    Molecular psychiatry 2004;9;8;801-10

  • Polymorphism of the 5'-upstream region of the human SNAP-25 gene: an association analysis with schizophrenia.

    Tachikawa H, Harada S, Kawanishi Y, Okubo T and Suzuki T

    Department of Psychiatry, Kurita Hospital, Ibaraki, Japan. mg8@ea.mbn.or.jp

    Recent studies have suggested that synaptic abnormalities may be part of the pathophysiology of schizophrenia. SNAP-25 (synaptosomal-associated protein of 25 kD) is one of the synaptic proteins responsible for presynaptic neurotransmission, axonal elongation and synaptogenesis. Genetic variation in the 5'-upstream region of the SNAP-25 gene was analyzed in 87 unrelated schizophrenic patients and 100 healthy controls. A novel polymorphic (TAAA)(n) tandem repeat was identified in the 5'-upstream region. There were no significant differences between the patient and the control groups in the distribution of repeat numbers of alleles or genotypes. In addition, no associations were found between the polymorphism for subtypes, longitudinal courses or positive family history of the patients. Our results suggest that polymorphisms in the 5'-upstream region of the SNAP-25 gene have no association with schizophrenia.

    Neuropsychobiology 2001;43;3;131-3

  • Construction of an integrated physical and gene map of human chromosome 20p12 providing candidate genes for Alagille syndrome.

    Pollet N, Boccaccio C, Dhorne-Pollet S, Driancourt C, Raynaud N, Auffray C, Hadchouel M and Meunier-Rotival M

    INSERM U347 affiliée au CNRS, Le Kremlin-Bicêtre, France.

    Physical mapping and localization of eSTS markers were used to generate an integrated physical and gene map covering a approximately 10-Mb region of human chromosome 20p12 containing the Alagille syndrome (AGS) locus. Seventy-four STSs, 28 of which were derived from cDNA sequences, mapped with an average resolution of 135 kb. The 28 eSTS markers define 20 genes. Six known genes, namely CHGB, BMP2, PLCB1, PLCB4, SNAP, and HJ1, were precisely mapped. Among the genes identified, one maps in the smallest region of overlap of the deletions associated with AGS and could therefore be regarded as a candidate gene for Alagille syndrome.

    Genomics 1997;42;3;489-98

Literature (132)

Pubmed - human_disease

  • Netherton syndrome: report of identical twins presenting with severe atopic dermatitis.

    Kilic G, Guler N, Ones U, Tamay Z and Guzel P

    Department of Pediatrics, Division of Pediatric Allergy and Chest Diseases, Istanbul University, Istanbul Medical School, Istanbul, Turkey. kilicgurkan@yahoo.com

    We report the cases of 4-year-old identical twin sisters who presented with severe atopic dermatitis with intractable skin manifestations and multiple food allergies. Netherton syndrome (NS) (OMIM 256500) was suspected due to very high serum IgE levels, growth retardation, severe food allergies and typical hair finding (trichorrhexis invaginata). A definite diagnosis was made by genetic analysis. Our cases are unique in being the first identical twins with NS diagnosed by a novel mutation in the SPINK5 gene. NS should be considered in differential diagnosis in children who have generalized erythema with intractable eczematous lesions and elevated levels of IgE.

    European journal of pediatrics 2006;165;9;594-7

  • Analysis of polymorphisms in 16 genes in type 1 diabetes that have been associated with other immune-mediated diseases.

    Smyth DJ, Howson JM, Payne F, Maier LM, Bailey R, Holland K, Lowe CE, Cooper JD, Hulme JS, Vella A, Dahlman I, Lam AC, Nutland S, Walker NM, Twells RC and Todd JA

    Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Rd, Cambridge, CB2 2XY, UK. debbie.smyth@cimr.cam.ac.uk

    Background: The identification of the HLA class II, insulin (INS), CTLA-4 and PTPN22 genes as determinants of type 1 diabetes (T1D) susceptibility indicates that fine tuning of the immune system is centrally involved in disease development. Some genes have been shown to affect several immune-mediated diseases. Therefore, we tested the hypothesis that alleles of susceptibility genes previously associated with other immune-mediated diseases might perturb immune homeostasis, and hence also associate with predisposition to T1D.

    Methods: We resequenced and genotyped tag single nucleotide polymorphisms (SNPs) from two genes, CRP and FCER1B, and genotyped 27 disease-associated polymorphisms from thirteen gene regions, namely FCRL3, CFH, SLC9A3R1, PADI4, RUNX1, SPINK5, IL1RN, IL1RA, CARD15, IBD5-locus (including SLC22A4), LAG3, ADAM33 and NFKB1. These genes have been associated previously with susceptibility to a range of immune-mediated diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Graves' disease (GD), psoriasis, psoriatic arthritis (PA), atopy, asthma, Crohn disease and multiple sclerosis (MS). Our T1D collections are divided into three sample subsets, consisting of set 1 families (up to 754 families), set 2 families (up to 743 families), and a case-control collection (ranging from 1,500 to 4,400 cases and 1,500 to 4,600 controls). Each SNP was genotyped in one or more of these subsets. Our study typically had approximately 80% statistical power for a minor allele frequency (MAF) >5% and odds ratios (OR) of 1.5 with the type 1 error rate, alpha = 0.05.

    Results: We found no evidence of association with T1D at most of the loci studied 0.02 <P < 1.0. Only a SNP in ADAM33, rs2787094, was any evidence of association obtained, P = 0.0004 in set 1 families (relative risk (RR) = 0.78), but further support was not observed in the 4,326 cases and 4,610 controls, P = 0.57 (OR = 1.02).

    Conclusion: Polymorphisms in a variety of genes previously associated with immune-mediated disease susceptibility and/or having effects on gene function and the immune system, are unlikely to be affecting T1D susceptibility in a major way, even though some of the genes tested encode proteins of immune pathways that are believed to be central to the development of T1D. We cannot, however, rule out effect sizes smaller than OR 1.5.

    Funded by: Medical Research Council: G0000934; Wellcome Trust: 068545/Z/02

    BMC medical genetics 2006;7;20

  • Netherton syndrome in two Japanese siblings with a novel mutation in the SPINK5 gene: immunohistochemical studies of LEKTI and other epidermal molecules.

    Shimomura Y, Sato N, Kariya N, Takatsuka S and Ito M

    Department of Dermatology, Niigata University School of Medicine, Asahimachi-dori, Niigata 951-8510, Japan. yshimo@med.niigata-u.ac.jp

    Background: Netherton syndrome (NS) is a severe autosomal recessive disorder characterized by ichthyosiform erythroderma, bamboo hair and atopy. The disease is caused by mutations in the SPINK5 gene, which encodes a putative serine protease inhibitor, LEKTI (lymphoepithelial Kazal-type-related inhibitor). Previous studies have clearly shown a crucial role for LEKTI in skin barrier formation.

    Objectives: To identify pathogenic mutations in two Japanese siblings with NS, and further to investigate the consequences of the mutations at the protein level.

    Methods: To screen for mutations in the SPINK5 gene, all of its exons and splice junctions were amplified by polymerase chain reaction and directly sequenced. In addition, immunohistochemical staining of LEKTI, desmoglein (Dsg) 1 and elafin was performed with their specific antibodies.

    Results: Mutation analysis resulted in the identification of compound heterozygous mutations, Q713X and R790X, in the SPINK5 gene of both patients. The former one is a novel mutation. Immunohistochemical studies in one patient demonstrated a complete absence of LEKTI and a strong expression of elafin in the patient's skin. Dsg1 was normally expressed in our patient.

    Conclusions: In this report, we describe compound heterozygous mutations in the SPINK5 gene in two Japanese siblings with NS. The result of immunohistochemistry shows LEKTI deficiency and upregulation of elafin in the skin of one patient. Furthermore, our data indicate that degradation of Dsg1 does not always occur in NS.

    The British journal of dermatology 2005;153;5;1026-30

  • A Japanese infant with localized ichthyosis linearis circumflexa on the palms and soles harbouring a compound heterozygous mutation in the SPINK5 gene.

    Mizuno Y, Suga Y, Muramatsu S, Hasegawa T, Shimizu T and Ogawa H

    Department of Dermatology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, Japan.

    We report a 6-month-old Japanese boy showing ichthyosis linearis circumflexa localized on the palms and soles. He showed bamboo hairs and aminoaciduria, and was positive for cow's milk and egg IgE antibodies by radioallergosorbent tests. Trypsin-like hydrolytic activity in the patient's lesional stratum corneum showed an activity seven times higher than that in age-matched controls. DNA analysis showed that the patient harboured the compound heterozygous mutations R790X and 1220+1 G-->C in the SPINK5 gene, compatible with the diagnosis of Netherton syndrome (NS). As the genotype/phenotype correlations in NS have not yet been fully clarified, the position of the premature termination codon in the SPINK5 gene may contribute to explain such a mild form of NS in our patient.

    The British journal of dermatology 2005;153;3;661-3

  • Lack of association of SPINK5 polymorphisms with nonsyndromic atopic dermatitis in the population of Northern Germany.

    Fölster-Holst R, Stoll M, Koch WA, Hampe J, Christophers E and Schreiber S

    The British journal of dermatology 2005;152;6;1365-7

  • The SNAP-25 gene may be associated with clinical response and weight gain in antipsychotic treatment of schizophrenia.

    Müller DJ, Klempan TA, De Luca V, Sicard T, Volavka J, Czobor P, Sheitman BB, Lindenmayer JP, Citrome L, McEvoy JP, Lieberman JA, Honer WG and Kennedy JL

    Neurogenetics Section, Centre for Addiction and Mental Health, Department of Psychiatry, University of Toronto, 250 College Street R30, Toronto, Ont. M5T 1R8, Canada. daniel.mueller@charite.de

    The synaptosomal-associated protein of 25 kDa (SNAP-25) is an essential component of the core complex that mediates presynaptic vesicle trafficking. Thus, SNAP-25 is directly involved in the release of neurotransmitters. Quantitative alterations of SNAP-25 expression have been reported in brain regions and cerebrospinal fluid (CSF) of schizophrenics and in haloperidol treated rats. This observed altered expression may be influenced by genetic variants of SNAP-25. We hypothesized that polymorphisms of the SNAP-25 gene (sites DdeI, MnlI and TaiI in the 3'UTR) are associated with antipsychotic drug response and induced weight gain. A sample of 59 patients with prior suboptimal response to antipsychotic treatment and diagnosed with DSM-IV schizophrenia or schizoaffective disorder was examined. Patients were administered clozapine, haloperidol, olanzapine or risperidone for up to 14 weeks. Clinical response was defined as the difference between the baseline and the endpoint total scores on the Positive and Negative Syndrome Scale (PANSS). Weight was assessed at baseline and at study endpoint. ANOVA revealed that the MnlI and TaiI polymorphisms were associated with response (F[2,53] = 4.57, p = 0.01 and F[2,52] = 3.53, p = 0.03) and with weight gain (F[2,52] = 4.28, p = 0.01 and F[2,51] = 3.38, p = 0.04). When covariates were included, the MnlI polymorphism remained significantly associated with changes of PANSS scores, but not with weight gain. The DdeI polymorphism was not associated with response or weight gain. These findings suggest that SNAP-25 gene variants affect clinical response in patients with prior poor response to antipsychotics. Weight changes do not seem to be associated with polymorphism of the SNAP-25 gene, however, replication in independent samples is warranted.

    Funded by: NIMH NIH HHS: MH MH33127, R10 MH53550

    Neuroscience letters 2005;379;2;81-9

  • Polymorphisms in SPINK5 are not associated with asthma in a Dutch population.

    Jongepier H, Koppelman GH, Nolte IM, Bruinenberg M, Bleecker ER, Meyers DA, te Meerman GJ and Postma DS

    Department of Pulmonary Rehabilitation, Beatrixoord, The Netherlands.

    Background: Asthma and allergic phenotypes are complex genetic diseases with known linkage to chromosome 5q. This region has many candidate genes, including serine protease inhibitor Kazal type 5 (SPINK5), which has been associated with asthma and atopic dermatitis in family-based studies of children with atopic dermatitis.

    Objective: We sought to investigate whether single nucleotide polymorphisms in SPINK5 are associated with asthma, atopic phenotypes, and atopic dermatitis.

    Methods: We investigated whether single nucleotide polymorphisms in SPINK5 (ie, -785 A/G, Asn368Ser, and Lys420Glu) are associated with asthma, atopic phenotypes, and atopic dermatitis in 200 families ascertained by a proband with asthma (nonaffected spouses served as a matched control population) and an independent set of 252 trios with asthma.

    Results: We found no association with asthma, atopic phenotypes, and atopic dermatitis after correction for multiple testing.

    Conclusion: The negative results in this study suggest that SPINK5 is not associated with asthma or atopic phenotypes in individuals ascertained by a proband with asthma. This is consistent with the finding that SPINK5 is not expressed in the lung. Because our patients were ascertained for asthma, a role of SPINK5 in atopic dermatitis cannot be excluded.

    The Journal of allergy and clinical immunology 2005;115;3;486-92

  • SPINK5 polymorphism is associated with disease severity and food allergy in children with atopic dermatitis.

    Kusunoki T, Okafuji I, Yoshioka T, Saito M, Nishikomori R, Heike T, Sugai M, Shimizu A and Nakahata T

    The Journal of allergy and clinical immunology 2005;115;3;636-8

  • Netherton syndrome: report of two Taiwanese siblings with staphylococcal scalded skin syndrome and mutation of SPINK5.

    Chao SC, Richard G and Lee JY

    Department of Dermatology, School of Medicine, National Cheng Kung University, 138 Sheng-Li Road, Tainan, Taiwan.

    Netherton syndrome (NS) is a severe autosomal recessive ichthyosis. It is characterized by congenital ichthyosiform erythroderma, trichorrhexis invaginata, ichthyosis linearis circumflexa, atopic diathesis and frequent bacterial infections. Pathogenic mutations in SPINK5 have recently been identified in NS. SPINK5 encodes lymphoepithelial Kazal-type-related inhibitor (LEKTI), a new type of serine protease inhibitor involved in the regulation of skin barrier formation and immunity. We report two Taiwanese brothers with NS. The patients had typical manifestations of NS with an atopic diathesis and recurrent staphylococcal infections, including staphylococcal scalded skin syndrome (SSSS) since birth. Horny layers were obtained by skin surface biopsy for electron microscopy from lesional skin of both patients and from normal controls. All 33 exons and flanking intron boundaries of SPINK5 were amplified for direct sequencing. The ultrastructure of the stratum corneum (SC) was characterized by premature degradation of corneodesmosomes (CDs) with separation of corneocytes. A homozygous 2260A --> T (K754X) mutation of SPINK5 was found in both patients. Staphylococcal exfoliative toxin A (ETA) is a serine protease capable of cleaving desmoglein 1, an important adhesive molecule of CDs, and can cause separation of the SC, resulting in SSSS. The premature degradation of CDs found in our patients may be attributable to insufficient LEKTI, and possibly also to colonization/infection of ETA-producing Staphylococcus aureus. Mechanisms involved in the pathogenesis of the skin barrier defect in NS are proposed. Further study is needed to prove this hypothesis.

    The British journal of dermatology 2005;152;1;159-65

  • Polymorphism of the 5'-upstream region of the human SNAP-25 gene: an association analysis with schizophrenia.

    Tachikawa H, Harada S, Kawanishi Y, Okubo T and Suzuki T

    Department of Psychiatry, Kurita Hospital, Ibaraki, Japan. mg8@ea.mbn.or.jp

    Recent studies have suggested that synaptic abnormalities may be part of the pathophysiology of schizophrenia. SNAP-25 (synaptosomal-associated protein of 25 kD) is one of the synaptic proteins responsible for presynaptic neurotransmission, axonal elongation and synaptogenesis. Genetic variation in the 5'-upstream region of the SNAP-25 gene was analyzed in 87 unrelated schizophrenic patients and 100 healthy controls. A novel polymorphic (TAAA)(n) tandem repeat was identified in the 5'-upstream region. There were no significant differences between the patient and the control groups in the distribution of repeat numbers of alleles or genotypes. In addition, no associations were found between the polymorphism for subtypes, longitudinal courses or positive family history of the patients. Our results suggest that polymorphisms in the 5'-upstream region of the SNAP-25 gene have no association with schizophrenia.

    Neuropsychobiology 2001;43;3;131-3

  • Construction of an integrated physical and gene map of human chromosome 20p12 providing candidate genes for Alagille syndrome.

    Pollet N, Boccaccio C, Dhorne-Pollet S, Driancourt C, Raynaud N, Auffray C, Hadchouel M and Meunier-Rotival M

    INSERM U347 affiliée au CNRS, Le Kremlin-Bicêtre, France.

    Physical mapping and localization of eSTS markers were used to generate an integrated physical and gene map covering a approximately 10-Mb region of human chromosome 20p12 containing the Alagille syndrome (AGS) locus. Seventy-four STSs, 28 of which were derived from cDNA sequences, mapped with an average resolution of 135 kb. The 28 eSTS markers define 20 genes. Six known genes, namely CHGB, BMP2, PLCB1, PLCB4, SNAP, and HJ1, were precisely mapped. Among the genes identified, one maps in the smallest region of overlap of the deletions associated with AGS and could therefore be regarded as a candidate gene for Alagille syndrome.

    Genomics 1997;42;3;489-98

Pubmed - other

  • A SNAP25 promoter variant is associated with early-onset bipolar disorder and a high expression level in brain.

    Etain B, Dumaine A, Mathieu F, Chevalier F, Henry C, Kahn JP, Deshommes J, Bellivier F, Leboyer M and Jamain S

    INSERM, U 955, IMRB, Department of Genetics, Psychiatry Genetics, Creteil, F-94010, France.

    Bipolar disorder (BD) is one of the most common and persistent psychiatric disorders. Early-onset BD has been shown to be the most severe and familial form. We recently carried out a whole-genome linkage analysis on sibpairs affected by early-onset BD and showed that the 20p12 region was more frequently shared in our families than expected by chance. The synaptosomal-associated protein SNAP25 is a presynaptic plasma membrane protein essential for the triggering of vesicular fusion and neurotransmitter release, and for which abnormal protein levels have been reported in postmortem studies of bipolar patients. We hypothesised that variations in the gene encoding SNAP25, located on chromosome 20p12, might influence the susceptibility to early-onset BD. We screened SNAP25 for mutations and performed a case-control association study in 197 patients with early-onset BD, 202 patients with late-onset BD and 136 unaffected subjects. In addition, we analysed the expression level of the two SNAP25 isoforms in 60 brains. We showed that one variant, located in the promoter region, was associated with early-onset BD but not with the late-onset subgroup. In addition, individuals homozygous for this variant showed a significant higher SNAP25b expression level in prefrontal cortex. These results show that variations in SNAP25, associated with an increased gene expression level in prefrontal cortex, might predispose to early-onset BD. Further analyses of this gene, as well as analysis of genes encoding for the SNAP25 protein partners, are required to understand the impact of such molecular mechanisms in BD.

    Molecular psychiatry 2010;15;7;748-55

  • Genome-wide association scan for five major dimensions of personality.

    Terracciano A, Sanna S, Uda M, Deiana B, Usala G, Busonero F, Maschio A, Scally M, Patriciu N, Chen WM, Distel MA, Slagboom EP, Boomsma DI, Villafuerte S, Sliwerska E, Burmeister M, Amin N, Janssens AC, van Duijn CM, Schlessinger D, Abecasis GR and Costa PT

    National Institute on Aging, NIH, Baltimore, MD 21224, USA. terraccianoa@mail.nih.gov

    Personality traits are summarized by five broad dimensions with pervasive influences on major life outcomes, strong links to psychiatric disorders and clear heritable components. To identify genetic variants associated with each of the five dimensions of personality we performed a genome-wide association (GWA) scan of 3972 individuals from a genetically isolated population within Sardinia, Italy. On the basis of the analyses of 362 129 single-nucleotide polymorphisms we found several strong signals within or near genes previously implicated in psychiatric disorders. They include the association of neuroticism with SNAP25 (rs362584, P=5 x 10(-5)), extraversion with BDNF and two cadherin genes (CDH13 and CDH23; Ps<5 x 10(-5)), openness with CNTNAP2 (rs10251794, P=3 x 10(-5)), agreeableness with CLOCK (rs6832769, P=9 x 10(-6)) and conscientiousness with DYRK1A (rs2835731, P=3 x 10(-5)). Effect sizes were small (less than 1% of variance), and most failed to replicate in the follow-up independent samples (N up to 3903), though the association between agreeableness and CLOCK was supported in two of three replication samples (overall P=2 x 10(-5)). We infer that a large number of loci may influence personality traits and disorders, requiring larger sample sizes for the GWA approach to confidently identify associated genetic variants.

    Funded by: Intramural NIH HHS: Z99 AG999999, ZIA AG000180-25, ZIA AG000180-26, ZIA AG000196-03, ZIA AG000196-04; NIMH NIH HHS: R21 MH070793; PHS HHS: NIMH R21 MH070793

    Molecular psychiatry 2010;15;6;647-56

  • Kinesin-1 plays a role in transport of SNAP-25 to the plasma membrane.

    Morton AM, Cunningham AL and Diefenbach RJ

    Centre for Virus Research, Westmead Millennium Institute, The University of Sydney and Westmead Hospital, Westmead, NSW 2145, Australia.

    The cellular molecular motor kinesin-1 mediates the microtubule-dependent transport of a range of cargo. We have previously identified an interaction between the cargo-binding domain of kinesin-1 heavy chain KIF5B and the membrane-associated SNARE proteins SNAP-25 and SNAP-23. In this study we further defined the minimal SNAP-25 binding domain in KIF5B to residues 874-894. Overexpression of a fragment of KIF5B (residues 594-910) resulted in significant colocalization with SNAP-25 with resulting blockage of the trafficking of SNAP-25 to the periphery of cells. This indicates that kinesin-1 facilitates the transport of SNAP-25 containing vesicles as a prerequisite to SNAP-25 driven membrane fusion events.

    Biochemical and biophysical research communications 2010;391;1;388-93

  • A candidate gene analysis of methylphenidate response in attention-deficit/hyperactivity disorder.

    McGough JJ, McCracken JT, Loo SK, Manganiello M, Leung MC, Tietjens JR, Trinh T, Baweja S, Suddath R, Smalley SL, Hellemann G and Sugar CA

    Semel Institute for Neuroscience & Human Behavior and David Geffen School of Medicine, University of California, Los Angeles, USA. jmcgough@mednet.ucla.edu

    Objective: This study examines the potential role of candidate genes in moderating treatment effects of methylphenidate (MPH) in attention-deficit/hyperactivity disorder (ADHD).

    Method: Eighty-two subjects with ADHD aged 6 to 17 years participated in a prospective, double-blind, placebo-controlled, multiple-dose, crossover titration trial of immediate release MPH three times daily. The subjects were assessed on a variety of parent and clinician ratings and a laboratory math test. Data reduction based on principal components analysis identified statistically derived efficacy and side effect outcomes.

    Results: Attention-deficit/hyperactivity disorder symptom response was predicted by polymorphisms at the serotonin transporter (SLC6A4) intron 2 VNTR (p = .01), with a suggested trend for catechol-O-methyltransferase (COMT) (p = .04). Gene × dose interactions were noted on math test outcomes for the dopamine D4 receptor (DRD4) promoter (p = .008), DRD4 exon 3 VNTR (p = .006), and SLC6A4 promoter insertion/deletion polymorphism (5HTTLPR) (p = .02). Irritability was predicted by COMT (p = .02). Vegetative symptoms were predicted by 5HTTLPR (p = .003). No significant effects were noted for the dopamine transporter (SLC6A3) or synaptosomal-associated protein 25 (SNAP25).

    Conclusions: This article confirms and expands previous studies suggesting that genes moderate ADHD treatment response. The ADHD outcomes are not unitary but reflect both behavioral and learning domains that are likely influenced by different genes. Future research should emphasize candidate gene and genome-wide association studies in larger samples, symptom reduction as well as side effects outcomes, and responses over full therapeutic dose ranges to assess differences in both gene and gene × dose interactive effects.

    Funded by: NIMH NIH HHS: K23 MH001966, K23 MH001966-01, K23MH01966, K24 MH001805, K24 MH001805-01A2, K24MH01805

    Journal of the American Academy of Child and Adolescent Psychiatry 2009;48;12;1155-64

  • Neuropsycological gender differences in healthy individuals and in pediatric neurodevelopmental disorders. A role for SNAP-25.

    Ghezzo A, Guerini FR, Bolognesi E, Matteoli M, Manca S, Sotgiu S, Bejor M, Clerici M and Chiappedi M

    Don Carlo Gnocchi ONLUS Foundation, Centro Bignamini, Falconara, AN, Italy.

    Synaptosomal-associated protein of 25 KD (SNAP-25) is a protein that participates in synaptic vesicle exocytosis through the formation of a SNARE complex; SNAP-25 also plays a pivotal role in modulating calcium homeostasis through negative regulation of voltage-gated calcium channels. SNAP-25 has been involved in different neuropsychiatric disorders, including attention deficit hyperactivity disorder. There are well known physiological gender differences in many neuropsychological skills, and there are even more striking gender differences in patients with attention deficit hyperactivity disorder and autism spectrum disorders. We hypothesize that these differences are the result of a mechanism involving SNAP-25 polymorphisms and its differential expression in specific brain areas.

    Medical hypotheses 2009;73;6;978-80

  • Evidence that putative ADHD low risk alleles at SNAP25 may increase the risk of schizophrenia.

    Carroll LS, Kendall K, O'Donovan MC, Owen MJ and Williams NM

    Department of Psychological Medicine, School of Medicine, Cardiff University, Henry Wellcome Building for Biomedical Research in Wales, Heath Park, Cardiff, UK.

    Synaptosomal Associated Protein 25 kDa (SNAP25) has been implicated in the pathogenesis of schizophrenia by numerous neuropathological studies and genetic variation at SNAP25 has been reported to be associated with ADHD. Expression levels of the putative schizophrenia susceptibility gene DTNBP1 has been shown to influence the levels of SNAP25 in vitro. We undertook directed mutation screening of SNAP25 in UK schizophrenic cases followed by direct association analysis of all variants identified and identified known exonic SNPs that showed evidence for association (rs3746544 P = 0.004 OR = 1.26, rs8636 P = 0.003 OR = 1.27), although these SNPs are highly correlated (r(2) > 0.99). We additionally genotyped a further 31 tag SNPs spanning the SNAP25 locus and identified several independent SNPs that were nominally associated with schizophrenia (strongest association at rs3787283, P = 0.006, OR = 1.25) however, due to the number of tests performed no SNP met experiment-wise significance (minimum permuted P-value = 0.1). Post hoc analysis revealed that the SNPs nominally associated with schizophrenia (rs3787283, rs3746544) were the same as those previously demonstrated to be associated with ADHD but with the opposite alleles, allowing the intriguing hypothesis that genetic variation at SNAP25 may be differentially associated with both schizophrenia and ADHD.

    Funded by: Medical Research Council: G0800509; Wellcome Trust

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2009;150B;7;893-9

  • Identification of sex hormone binding globulin-interacting proteins in the brain using phage display screening.

    Gnanasekar M, Suleman FG, Ramaswamy K and Caldwell JD

    Department of Biomedical Sciences, University of Illinois College of Medicine, Rockford, IL 61107, USA. mgnanas@uic.edu

    The present study reports the identification of human sex hormone binding globulin (SHBG)-interacting proteins in the brain using a phage display-based screening technology. Phage display is a system in which a foreign protein is displayed on the surface of a bacteriophage as a fusion protein with one of the coat proteins of the bacteriophage. T7 phage clones expressing normal human brain proteins (human normal brain phage-display cDNA expression library) were screened using SHBG as bait. The bound phage clones were then identified by DNA sequencing and by BLAST search analysis. Of the twenty binding proteins analyzed, three were found to be membrane-associated proteins: synaptosomal associated protein 25 (SNAP25), Thy-1 cell surface antigen and zonadhesin. Further studies will determine if the interactions of SHBG with these proteins have any role in the internalization and cell signaling events or whether they contribute to steroid delivery to specific cells.

    Funded by: NIMH NIH HHS: R21-MH069810

    International journal of molecular medicine 2009;24;4;421-6

  • Identification of new putative susceptibility genes for several psychiatric disorders by association analysis of regulatory and non-synonymous SNPs of 306 genes involved in neurotransmission and neurodevelopment.

    Gratacòs M, Costas J, de Cid R, Bayés M, González JR, Baca-García E, de Diego Y, Fernández-Aranda F, Fernández-Piqueras J, Guitart M, Martín-Santos R, Martorell L, Menchón JM, Roca M, Sáiz-Ruiz J, Sanjuán J, Torrens M, Urretavizcaya M, Valero J, Vilella E, Estivill X, Carracedo A and Psychiatric Genetics Network Group

    CIBER en Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, Madrid, Spain.

    A fundamental difficulty in human genetics research is the identification of the spectrum of genetic variants that contribute to the susceptibility to common/complex disorders. We tested here the hypothesis that functional genetic variants may confer susceptibility to several related common disorders. We analyzed five main psychiatric diagnostic categories (substance-abuse, anxiety, eating, psychotic, and mood disorders) and two different control groups, representing a total of 3,214 samples, for 748 promoter and non-synonymous single nucleotide polymorphisms (SNPs) at 306 genes involved in neurotransmission and/or neurodevelopment. We identified strong associations to individual disorders, such as growth hormone releasing hormone (GHRH) with anxiety disorders, prolactin regulatory element (PREB) with eating disorders, ionotropic kainate glutamate receptor 5 (GRIK5) with bipolar disorder and several SNPs associated to several disorders, that may represent individual and related disease susceptibility factors. Remarkably, a functional SNP, rs945032, located in the promoter region of the bradykinin receptor B2 gene (BDKRB2) was associated to three disorders (panic disorder, substance abuse, and bipolar disorder), and two additional BDKRB2 SNPs to obsessive-compulsive disorder and major depression, providing evidence for common variants of susceptibility to several related psychiatric disorders. The association of BDKRB2 (odd ratios between 1.65 and 3.06) to several psychiatric disorders supports the view that a common genetic variant could confer susceptibility to clinically related phenotypes, and defines a new functional hint in the pathophysiology of psychiatric diseases.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2009;150B;6;808-16

  • Candidate genes involved in neural plasticity and the risk for attention-deficit hyperactivity disorder: a meta-analysis of 8 common variants.

    Forero DA, Arboleda GH, Vasquez R and Arboleda H

    Neurosciences Research Group, School of Medicine, Universidad Nacional de Colombia, Bogotá, Colombia. daforerog@unal.edu.co

    Background: Attention-deficit hyperactivity disorder (ADHD) is an important psychiatric condition in terms of its prevalence and impact on quality of life. It has one of the highest heritabilities found in psychiatric disorders. A number of association studies exploring several candidate genes in different populations around the world have been carried out. The objective of the present study was to carry out a meta-analysis for 8 common variants located in 5 top candidate genes for ADHD (BDNF, HTR1B, SLC6A2, SLC6A4 and SNAP25); these genes are known to be involved in synaptic transmission and plasticity.

    Methods: We performed a search for published genetic association studies that analyzed the candidate polymorphisms in different populations, and we applied state-of-the-art meta-analytical procedures to obtain pooled odds ratios (ORs) and to evaluate potential basis of heterogeneity. We included 75 genetic association studies in these meta-analyses.

    Results: A major part of the previously postulated associations were nonconsistent in the pooled odds ratios. We observed a weak significant association with a single nucleotide polymorphism (SNP) located in the 3' UTR region of the SNAP25 gene (rs3746544, T allele, OR 1.15, 95% confidence interval 1.01-1.31, p = 0.028, I(2) = 0%). In addition to the low coverage of genetic variability given by these variants, phenotypic heterogeneity between samples (ADHD subtypes, comorbidities) and genetic background may explain these differences.

    Limitations: Limitations of our study include the retrospective nature of our meta-analysis with the incorporation of study-level data from published articles.

    Conclusion: To our knowledge, the present study is the largest meta-analysis carried out for ADHD genetics; previously proposed cumulative associations with common polymorphisms in SLC6A4 and HTR1B genes were not supported. We identified a weak consistent association with a common SNP in the SNAP25 gene, a molecule that is known to be central for synaptic transmission and plasticity mechanisms.

    Journal of psychiatry & neuroscience : JPN 2009;34;5;361-6

  • [Association of 14 polymorphisms in the five candidate genes and attention deficit hyperactivity disorder].

    Gao XP, Su LY, Zhao AL, Luo XR and Xia K

    Institute of Mental Health, Second Xiangya Hospital, Central South University, Changsha, China.

    Objective: Attention deficit hyperactivity disorder (ADHD) is one of the most common behavior disorders in childhood and adolescent. The etiology of ADHD is unknown. The aim of this study was to investigate the relationship between each of the 14 polymorphisms in the five candidate genes and ADHD, and between the combination of some polymorphisms in those genes and ADHD, in attempting to examine whether combinations of genotypes would confer a significant susceptibility to ADHD.

    Methods: One hundred and thirty-nine children with ADHD and one hundred and nineteen normal children were enrolled. Eight single nucleotide polymorphisms (SNP) of three candidate genes were examined with PCR and RFLP techniques. 48 bp VNTR in DRD4 gene was examined with PCR, nondenaturing polyacrylamide gel electrophoresis and silver staining. Five microsatellites (MS) of three candidate genes were examined with genotyping. The relationship between the combinations of 12 polymorphisms and ADHD was examined with logistic regression analysis.

    Results: 1.The frequency of 1065T/1065T genotype and the 1065T allele were significantly higher in ADHD children than that in normal controls (P<0.05). The frequency of -48G/-48G genotype of the A-48G polymorphism of DRD1 gene was significantly lower in ADHD children than that in normal controls (P<0.05). 2. A specific combination of three polymorphisms in the two genes showing an association with ADHD gave a prediction level of 77.5%.

    Conclusions: The T1065G polymorphism in the SNAP-25 may be associated with ADHD. The 1065T/1065T genotype and the 1065T allele may be a risk factor for ADHD. The A-48G polymorphism of DRDI may be associated with ADHD. The -48G/-48G genotype may be a protective factor for ADHD. The specific combination of three sites of SNP in SNAP-25 gene and DRDI gene is found and shows an association with ADHD in 12 polymorphisms of the five candidate genes on glutamatergic/dopaminergic pathway.

    Zhongguo dang dai er ke za zhi = Chinese journal of contemporary pediatrics 2009;11;8;617-22

  • Candidate gene studies of ADHD: a meta-analytic review.

    Gizer IR, Ficks C and Waldman ID

    Department of Genetics, University of North Carolina at Chapel Hill, 120 Mason Farm Road, Room 5015 Genetic Medicine Building CB 7264, Chapel Hill, NC 27599-7264, USA. igizer@unc.edu

    Quantitative genetic studies (i.e., twin and adoption studies) suggest that genetic influences contribute substantially to the development of attention deficit hyperactivity disorder (ADHD). Over the past 15 years, considerable efforts have been made to identify genes involved in the etiology of this disorder resulting in a large and often conflicting literature of candidate gene associations for ADHD. The first aim of the present study was to conduct a comprehensive meta-analytic review of this literature to determine which candidate genes show consistent evidence of association with childhood ADHD across studies. The second aim was to test for heterogeneity across studies in the effect sizes for each candidate gene as its presence might suggest moderating variables that could explain inconsistent results. Significant associations were identified for several candidate genes including DAT1, DRD4, DRD5, 5HTT, HTR1B, and SNAP25. Further, significant heterogeneity was observed for the associations between ADHD and DAT1, DRD4, DRD5, DBH, ADRA2A, 5HTT, TPH2, MAOA, and SNAP25, suggesting that future studies should explore potential moderators of these associations (e.g., ADHD subtype diagnoses, gender, exposure to environmental risk factors). We conclude with a discussion of these findings in relation to emerging themes relevant to future studies of the genetics of ADHD.

    Funded by: NIAAA NIH HHS: T32 AA007573; NIMH NIH HHS: R13MH59126-0641

    Human genetics 2009;126;1;51-90

  • Pharmacogenetics of antipsychotic response in the CATIE trial: a candidate gene analysis.

    Need AC, Keefe RS, Ge D, Grossman I, Dickson S, McEvoy JP and Goldstein DB

    Center for Human Genome Variation, Institute for Genome Sciences & Policy, Duke University, Durham, NC 27708, USA.

    The Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) Phase 1 Schizophrenia trial compared the effectiveness of one typical and four atypical antipsychotic medications. Although trials such as CATIE present important opportunities for pharmacogenetics research, the very richness of the clinical data presents challenges for statistical interpretation, and in particular the risk that data mining will lead to false-positive discoveries. For this reason, it is both misleading and unhelpful to perpetuate the current practice of reporting association results for these trials one gene at a time, ignoring the fact that multiple gene-by-phenotype tests are being carried out on the same data set. On the other hand, suggestive associations in such trials may lead to new hypotheses that can be tested through both replication efforts and biological experimentation. The appropriate handling of these forms of data therefore requires dissemination of association statistics without undue emphasis on select findings. Here we attempt to illustrate this approach by presenting association statistics for 2769 polymorphisms in 118 candidate genes evaluated for 21 pharmacogenetic phenotypes. On current evidence it is impossible to know which of these associations may be real, although in total they form a valuable resource that is immediately available to the scientific community.

    Funded by: NIMH NIH HHS: N01 MH90001

    European journal of human genetics : EJHG 2009;17;7;946-57

  • Candidate gene analysis in an on-going genome-wide association study of attention-deficit hyperactivity disorder: suggestive association signals in ADRA1A.

    Elia J, Capasso M, Zaheer Z, Lantieri F, Ambrosini P, Berrettini W, Devoto M and Hakonarson H

    Department of Pediatrics, Division of Pulmonary Medicine, Center for Applied Genomics, The University of Pennsylvania, Philadelphia, Pennsylvania, USA. elia@email.chop.edu

    Objectives: Attention-deficit hyperactivity disorder (ADHD) is a highly heritable, common developmental disorder. Although a few confirmed associations have emerged from candidate gene studies, these have shown the same limitations that have become evident in the study of other complex diseases, often with inconsistent and nonreplicated results across different studies.

    Methods: In this report, 27 ADHD candidate genes were explored in greater depth using high-density tag single nucleotide polymorphism (SNP) genotyping. Association with 557 SNPs was tested using the transmission disequilibrium test in 270 nuclear pedigrees selected from an ongoing ADHD genetic study that includes all disease subtypes.

    Results: SNPs in seven genes including SLC1A3, SLC6A3, HTR4, ADRA1A, HTR2A, SNAP25, and COMT showed a nominal level of association with ADHD (P values <0.05), but none remained significant after a stringent correction for the total number of tests performed.

    Conclusion: The strongest signal emerged from SNPs in the promoter region (rs3808585) and in an intron (rs17426222, rs4732682, rs573514) of ADRA1A, all located within the same haplotype block. Some of the SNPs in HTR2A and COMT have already been reported by others, whereas other SNPs will need confirmation in independent samples.

    Funded by: NCRR NIH HHS: UL1-RR-024134; NIMH NIH HHS: K23MH066275-01

    Psychiatric genetics 2009;19;3;134-41

  • De novo STXBP1 mutations in mental retardation and nonsyndromic epilepsy.

    Hamdan FF, Piton A, Gauthier J, Lortie A, Dubeau F, Dobrzeniecka S, Spiegelman D, Noreau A, Pellerin S, Côté M, Henrion E, Fombonne E, Mottron L, Marineau C, Drapeau P, Lafrenière RG, Lacaille JC, Rouleau GA and Michaud JL

    Centre of Excellence in Neuromics of Université de Montréal and Synapse to Disease Group, Centre Hospitalier Universitaire Sainte-Justine Research Center, Université de Montréal, Montreal, Quebec, Canada.

    We sequenced genes coding for components of the SNARE complex (STX1A, VAMP2, SNAP25) and their regulatory proteins (STXBP1/Munc18-1, SYT1), which are essential for neurotransmission, in 95 patients with idiopathic mental retardation. We identified de novo mutations in STXBP1 (nonsense, p.R388X; splicing, c.169+1G>A) in two patients with severe mental retardation and nonsyndromic epilepsy. Reverse transcriptase polymerase chain reaction and sequencing showed that the splicing mutation creates a stop codon downstream of exon-3. No de novo or deleterious mutations in STXBP1 were found in 190 control subjects, or in 142 autistic patients. These results suggest that STXBP1 disruption is associated with autosomal dominant mental retardation and nonsyndromic epilepsy.

    Annals of neurology 2009;65;6;748-53

  • A common variant in DRD3 receptor is associated with autism spectrum disorder.

    de Krom M, Staal WG, Ophoff RA, Hendriks J, Buitelaar J, Franke B, de Jonge MV, Bolton P, Collier D, Curran S, van Engeland H and van Ree JM

    Department of Neuroscience and Pharmacology and Department of Child and Adolescent Psychiatry, Rudolph Magnus Institute of Neuroscience, University Medical Center Utrecht, Heidelberglaan 100, Utrecht, The Netherlands.

    Background: The presence of specific and common genetic etiologies for autism spectrum disorders (ASD) and attention-deficit/hyperactivity disorder (ADHD) was investigated for 132 candidate genes in a two-stage design-association study.

    Methods: 1,536 single nucleotide polymorphisms (SNPs) covering these candidate genes were tested in ASD (n = 144) and ADHD (n = 110) patients and control subjects (n = 404) from The Netherlands. A second stage was performed with those SNPs from Stage I reaching a significance threshold for association of p < .01 in an independent sample of ASD patients (n = 128) and controls (n = 124) from the United Kingdom and a Dutch ADHD (n = 150) and control (n = 149) sample.

    Results: No shared association was found between ASD and ADHD. However, in the first and second ASD samples and in a joint statistical analysis, a significant association between SNP rs167771 located in the DRD3 gene was found (joint analysis uncorrected: p = 3.11 x 10(-6); corrected for multiple testing and potential stratification: p = .00162).

    Conclusions: The DRD3 gene is related to stereotyped behavior, liability to side effects of antipsychotic medication, and movement disorders and may therefore have important clinical implications for ASD.

    Biological psychiatry 2009;65;7;625-30

  • Direct interaction of otoferlin with syntaxin 1A, SNAP-25, and the L-type voltage-gated calcium channel Cav1.3.

    Ramakrishnan NA, Drescher MJ and Drescher DG

    Department of Otolaryngology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

    The molecular mechanisms underlying synaptic exocytosis in the hair cell, the auditory and vestibular receptor cell, are not well understood. Otoferlin, a C2 domain-containing Ca2+-binding protein, has been implicated as having a role in vesicular release. Mutations in the OTOF gene cause nonsyndromic deafness in humans, and OTOF knock-out mice are deaf. In the present study, we generated otoferlin fusion proteins containing two of the same amino acid substitutions detected in DFNB9 patients (P1825A in C2F and L1011P in C2D). The native otoferlin C2F domain bound syntaxin 1A and SNAP-25 in a Ca2+-dependent manner (with optimal 61 microm free Ca2+ required for binding). These interactions were greatly diminished for C2F with the P1825A mutation, possibly because of a reduction in tertiary structural change, induced by Ca2+, for the mutated C2F compared with the native C2F. The otoferlin C2D domain also bound syntaxin 1A, but with weaker affinity (Kd = 1.7 x 10(-5) m) than for the C2F interaction (Kd = 2.6 x 10(-9) m). In contrast, it was the otoferlin C2D domain that bound the Cav1.3 II-III loop, in a Ca2+-dependent manner. The L1011P mutation in C2D rendered this binding insensitive to Ca2+ and considerably diminished. Overall, we demonstrated that otoferlin interacts with two main target-SNARE proteins of the hair-cell synaptic complex, syntaxin 1A and SNAP-25, as well as the calcium channel, with the otoferlin C2F and C2D domains of central importance for binding. Because mutations in the otoferlin C2 domains that cause deafness in humans impair the ability of otoferlin to bind syntaxin, SNAP-25, and the Cav1.3 calcium channel, it is these interactions that may mediate regulation by otoferlin of hair cell synaptic exocytosis critical to inner ear hair cell function.

    Funded by: NIDCD NIH HHS: DC000156, DC004076, R01 DC000156, R01 DC000156-27, R01 DC004076

    The Journal of biological chemistry 2009;284;3;1364-72

  • [The association of the SNAP-25 gene polymorphism with verbal memory and attention in patients with major psychosis and healthy people].

    Golimbet VE, Alfimova MV, Gritsenko IK, Lezheĭko TV, Lavrushina OM, Abramova LI, Kaleda VG, Barkhatova AN, Sokolov AV and Ebshteĭn RP

    The synaptosomal-associated protein (SNAP-25) plays an integral role in synaptic transmission and in memory consolidation in the hippocampus. Recently an association between SNAP-25 gene polymorphism and cognitive ability has been reported in two independent studies of healthy people and patients with schizophrenia. Authors carried out an association study of MnlI SNAP-25 polymorphism and performance on neurocognitive tests measuring verbal memory, attention/executive functions in 66 patients with major psychosis, 75 their relatives and 136 controls. MANCOVA revealed a significant effect of group (small er, Cyrillic=0,00001) and genotype (small er, Cyrillic=0,012) as well as an interaction effect between group and genotype (small er, Cyrillic=0,02) on the performance on neurocognitive tests. Carriers of the TT genotype performed worse on the most tasks compared to other genotypes. The similar character of the MnlI SNAP-25 polymorphism effect on the variability of neurocognitive traits in all groups suggests the relationship between this gene and general cognitive ability.

    Zhurnal nevrologii i psikhiatrii imeni S.S. Korsakova 2009;109;1;59-63

  • Genome-wide association scan of quantitative traits for attention deficit hyperactivity disorder identifies novel associations and confirms candidate gene associations.

    Lasky-Su J, Neale BM, Franke B, Anney RJ, Zhou K, Maller JB, Vasquez AA, Chen W, Asherson P, Buitelaar J, Banaschewski T, Ebstein R, Gill M, Miranda A, Mulas F, Oades RD, Roeyers H, Rothenberger A, Sergeant J, Sonuga-Barke E, Steinhausen HC, Taylor E, Daly M, Laird N, Lange C and Faraone SV

    Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    Attention deficit hyperactivity disorder (ADHD) is a complex condition with environmental and genetic etiologies. Up to this point, research has identified genetic associations with candidate genes from known biological pathways. In order to identify novel ADHD susceptibility genes, 600,000 SNPs were genotyped in 958 ADHD proband-parent trios. After applying data cleaning procedures we examined 429,981 autosomal SNPs in 909 family trios. We generated six quantitative phenotypes from 18 ADHD symptoms to be used in genome-wide association analyses. With the PBAT screening algorithm, we identified 2 SNPs, rs6565113 and rs552655 that met the criteria for significance within a specified phenotype. These SNPs are located in intronic regions of genes CDH13 and GFOD1, respectively. CDH13 has been implicated previously in substance use disorders. We also evaluated the association of SNPs from a list of 37 ADHD candidate genes that was specified a priori. These findings, along with association P-values with a magnitude less than 10(-5), are discussed in this manuscript. Seventeen of these candidate genes had association P-values lower then 0.01: SLC6A1, SLC9A9, HES1, ADRB2, HTR1E, DDC, ADRA1A, DBH, DRD2, BDNF, TPH2, HTR2A, SLC6A2, PER1, CHRNA4, SNAP25, and COMT. Among the candidate genes, SLC9A9 had the strongest overall associations with 58 association test P-values lower than 0.01 and multiple association P-values at a magnitude of 10(-5) in this gene. In sum, these findings identify novel genetic associations at viable ADHD candidate genes and provide confirmatory evidence for associations at previous candidate genes. Replication of these results is necessary in order to confirm the proposed genetic variants for ADHD.

    Funded by: NHLBI NIH HHS: R00 HL096840; NIMH NIH HHS: R01 MH062873, R01 MH081803, R01MH081803, R01MH62873

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2008;147B;8;1345-54

  • SNPs in dopamine D2 receptor gene (DRD2) and norepinephrine transporter gene (NET) are associated with continuous performance task (CPT) phenotypes in ADHD children and their families.

    Kollins SH, Anastopoulos AD, Lachiewicz AM, FitzGerald D, Morrissey-Kane E, Garrett ME, Keatts SL and Ashley-Koch AE

    Department of Psychiatry, Duke University Medical Center, Durham, North Carolina, USA . kolli001@mc.duke.edu

    Haplotype-tagging SNP analyses were conducted to identify molecular genetic substrates of quantitative phenotypes derived from performance on a Continuous Performance Task (CPT). Three hundred sixty-four individuals were sampled from 152 families ascertained on the basis of at least one child having ADHD. Probands, their affected and unaffected siblings, and parents were administered a CPT. Four different components of performance were analyzed and tested for association with SNPs from 10 candidate genes involved in monoaminergic function. After correcting for multiple comparisons and controlling for multiple individuals from the same family, significant associations were identified between commission errors and SNPs in the DRD2 gene (rs2075654, rs1079596), and between reaction time variability and a SNP in the NET gene (rs3785155). These findings suggest that commission errors and reaction time variability are excellent candidates as ADHD endophenotypes based on previously published criteria. Results also shed light on the molecular genetic basis of specific processes that may underlie the disorder.

    Funded by: NIDA NIH HHS: K24 DA023464, K24DA023464; NIEHS NIH HHS: P30 ES011961, P30 ES011961-01A1; NINDS NIH HHS: 1R01NS049067, R01 NS049067, R01 NS049067-01, R01 NS049067-02, R01 NS049067-03, R01 NS049067-04, R01 NS049067-05

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2008;147B;8;1580-8

  • SNAP-25 genotype influences NAA/Cho in left hippocampus.

    Scherk H, Backens M, Zill P, Schneider-Axmann T, Wobrock T, Usher J, Reith W, Falkai P, Möller HJ, Bondy B and Gruber O

    Department of Psychiatry and Psychotherapy, Georg-August-University Goettingen, von-Siebold-Str. 5, 37075, Göttingen, Germany. hscherk@uni-goettingen.de

    The SNAP-25 gene is an integral part of the vesicle docking and fusion machinery that controls neurotransmitter release. Several post mortem studies revealed a reduction of SNAP-25 protein in the hippocampus of patients with schizophrenia and bipolar disorder (BD). Thirty-eight patients with schizophrenia, BD or obsessive-compulsive disorder and 17 healthy controls participated in the study. Proton magnetic resonance spectroscopy in left hippocampus was performed in each individual. Three single nucleotide polymorphisms (SNP) of the SNAP-25 gene were genotyped. Individuals with the homozygous CC genotype of the DdeI SNP presented a significantly higher ratio of N-acetyl-aspartate (NAA)/choline-containing compounds (Cho) in the left hippocampus compared to the group of individuals with the homozygous TT genotype. The SNAP-25 genotype may modulate synaptic plasticity and neurogenesis in the left hippocampus, and altered NAA/Cho ratio may be an indicator for this genetic modulation of neuronal function in the hippocampus.

    Journal of neural transmission (Vienna, Austria : 1996) 2008;115;11;1513-8

  • No association between tagging SNPs of SNARE complex genes (STX1A, VAMP2 and SNAP25) and schizophrenia in a Japanese population.

    Kawashima K, Kishi T, Ikeda M, Kitajima T, Yamanouchi Y, Kinoshita Y, Takahashi N, Saito S, Ohi K, Yasuda Y, Hashimoto R, Takeda M, Inada T, Ozaki N and Iwata N

    Department of Psychiatry, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

    Abnormalities in neural connections and the neurotransmitter system appear to be involved in the pathophysiology of schizophrenia. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which consists of Syntaxin1A, vesicle-associated membrane protein 2 (VAMP2) and synaptosomal-associated protein 25 kDa (SNAP25), plays an important role in the neurotransmitter system, and is therefore an attractive place to search for candidate genes for schizophrenia. We conducted a two-stage genetic association analysis of Syntaxin1A (STX1A), VAMP2 and SNAP25 genes with schizophrenia (first-set screening samples: 377 cases and 377 controls, second-set confirmation samples: 657 cases and 527 controls). Based on the linkage disequilibrium, 40 SNPs (STX1A, 8 SNPs; VAMP2, 3 SNPs; SNAP25, 29 SNPs) were selected as 'tagging SNPs'. Only nominally significant associations of an SNP (rs12626080) and haplotype (rs363014 and rs12626080) in SNAP25 were detected in the first-set screening scan. To validate this significance, we carried out a replication analysis of these SNP and haplotype associations in second-set samples with a denser set of markers (including five additional SNPs). However, these associations could not be confirmed in the second-set analysis. These results suggest that the SNARE complex-related genes do not play a major role in susceptibility to schizophrenia in the Japanese population.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2008;147B;7;1327-31

  • Immunohistochemical expression of SNAP-25 protein in adenomas of the human pituitary.

    Rotondo F, Kovacs K, Scheithauer BW, Horvath E, Bell CD, Lloyd RV and Cusimano M

    Department of Laboratory Medicine, St Michael's Hospital, University of Toronto, Toronto, ON, Canada. rotondof@smh.toronto.on.ca

    SNAP-25, a synaptosome-associated exocytosis protein of 25 kd mw, plays an important role in the secretory activity of several endocrine cells. In the present study, we investigated surgically removed pituitary adenomas including 40 prolactin (PRL), 31 growth hormone, 5 adrenocorticotropic hormone, 5 thyroid-stimulating hormone, 14 follicle-stimulating hormone/luteinizing hormone/alpha-subunit-producing tumors, and 5 null cell adenomas. Among the 40 patients with PRL-producing pituitary adenoma, 16 had been preoperatively treated with the dopamine agonist bromocriptine. Similarly, of the 31 patients with GH-producing pituitary adenomas, 15 had been treated with the long-acting somatostatin analog, octreotide. All tumors were subjected to transsphenoidal surgery, formalin-fixed, routinely processed, and paraffin-embedded. Sections of 4 to 6-microm thickness were stained with hematoxylin and eosin and the periodic acid-Schiff as well as the Gordon-Sweet silver methods. Immunostaining for SNAP-25 (streptavidin-biotin peroxidase complex method) showed that 10 PRL-producing adenomas were strongly immunoreactive. Immunopositivity was mainly cell membrane in distribution but several cells showed mild cytoplasmic staining. Nuclei were immunonegative. Preoperative bromocriptine treatment markedly decreased SNAP-25 immunopositivity. Among GH-producing adenomas, SNAP-25 was seen in 5 cases; reactivity being mild-to-moderate, membrane-bound, and cytoplasmic. Octreotide caused no significant reduction in immunopositivity. Other adenoma types were virtually immunonegative. Six autopsy-derived human pituitaries and 4 surgically obtained nontumorous pituitaries were also immunonegative for SNAP-25. It is conceivable that SNAP-25 plays an important role in PRL release and is involved in the bromocriptine-induced suppression of PRL secretion from PRL-producing adenoma cells.

    Applied immunohistochemistry & molecular morphology : AIMM 2008;16;5;477-81

  • SNAP-25 gene polymorphisms and weight gain in schizophrenic patients.

    Musil R, Spellmann I, Riedel M, Dehning S, Douhet A, Maino K, Zill P, Müller N, Möller HJ and Bondy B

    Psychiatric Clinic of University Munich, Nussbaumstrasse 7, 80336 Munich, Germany. Richard.musil@med.uni-muenchen.de

    Drug induced weight gain is a serious side effect of several atypical antipsychotics. As genetic factors play an important role in the homeostasis of hunger/satiety we tried to replicate a preliminary previous finding about an impact of three polymorphisms in the synaptosomal-associated protein of 25kDa (SNAP-25; sites MnlI, TaiI and DdelI in the 3(')-UTR) on clinical response and antipsychotic induced weight gain. We genotyped 162 schizophrenic patients being treated in monotherapy with atypical antipsychotics and 312 healthy control subjects for the three polymorphisms in the SNAP-25 gene using PCR. PANSS scores and weight were measured weekly for a minimum of five weeks. We found significant associations between the TaiI and MnlI polymorphisms and serum triglyceride levels at baseline and for the DdelI polymorphism and weight gain. In conclusion our study can at least partly replicate the previous findings concerning the impact of SNAP-25 gene polymorphisms on weight gain during antipsychotic treatment.

    Journal of psychiatric research 2008;42;12;963-70

  • Vasoactive intestinal peptide-induced neuritogenesis in neuroblastoma SH-SY5Y cells involves SNAP-25.

    Héraud C, Chevrier L, Meunier AC, Muller JM and Chadéneau C

    Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, CNRS UMR 6187, Pôle Biologie Santé, Faculté des Sciences Fondamentales et Appliquées, 40 Avenue du Recteur Pineau, Poitiers Cedex F-86022, France.

    Vasoactive intestinal peptide (VIP) is a neuropeptide known to regulate proliferation and differentiation in normal and tumoral cells. We previously reported that VIP induced neuritogenesis in human neuroblastoma SH-SY5Y cells cultured in serum-free medium. This neuritogenesis was associated with a regulated expression of neuronal cytoskeleton markers. To further characterize the neuroblastic cell differentiation induced by VIP in human SH-SY5Y cells, we investigated expression of synaptosomal-associated protein of 25 kDa (SNAP-25), a protein implicated in exocytosis associated with different processes, including neurite outgrowth. Western immunoblotting and real-time RT-PCR analyses revealed that VIP increased expression of the SNAP-25 protein and the level of both SNAP-25a and SNAP-25b mRNA isoforms. Immunofluorescence experiments indicated that SNAP-25 was mainly located in neurites and at the plasma membrane in SH-SY5Y cells treated with VIP. RNA interference experiments demonstrated that SNAP-25 was involved in VIP-induced neuritogenesis. In conclusion, SNAP-25 is up-regulated and implicated in neuritogenesis in human neuroblastoma SH-SY5Y cells treated with the neuropeptide VIP.

    Neuropeptides 2008;42;5-6;611-21

  • SNAP-25 substrate peptide (residues 180-183) binds to but bypasses cleavage by catalytically active Clostridium botulinum neurotoxin E.

    Agarwal R and Swaminathan S

    Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.

    Clostridium botulinum neurotoxins are the most potent toxins to humans. The recognition and cleavage of SNAREs are prime evente in exhibiting their toxicity. We report here the crystal structure of the catalytically active full-length botulinum serotype E catalytic domain (BoNT E) in complex with SNAP-25 (a SNARE protein) substrate peptide Arg(180)-Ile(181)-Met(182)-Glu(183) (P1-P3'). It is remarkable that the peptide spanning the scissile bond binds to but bypasses cleavage by the enzyme and inhibits the catalysis fairly with K(i) approximately 69 microm. The inhibitory peptide occupies the active site of BoNT E and shows well defined electron density. The catalytic zinc and the conserved key residue Tyr(350) of the enzyme facilitate the docking of Arg(180) (P1) by interacting with its carbonyl oxygen that displaces the nucleophilic water. The general base Glu(212) side chain interacts with the main chain amino group of P1 and P1'. Conserved Arg(347) of BoNT E stabilizes the proper docking of the Ile(181) (P1') main chain, whereas the hydrophobic pockets stabilize the side chains of Ile(181) (P1') and Met(182) (P2'), and the 250 loop stabilizes Glu(183) (P3'). Structural and functional analysis revealed an important role for the P1' residue and S1' pocket in driving substrate recognition and docking at the active site. This study is the first of its kind and rationalizes the substrate cleavage strategy of BoNT E. Also, our complex structure opens up an excellent opportunity of structure-based drug design for this fast acting and extremely toxic high priority BoNT E.

    Funded by: PHS HHS: 1R56AL058175

    The Journal of biological chemistry 2008;283;38;25944-51

  • The structural and functional implications of linked SNARE motifs in SNAP25.

    Wang L, Bittner MA, Axelrod D and Holz RW

    Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109-5632, USA.

    We investigated the functional and structural implications of SNAP25 having two SNARE motifs (SN1 and SN2). A membrane-bound, intramolecular FRET probe was constructed to report on the folding of N-terminal SN1 and C-terminal SN2 in living cells. Membrane-bound constructs containing either or both SNARE motifs were also singly labeled with donor or acceptor fluorophores. Interaction of probes with other SNAREs was monitored by the formation of SDS-resistant complexes and by changes in FRET measured in vitro using spectroscopy and in the plasma membrane of living cells using TIRF microscopy. The probes formed the predicted SDS-resistant SNARE complexes. FRET measurements revealed that syntaxin induced a close association of the N-termini of SN1 and SN2. This association required that the SNARE motifs reside in the same molecule. Unexpectedly, the syntaxin-induced FRET was prevented by VAMP. Both full-length SNAP25 constructs and the combination of its separated, membrane-bound constituent chains supported secretion in permeabilized chromaffin cells that had been allowed to rundown. However, only full-length SNAP25 constructs enabled robust secretion from intact cells or permeabilized cells before rundown. The experiments suggest that the bidentate structure permits specific conformations in complexes with syntaxin and VAMP and facilitates the function of SN1 and SN2 in exocytosis.

    Funded by: NIDDK NIH HHS: R01 DK050127, R01-DK050127; NINDS NIH HHS: R01 NS038129, R01-NS38129

    Molecular biology of the cell 2008;19;9;3944-55

  • Associations of SNAP-25 polymorphisms with cognitive dysfunctions in Caucasian patients with schizophrenia during a brief trail of treatment with atypical antipsychotics.

    Spellmann I, Müller N, Musil R, Zill P, Douhet A, Dehning S, Cerovecki A, Bondy B, Möller HJ and Riedel M

    Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-University of Munich, Nussbaumstrasse 7, 80336, Munich, Germany. ilja.spellmann@med.uni-muenchen.de

    The synaptosomal-associated protein of 25 kDa (SNAP-25) is part of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment receptor (SNARE), which mediates synaptic neurotransmission. In earlier studies a possible involvement of this protein in schizophrenia has been shown. As neurocognitive impairment is a core feature in the pathology of schizophrenia and considered to be a putative endophenotype according to genetic studies we investigated the influences of different SNAP-25 polymorphisms on neuropsychological test results before and during treatment with atypical antipsychotics. A total of 104 schizophrenic patients treated with atypical antipsychotics were genotyped for three different polymorphisms of the SNAP-25 gene (MnlI, TaiI and DdeI in the 3'-UTR). Cognitive function was assessed at baseline, week 4 or 6 and week 8 or 12. Results of individual neuropsychological tests were assigned to six cognitive domains (reaction time and quality; executive function; working, verbal and visual memory) and a general cognitive index. The MnlI and TaiI polymorphisms showed no associations to deficits on neuropsychological test results. In contrast, we observed a significant relation between the DdeI polymorphism of the SNAP-25 gene and cognitive dysfunctions. Homozygote T/T allele carriers of the DdeI polymorphism showed significant better neuropsychological test results in cognitive domains verbal memory and executive functions than those with the combined T/C and C/C genotypes (P < 0.01) at all three time points, but no differences in response to treatment with atypical antipsychotics. Additionally, TT carriers exhibited significantly better results in a general cognitive index (P < 0.05). As we observed an association between the DdeI polymorphism of the SNAP-25 gene and cognitive dysfunctions of schizophrenic patients our finding suggests that the SNAP-25 gene could play a role in the pathophysiology of neurocognitive dysfunctions in schizophrenia but is not predictive for treatment response with atypical antipsychotics.

    European archives of psychiatry and clinical neuroscience 2008;258;6;335-44

  • Multiple genetic factors in olanzapine-induced weight gain in schizophrenia patients: a cohort study.

    Ujike H, Nomura A, Morita Y, Morio A, Okahisa Y, Kotaka T, Kodama M, Ishihara T and Kuroda S

    Department of Neuropsychiatry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. hujike@cc.okayama-u.ac.jp

    Objective: One of the clinically significant adverse effects of olanzapine treatment is weight gain, which shows substantial inter-individual differences and may be influenced by genetic variation. The aim of this investigation was identification of genetic risk factors associated with olanzapine-induced weight gain.

    Method: Inpatients with DSM-IV-TR schizophrenia (N = 164) were administered olanzapine for 8 to 24 (mean +/- SD = 17.9 +/- 9.4) weeks. The clinical background, body mass index (BMI), and clinical response to olanzapine were investigated. Twenty-one loci of diverse candidate genes encoding dopamine, serotonin (5-HT), histamine, and adrenergic receptors, tumor necrosis factor-alpha, ghrelin, adiponectin, and peroxisome proliferator-activated receptor gamma-2, were analyzed. The study was conducted from June 2001 to June 2003 at 4 psychiatric hospitals in Japan.

    Results: BMI increased by a mean +/- SD 4.3 +/- 10.7% after treatment with olanzapine (mean +/- SD dose = 15.5 +/- 5.8 mg/day). Olanzapine-induced weight gain correlated negatively with baseline BMI and positively with clinical global improvement and the length of olanzapine treatment (p < .0001), but it did not correlate with the daily dose of olanzapine, concomitant antipsychotics, sex, age, or smoking. Four genetic variants, the 102T allele of HTR2A, the 825T allele of GNB3, the 23Cys allele of HTR2C, and the 64Arg/Arg genotype of ADRB3, were significantly associated with olanzapine-induced weight gain. Stepwise regression analysis revealed that the baseline BMI predicted 12.5% of the weight gain, and the 2 latter genetic factors added 6.8%. The patients with double and triple genetic risk factors showed 5.1% and 8.8% BMI increases, respectively, during olanzapine treatment, whereas the patients with a single or no risk factor showed approximately a 1% BMI increase.

    Conclusions: We identified genetic variants of 5-HT(2A) and 5-HT(2C) receptors, the G-protein beta-3 subunit, and the adrenergic receptor beta-3, as genetic risk factors for olanzapine-induced weight gain, and they showed additive genetic effects on weight gain.

    The Journal of clinical psychiatry 2008;69;9;1416-22

  • [Association between SNAP-25 gene polymorphism and attention deficit hyperactivity disorder].

    Zhang HY, Zhu YB, Chang H and Chen JC

    Department of Pediatrics, the First Affiliated Hospital, SUN Yat-sen University, Guangzhou, China.

    Objective: To analyse the association between polymorphism in human synaptosomal-associated protein of 25 000 (SNAP-25) gene and attention deficit hyperactivity disorder (ADHD) in Han Chinese children.

    Methods: The study samples were comprised of 100 integrated ADHD trios (included proband and biological parents) and 97 unrelated controls. Association of polymorphism with ADHD and its subtype was examined by: (1) comparing cases and controls; (2) using family-based association study in transmission disequilibrium test (TDT).

    Results: Case-control analysis of short tandem repeat (STR) showed that there was no significant difference between the two groups in allele gene frequency and genotype frequency (P > 0.05); TDT analysis of the rs363006 SNP and the rs362549 SNP revealed no association between SNAP-25 polymorphisms and ADHD (P > 0.05). But after a stratification by ADHD subtype, the rs362549 SNP A allele showed a tendency to preferentially transmitted to ADHD-I subtype (chi(2) = 8.00, P < 0.01); and the rs362549 SNP G allele had a tendency to preferentially transmitted to ADHD-C subtype (chi(2) = 4.122, P < 0.05).

    Conclusions: No association was found between SNAP-25 polymorphisms and ADHD. There was a possible association between rs362549 SNP polymorphism and ADHD subtypes, but the findings require replication before drawing a definitive conclusion.

    Zhonghua er ke za zhi = Chinese journal of pediatrics 2008;46;8;564-9

  • Heterogeneous expression of SNARE proteins SNAP-23, SNAP-25, Syntaxin1 and VAMP in human parathyroid tissue.

    Lu M, Forsberg L, Höög A, Juhlin CC, Vukojević V, Larsson C, Conigrave AD, Delbridge LW, Gill A, Bark C, Farnebo LO and Bränström R

    Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.

    In regulated exocytosis synaptosomal-associated protein of 25kDa (SNAP-25) is one of the key-players in the formation of SNARE (soluble N-ethylmaleimide-sensitive fusion attachment protein receptor) complex and membrane fusion. SNARE proteins are essentially expressed in neurons, neuroendocrine and endocrine cells. Whether parathyroid cells express these proteins is not known. In this study, we have examined the expression of the SNARE protein SNAP-25 and its cellular homologue SNAP-23, as well as syntaxin1 and VAMP (vesicle-associated membrane protein) in samples of normal parathyroid tissue, chief cell adenoma, and parathyroid carcinoma, using immunohistochemistry and Western blot analysis. SNAP-23 and VAMP were evenly expressed in all studied parathyroid tissues using immunohistochemistry and/or Western blot analysis. SNAP-25 (and Syntaxin1) was not expressed in normal parathyroid tissue, but in approximately 20% of chief cell adenomas, and in approximately 45% of parathyroid carcinoma samples. It is likely that the SNARE proteins SNAP-23 and VAMP play a role in the stimulus-secretion coupling and exocytosis of parathyroid hormone as these proteins were expressed in all of the parathyroid samples we studied. In particular, preferential expression of SNAP-23 rather than SNAP-25 provides an explanation of the high level of PTH secretion that occurs under conditions of low cytoplasmic free Ca(2+) concentration (around 0.1micromol/l). SNAP-25 (and Syntaxin1) appears to be a tumour-specific protein(s) in parathyroid tissues since its expression was restricted to pathological tissues.

    Molecular and cellular endocrinology 2008;287;1-2;72-80

  • Molecular genetics of successful smoking cessation: convergent genome-wide association study results.

    Uhl GR, Liu QR, Drgon T, Johnson C, Walther D, Rose JE, David SP, Niaura R and Lerman C

    Molecular Neurobiology Research Branch, National Institutes of Health-Intramural Research Program, National Institute on Drug Abuse, 333 Cassell Dr, Ste 3510, Baltimore, MD 21224, USA. guhl@intra.nida.nih.gov

    Context: Smoking remains a major public health problem. Twin studies indicate that the ability to quit smoking is substantially heritable, with genetics that overlap modestly with the genetics of vulnerability to dependence on addictive substances.

    Objectives: To identify replicated genes that facilitate smokers' abilities to achieve and sustain abstinence from smoking (herein after referred to as quit-success genes) found in more than 2 genome-wide association (GWA) studies of successful vs unsuccessful abstainers, and, secondarily, to nominate genes for selective involvement in smoking cessation success with bupropion hydrochloride vs nicotine replacement therapy (NRT).

    Design: The GWA results in subjects from 3 centers, with secondary analyses of NRT vs bupropion responders.

    Setting: Outpatient smoking cessation trial participants from 3 centers.

    Participants: European American smokers who successfully vs unsuccessfully abstain from smoking with biochemical confirmation in a smoking cessation trial using NRT, bupropion, or placebo (N = 550).

    Quit-success genes, reproducibly identified by clustered nominally positive single-nucleotide polymorphisms (SNPs) in more than 2 independent samples with significant P values based on Monte Carlo simulation trials. The NRT-selective genes were nominated by clustered SNPs that display much larger t values for NRT vs placebo comparisons. The bupropion-selective genes were nominated by bupropion-selective results.

    Results: Variants in quit-success genes are likely to alter cell adhesion, enzymatic, transcriptional, structural, and DNA, RNA, and/or protein-handling functions. Quit-success genes are identified by clustered nominally positive SNPs from more than 2 samples and are unlikely to represent chance observations (Monte Carlo P< .0003). These genes display modest overlap with genes identified in GWA studies of dependence on addictive substances and memory.

    Conclusions: These results support polygenic genetics for success in abstaining from smoking, overlap with genetics of substance dependence and memory, and nominate gene variants for selective influences on therapeutic responses to bupropion vs NRT. Molecular genetics should help match the types and/or intensity of antismoking treatments with the smokers most likely to benefit from them.

    Funded by: Intramural NIH HHS; NCI NIH HHS: P50 CA084719, P50CA/DA84718, P50CA84719, R01 CA063562, R01CA 63562; NHLBI NIH HHS: HL32318; NIDA NIH HHS: 1K08 DA14276-05, DA08511, K08 DA014276, K08 DA014276-01A2, K08 DA014276-02, K08 DA014276-03, K08 DA014276-04, K08 DA014276-05

    Archives of general psychiatry 2008;65;6;683-93

  • Common variants underlying cognitive ability: further evidence for association between the SNAP-25 gene and cognition using a family-based study in two independent Dutch cohorts.

    Gosso MF, de Geus EJ, Polderman TJ, Boomsma DI, Heutink P and Posthuma D

    Department of Biological Psychology, Vrije Universiteit, Amsterdam, The Netherlands. mf.gosso@vumc.nl

    The synaptosomal associated protein of 25 kDa (SNAP-25) gene, located on chromosome 20 p12-12p11.2 encodes a presynaptic terminal protein. SNAP-25 is differentially expressed in the brain, and primarily present in the neocortex, hippocampus, anterior thalamic nuclei, substantia nigra and cerebellar granular cells. Recently, a family-based genetic association was reported between variation in intelligence quotient (IQ) phenotypes and two intronic variants on the SNAP-25 gene. The present study is a follow-up association study in two Dutch cohorts of 371 children (mean age 12.4 years) and 391 adults (mean age 36.2 years). It examines the complete genomic region of the SNAP-25 gene to narrow down the location of causative genetic variant underlying the association. Two new variants in intron 1 (rs363043 and rs353016), close to the two previous reported variants (rs363039 and rs363050) showed association with variation in IQ phenotypes across both cohorts. All four single nucleotide polymorphisms were located in intron 1, within a region of about 13.8 kbp, and are known to affect transcription factor-binding sites. Contrary to what is expected in monogenic traits, subtle changes are postulated to influence the phenotypic outcome of complex (common) traits. As a result, functional polymorphisms in (non)coding regulatory sequences may affect spatial and temporal regulation of gene expression underlying normal cognitive variation.

    Genes, brain, and behavior 2008;7;3;355-64

  • Heterogeneous expression of SNAP-25 in rat and human brain.

    Garbelli R, Inverardi F, Medici V, Amadeo A, Verderio C, Matteoli M and Frassoni C

    Clinical Epileptology and Experimental Neurophysiology Unit, Fondazione IRCCS, Istituto Neurologico C Besta, via Celoria 11, Milano, Italy.

    Synaptosomal associated protein of 25 kDa (SNAP-25) is a SNARE component of the exocytotic apparatus involved in the release of neurotransmitter. We used multiple-labeling immunofluorescence, confocal microscopy, and ultrastructural immunocytochemistry to examine the expression of SNAP-25 in excitatory and inhibitory terminals from different rat and human brain areas. Glutamatergic and GABAergic terminals were identified by staining for the vesicular glutamate transporter (vGLUT1), glutamic acid decarboxylase (GAD67), or the vesicular GABA transporter (vGAT). In all examined areas GABAergic terminals did not display detectable levels of SNAP-25, whereas glutamatergic terminals expressed the protein to a variable extent. Codistribution analysis revealed a high colocalization between pixels detecting SNAP-25 labeling and pixels detecting vGLUT1 immunoreactivity. On the contrary, a low degree of pixel colocalization, comparable to that between two unrelated antigens, was detected between SNAP-25 and vGAT, thus suggesting a random overlap of immunofluorescence signals. Our immunofluorescence evidence was supported by ultrastructural data, which clearly confirmed that SNAP-25 was undetectable in GABAergic terminals identified by both their typical morphology and specific staining for GABA. Interestingly, our ultrastructural results confirmed that a subset of glutamatergic synapses do not contain detectable levels of SNAP-25. The present study extends our previous findings obtained in rodent hippocampus and provides evidence that SNAP-25 expression is highly variable between different axon terminals both in rat and human brain. The heterogeneous distribution of SNAP-25 may have important implications not only in relation to the function of the protein as a SNARE but also in the control of network excitability.

    Funded by: Telethon: GGP04196

    The Journal of comparative neurology 2008;506;3;373-86

  • Allelic variants of SNAP25 in a family-based sample of ADHD.

    Renner TJ, Walitza S, Dempfle A, Eckert L, Romanos M, Gerlach M, Schäfer H, Warnke A, Lesch KP and Jacob C

    Department of Child and Adolescent Psychiatry and Psychotherapy, University of Würzburg, Würzburg, Germany. renner@kjp.uni-wuerzburg.de

    Altered neurotransmission has been suggested to be a crucial factor in the pathophysiology of attention-deficit/hyperactivity disorder ADHD. Subsequently genes encoding for synaptic proteins have been investigated in candidate gene studies. These proteins mediate the release of neurotransmitters into the synaptic cleft in the process of signal transduction by forming a transient complex, enabling the junction of vesicle and synaptic membrane. One of the core proteins of this complex is the synaptosomal-associated protein 25 (SNAP25). It is one of the most validated candidate genes in ADHD according to meta-analyses. However, differing results were observed in previous studies, some of which were not able to observe association with ADHD. In this study we aimed to investigate association of genetic variants of SNAP25 located in the putative promoter region of SNAP25 and a SNP in intron 8, previously reported to associated with ADHD. A family based design was applied to detect preferential transmission of genetic variants. In our German ADHD sample no preferential transmission of either variant could be observed. Further investigation considering sub-sample analysis regarding response to D-amphetamine could enlight the role of SNAP25 in ADHD.

    Journal of neural transmission (Vienna, Austria : 1996) 2008;115;2;317-21

  • Loss of SNAP-25 and rabphilin 3a in sensory-motor cortex in Huntington's disease.

    Smith R, Klein P, Koc-Schmitz Y, Waldvogel HJ, Faull RL, Brundin P, Plomann M and Li JY

    Neuronal Survival Unit, Wallenberg Neuroscience Center, Lund University, Lund, Sweden. ruben.smith@med.lu.se

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG-expansion in the gene encoding the protein huntingtin. The disease is characterized by progressive motor disturbances, cognitive defects, dementia, and weight loss. Using western blotting and immunohistochemistry we have assessed the expression levels and patterns of a number of proteins involved in neurotransmitter release in post-mortem frontal cortex samples from 10 HD cases with different disease grades. We report a loss of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, synaptosome-associated protein 25 (SNAP 25) in HD brains of grades I-IV. Moreover, in brains of grade III and IV we found a reduction in rabphilin 3a, a protein involved in vesicle docking and recycling. These losses appear to be specific and not due to a general loss of synapses in the HD cortex. Thus, levels of synaptobrevin II, syntaxin 1, rab3a or synaptophysin are unaltered in the same patient samples. SNAP 25 and rabphilin 3a are crucial for neurotransmitter release. Therefore, we suggest that a deficient pre-synaptic transmitter release may underlie some of the symptoms of HD.

    Journal of neurochemistry 2007;103;1;115-23

  • Investigation of parent-of-origin effects in ADHD candidate genes.

    Kim JW, Waldman ID, Faraone SV, Biederman J, Doyle AE, Purcell S, Arbeitman L, Fagerness J, Sklar P and Smoller JW

    Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA. jwkim@partners.org

    Attention deficit hyperactivity disorder (ADHD) is a common early-onset childhood disorder with a strong genetic component. Results from previous studies have suggested that there may be a parent-of-origin effect for ADHD candidate genes. In particular, a recent investigation identified a pattern of paternal over-transmission of risk alleles for nine ADHD candidate genes. We examined this phenomenon in a sample of 291 trios for five genes previously associated with ADHD (HTR1B, SNAP-25, DRD5, DAT1, and BDNF). Using a dense map of markers and two analytic methods in this relatively large family-based sample, we do not find any evidence for significant paternal over-transmission of risk alleles in these candidate loci. Thus, we conclude that a substantial parent-of-origin effect is unlikely for these leading ADHD candidate genes.

    Funded by: NICHD NIH HHS: HD37694, HD37999; NIMH NIH HHS: MH66877

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2007;144B;6;776-80

  • Investigation of variation in SNAP-25 and ADHD and relationship to co-morbid major depressive disorder.

    Kim JW, Biederman J, Arbeitman L, Fagerness J, Doyle AE, Petty C, Perlis RH, Purcell S, Smoller JW, Faraone SV and Sklar P

    Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.

    Synaptosomal-associated protein of 25 kDa (SNAP-25), a protein involved in presynaptic neurotransmitter release, is a candidate gene for attention deficit/hyperactivity disorder (ADHD). Previous investigators have reported association initially with two single nucleotide polymorphisms (SNPs) (rs3746544, rs1051312) and their associated haplotypes. Subsequently, additional SNPs across the region were also reported to be associated with ADHD. We attempted to replicate these observations in a sample of 229 families with ADHD offspring by genotyping 61 SNPs spanning the region containing SNAP-25. A single SNP (rs3787283) which is in strong linkage disequilibrium (LD) with rs3746544 and rs1051312 (D' = 0.89-0.94) resulted in a nominally significant association (P = 0.002). When we pooled our data with those from prior studies, results were modestly significant for rs3746544 (P = 0.048) and rs6077690 (P = 0.031). As an attempt to determine if specific ADHD-related phenotypes may be more relevant to SNAP-25 than the categorical diagnosis, we carried out exploratory subgroup analysis in our ADHD sample according to co-morbid status. We found the strongest association result in the ADHD patients with co-morbid major depressive disorder (MDD). Six SNPs were nominally associated with the ADHD and co-morbid MDD cases (P = 0.012-0.045). Furthermore, a haplotype block located 11 kb 3' of the gene showed positive evidence for association with this phenotype (global P = 0.013). In conclusion, we report some evidence supporting the association of previously implicated SNPs (rs3746544, rs1051312) of SNAP-25 to ADHD. We further suggest that co-morbidity with MDD may enhance detection of the association between SNAP-25 and ADHD.

    Funded by: NICHD NIH HHS: HD37694; NIDDK NIH HHS: DK37999; NIMH NIH HHS: MH66877

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2007;144B;6;781-90

  • Evidence for an association of the dopamine D5 receptor gene on age at onset of attention deficit hyperactivity disorder.

    Lasky-Su J, Biederman J, Laird N, Tsuang M, Doyle AE, Smoller JW, Lange C and Faraone SV

    Department of Epidemiology, Harvard School of Public Health, Boston, MA, USA.

    The purpose of this study was to determine whether the single nucleotide polymorphisms (SNPs) within candidate genes for attention deficit hyperactivity disorder (ADHD) are associated with the age at onset for ADHD. One hundred and forty-three SNPs were genotyped across five candidate genes (DRD5, SLC6A3, HTR1B, SNAP25, DRD4) for ADHD in 229 families with at least one affected offspring. SNPs with the highest estimated power to detect an association with age at onset were selected for each candidate gene, using a power-based screening procedure that does not compromise the nominal significance level. A time-to-onset analysis for family-based samples was performed on these SNPs to determine if an association exists with age at onset for ADHD. Seven consecutive SNPs surrounding the D5 dopamine receptor gene (DRD5), were associated with the age at onset for ADHD; FDR adjusted q-values ranged from 0.008 to 0.023. This analysis indicates that individuals with the risk genotype develop ADHD earlier than individuals with any other genotype. A haplotype analysis across the 6 significant SNPs that were in linkage disequilibrium with one another, CTCATA, was also found to be significant (p-value = 0.02). We did not observe significant associations with age at onset for the other candidate loci tested. Although definitive conclusions await independent replication, these results suggest that a variant in DRD5 may affect age at onset for ADHD.

    Funded by: NICHD NIH HHS: R01HD37694, R01HD37999; NIMH NIH HHS: MH059532, R01MH66877, T32-MH017119

    Annals of human genetics 2007;71;Pt 5;648-59

  • Mechanism of arachidonic acid action on syntaxin-Munc18.

    Connell E, Darios F, Broersen K, Gatsby N, Peak-Chew SY, Rickman C and Davletov B

    Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

    Syntaxin and Munc18 are, in tandem, essential for exocytosis in all eukaryotes. Recently, it was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by arachidonic acid, indicating that this common second messenger acts to disrupt the syntaxin-Munc18 interaction. Here, we show that arachidonic acid can stimulate syntaxin 1 alone, indicating that it is syntaxin 1 that undergoes a structural change in the syntaxin 1-Munc18 complex. Arachidonic acid is incapable of dissociating Munc18 from syntaxin 1 and, crucially, Munc18 remains associated with syntaxin 1 after arachidonic-acid-induced syntaxin 1 binding to synaptosomal-associated protein 25 kDa (SNAP25). We also show that the same principle operates in the case of the ubiquitous syntaxin 3 isoform, highlighting the conserved nature of the mechanism of arachidonic acid action. Neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) can be isolated from brain membranes in a complex with endogenous Munc18, consistent with a proposed function of Munc18 in vesicle docking and fusion.

    Funded by: Medical Research Council: MC_U105178791; Parkinson's UK: G-4066

    EMBO reports 2007;8;4;414-9

  • No preferential transmission of paternal alleles at risk genes in attention-deficit hyperactivity disorder.

    Laurin N, Feng Y, Ickowicz A, Pathare T, Malone M, Tannock R, Schachar R, Kennedy JL and Barr CL

    Molecular psychiatry 2007;12;3;226-9

  • Support for the MnlI polymorphism of SNAP25; a Korean ADHD case-control study.

    Choi TK, Lee HS, Kim JW, Park TW, Song DH, Yook KW, Lee SH, Kim JI and Suh SY

    Molecular psychiatry 2007;12;3;224-6

  • Binding of calcium ions and SNAP-25 to the hexa EF-hand protein secretagogin.

    Rogstam A, Linse S, Lindqvist A, James P, Wagner L and Berggård T

    Department of Biophysical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.

    Secretagogin is a hexa EF-hand protein, which has been identified as a novel potential tumour marker. In the present study, we show that secretagogin binds four Ca2+ ions (log K1=7.1+/-0.4, log K2=4.7+/-0.6, log K3=3.6+/-0.7 and log K4=4.6+/-0.6 in physiological salt buffers) with a [Ca2+](0.5) of approx. 25 microM. The tertiary structure of secretagogin changes significantly upon Ca2+ binding, but not upon Mg2+ binding, and the amount of exposed hydrophobic surface in secretagogin increases upon Ca2+ binding, but not upon Mg2+ binding. These properties suggest that secretagogin belongs to the 'sensor' family of Ca2+-binding proteins. However, in contrast with the prototypical Ca2+ sensor calmodulin, which interacts with a very large number of proteins, secretagogin is significantly less promiscuous. Only one secretagogin-interacting protein was reproducibly identified from insulinoma cell lysates and from bovine and mouse brain homogenates. This protein was identified as SNAP-25 (25 kDa synaptosome-associated protein), a protein involved in Ca2+-induced exocytosis in neurons and in neuroendocrine cells. K(d) was determined to be 1.2x10(-7) M in the presence of Ca2+ and 1.5x10(-6) M in the absence of Ca2+. The comparatively low Ca2+ affinity for secretagogin and the fact that it undergoes Ca2+-induced conformational changes and interacts with SNAP-25 raise the possibility that secretagogin may link Ca2+ signalling to exocytotic processes.

    The Biochemical journal 2007;401;1;353-63

  • The SNAP-25 gene is associated with cognitive ability: evidence from a family-based study in two independent Dutch cohorts.

    Gosso MF, de Geus EJ, van Belzen MJ, Polderman TJ, Heutink P, Boomsma DI and Posthuma D

    Department of Biological Psychology, Vrije Universiteit, Amsterdam, The Netherlands. mf.gosso@vumc.nl

    The synaptosomal-associated protein of 25 kDa (SNAP-25) gene plays an integral role in synaptic transmission, and is differentially expressed in the mammalian brain in the neocortex, hippocampus, anterior thalamic nuclei, substantia nigra and cerebellar granular cells. Recent studies have suggested a possible involvement of SNAP-25 in learning and memory, both of which are key components of human intelligence. In addition, the SNAP-25 gene lies in a linkage area implicated previously in human intelligence. In two independent family-based Dutch samples of 391 (mean age 12.4 years) and 276 (mean age 37.3 years) subjects, respectively, we genotyped 12 single-nucleotide polymorphisms (SNPs) in the SNAP-25 gene on 20p12-20p11.2. From all individuals, standardized intelligence measures were available. Using a family-based association test, a strong association was found between three SNPs in the SNAP-25 gene and intelligence, two of which showed association in both independent samples. The strongest, replicated association was found between SNP rs363050 and performance IQ (PIQ), where the A allele was associated with an increase of 2.84 PIQ points (P=0.0002). Variance in this SNP accounts for 3.4% of the phenotypic variance in PIQ.

    Molecular psychiatry 2006;11;9;878-86

  • CytLEK1 is a regulator of plasma membrane recycling through its interaction with SNAP-25.

    Pooley RD, Reddy S, Soukoulis V, Roland JT, Goldenring JR and Bader DM

    Stahlman Cardiovascular Research Laboratories, Program for Developmental Biology, and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232-6300, USA.

    SNAP-25 is a component of the SNARE complex that is involved in membrane docking and fusion. Using a yeast two-hybrid screen, we identify a novel interaction between SNAP-25 and cytoplasmic Lek1 (cytLEK1), a protein previously demonstrated to associate with the microtubule network. The binding domains within each protein were defined by yeast two-hybrid, coimmunoprecipitation, and colocalization studies. Confocal analyses reveal a high degree of colocalization between the proteins. In addition, the endogenous proteins can be isolated as a complex by immunoprecipitation. Further analyses demonstrate that cytLEK1 and SNAP-25 colocalize and coprecipitate with Rab11a, myosin Vb, VAMP2, and syntaxin 4, components of the plasma membrane recycling pathway. Overexpression of the SNAP-25-binding domain of cytLEK1, and depletion of endogenous Lek1 alters transferrin trafficking, consistent with a function in vesicle recycling. Taken together, our studies indicate that cytLEK1 is a link between recycling vesicles and the microtubule network through its association with SNAP-25. This interaction may play a key role in the regulation of the recycling endosome pathway.

    Funded by: NHLBI NIH HHS: HL37675, R01 HL037675; NIDDK NIH HHS: 1F32DK072789A, DK070856, DK48370, F32 DK072789, R01 DK048370, R01 DK070856, R56 DK070856

    Molecular biology of the cell 2006;17;7;3176-86

  • The LIFEdb database in 2006.

    Mehrle A, Rosenfelder H, Schupp I, del Val C, Arlt D, Hahne F, Bechtel S, Simpson J, Hofmann O, Hide W, Glatting KH, Huber W, Pepperkok R, Poustka A and Wiemann S

    Division Molecular Genome Analysis, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany. a.mehrle@dkfz.de

    LIFEdb (http://www.LIFEdb.de) integrates data from large-scale functional genomics assays and manual cDNA annotation with bioinformatics gene expression and protein analysis. New features of LIFEdb include (i) an updated user interface with enhanced query capabilities, (ii) a configurable output table and the option to download search results in XML, (iii) the integration of data from cell-based screening assays addressing the influence of protein-overexpression on cell proliferation and (iv) the display of the relative expression ('Electronic Northern') of the genes under investigation using curated gene expression ontology information. LIFEdb enables researchers to systematically select and characterize genes and proteins of interest, and presents data and information via its user-friendly web-based interface.

    Nucleic acids research 2006;34;Database issue;D415-8

  • The SNAP25 gene as a susceptibility gene contributing to attention-deficit hyperactivity disorder.

    Feng Y, Crosbie J, Wigg K, Pathare T, Ickowicz A, Schachar R, Tannock R, Roberts W, Malone M, Swanson J, Kennedy JL and Barr CL

    Department of Psychiatry, Cellular and Molecular Division, The Toronto Western Research Institute, University Health Network, Toronto, ON, Canada.

    The synaptosomal-associated protein of 25 kDa gene (SNAP25) has been suggested as a genetic susceptibility factor in attention-deficit hyperactivity disorder (ADHD) based on the mouse strain coloboma. This strain is hemizygous for the SNAP25 gene and displays hyperactivity that responds to dextroamphetamine, but not to methylphenidate. Previously, we reported association of SNAP25 and ADHD using two polymorphisms. To further investigate this gene, we screened the exons for DNA variation and genotyped ten additional polymorphisms in an expanded sample of families from Toronto and a second sample of families collected in Irvine, CA. Significant results were observed in the Toronto sample for four markers, although not in the Irvine sample. The paper discusses the possible influence of the selection criteria on these differential results. The Irvine sample selected subjects that met the DSM-IV combined subtype diagnosis, whereas the Toronto sample included all subtypes. Analysis of the DSM-IV subtypes in the Toronto sample indicated that the differential results were not attributable to ADHD subtype. Differences in ethnicity, differential medication response, and other clinical characteristics of the samples cannot be ruled out at this time. Quantitative analysis of the dimensions of hyperactivity/impulsivity and inattention in the Toronto sample found that both behavioral traits were associated with SNAP25. Our findings continue to support SNAP25 in the susceptibility to ADHD.

    Funded by: NICHD NIH HHS: HD99-004; NIMH NIH HHS: U01:MH50440

    Molecular psychiatry 2005;10;11;998-1005, 973

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • Evidence that insulin secretion influences SNAP-25 through proteasomal activation.

    Ma Z, Portwood N, Foss A, Grill V and Björklund A

    Department of Molecular Medicine, Endocrine and Diabetes Unit, Rolf Luft Center of Diabetes Research, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden.

    Regulation of SNARE proteins by glucose in pancreatic islets is complex and insufficiently clarified. We aimed to study effects of glucose per se separate from enhancing effects on exocytosis. A 24h culture of rat islets at elevated glucose (27 mmol/L) increased t-SNARES (SNAP-25, syntaxin) (Western blotting). Co-culture with diazoxide, which inhibits glucose-induced insulin secretion, reversed these effects. Effects on SNAP-25 were similar in human and rat islets. Effects of diazoxide were mimicked by blocking secretion with somatostatin (rat islets). Blocking secretion by cooling abolished both glucose and diazoxide effects on SNAP-25. Total SNAP-25 mRNA as well as isoforms alpha and beta were increased by 24-h elevated glucose. Diazoxide failed to reverse the glucose effects on mRNA. However, effects of diazoxide on SNAP-25 protein were nullified by proteasome inhibitors (ALLN, MG-132, and epoxomicin) but not by lysosomal inhibition (NH(4)Cl). Exocytosis per se modifies SNAREs by a process linked to proteasomal activation.

    Biochemical and biophysical research communications 2005;329;3;1118-26

  • Support for association between ADHD and two candidate genes: NET1 and DRD1.

    Bobb AJ, Addington AM, Sidransky E, Gornick MC, Lerch JP, Greenstein DK, Clasen LS, Sharp WS, Inoff-Germain G, Wavrant-De Vrièze F, Arcos-Burgos M, Straub RE, Hardy JA, Castellanos FX and Rapoport JL

    Child Psychiatry Branch, NIMH, NIH, Bethesda, Maryland 20892-1600, USA.

    Attention deficit hyperactivity disorder (ADHD) is a common, multifactorial disorder with significant genetic contribution. Multiple candidate genes have been studied in ADHD, including the norepinephrine transporter (NET1) and dopamine D1 receptor (DRD1). NET1 is implicated in ADHD because of the efficacy of atomoxetine, a selective noradrenergic reuptake inhibitor, in the treatment of ADHD. DRD1 is primarily implicated through mouse models of ADHD. DNA from 163 ADHD probands, 192 parents, and 129 healthy controls was used to investigate possible associations between ADHD and polymorphisms in 12 previously studied candidate genes (5-HT1B, 5-HT2A, 5-HT2C, ADRA2A, CHRNA4, COMT, DAT1, DRD1, DRD4, DRD5, NET1, and SNAP-25). Analyses included case-control and family-based methods, and dimensional measures of behavior, cognition, and anatomic brain magnetic resonance imaging (MRI). Of the 12 genes examined, two showed a significant association with ADHD. Transmission disequilibrium test (TDT) analysis revealed significant association of two NET1 single nucleotide polymorphisms (SNPs) with ADHD (P < or = 0.009); case-control analysis revealed significant association of two DRD1 SNPs with ADHD (P < or = 0.008). No behavioral, cognitive, or brain MRI volume measurement significantly differed across NET1 or DRD1 genotypes at an alpha of 0.01. This study provides support for an association between ADHD and polymorphisms in both NET1 and DRD1; polymorphisms in ten other candidate genes were not associated with ADHD. Because family-based and case-control methods gave divergent results, both should be used in genetic studies of ADHD.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2005;134B;1;67-72

  • EBAG9 adds a new layer of control on large dense-core vesicle exocytosis via interaction with Snapin.

    Rüder C, Reimer T, Delgado-Martinez I, Hermosilla R, Engelsberg A, Nehring R, Dörken B and Rehm A

    Department of Hematology, Oncology, and Tumor Immunology, Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany.

    Regulated exocytosis is subject to several modulatory steps that include phosphorylation events and transient protein-protein interactions. The estrogen receptor-binding fragment-associated gene9 (EBAG9) gene product was recently identified as a modulator of tumor-associated O-linked glycan expression in nonneuronal cells; however, this molecule is expressed physiologically in essentially all mammalian tissues. Particular interest has developed toward this molecule because in some human tumor entities high expression levels correlated with clinical prognosis. To gain insight into the cellular function of EBAG9, we scored for interaction partners by using the yeast two-hybrid system. Here, we demonstrate that EBAG9 interacts with Snapin, which is likely to be a modulator of Synaptotagmin-associated regulated exocytosis. Strengthening of this interaction inhibited regulated secretion of neuropeptide Y from PC12 cells, whereas evoked neurotransmitter release from hippocampal neurons remained unaltered. Mechanistically, EBAG9 decreased phosphorylation of Snapin; subsequently, association of Snapin with synaptosome-associated protein of 25 kDa (SNAP25) and SNAP23 was diminished. We suggest that the occurrence of SNAP23, Snapin, and EBAG9 also in nonneuronal cells might extend the modulatory role of EBAG9 to a broad range of secretory cells. The conjunction between EBAG9 and Snapin adds an additional layer of control on exocytosis processes; in addition, mechanistic evidence is provided that inhibition of phosphorylation has a regulatory function in exocytosis.

    Molecular biology of the cell 2005;16;3;1245-57

  • A direct interaction between Cdc42 and vesicle-associated membrane protein 2 regulates SNARE-dependent insulin exocytosis.

    Nevins AK and Thurmond DC

    Department of Biochemistry and Molecular Biology and the Center for Diabetes Research, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.

    In pancreatic beta cells, insulin granule exocytosis is regulated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein (SNAP) receptor) proteins, and this is coupled to cortical F-actin reorganization via the Rho family GTPase Cdc42 by an unknown mechanism. We investigated interactions among the target SNARE protein Syntaxin 1A and the vesicle-associated membrane SNARE protein (VAMP2) with Cdc42 and compared these structural interactions with their functional importance to glucose-stimulated insulin secretion in MIN6 beta cells. Subcellular fractionation analyses revealed a parallel redistribution of Cdc42 and VAMP2 from the granule fraction to the plasma membrane in response to glucose that temporally corresponded with the glucose-induced activation of Cdc42. Moreover, within these fractions Cdc42 and VAMP2 were found to co-immunoprecipitate under basal and glucose-stimulated conditions, suggesting that they moved as a complex. Furthermore, VAMP2 bound both GST-Cdc42-GTPgammaS and GST-Cdc42-GDP, indicating that the Cdc42-VAMP2 complex could form under both cytosolic GDP-bound Cdc42 and plasma membrane GTP-bound Cdc42 conformational conditions. In vitro binding analyses showed that VAMP2 bound directly to Cdc42 and that a heterotrimeric complex with Syntaxin 1A could also be formed. Deletion analyses of VAMP2 revealed that only the N-terminal 28 residues were required for Cdc42 binding. Expression of this 28-residue VAMP2 peptide in MIN6 beta cells resulted in the specific impairment of glucose-stimulated insulin secretion, indicating a functional importance for the Cdc42-VAMP2 interaction. Taken together, these data suggest a mechanism whereby glucose activates Cdc42 to induce the targeting of intracellular Cdc42-VAMP2-insulin granule complexes to Syntaxin 1A at the plasma membrane.

    Funded by: NIDDK NIH HHS: T32 DK064466-01

    The Journal of biological chemistry 2005;280;3;1944-52

  • Netherton syndrome with extensive skin peeling and failure to thrive due to a homozygous frameshift mutation in SPINK5.

    Geyer AS, Ratajczak P, Pol-Rodriguez M, Millar WS, Garzon M and Richard G

    Department of Dermatology, Columbia University Medical Center, Columbia University, New York, NY, USA.

    Background: Netherton syndrome (NTS) is a rare autosomal recessive multisystem disorder characterized by congenital erythroderma and ichthyosis, hair shaft abnormalities and immune dysregulation. The disorder is caused by deleterious mutations in the SPINK5 gene, encoding the serine protease inhibitor LEKTI.

    Objective: Our objective was to investigate if the erythrodermic variant of peeling skin syndrome is also caused by SPINK5 mutations and to study the consequences of the disease on infantile brain development.

    Methods: In an infant with extensive erythroderma, peeling skin and failure to thrive, we analyzed the SPINK5 gene for pathogenic mutations by direct DNA sequencing and performed repeated brain MRI studies with diffusion-weighted imaging.

    Results: We identified a homozygous 4-base-pair insertion in exon 5 of SPINK5, which introduces a premature termination codon and appears to be a common mutation among West Indies islanders. MRI analyses revealed a persistent diffuse volume loss.

    Conclusion: Our results confirm that early truncation mutations of the coding sequence of SPINK5 produce a severe phenotype and that generalized peeling skin is one of the manifestations of NTS. We further demonstrate for the first time that NTS may be associated with MRI abnormalities indicative of a permanent tissue injury of the brain.

    Funded by: NIAMS NIH HHS: AR02141, AR38923, AR47157

    Dermatology (Basel, Switzerland) 2005;210;4;308-14

  • Huntingtin-interacting protein HIP14 is a palmitoyl transferase involved in palmitoylation and trafficking of multiple neuronal proteins.

    Huang K, Yanai A, Kang R, Arstikaitis P, Singaraja RR, Metzler M, Mullard A, Haigh B, Gauthier-Campbell C, Gutekunst CA, Hayden MR and El-Husseini A

    Department of Psychiatry, Brain Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

    In neurons, posttranslational modification by palmitate regulates the trafficking and function of signaling molecules, neurotransmitter receptors, and associated synaptic scaffolding proteins. However, the enzymatic machinery involved in protein palmitoylation has remained elusive. Here, using biochemical assays, we show that huntingtin (htt) interacting protein, HIP14, is a neuronal palmitoyl transferase (PAT). HIP14 shows remarkable substrate specificity for neuronal proteins, including SNAP-25, PSD-95, GAD65, synaptotagmin I, and htt. Conversely, HIP14 is catalytically invariant toward paralemmin and synaptotagmin VII. Exogenous HIP14 enhances palmitoylation-dependent vesicular trafficking of several acylated proteins in both heterologous cells and neurons. Moreover, interference with endogenous expression of HIP14 reduces clustering of PSD-95 and GAD65 in neurons. These findings define HIP14 as a mammalian palmitoyl transferase involved in the palmitoylation and trafficking of multiple neuronal proteins.

    Neuron 2004;44;6;977-86

  • Substrate recognition strategy for botulinum neurotoxin serotype A.

    Breidenbach MA and Brunger AT

    Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305, USA.

    Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus. CNTs impair neuronal exocytosis through specific proteolysis of essential proteins called SNAREs. SNARE assembly into a low-energy ternary complex is believed to catalyse membrane fusion, precipitating neurotransmitter release; this process is attenuated in response to SNARE proteolysis. Site-specific SNARE hydrolysis is catalysed by the CNT light chains, a unique group of zinc-dependent endopeptidases. The means by which a CNT properly identifies and cleaves its target SNARE has been a subject of much speculation; it is thought to use one or more regions of enzyme-substrate interaction remote from the active site (exosites). Here we report the first structure of a CNT endopeptidase in complex with its target SNARE at a resolution of 2.1 A: botulinum neurotoxin serotype A (BoNT/A) protease bound to human SNAP-25. The structure, together with enzyme kinetic data, reveals an array of exosites that determine substrate specificity. Substrate orientation is similar to that of the general zinc-dependent metalloprotease thermolysin. We observe significant structural changes near the toxin's catalytic pocket upon substrate binding, probably serving to render the protease competent for catalysis. The novel structures of the substrate-recognition exosites could be used for designing inhibitors specific to BoNT/A.

    Nature 2004;432;7019;925-9

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • From ORFeome to biology: a functional genomics pipeline.

    Wiemann S, Arlt D, Huber W, Wellenreuther R, Schleeger S, Mehrle A, Bechtel S, Sauermann M, Korf U, Pepperkok R, Sültmann H and Poustka A

    Molecular Genome Analysis, German Cancer Research Center, 69120 Heidelberg, Germany. s.wiemann@dkfz.de

    As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease. We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction. Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network. We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information. All information is presented in a Web database (http://www.dkfz.de/LIFEdb). It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy.

    Genome research 2004;14;10B;2136-44

  • Haplotype analysis of SNAP-25 suggests a role in the aetiology of ADHD.

    Mill J, Richards S, Knight J, Curran S, Taylor E and Asherson P

    Social, Genetic, and Developmental Psychiatry Research Centre, Institute of Psychiatry, Kings College, London, UK. spjgjsm@iop.kcl.ac.uk

    Several lines of evidence suggest a role for SNAP-25 (synaptosomal-associated protein of 25 kDa) in the genetic aetiology of ADHD. Most notable is the coloboma mouse mutant, which displays spontaneous hyperactivity and is hemizygous for a deletion spanning this gene. We have screened the SNAP-25 gene using denaturing high-performance liquid chromatography and sequencing, and genotyped six polymorphic single-nucleotide polymorphisms and two microsatellites in a clinically ascertained sample of 188 probands. Several markers were found to show association with ADHD, both individually and in combination with other markers to form multimarker haplotypes. Analyses of transmission by parental sex suggested that the association of SNAP-25 with ADHD is largely due to transmission of alleles from paternal chromosomes to affected probands, suggesting that this locus may be subject to genomic imprinting. Overall our data provide some evidence for a role of this gene in ADHD, although the precise causal functional variant is yet to be ascertained.

    Molecular psychiatry 2004;9;8;801-10

  • A role for SNAP-25 but not VAMPs in store-mediated Ca2+ entry in human platelets.

    Redondo PC, Harper AG, Salido GM, Pariente JA, Sage SO and Rosado JA

    Department of Physiology, University of Extremadura, Cáceres, Spain.

    Store-mediated Ca2+ entry (SMCE) is a major mechanism for Ca2+ influx in non-excitable cells. Recently, a conformational coupling mechanism allowing coupling between transient receptor potential channels (TRPCs) and IP3 receptors has been proposed to activate SMCE. Here we have investigated the role of two soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs), which are involved in membrane trafficking and docking, in SMCE in human platelets. We found that the synaptosome-associated protein (SNAP-25) and the vesicle-associated membrane proteins (VAMP) coimmunoprecipitate with hTRPC1 in platelets. Treatment with botulinum toxin (BoNT) E or with tetanus toxin (TeTx), induced cleavage and inactivation of SNAP-25 and VAMPs, respectively. BoNTs significantly reduced thapsigargin- (TG) and agonist-evoked SMCE. Treatment with BoNTs once SMCE had been activated decreased Ca2+ entry, indicating that SNAP-25 is required for the activation and maintenance of SMCE. In contrast, treatment with TeTx had no effect on either the activation or the maintenance of SMCE in platelets. Finally, treatment with BoNT E impaired the coupling between naturally expressed hTRPC1 and IP3 receptor type II in platelets. From these findings we suggest SNAP-25 has a role in SMCE in human platelets.

    The Journal of physiology 2004;558;Pt 1;99-109

  • Synaptotagmin interaction with the syntaxin/SNAP-25 dimer is mediated by an evolutionarily conserved motif and is sensitive to inositol hexakisphosphate.

    Rickman C, Archer DA, Meunier FA, Craxton M, Fukuda M, Burgoyne RD and Davletov B

    Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.

    Synaptotagmins are membrane proteins that possess tandem C2 domains and play an important role in regulated membrane fusion in metazoan organisms. Here we show that both synaptotagmins I and II, the two major neuronal isoforms, can interact with the syntaxin/synaptosomal-associated protein of 25 kDa (SNAP-25) dimer, the immediate precursor of the soluble NSF attachment protein receptor (SNARE) fusion complex. A stretch of basic amino acids highly conserved throughout the animal kingdom is responsible for this calcium-independent interaction. Inositol hexakisphosphate modulates synaptotagmin coupling to the syntaxin/SNAP-25 dimer, which is mirrored by changes in chromaffin cell exocytosis. Our results shed new light on the functional importance of the conserved polybasic synaptotagmin motif, suggesting that synaptotagmin interacts with the t-SNARE dimer to up-regulate the probability of SNARE-mediated membrane fusion.

    The Journal of biological chemistry 2004;279;13;12574-9

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • A dual mechanism controlling the localization and function of exocytic v-SNAREs.

    Martinez-Arca S, Rudge R, Vacca M, Raposo G, Camonis J, Proux-Gillardeaux V, Daviet L, Formstecher E, Hamburger A, Filippini F, D'Esposito M and Galli T

    Membrane Traffic and Neuronal Plasticity, Institut National de la Santé et de la Recherche Médicale U536, Institut du Fer-à-Moulin, 75005 Paris, France.

    SNARE [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor] proteins are essential for membrane fusion but their regulation is not yet fully understood. We have previously shown that the amino-terminal Longin domain of the v-SNARE TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein)/VAMP7 plays an inhibitory role in neurite outgrowth. The goal of this study was to investigate the regulation of TI-VAMP as a model of v-SNARE regulation. We show here that the Longin domain (LD) plays a dual role. First, it negatively regulates the ability of TI-VAMP and of a Longin/Synaptobrevin chimera to participate in SNARE complexes. Second, it interacts with the adaptor complex AP-3 and this interaction targets TI-VAMP to late endosomes. Accordingly, in mocha cells lacking AP-3 delta, TI-VAMP is retained in an early endosomal compartment. Furthermore, TI-VAMPc, an isoform of TI-VAMP lacking part of the LD, does not interact with AP-3, and therefore is not targeted to late endosomes; however, this shorter LD still inhibits SNARE-complex formation. These findings support a mechanism controlling both localization and function of TI-VAMP through the LD and clathrin adaptors. Moreover, they point to the amino-terminal domains of SNARE proteins as multifunctional modules responsible for the fine tuning of SNARE function.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;15;9011-6

  • Hrs regulates early endosome fusion by inhibiting formation of an endosomal SNARE complex.

    Sun W, Yan Q, Vida TA and Bean AJ

    The University of Texas Health Science Center, Dept. of Neurobiology and Anatomy, 6431 Fannin Street, MSB 7.208, Houston, TX 77030, USA.

    Movement through the endocytic pathway occurs principally via a series of membrane fusion and fission reactions that allow sorting of molecules to be recycled from those to be degraded. Endosome fusion is dependent on SNARE proteins, although the nature of the proteins involved and their regulation has not been fully elucidated. We found that the endosome-associated hepatocyte responsive serum phosphoprotein (Hrs) inhibited the homotypic fusion of early endosomes. A region of Hrs predicted to form a coiled coil required for binding the Q-SNARE, SNAP-25, mimicked the inhibition of endosome fusion produced by full-length Hrs, and was sufficient for endosome binding. SNAP-25, syntaxin 13, and VAMP2 were bound from rat brain membranes to the Hrs coiled-coil domain. Syntaxin 13 inhibited early endosomal fusion and botulinum toxin/E inhibition of early endosomal fusion was reversed by addition of SNAP-25(150-206), confirming a role for syntaxin 13, and establishing a role for SNAP-25 in endosomal fusion. Hrs inhibited formation of the syntaxin 13-SNAP-25-VAMP2 complex by displacing VAMP2 from the complex. These data suggest that SNAP-25 is a receptor for Hrs on early endosomal membranes and that the binding of Hrs to SNAP-25 on endosomal membranes inhibits formation of a SNARE complex required for homotypic endosome fusion.

    Funded by: NCI NIH HHS: CA 16672, P30 CA016672; NIGMS NIH HHS: GM 52092, R01 GM052092; NIMH NIH HHS: MH 58920, R01 MH058920

    The Journal of cell biology 2003;162;1;125-37

  • Elevated cerebrospinal fluid SNAP-25 in schizophrenia.

    Thompson PM, Kelley M, Yao J, Tsai G and van Kammen DP

    Department of Psychiatry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA.

    Background: Research suggests an association between abnormal exocytosis and schizophrenia. We previously demonstrated increased synaptosomal associated protein, 25 kDa (SNAP-25), a member of the exocytotic mechanism, in the cerebrospinal fluid (CSF) of schizophrenia subjects. In this study, we explored SNAP-25 level and clinical variables in a new group of subjects.

    Methods: Twenty-five haloperidol-treated subjects with chronic schizophrenia and twenty-five healthy control subjects participated in the study. Subjects received haloperidol treatment for at least 3 months and then had a lumbar puncture (n = 19). Medication was replaced by placebo, and the lumbar puncture was repeated (n = 25) after 6 weeks or sooner if limited psychotic symptoms occurred. We measured the level of SNAP-25 in the CSF and symptoms with the Brief Psychiatric Rating Scale (BPRS).

    Results: In both haloperidol (p =.001) and placebo (p =.001) treatment conditions, SNAP-25 was elevated. There was no significant difference in SNAP-25 level between conditions. We identified significant positive correlations among SNAP-25 and the BPRS total score and psychosis and thinking disturbance subscales in subjects on haloperidol.

    Conclusions: These observations confirm our previous report of elevated CSF SNAP-25 and suggest that synaptic pathology may be linked with the pathophysiology of schizophrenia.

    Biological psychiatry 2003;53;12;1132-7

  • SNAP-25 reduction in the hippocampus of patients with schizophrenia.

    Thompson PM, Egbufoama S and Vawter MP

    Mood and Anxiety Disorders Division, Department of Psychiatry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, Mail Code 7792, San Antonio, TX 78229-3900, USA. Thompsonp@uthscsa.edu

    In this study, the authors sought to replicate the findings of reduced synaptosomal associated protein 25 kDa (SNAP-25) immunoreactivity in the hippocampus of patients with schizophrenia. The authors also measured N-methyl-D-aspartate (NMDA) receptor 1 (NR1) receptor subunit to determine if glutamatergic synapses were involved with the loss of SNAP-25. We found 49% less SNAP-25 immunointensity in the schizophrenic group (n=7) compared to the control (n=8) or bipolar groups (n=4) (P=.004). There was no change in NMDA NR1 levels in the three groups. The authors confirm the previous report of less SNAP-25 immunoreactivity in the hippocampus using a different cohort of patients with schizophrenia. It also appears that NMDA NR1 was unchanged, indicating that the overall level of NMDA glutamatergic synapses in hippocampus is normal. These data add to evidence suggesting that in schizophrenia the molecular pathology of the hippocampus involves presynaptic components.

    Progress in neuro-psychopharmacology & biological psychiatry 2003;27;3;411-7

  • Biased paternal transmission of SNAP-25 risk alleles in attention-deficit hyperactivity disorder.

    Kustanovich V, Merriman B, McGough J, McCracken JT, Smalley SL and Nelson SF

    Department of Human Genetics, UCLA School of Medicine, University of California, Los Angeles, USA.

    Attention-deficit hyperactivity disorder (ADHD) is the most common childhood psychiatric disorder, affecting 5-10% of school-age children. Although the biological basis of this disorder is unknown, twin and family studies provide strong evidence that ADHD has a genetic basis involving multiple genes. A previous study found an association between ADHD and two polymorphisms in the 3' untranslated region (UTR) of SNAP-25, a gene encoding a synaptic vesicle docking protein known to play a role in the hyperactivity observed in the Coloboma mouse strain. In this paper, we test biased transmission of the 3' UTR SNAP-25 haplotype using a larger ADHD sample of 113 families with 207 affected children. Using the transmission disequilibrium test (TDT), we found a trend consistent with biased transmission of the TC haplotype of SNAP-25 in all transmissions and detected a significant distortion (P=0.027) when paternal transmissions were evaluated.

    Funded by: NIGMS NIH HHS: GM07104; NIMH NIH HHS: MH58277

    Molecular psychiatry 2003;8;3;309-15

  • Increased RNA levels of the 25 kDa synaptosomal associated protein in brain samples of adult patients with Down Syndrome.

    Greber-Platzer S, Fleischmann C, Nussbaumer C, Cairns N and Lubec G

    Department of Pediatrics, University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. susanne.greber-platzer@akh-wien.ac.at

    The synaptosomal associated protein of 25kDa (SNAP-25) is widely distributed in the brain and reduced in neurodegenerative diseases. In a previous paper we have shown reduced amounts of SNAP-25 protein in adult Down Syndrome (DS) brain. Neuronal cell death and downregulation at the transcriptional level may be responsible for the decrease. Therefore SNAP-25 mRNA levels were determined in frontal cortex and cerebellum of adult DS by a competitive reverse transcription-polymerase chain reaction. We found significantly increased mRNA levels in DS either related to 10 ng total RNA (P < 0.05 level in cerebellum: DS 2622 +/- 1081 attogr mean +/- SEM and controls 154 +/- 37 attogr. mean +/- SEM) or normalized versus the house keeping gene beta-actin (P < 0.05 level in frontal cortex: DS 1324 +/- 504 attogr. mean +/- SEM and control 131 +/- 32 attogr. mean +/- SEM; P<0.01 in cerebellum: DS 632 +/- 189 attogr. mean +/- SEM and control 21 +/- 2 attogr. mean +/- SEM). The main finding of this study shows elevated mRNA levels of SNAP-25 in adult DS brain whereas histological and protein-chemical evidence for decreased synaptosomal structures including SNAP-25 in a comparable cohort has been reported. We suggest compensatory mechanisms for the upregulation at the transcriptional level. We propose that SNAP-25 as many other brain proteins are regulated by protein stability rather than at the mRNA level.

    Neuroscience letters 2003;336;2;77-80

  • The heavy chain of conventional kinesin interacts with the SNARE proteins SNAP25 and SNAP23.

    Diefenbach RJ, Diefenbach E, Douglas MW and Cunningham AL

    Centre For Virus Research, Westmead Millennium Institute, Westmead Hospital and The University of Sydney, NSW 2145, Australia. russell_diefenbach@wmi.usyd.edu.au

    Recent studies on the conventional motor protein kinesin have identified a putative cargo-binding domain (residues 827-906) within the heavy chain. To identify possible cargo proteins which bind to this kinesin domain, we employed a yeast two-hybrid assay. A human brain cDNA library was screened, using as bait residues 814-963 of human ubiquitous kinesin heavy chain. This screen initially identified synaptosome-associated protein of 25 kDa (SNAP25) as a kinesin-binding protein. Subsequently, synaptosome-associated protein of 23 kDa (SNAP23), the nonneuronal homologue of SNAP25, was also confirmed to interact with kinesin. The sites of interaction, determined from in vivo and in vitro assays, are the N-terminus of SNAP25 (residues 1-84) and the cargo-binding domain of kinesin heavy chain (residues 814-907). Both regions are composed almost entirely of heptad repeats, suggesting the interaction between heavy chain and SNAP25 is that of a coiled-coil. The observation that SNAP23 also binds to residues 814-907 of heavy chain would indicate that the minimal kinesin-binding domain of SNAP23 and SNAP25 is most likely residues 45-84 (SNAP25 numbering), a heptad-repeat region in both proteins. The major binding site for kinesin light chain in kinesin heavy chain was mapped to residues 789-813 at the C-terminal end of the heavy chain stalk domain. Weak binding of light chain was also detected at the N-terminus of the heavy chain tail domain (residues 814-854). In support of separate binding sites on heavy chain for light chain and SNAPs, a complex of heavy and light chains was observed to interact with SNAP25 and SNAP23.

    Biochemistry 2002;41;50;14906-15

  • Differential phosphorylation of SNAP-25 in vivo by protein kinase C and protein kinase A.

    Hepp R, Cabaniols JP and Roche PA

    Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bldg. 10, Room 4B36, Bethesda, MD 20892, USA.

    SNAP-25 is a key protein required for the fusion of synaptic vesicles with the plasma membrane during exocytosis. This study establishes that SNAP-25 is differentially phosphorylated by protein kinase C and protein kinase A in neuroendocrine PC12 cells. Using phosphopeptide mapping and site-directed mutagenesis we identified both Thr138 and Ser187 as the targets of SNAP-25 phosphorylation by protein kinase C and Thr138 as the exclusive site of SNAP-25 phosphorylation by protein kinase A in vivo. Finally, despite published data to the contrary, we demonstrate that stimulation of regulated exocytosis under physiological conditions is independent of a measurable increase in SNAP-25 phosphorylation in PC12 cells.

    FEBS letters 2002;532;1-2;52-6

  • Action of complexin on SNARE complex.

    Hu K, Carroll J, Rickman C and Davletov B

    MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.

    Calcium-dependent synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: synaptobrevin/vesicle-associated membrane protein in the vesicular membrane and syntaxin and SNAP-25 in the presynaptic membrane. The SNAREs form a thermodynamically stable complex that is believed to drive fusion of vesicular and presynaptic membranes. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a positive regulator of synaptic vesicle exocytosis. Complexin binds selectively to the neuronal SNARE complex, but how this promotes exocytosis remains unknown. Here we used purified full-length and truncated SNARE proteins and a gel shift assay to show that the action of complexin on SNARE complex depends strictly on the transmembrane regions of syntaxin and synaptobrevin. By means of a preparative immunoaffinity procedure to achieve total extraction of SNARE complex from brain, we demonstrated that complexin is the only neuronal protein that tightly associates with it. Our data indicated that, in the presence of complexin, the neuronal SNARE proteins assemble directly into a complex in which the transmembrane regions interact. We propose that complexin facilitates neuronal exocytosis by promoting interaction between the complementary syntaxin and synaptobrevin transmembrane regions that reside in opposing membranes prior to fusion.

    The Journal of biological chemistry 2002;277;44;41652-6

  • Synaptosome-associated protein of 25 kilodaltons modulates Kv2.1 voltage-dependent K(+) channels in neuroendocrine islet beta-cells through an interaction with the channel N terminus.

    MacDonald PE, Wang G, Tsuk S, Dodo C, Kang Y, Tang L, Wheeler MB, Cattral MS, Lakey JR, Salapatek AM, Lotan I and Gaisano HY

    Department of Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8.

    Insulin secretion is initiated by ionic events involving membrane depolarization and Ca(2+) entry, whereas exocytic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate exocytosis itself. In the present study, we characterize the interaction of the SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) with the beta-cell voltage-dependent K(+) channel Kv2.1. Expression of Kv2.1, SNAP-25, and syntaxin 1A was detected in human islet lysates by Western blot, and coimmunoprecipitation studies showed that heterologously expressed SNAP-25 and syntaxin 1A associate with Kv2.1. SNAP-25 reduced currents from recombinant Kv2.1 channels by approximately 70% without affecting channel localization. This inhibitory effect could be partially alleviated by codialysis of a Kv2.1N-terminal peptide that can bind in vitro SNAP-25, but not the Kv2.1C-terminal peptide. Similarly, SNAP-25 blocked voltage-dependent outward K(+) currents from rat beta-cells by approximately 40%, an effect that was completely reversed by codialysis of the Kv2.1N fragment. Finally, SNAP-25 had no effect on outward K(+) currents in beta-cells where Kv2.1 channels had been functionally knocked out using a dominant-negative approach, indicating that the interaction is specific to Kv2.1 channels as compared with other beta-cell Kv channels. This study demonstrates that SNAP-25 can regulate Kv2.1 through an interaction at the channel N terminus and supports the hypothesis that SNARE proteins modulate secretion through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.

    Funded by: NIDDK NIH HHS: DK-55160

    Molecular endocrinology (Baltimore, Md.) 2002;16;11;2452-61

  • SNAP-25 traffics to the plasma membrane by a syntaxin-independent mechanism.

    Loranger SS and Linder ME

    Department of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110-1093, USA.

    SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are essential for vesicle docking and fusion. SNAP-25, syntaxin 1A, and synaptobrevin/vesicle-associated membrane protein (VAMP) are SNARE proteins that mediate fusion of synaptic vesicles with the plasma membrane. It has been proposed that interactions of SNAP-25 with syntaxin 1A are required for initial membrane attachment of SNAP-25 (Vogel, K., Cabaniols, J.-P., and Roche, P. (2000) J. Biol. Chem. 275, 2959-2965). However, we have shown previously that residues 85-120 of the SNAP-25 interhelical domain, which do not interact with syntaxin, are necessary and sufficient for palmitoylation and plasma membrane localization of a green fluorescent protein reporter molecule (Gonzalo, S., Greentree, W. K., and Linder, M. E. (1999) J. Biol. Chem. 274, 21313-21318). To clarify the role of syntaxin in membrane targeting of SNAP-25, we studied a SNAP-25 point mutant (G43D) that does not interact with syntaxin. SNAP-25 G43D/green fluorescent protein was palmitoylated and localized at the plasma membrane. Newly synthesized SNAP-25 G43D had the same kinetics of membrane association as the wild-type protein. Furthermore, expression of a cytosolic mutant syntaxin 1A did not interfere with SNAP-25 membrane interactions or palmitoylation in the neuronal cell line NG108-15. Exogenously expressed SNAP-25 targets efficiently to the plasma membrane in cells of neuronal origin but only partially in HeLa cells, a neurosecretion-incompetent line. This phenotype was not rescued when syntaxin 1A was co-expressed with SNAP-25. Our data support a syntaxin-independent mechanism of membrane targeting for SNAP-25.

    Funded by: NIGMS NIH HHS: T32 GM 07067

    The Journal of biological chemistry 2002;277;37;34303-9

  • Amisyn, a novel syntaxin-binding protein that may regulate SNARE complex assembly.

    Scales SJ, Hesser BA, Masuda ES and Scheller RH

    Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5345, USA.

    The regulation of SNARE complex assembly likely plays an important role in governing the specificity as well as the timing of membrane fusion. Here we identify a novel brain-enriched protein, amisyn, with a tomosyn- and VAMP-like coiled-coil-forming domain that binds specifically to syntaxin 1a and syntaxin 4 both in vitro and in vivo, as assessed by co-immunoprecipitation from rat brain. Amisyn is mostly cytosolic, but a fraction co-sediments with membranes. The amisyn coil domain can form SNARE complexes of greater thermostability than can VAMP2 with syntaxin 1a and SNAP-25 in vitro, but it lacks a transmembrane anchor and so cannot act as a v-SNARE in this complex. The amisyn coil domain prevents the SNAP-25 C-terminally mediated rescue of botulinum neurotoxin E inhibition of norepinephrine exocytosis in permeabilized PC12 cells to a greater extent than it prevents the regular exocytosis of these vesicles. We propose that amisyn forms nonfusogenic complexes with syntaxin 1a and SNAP-25, holding them in a conformation ready for VAMP2 to replace it to mediate the membrane fusion event, thereby contributing to the regulation of SNARE complex formation.

    The Journal of biological chemistry 2002;277;31;28271-9

  • Ca2+-dependent synaptotagmin binding to SNAP-25 is essential for Ca2+-triggered exocytosis.

    Zhang X, Kim-Miller MJ, Fukuda M, Kowalchyk JA and Martin TF

    Department of Biochemistry, University of Wisconsin, Madison 53706, USA.

    Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but the mechanism by which Ca2+ triggers membrane fusion is uncertain. Previous studies suggested that SNAP-25 plays a role in the Ca2+ regulation of secretion. We found that synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for Ca2+-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion.

    Funded by: NIDDK NIH HHS: DK25861; NIGMS NIH HHS: T32 GM07507; NIMH NIH HHS: MH12694

    Neuron 2002;34;4;599-611

  • Association study of a SNAP-25 microsatellite and attention deficit hyperactivity disorder.

    Mill J, Curran S, Kent L, Gould A, Huckett L, Richards S, Taylor E and Asherson P

    Social, Genetic, and Developmental Psychiatry Research Centre, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London, United Kingdom. j.mill@iop.kcl.ac.uk

    Several lines of evidence implicate synaptosomal-associated protein of 25 kDa (SNAP-25) in the etiology of attention deficit hyperactivity disorder (ADHD). Most notably, the coloboma mouse mutant, considered to be a good animal model of hyperactivity, has a deletion spanning this gene. Introducing a SNAP-25 transgene into these animals alleviates hyperlocomotion. We have identified a novel microsatellite repeat in SNAP-25 located between the 5'UTR and the first coding exon, and tested for association with ADHD. Case-control analyses suggest there may be a role of this polymorphism in ADHD, with one allele over-represented in controls and another over-represented in probands. Within-family tests of linkage and association confirmed these findings. Further work is needed to ascertain the role of SNAP-25 in ADHD and assess the functional significance of this polymorphism.

    American journal of medical genetics 2002;114;3;269-71

  • Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion.

    Hu K, Carroll J, Fedorovich S, Rickman C, Sukhodub A and Davletov B

    MRC Laboratory of Molecular Biology, Cambridge, UK.

    Release of neurotransmitter occurs when synaptic vesicles fuse with the plasma membrane. This neuronal exocytosis is triggered by calcium and requires three SNARE (soluble-N-ethylmaleimide-sensitive factor attachment protein receptors) proteins: synaptobrevin (also known as VAMP) on the synaptic vesicle, and syntaxin and SNAP-25 on the plasma membrane. Neuronal SNARE proteins form a parallel four-helix bundle that is thought to drive the fusion of opposing membranes. As formation of this SNARE complex in solution does not require calcium, it is not clear what function calcium has in triggering SNARE-mediated membrane fusion. We now demonstrate that whereas syntaxin and SNAP-25 in target membranes are freely available for SNARE complex formation, availability of synaptobrevin on synaptic vesicles is very limited. Calcium at micromolar concentrations triggers SNARE complex formation and fusion between synaptic vesicles and reconstituted target membranes. Although calcium does promote interaction of SNARE proteins between opposing membranes, it does not act by releasing synaptobrevin from synaptic vesicle restriction. Rather, our data suggest a mechanism in which calcium-triggered membrane apposition enables syntaxin and SNAP-25 to engage synaptobrevin, leading to membrane fusion.

    Nature 2002;415;6872;646-50

  • Three-dimensional structure of the complexin/SNARE complex.

    Chen X, Tomchick DR, Kovrigin E, Araç D, Machius M, Südhof TC and Rizo J

    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

    During neurotransmitter release, the neuronal SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 form a four-helix bundle, the SNARE complex, that pulls the synaptic vesicle and plasma membranes together possibly causing membrane fusion. Complexin binds tightly to the SNARE complex and is essential for efficient Ca(2+)-evoked neurotransmitter release. A combined X-ray and TROSY-based NMR study now reveals the atomic structure of the complexin/SNARE complex. Complexin binds in an antiparallel alpha-helical conformation to the groove between the synaptobrevin and syntaxin helices. This interaction stabilizes the interface between these two helices, which bears the repulsive forces between the apposed membranes. These results suggest that complexin stabilizes the fully assembled SNARE complex as a key step that enables the exquisitely high speed of Ca(2+)-evoked neurotransmitter release.

    Funded by: NINDS NIH HHS: NS37200

    Neuron 2002;33;3;397-409

  • The DNA sequence and comparative analysis of human chromosome 20.

    Deloukas P, Matthews LH, Ashurst J, Burton J, Gilbert JG, Jones M, Stavrides G, Almeida JP, Babbage AK, Bagguley CL, Bailey J, Barlow KF, Bates KN, Beard LM, Beare DM, Beasley OP, Bird CP, Blakey SE, Bridgeman AM, Brown AJ, Buck D, Burrill W, Butler AP, Carder C, Carter NP, Chapman JC, Clamp M, Clark G, Clark LN, Clark SY, Clee CM, Clegg S, Cobley VE, Collier RE, Connor R, Corby NR, Coulson A, Coville GJ, Deadman R, Dhami P, Dunn M, Ellington AG, Frankland JA, Fraser A, French L, Garner P, Grafham DV, Griffiths C, Griffiths MN, Gwilliam R, Hall RE, Hammond S, Harley JL, Heath PD, Ho S, Holden JL, Howden PJ, Huckle E, Hunt AR, Hunt SE, Jekosch K, Johnson CM, Johnson D, Kay MP, Kimberley AM, King A, Knights A, Laird GK, Lawlor S, Lehvaslaiho MH, Leversha M, Lloyd C, Lloyd DM, Lovell JD, Marsh VL, Martin SL, McConnachie LJ, McLay K, McMurray AA, Milne S, Mistry D, Moore MJ, Mullikin JC, Nickerson T, Oliver K, Parker A, Patel R, Pearce TA, Peck AI, Phillimore BJ, Prathalingam SR, Plumb RW, Ramsay H, Rice CM, Ross MT, Scott CE, Sehra HK, Shownkeen R, Sims S, Skuce CD, Smith ML, Soderlund C, Steward CA, Sulston JE, Swann M, Sycamore N, Taylor R, Tee L, Thomas DW, Thorpe A, Tracey A, Tromans AC, Vaudin M, Wall M, Wallis JM, Whitehead SL, Whittaker P, Willey DL, Williams L, Williams SA, Wilming L, Wray PW, Hubbard T, Durbin RM, Bentley DR, Beck S and Rogers J

    The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. panos@sanger.ac.uk

    The finished sequence of human chromosome 20 comprises 59,187,298 base pairs (bp) and represents 99.4% of the euchromatic DNA. A single contig of 26 megabases (Mb) spans the entire short arm, and five contigs separated by gaps totalling 320 kb span the long arm of this metacentric chromosome. An additional 234,339 bp of sequence has been determined within the pericentromeric region of the long arm. We annotated 727 genes and 168 pseudogenes in the sequence. About 64% of these genes have a 5' and a 3' untranslated region and a complete open reading frame. Comparative analysis of the sequence of chromosome 20 to whole-genome shotgun-sequence data of two other vertebrates, the mouse Mus musculus and the puffer fish Tetraodon nigroviridis, provides an independent measure of the efficiency of gene annotation, and indicates that this analysis may account for more than 95% of all coding exons and almost all genes.

    Nature 2001;414;6866;865-71

  • Spring, a novel RING finger protein that regulates synaptic vesicle exocytosis.

    Li Y, Chin LS, Weigel C and Li L

    Department of Pharmacology and Cell and Molecular Physiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.

    The synaptosome-associated protein of 25 kDa (SNAP-25) interacts with syntaxin 1 and vesicle-associated membrane protein 2 (VAMP2) to form a ternary soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex that is essential for synaptic vesicle exocytosis. We report a novel RING finger protein, Spring, that specifically interacts with SNAP-25. Spring is exclusively expressed in brain and is concentrated at synapses. The association of Spring with SNAP-25 abolishes the ability of SNAP-25 to interact with syntaxin 1 and VAMP2 and prevents the assembly of the SNARE complex. Overexpression of Spring or its SNAP-25-interacting domain reduces Ca(2+)-dependent exocytosis from PC12 cells. These results indicate that Spring may act as a regulator of synaptic vesicle exocytosis by controlling the availability of SNAP-25 for the SNARE complex formation.

    Funded by: NINDS NIH HHS: NS37939

    The Journal of biological chemistry 2001;276;44;40824-33

  • Direct interaction of the Rab3 effector RIM with Ca2+ channels, SNAP-25, and synaptotagmin.

    Coppola T, Magnin-Luthi S, Perret-Menoud V, Gattesco S, Schiavo G and Regazzi R

    Institut de Biologie Cellulaire et de Morphologie, University of Lausanne, 1005 Lausanne, Switzerland.

    To define the role of the Rab3-interacting molecule RIM in exocytosis we searched for additional binding partners of the protein. We found that the two C(2) domains of RIM display properties analogous to those of the C(2)B domain of synaptotagmin-I. Thus, RIM-C(2)A and RIM-C(2)B bind in a Ca(2+)-independent manner to alpha1B, the pore-forming subunit of N-type Ca(2+) channels (EC(50) = approximately 20 nm). They also weakly interact with the alpha1C but not the alpha1D subunit of L-type Ca(2+) channels. In addition, the C(2) domains of RIM associate with SNAP-25 and synaptotagmin-I. The binding affinities for these two proteins are 203 and 24 nm, respectively, for RIM-C(2)A and 224 and 16 nm for RIM-C(2)B. The interactions of the C(2) domains of RIM with SNAP-25 and synaptotagmin-I are modulated by Ca(2+). Thus, in the presence of Ca(2+) (EC(50) = approximately 75 microm) the interaction with synaptotagmin-I is increased, whereas SNAP-25 binding is reduced. Synaptotagmin-I binding is abolished by mutations in two positively charged amino acids in the C(2) domains of RIM and by the addition of inositol polyphosphates. We propose that the Rab3 effector RIM is a scaffold protein that participates through its multiple binding partners in the docking and fusion of secretory vesicles at the release sites.

    The Journal of biological chemistry 2001;276;35;32756-62

  • Induction of neurite outgrowth in PC12 cells by alpha -phenyl-N-tert-butylnitron through activation of protein kinase C and the Ras-extracellular signal-regulated kinase pathway.

    Tsuji M, Inanami O and Kuwabara M

    Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.

    The spin trap alpha-phenyl-N-tert-butylnitron (PBN) is widely used for studies of the biological effects of free radicals. We previously reported the protective effects of PBN against ischemia-reperfusion injury in gerbil hippocampus by its activation of extracellular signal-regulated kinase (ERK) and suppression of both stress-activated protein kinase and p38 mitogen-activated protein kinase. In the present study, we found that PBN induced neurite outgrowth accompanied by ERK activation in PC12 cells in a dose-dependent manner. The induction of neurite outgrowth was inhibited significantly not only by transient transfection of PC12 cells with dominant negative Ras, but also by treatment with mitogen-activated protein kinase/ERK kinase inhibitor PD98059. The activation of receptor tyrosine kinase TrkA was not involved in PBN-induced neurite outgrowth. A protein kinase C (PKC) inhibitor, GF109203X, was found to inhibit neurite outgrowth. The activation of PKCepsilon was observed after PBN stimulation. PBN-induced neurite outgrowth and ERK activation were counteracted by the thiol-based antioxidant N-acetylcysteine. From these results, it was concluded that PBN induced neurite outgrowth in PC12 cells through activation of the Ras-ERK pathway and PKC.

    The Journal of biological chemistry 2001;276;35;32779-85

  • The survival of motor neurons (SMN) protein interacts with the snoRNP proteins fibrillarin and GAR1.

    Pellizzoni L, Baccon J, Charroux B and Dreyfuss G

    Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

    Background: The survival of motor neurons (SMN) protein is the protein product of the spinal muscular atrophy (SMA) disease gene. SMN and its associated proteins Gemin2, Gemin3, and Gemin4 form a large complex that plays a role in snRNP assembly, pre-mRNA splicing, and transcription. The functions of SMN in these processes are mediated by a direct interaction of SMN with components of these machineries, such as Sm proteins and RNA helicase A.

    Results: We show that SMN binds directly to fibrillarin and GAR1. Fibrillarin and GAR1 are specific markers of the two classes of small nucleolar ribonucleoprotein particles (snoRNPs) that are involved in posttranscriptional processing and modification of ribosomal RNA. SMN interaction requires the arginine- and glycine-rich domains of both fibrillarin and GAR1 and is defective in SMN mutants found in some SMA patients. Coimmunoprecipitations demonstrate that the SMN complex associates with fibrillarin and with GAR1 in vivo. The inhibition of RNA polymerase I transcription causes a transient redistribution of SMN to the nucleolar periphery and loss of fibrillarin and GAR1 colocalization with SMN in gems. Furthermore, the expression of a dominant-negative mutant of SMN (SMNDeltaN27) causes snoRNPs to accumulate outside of the nucleolus in structures that also contain components of gems and coiled (Cajal) bodies.

    Conclusions: These findings identify fibrillarin and GAR1 as novel interactors of SMN and suggest a function for the SMN complex in the assembly and metabolism of snoRNPs. We propose that the SMN complex performs functions necessary for the biogenesis and function of diverse ribonucleoprotein complexes.

    Current biology : CB 2001;11;14;1079-88

  • FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes.

    Raiborg C, Bremnes B, Mehlum A, Gillooly DJ, D'Arrigo A, Stang E and Stenmark H

    Department of Biochemistry, Institute for Cancer Research, the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.

    Hrs, an essential tyrosine kinase substrate, has been implicated in intracellular trafficking and signal transduction pathways. The protein contains several distinctive domains, including an N-terminal VHS domain, a phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding FYVE domain and two coiled-coil domains. Here we have investigated the roles of these domains in the subcellular localisation of Hrs. Hrs was found to colocalise extensively with EEA1, an established marker of early endosomes. While the membrane association of EEA1 was abolished in the presence of a dominant negative mutant of the endosomal GTPase Rab5, the localisation of Hrs to early endosomes was Rab5 independent. The VHS-domain was nonessential for the subcellular targeting of Hrs. In contrast, the FYVE domain as well as the second coiled-coil domain, which has been shown to bind to SNAP-25, were required for targeting of Hrs to early endosomes. A small construct consisting of only these two domains was correctly localised to early endosomes, whereas a point mutation (R183A) in the PtdIns(3)P-binding pocket of the FYVE domain inhibited the membrane targeting of Hrs. Thus, like EEA1, the endosomal targeting of Hrs is mediated by a PtdIns(3)P-binding FYVE domain in cooperation with an additional domain. We speculate that binding to PtdIns(3)P and a SNAP-25-related molecule may target Hrs specifically to early endosomes.

    Journal of cell science 2001;114;Pt 12;2255-63

  • DNA cloning using in vitro site-specific recombination.

    Hartley JL, Temple GF and Brasch MA

    Life Technologies, Inc., Rockville, Maryland 20850, USA. jhartley@lifetech.com

    As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function. Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis. We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. Numerous DNA segments can be transferred in parallel into many different vector backgrounds, providing an approach to high-throughput, in-depth functional analysis of genes and rapid optimization of protein expression. The resulting subclones maintain orientation and reading frame register, allowing amino- and carboxy-terminal translation fusions to be generated. In this paper, we outline the concepts of this approach and provide several examples that highlight some of its potential.

    Genome research 2000;10;11;1788-95

  • Membrane localization and biological activity of SNAP-25 cysteine mutants in insulin-secreting cells.

    Gonelle-Gispert C, Molinete M, Halban PA and Sadoul K

    Louis-Jeantet Research Laboratories, University Medical Center, 1211 Geneva 4, Switzerland. carmen.gonelle@medecine.unige.ch

    The tSNARE SNAP-25 is expressed in pancreatic (beta)-cells and is involved in the regulated release of insulin. It has been shown previously that SNAP-25 associates with the plasma membrane consequent to palmitoylation of one or more cysteines in the central region of the molecule. The importance of palmitolyation in the biological function of SNAP-25 in exocytosis was not addressed. Furthermore, studies on both SNAP-25 and its non-palmitoylated homologues SNAP-29 and sec9, have suggested an alternative or complementary mechanism for membrane association involving interaction with syntaxin. To address these issues, we have now studied the behavior and biological activity of cysteine mutant SNAP-25 in insulin-secreting (HIT) cells. While 91% of native SNAP-25 was associated with the membrane, this value decreased to 56% for the single cysteine mutant C85/A and to 10% for the double (C85,88/A) and quadruple (C85,88,90,92/A) mutants. The mutant SNAP-25 forms were all found to bind syntaxin 1A with equal efficacy. Over-expression of syntaxin 1A in HIT cells allowed for partial relocalization of both the double and quadruple SNAP-25 cys mutants to the membrane. By introducing a further mutation to the SNAP-25 molecules to render them resistant to botulinum neurotoxin E, it was possible to study their ability to reconstitute regulated insulin secretion in toxin-treated HIT cells. Native SNAP-25 was able to fully reconstitute secretory activity in such cells. Despite the fact that the single cysteine mutant was significantly displaced to the cytosol, it still displayed 82% activity in the secretion reconstitution assay, and a similar discrepancy was seen for the double mutant. Even the quadruple mutant with no remaining cysteines was able to support a minimal level of secretion. It is concluded that both palmitoylation and binding to syntaxin are implicated in membrane association of SNAP-25. This as well as the discrepancy between membrane localization and biological activity of the cysteine mutants, suggests a complex, multi-component process for association of SNAP-25 with the membrane and its recruitment to a biologically productive state.

    Journal of cell science 2000;113 ( Pt 18);3197-205

  • Synaptosome-associated protein of 25 kilodaltons in oocytes and steroid-producing cells of rat and human ovary: molecular analysis and regulation by gonadotropins.

    Grosse J, Bulling A, Brucker C, Berg U, Amsterdam A, Mayerhofer A and Gratzl M

    Anatomisches Institut, Technische Universität München, D-80802 München, Germany.

    The synaptosome-associated protein of 25 kDa (SNAP-25) is crucially involved in exocytosis in neurons. The aim of this study was to investigate whether it is present in the ovary. We found SNAP-25 to be expressed in nonneuronal cells of the rat and human ovary, namely in all oocytes and in steroidogenic cells, including granulosa cells (GC) of large antral follicles and luteal cells. Both isoforms, SNAP-25a and b, were found in the ovary. Oocytes obtained by laser capture microdissection were shown to express SNAP-25b, whereas SNAP-25a was found in rat GC and human luteinized GC. Immunohistochemical observations of strong SNAP-25 staining in GC of large growing antral follicles compared with absent or weak staining in small follicles suggested a role in folliculogenesis. To study a presumed regulation of SNAP-25, we used a rat GC line (GFSHR-17), which expresses FSH receptors, and luteinizing human GC, which express LH receptors. FSH elevated SNAP-25 mRNA and protein levels about fivefold within 24 h in GFSHR-17 cells. The cAMP analogue dibutyryl-cAMP (db-cAMP) mimicked this action of FSH. The effects of both db-cAMP and FSH were inhibited by the protein kinase A (PKA) inhibitor H89. In contrast, SNAP-25 protein and mRNA-levels were not altered by LH/hCG in luteinized human GC. Our results for the first time identify SNAP-25b in oocytes and SNAP-25a in steroidogenic cells of the mammalian ovary. SNAP-25a and b may be involved in different exocytotic processes in these cell types.

    Biology of reproduction 2000;63;2;643-50

  • Hrs interacts with SNAP-25 and regulates Ca(2+)-dependent exocytosis.

    Kwong J, Roundabush FL, Hutton Moore P, Montague M, Oldham W, Li Y, Chin LS and Li L

    Department of Pharmacology, Bowles Center for Alcohol Studies, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA.

    Synaptosome-associated protein of 25 kDa (SNAP-25) is a neuronal membrane protein essential for synaptic vesicle exocytosis. To investigate the mechanisms by which SNAP-25 mediates neurosecretion, we performed a search for proteins that interact with SNAP-25 using a yeast two-hybrid screen. Here, we report the isolation and characterization of a SNAP-25-interacting protein that is the rat homologue of mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Hrs specifically interacts with SNAP-25, but not SNAP-23/syndet. The association of Hrs and SNAP-25 is mediated via coiled-coil interactions. Using an Hrs-specific antibody, we have shown that Hrs is highly enriched in brain, where it codistributes with SNAP-25 in most brain regions. Subcellular fractionation studies demonstrate that in brain, Hrs exists in both cytosolic and membrane-associated pools. Studies using indirect immunofluorescence and confocal microscopy reveal that, in addition to early endosomes, Hrs is also localized to large dense-core secretory granules and synaptic-like microvesicles in nerve growth factor-differentiated PC12 cells. Moreover, overexpression of Hrs in PC12 cells inhibits Ca(2+)-dependent exocytosis. These results suggest that Hrs is involved in regulation of neurosecretion through interaction with SNAP-25.

    Funded by: NINDS NIH HHS: NS37939

    Journal of cell science 2000;113 ( Pt 12);2273-84

  • Syntaxin 13 is a developmentally regulated SNARE involved in neurite outgrowth and endosomal trafficking.

    Hirling H, Steiner P, Chaperon C, Marsault R, Regazzi R and Catsicas S

    Institut de Biologie Cellulaire et de Morphologie (IBCM), Rue du Bugnon 9, 1005 Lausanne, Switzerland. harald.hirling@ibcm.unil.ch

    In addition to its role in exocytosis, SNAP-25 is essential for axonal outgrowth. In order to identify SNARE proteins involved in neurite growth we have used SNAP-25 antibodies to affinity-purify protein complexes enriched in developing rat brain membrane extracts. We have identified a complex between SNAP-25 and syntaxin 13 predominantly present in brain at embryonic or early postnatal stages. We show that syntaxin 13 is developmentally regulated with a decrease in adult brain. In differentiated neuroendocrine PC12 cells as well as primary cortical neurons the protein is localized to a punctated and tubular staining in the perinuclear region and along processes with high levels in the central region of growth cones. Carboxy-terminally tagged syntaxin 13 was also detected on the plasma membrane by in vivo surface-labelling where it colocalized with SNAP-25. Syntaxin 13 has recently been shown to be implicated in early endosomal trafficking. In our study, colocalization with internalized transferrin in the cell body and along neurites confirmed endosomal location in both compartments. Finally, overexpression of full-length syntaxin 13 enhanced neurite outgrowth in NGF-stimulated PC12 cells, whilst it had no effect on regulated secretion. The data suggest that a syntaxin 13-dependent endocytic trafficking step plays a limiting role in membrane expansion during neuronal development.

    The European journal of neuroscience 2000;12;6;1913-23

  • SCAMP1 function in endocytosis.

    Fernández-Chacón R, Achiriloaie M, Janz R, Albanesi JP and Südhof TC

    Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.

    Secretory carrier membrane proteins (SCAMPs) are ubiquitous components of recycling vesicles that shuttle between the plasma membrane, endosomes, and the trans-Golgi complex. SCAMPs contain multiple N-terminal NPF repeats and four highly conserved transmembrane regions. NPF repeats often interact with EH domain proteins that function in budding of transport vesicles from the plasma membrane or the Golgi complex. We now show that the NPF repeats of SCAMP1 bind to two EH domain proteins, intersectin 1, which is involved in endocytic budding at the plasma membrane, and gamma-synergin, which may mediate the budding of vesicles in the trans-Golgi complex. Expression of SCAMP1 lacking the N-terminal NPF repeats potently inhibited transferrin uptake by endocytosis. Our data suggest that one of the functions of SCAMPs is to participate in endocytosis via a mechanism which may involve the recruitment of clathrin coats to the plasma membrane and the trans-Golgi network.

    Funded by: NIGMS NIH HHS: R01-GM55562

    The Journal of biological chemistry 2000;275;17;12752-6

  • The C terminus of SNAP25 is essential for Ca(2+)-dependent binding of synaptotagmin to SNARE complexes.

    Gerona RR, Larsen EC, Kowalchyk JA and Martin TF

    Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.

    The plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) and the vesicle SNARE protein vesicle-associated membrane protein (VAMP) are essential for a late Ca(2+)-dependent step in regulated exocytosis, but their precise roles and regulation by Ca(2+) are poorly understood. Botulinum neurotoxin (BoNT) E, a protease that cleaves SNAP25 at Arg(180)-Ile(181), completely inhibits this late step in PC12 cell membranes, whereas BoNT A, which cleaves SNAP25 at Gln(197)-Arg(198), is only partially inhibitory. The difference in toxin effectiveness was found to result from a reversal of BoNT A but not BoNT E inhibition by elevated Ca(2+) concentrations. BoNT A treatment essentially increased the Ca(2+) concentration required to activate exocytosis, which suggested a role for the C terminus of SNAP25 in the Ca(2+) regulation of exocytosis. Synaptotagmin, a proposed Ca(2+) sensor for exocytosis, was found to bind SNAP25 in a Ca(2+)-stimulated manner. Ca(2+)-dependent binding was abolished by BoNT E treatment, whereas BoNT A treatment increased the Ca(2+) concentration required for binding. The C terminus of SNAP25 was also essential for Ca(2+)-dependent synaptotagmin binding to SNAP25. syntaxin and SNAP25.syntaxin.VAMP SNARE complexes. These results clarify classical observations on the Ca(2+) reversal of BoNT A inhibition of neurosecretion, and they suggest that an essential role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca(2+)-dependent interactions between synaptotagmin and SNARE protein complexes.

    Funded by: NIDDK NIH HHS: DK25861, DK40428; NIGMS NIH HHS: GM07215

    The Journal of biological chemistry 2000;275;9;6328-36

  • Targeting of SNAP-25 to membranes is mediated by its association with the target SNARE syntaxin.

    Vogel K, Cabaniols JP and Roche PA

    Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.

    The docking and fusion of synaptic vesicles with the presynaptic plasma membrane require the interaction of the vesicle-associated membrane protein VAMP with the plasma membrane proteins syntaxin and SNAP-25. Both of these proteins behave as integral membrane proteins, although they are unusual in that they insert into membranes post-translationally. Whereas VAMP and syntaxin possess hydrophobic transmembrane domains, SNAP-25 does not, and it is widely believed that SNAP-25 traffics to and inserts into membranes by post-translational palmitoylation. In pulse-chase biosynthesis studies, we now show that SNAP-25 and syntaxin rapidly bind to each other while still in the cytosol of neuroendocrine and transfected heterologous cells. Cell fractionation studies revealed that cytosolic SNAP-25.syntaxin complexes then traffic to and insert into membranes. Furthermore, the association of SNAP-25 with membranes is dramatically enhanced by syntaxin, and the transmembrane domain of syntaxin is essential for this effect. Surprisingly, despite the importance of the SNAP-25 palmitoylation domain for membrane anchoring at steady state, removal of this domain did not inhibit the initial association of newly synthesized SNAP-25 with membranes in the presence of syntaxin. These data demonstrate that the initial attachment of newly synthesized SNAP-25 to membranes is a consequence of its association with syntaxin and that it is only after syntaxin-mediated membrane tethering that SNAP-25 is palmitoylated.

    The Journal of biological chemistry 2000;275;4;2959-65

  • SNIP, a novel SNAP-25-interacting protein implicated in regulated exocytosis.

    Chin LS, Nugent RD, Raynor MC, Vavalle JP and Li L

    Departments of Pharmacology and Physiology, Bowles Center for Alcohol Studies, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

    Synaptosome-associated protein of 25 kDa (SNAP-25) is a presynaptic membrane protein that has been clearly implicated in membrane fusion in both developing and mature neurons, although its mechanisms of action are unclear. We have now identified a novel SNAP-25-interacting protein named SNIP. SNIP is a hydrophilic, 145-kDa protein that comprises two predicted coiled-coil domains, two highly charged regions, and two proline-rich domains with multiple PPXY and PXXP motifs. SNIP is selectively expressed in brain where it co-distributes with SNAP-25 in most brain regions. Biochemical studies have revealed that SNIP is tightly associated with the brain cytoskeleton. Subcellular fractionation and immunofluorescence localization studies have demonstrated that SNIP co-localizes with SNAP-25 as well as the cortical actin cytoskeleton, suggesting that SNIP serves as a linker protein connecting SNAP-25 to the submembranous cytoskeleton. By using deletion analysis, we have mapped the binding domains of SNIP and SNAP-25, and we have demonstrated that the SNIP-SNAP-25 association is mediated via coiled-coil interactions. Moreover, we have shown that overexpression of SNIP or its SNAP-25-interacting domain inhibits Ca(2+)-dependent exocytosis from PC12 cells. These results indicate that SNIP is involved in regulation of neurosecretion, perhaps via its interaction with SNAP-25 and the cytoskeleton.

    Funded by: NINDS NIH HHS: NS37939

    The Journal of biological chemistry 2000;275;2;1191-200

  • A conformational switch in syntaxin during exocytosis: role of munc18.

    Dulubova I, Sugita S, Hill S, Hosaka M, Fernandez I, Südhof TC and Rizo J

    Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA.

    Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N-terminal domain, a C-terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP-25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18-1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a 'closed' conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an 'open' syntaxin conformation. Using site-directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18-1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18-1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin-munc18-1 complex and the core complex.

    Funded by: NINDS NIH HHS: NS37200

    The EMBO journal 1999;18;16;4372-82

  • EHSH1/intersectin, a protein that contains EH and SH3 domains and binds to dynamin and SNAP-25. A protein connection between exocytosis and endocytosis?

    Okamoto M, Schoch S and Südhof TC

    Center for Basic Neuroscience, Howard Hughes Medical Institute, and the Department of Molecular Genetics, The University of Texas Southwestern Medical School, Dallas Texas 75235, USA.

    In yeast two-hybrid screens for proteins that bind to SNAP-25 and may be involved in exocytosis, we isolated a protein called EHSH1 (for EH domain/SH3 domain-containing protein). Cloning of full-length cDNAs revealed that EHSH1 is composed of an N-terminal region with two EH domains, a central region that is enriched in lysine, leucine, glutamate, arginine, and glutamine (KLERQ domain), and a C-terminal region comprised of five SH3 domains. The third SH3 domain is alternatively spliced. Data bank searches demonstrated that EHSH1 is very similar to Xenopus and human intersectins and to human SH3P17. In addition, we identified expressed sequence tags that encode a second isoform of EHSH1, called EHSH2. EHSH1 is abundantly expressed in brain and at lower levels in all other tissues tested. In binding studies, we found that the central KLERQ domain of EHSH1 binds to recombinant or native brain SNAP-25 and SNAP-23. The C-terminal SH3 domains, by contrast, quantitatively interact with dynamin, a protein involved in endocytosis. Dynamin strongly binds to the alternatively spliced central SH3 domain (SH3C) and the two C-terminal SH3 domains (SH3D and SH3E) but not to the N-terminal SH3 domains (SH3A and SH3B). Immunoprecipitations confirmed that both dynamin and SNAP-25 are complexed to EHSH1 in brain. Our data suggest that EHSH1/intersectin may be a novel adaptor protein that couples endocytic membrane traffic to exocytosis. The ability of multiple SH3 domains in EHSH1 to bind to dynamin suggests that EHSH1 can cluster several dynamin molecules in a manner that is regulated by alternative splicing.

    The Journal of biological chemistry 1999;274;26;18446-54

  • Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properties.

    Fasshauer D, Antonin W, Margittai M, Pabst S and Jahn R

    Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.

    Assembly of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins between two opposing membranes is thought to be the key event that initiates membrane fusion. Many new SNARE proteins have recently been localized to distinct intracellular compartments, supporting the view that sets of specific SNAREs are specialized for distinct trafficking steps. We have now investigated whether other SNAREs can form complexes with components of the synaptic SNARE complex including synaptobrevin/VAMP 2, SNAP-25, and syntaxin 1. When the Q-SNAREs syntaxin 2, 3, and 4, and the R-SNARE endobrevin/VAMP 8 were used in various combinations, heat-resistant complexes were formed. Limited proteolysis revealed that these complexes contained a protease-resistant core similar to that of the synaptic complex. All complexes were disassembled by the ATPase N-ethylmaleimide-sensitive fusion protein and its cofactor alpha-SNAP. Circular dichroism spectroscopy showed that major conformational changes occur during assembly, which are associated with induction of structure from unstructured monomers. Furthermore, no preference for synaptobrevin was observed during the assembly of the synaptic complex when endobrevin/VAMP 8 was present in equal concentrations. We conclude that cognate and non-cognate SNARE complexes are very similar with respect to biophysical properties, assembly, and disassembly, suggesting that specificity of membrane fusion in intracellular membrane traffic is not due to intrinsic specificity of SNARE pairing.

    The Journal of biological chemistry 1999;274;22;15440-6

  • Human platelets contain SNARE proteins and a Sec1p homologue that interacts with syntaxin 4 and is phosphorylated after thrombin activation: implications for platelet secretion.

    Reed GL, Houng AK and Fitzgerald ML

    Cardiovascular Biology Laboratory, Harvard School of Public Health, Cardiac Unit, Massachusetts General Hospital, Boston, MA, USA. reed@cvlab.harvard.edu

    In response to thrombin and other extracellular activators, platelets secrete molecules from large intracellular vesicles (granules) to initiate thrombosis. Little is known about the molecular machinery responsible for vesicle docking and secretion in platelets and the linkage of that machinery to cell activation. We found that platelet membranes contain a full complement of interacting proteins-VAMP, SNAP-25, and syntaxin 4-that are necessary for vesicle docking and fusion with the plasma membrane. Platelets also contain an uncharacterized homologue of the Sec1p family that appears to regulate vesicle docking through its binding with a cognate syntaxin. This platelet Sec1 protein (PSP) bound to syntaxin 4 and thereby excluded the binding of SNAP-25 with syntaxin 4, an interaction critical to vesicle docking. As predicted by its sequence, PSP was detected predominantly in the platelet cytosol and was phosphorylated in vitro by protein kinase C (PKC), a secretion-linked kinase, incorporating 0.87 +/- 0.11 mol of PO4 per mole of protein. PSP was also specifically phosphorylated in permeabilized platelets after cellular stimulation by phorbol esters or thrombin and this phosphorylation was blocked by the PKC inhibitor Ro-31-8220. Phosphorylation by PKC in vitro inhibited PSP from binding to syntaxin 4. Taken together, these studies indicate that platelets, like neurons and other cells capable of regulated secretion, contain a unique complement of interacting vesicle docking proteins and PSP, a putative regulator of vesicle docking. The PKC-dependent phosphorylation of PSP in activated platelets and its inhibitory effects on syntaxin 4 binding provide a novel functional link that may be important in coupling the processes of cell activation, intracellular signaling, and secretion.

    Funded by: NHLBI NIH HHS: R01 HL57314-01

    Blood 1999;93;8;2617-26

  • SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion.

    Gonelle-Gispert C, Halban PA, Niemann H, Palmer M, Catsicas S and Sadoul K

    Laboratoires de Recherche Louis Jeantet, Centre Médical Universitaire, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland.

    The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N-ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in pancreatic B-cells and its cleavage by botulinum neurotoxin E (BoNT/E) abolishes stimulated secretion of insulin. In the nervous system, two SNAP-25 isoforms (a and b) have been described that are produced by alternative splicing. Here it is shown, using reverse transcriptase PCR, that messages for both SNAP-25 isoforms are expressed in primary pancreatic B and non-B cells as well as in insulin-secreting cell lines. After transfection, both isoforms can be detected at the plasma membrane as well as in an intracellular perinuclear region in the insulin-secreting cell line, HIT. To test for the functional role of the two isoforms in insulin secretion, mutant forms of SNAP-25a and b resistant against cleavage by BoNT/E were generated. Such mutant SNAP-25, when expressed in HIT cells, is not inactivated by BoNT/E and its ability to restore insulin secretion can thus be investigated. To obtain the toxin-resistant mutant isoforms, the sequence around the BoNT/E cleavage site (R176QIDRIM182) was changed to P176QIKRIT182. This is the sequence of the equivalent region of human SNAP-23 (P187-T194), which has been shown to be resistant to BoNT/E. The mutant SNAP-25 was resistant to BoNT/E in vitro and in vivo and both mutant isoforms were able to reconstitute insulin secretion from toxin-treated HIT cells.

    The Biochemical journal 1999;339 ( Pt 1);159-65

  • Snapin: a SNARE-associated protein implicated in synaptic transmission.

    Ilardi JM, Mochida S and Sheng ZH

    Synaptic Function Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Synaptic vesicle docking and fusion are mediated by the assembly of a stable SNARE core complex of proteins, which include the synaptic vesicle membrane protein VAMP/synaptobrevin and the plasmalemmal proteins syntaxin and SNAP-25. We have now identified another SNAP-25-binding protein, called Snapin. Snapin was enriched in neurons and exclusively located on synaptic vesicle membranes. It associated with the SNARE complex through direct interaction with SNAP-25. Binding of recombinant Snapin-CT to SNAP-25 blocked the association of the SNARE complex with synaptotagmin. Introduction of Snapin-CT and peptides containing the SNAP-25 binding sequence into presynaptic superior cervical ganglion neurons in culture reversibly inhibited synaptic transmission. These results suggest that Snapin is an important component of the neurotransmitter release process through its modulation of the sequential interactions between the SNAREs and synaptotagmin.

    Nature neuroscience 1999;2;2;119-24

  • Three novel proteins of the syntaxin/SNAP-25 family.

    Steegmaier M, Yang B, Yoo JS, Huang B, Shen M, Yu S, Luo Y and Scheller RH

    Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305-5345, USA.

    Intracellular membrane traffic is thought to be regulated in part by soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) through the formation of complexes between these proteins present on vesicle and target membranes. All known SNARE-mediated fusion events involve members of the syntaxin and vesicle-associated membrane protein families. The diversity of mammalian membrane compartments predicts the existence of a large number of different syntaxin and vesicle-associated membrane protein genes. To further investigate the spectrum of SNAREs and their roles in membrane trafficking we characterized three novel members of the syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) subfamilies. The proteins are broadly expressed, suggesting a general role in vesicle trafficking, and localize to distinct membrane compartments. Syntaxin 8 co-localizes with markers of the endoplasmic reticulum. Syntaxin 17, a divergent member of the syntaxin family, partially overlaps with endoplasmic reticulum markers, and SNAP-29 is broadly localized on multiple membranes. SNAP-29 does not contain a predicted membrane anchor characteristic of other SNAREs. In vitro studies established that SNAP-29 is capable of binding to a broad range of syntaxins.

    The Journal of biological chemistry 1998;273;51;34171-9

  • Syntaxin 13 mediates cycling of plasma membrane proteins via tubulovesicular recycling endosomes.

    Prekeris R, Klumperman J, Chen YA and Scheller RH

    Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5428, USA.

    Endocytosis-mediated recycling of plasma membrane is a critical vesicle trafficking step important in diverse biological processes. The membrane trafficking decisions and sorting events take place in a series of heterogeneous and highly dynamic organelles, the endosomes. Syntaxin 13, a recently discovered member of the syntaxin family, has been suggested to play a role in mediating endosomal trafficking. To better understand the function of syntaxin 13 we examined its intracellular distribution in nonpolarized cells. By confocal immunofluorescence and electron microscopy, syntaxin 13 is primarily found in tubular early and recycling endosomes, where it colocalizes with transferrin receptor. Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas. Furthermore, anti-syntaxin 13 antibody inhibits transferrin receptor recycling in permeabilized PC12 cells. Immunoprecipitation of syntaxin 13 revealed that, in Triton X-100 extracts, syntaxin 13 is present in a complex(es) comprised of betaSNAP, VAMP 2/3, and SNAP-25. This complex(es) binds exogenously added alphaSNAP and NSF and dissociates in the presence of ATP, but not ATPgammaS. These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.

    The Journal of cell biology 1998;143;4;957-71

  • Protease resistance of syntaxin.SNAP-25.VAMP complexes. Implications for assembly and structure.

    Poirier MA, Hao JC, Malkus PN, Chan C, Moore MF, King DS and Bennett MK

    Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.

    A stable ternary complex formed with vesicle-associated membrane protein 2 (VAMP2) and plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) is proposed to function in synaptic vesicle exocytosis. To analyze the structural characteristics of this synaptic protein complex, recombinant binary (syntaxin 1A.SNAP-25), recombinant ternary, and native ternary complexes were subjected to limited trypsin proteolysis. The protected fragments, defined by amino-terminal sequencing and mass spectrometry, included a carboxyl-terminal region of syntaxin 1A, the cytoplasmic domain of VAMP2, and amino- and carboxyl-terminal regions of SNAP-25. Furthermore, separate amino- and carboxyl-terminal fragments of SNAP-25, when combined with VAMP2 and syntaxin 1A, were sufficient for stable complex assembly. Analysis of ternary complexes formed with full-length proteins revealed that the carboxyl-terminal transmembrane anchors of both syntaxin 1A and VAMP2 were protected from trypsin digestion. Moreover, the stability of ternary complexes was increased by inclusion of these transmembrane domains. These results suggest that the transmembrane domains of VAMP2 and syntaxin 1A contribute to complex assembly and stability and that amino- and carboxyl-terminal regions of SNAP-25 may function as independent domains.

    Funded by: NIGMS NIH HHS: GM 51313

    The Journal of biological chemistry 1998;273;18;11370-7

  • Targeting of SNAP-23 and SNAP-25 in polarized epithelial cells.

    Low SH, Roche PA, Anderson HA, van Ijzendoorn SC, Zhang M, Mostov KE and Weimbs T

    Department of Anatomy, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0452, USA.

    SNAP-23 is the ubiquitously expressed homologue of the neuronal SNAP-25, which functions in synaptic vesicle fusion. We have investigated the subcellular localization of SNAP-23 in polarized epithelial cells. In hepatocyte-derived HepG2 cells and in Madin-Darby canine kidney (MDCK) cells, the majority of SNAP-23 was present at both the basolateral and apical plasma membrane domains with little intracellular localization. This suggests that SNAP-23 does not function in intracellular fusion events but rather as a general plasma membrane t-SNARE. Canine SNAP-23 is efficiently cleaved by the botulinum neurotoxin E, suggesting that it is the toxin-sensitive factor previously found to be involved in plasma membrane fusion in MDCK cells. The localization of SNAP-25 in transfected MDCK cells was studied for comparison and was found to be identical to SNAP-23 with the exception that SNAP-25 was transported to the primary cilia protruding from the apical plasma membrane, which suggests that subtle differences in the targeting signals of both proteins exist. In contrast to its behavior in neurons, the distribution of SNAP-25 in MDCK cells remained unaltered by treatment with dibutyryl cAMP or forskolin, which, however, caused an increased growth of the primary cilia. Finally, we found that SNAP-23/25 and syntaxin 1A, when co-expressed in MDCK cells, do not stably interact with each other but are independently targeted to the plasma membrane and lysosomes, respectively.

    Funded by: NIAID NIH HHS: AI36953, AI39161, R01 AI25144

    The Journal of biological chemistry 1998;273;6;3422-30

  • Mints, Munc18-interacting proteins in synaptic vesicle exocytosis.

    Okamoto M and Südhof TC

    Howard Hughes Medical Institute and the Department of Molecular Genetics, University of Texas Southwestern Medical School, Dallas, Texas 75235, USA.

    Munc18-1 is a neuronal protein that interacts with syntaxin 1 and is required for synaptic vesicle exocytosis. We have now identified two Munc18-1-interacting proteins called Mint1 and Mint2 that may mediate the function of Munc18-1. Mint proteins are detectable only in brain and are composed of an N-terminal sequence that binds Munc18-1, a middle phosphotyrosine-binding domain, and two C-terminal PDZ domains thought to attach proteins to the plasma membrane. In brain, Mint proteins are part of a multimeric complex containing Munc18-1 and syntaxin that likely functions as an intermediate in synaptic vesicle docking/fusion. The phosphotyrosine-binding domain specifically binds to phosphatidylinositol phosphates known to be produced during vesicle exocytosis (Hay, J. C., Fisette, P. L., Jenkins, G. H., Fukami, K., Takonawa, T., Anderson, R. A., and Martin, T. F. J. (1995) Nature 374, 173-177). Our data suggest a model whereby local production of phosphatidylinositol phosphates may trigger the binding of vesicles to the active zone via the Mint.Munc18-1 complex in conjunction with syntaxin 1.

    The Journal of biological chemistry 1997;272;50;31459-64

  • Characterization of the palmitoylation domain of SNAP-25.

    Lane SR and Liu Y

    Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, U.S.A.

    SNAP-25 (synaptosomal associated protein of 25 kDa) is a neural specific protein that has been implicated in the synaptic vesicle docking and fusion process. It is tightly associated with membranes, and it is one of the major palmitoylated proteins found in neurons. The functional role of palmitoylation for SNAP-25 is unclear. In this report, we show that the palmitate of SNAP-25 is rapidly turned over in PC12 cells, with a half-life of approximately 3 h, and the half-life for the protein is 8 h. Mutation of Cys to Ser at positions 85, 88, 90, and 92 reduced the palmitoylation to 9, 21, 42, and 35% of the wild-type protein, respectively. Additional mutations of either Cys(85,88) or Cys(90,92) nearly abolished palmitoylation of the protein. A similar effect on membrane binding for the mutant SNAP-25 was observed, which correlated with the degree of palmitoylation. These results suggest that all four Cys residues are involved in palmitoylation and that membrane association of SNAP-25 may be regulated through dynamic palmitoylation.

    Funded by: NINDS NIH HHS: NS35167

    Journal of neurochemistry 1997;69;5;1864-9

  • Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library.

    Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A and Sugano S

    International and Interdisciplinary Studies, The University of Tokyo, Japan.

    Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.

    Gene 1997;200;1-2;149-56

  • N-Ethylmaleimide-sensitive factor (NSF) and alpha-soluble NSF attachment proteins (SNAP) mediate dissociation of GS28-syntaxin 5 Golgi SNAP receptors (SNARE) complex.

    Subramaniam VN, Loh E and Hong W

    Membrane Biology Laboratory, Institute of Molecular and Cell Biology, 15 Lower Kent Ridge Road, Singapore 119076, Singapore.

    Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) GS28 and syntaxin 5 can be reciprocally coimmunoprecipitated from Golgi extracts, suggesting that they exist in a protein complex. When Golgi extract is preincubated with soluble NSF attachment proteins (alpha-SNAP) and N-ethylmaleimide-sensitive factor (NSF) under conditions that allow ATP hydrolysis by NSF, GS28 and syntaxin 5 become dissociated. GS28 and syntaxin 5 remain in a protein complex when Golgi extract is preincubated with similar amounts of alpha-SNAP and NSF under conditions that prevent ATP hydrolysis by NSF, suggesting that ATP hydrolysis by NSF is necessary for dissociating the GS28-syntaxin 5 complex. Since preincubation of Golgi extract with either alpha-SNAP or NSF alone has no effect on the GS28-syntaxin 5 complex, a concerted action of alpha-SNAP and NSF therefore mediates the dissociation of the GS28-syntaxin 5 complex. Furthermore, GS28 but not syntaxin 5 is capable of binding to immobilized alpha-SNAP when the GS28-syntaxin 5 complex is dissociated.

    The Journal of biological chemistry 1997;272;41;25441-4

  • Inhibition of the binding of SNAP-23 to syntaxin 4 by Munc18c.

    Araki S, Tamori Y, Kawanishi M, Shinoda H, Masugi J, Mori H, Niki T, Okazawa H, Kubota T and Kasuga M

    Second Department of Internal Medicine, Kobe University School of Medicine, Japan.

    SNARE proteins have been implicated in the insulin-induced translocation of vesicles containing the GLUT4 glucose transporter to the plasma membrane of adipocytes. The role of the target SNARE SNAP-25 or its homologs in this process was investigated by screening a mouse adipocyte cDNA library with rat SNAP-25 and human SNAP-23 cDNA probes. Both positive clones isolated encoded a protein with 87% sequence identity to human SNAP-23, and which was therefore designated mouse SNAP-23. Immunoblot and immunofluorescence analyses revealed that SNAP-23 is located predominantly in the plasma membrane of 3T3-L1 adipocytes incubated in the absence or presence of insulin. Of syntaxins 1 to 5, SNAP-23 bound with the highest affinity to syntaxins 1 and 4 in the yeast two-hybrid system. Expression of SNAP-23, syntaxin 4, and the syntaxin-binding protein Munc 18c in COS cells revealed that Munc18c reduced the amount of SNAP-23 bound to syntaxin 4 in a concentration-dependent manner. These results suggest that the binding of SNAP-23 to syntaxin 4 is inhibited by Munc18c in adipocytes.

    Biochemical and biophysical research communications 1997;234;1;257-62

  • Effect of mutations in vesicle-associated membrane protein (VAMP) on the assembly of multimeric protein complexes.

    Hao JC, Salem N, Peng XR, Kelly RB and Bennett MK

    Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.

    The assembly of multimeric protein complexes that include vesicle-associated membrane protein 2 (VAMP-2) and the plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) are thought to reflect the biochemical correlates of synaptic vesicle targeting, priming, or fusion. Using a variety of protein-protein interaction assays and a series of deletion and point mutations, we have investigated the domains of VAMP-2 required for the formation of binary complexes with either syntaxin 1A or SNAP-25 and ternary complexes with both syntaxin 1A and SNAP-25. Deletions within the central conserved domain of VAMP-2 eliminated binding to either syntaxin 1A or both syntaxin 1A and SNAP-25. Although all of the deletion mutants were able to form ternary complexes, only some of these complexes were resistant to denaturation in sodium dodecyl sulfate. These results demonstrate that cooperative interactions result in the formation of at least two biochemically distinct classes of ternary complex. Two point mutations previously shown to have effects on the intracellular trafficking of VAMP-2 (M46A, reduced endocytosis and sorting to synaptic vesicles; N49A, enhanced sorting to synaptic vesicles) lie within a domain required for both syntaxin 1A and SNAP-25 binding. Syntaxin 1A and SNAP-25 binding was reduced by the M46A mutation and enhanced by the N49A mutation, suggesting that a correlation exists between the membrane-trafficking phenotype of the two VAMP-2 point mutants and their competence to form complexes with either syntaxin 1A or SNAP-25.

    Funded by: NIDA NIH HHS: DA 10154; NIGMS NIH HHS: GM 51313; NINDS NIH HHS: NS 09878

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1997;17;5;1596-603

  • Direct interaction of the rat unc-13 homologue Munc13-1 with the N terminus of syntaxin.

    Betz A, Okamoto M, Benseler F and Brose N

    Max-Planck-Institut für experimentelle Medizin, Abteilung Molekulare Neurobiologie, Hermann-Rein-Strasse 3, D-37075 Göttingen, Federal Republic of Germany.

    unc-13 mutants in Caenorhabditis elegans are characterized by a severe deficit in neurotransmitter release. Their phenotype is similar to that of the C. elegans unc-18 mutation, which is thought to affect synaptic vesicle docking to the active zone. This suggests a crucial role for the unc-13 gene product in the mediation or regulation of synaptic vesicle exocytosis. Munc13-1 is one of three closely related rat homologues of unc-13. Based on the high degree of similarity between unc-13 and Munc13 proteins, it is thought that their essential function has been conserved from C. elegans to mammals. Munc13-1 is a brain-specific peripheral membrane protein with multiple regulatory domains that may mediate diacylglycerol, phospholipid, and calcium binding. In the present study, we demonstrate by three independent methods that the C terminus of Munc13-1 interacts directly with a putative coiled coil domain in the N-terminal part of syntaxin. Syntaxin is a component of the exocytotic synaptic core complex, a heterotrimeric protein complex with an essential role in transmitter release. Through this interaction, Munc13-1 binds to a subpopulation of the exocytotic core complex containing synaptobrevin, SNAP25 (synaptosomal-associated protein of 25 kDa), and syntaxin, but to no other tested syntaxin-interacting or core complex-interacting protein. The site of interaction in syntaxin is similar to the binding site for the unc-18 homologue Munc18, but different from that of all other known syntaxin interactors. These data indicate that unc-13-related proteins may indeed be involved in the mediation or regulation of synaptic vesicle exocytosis by modulating or regulating core complex formation. The similarity between the unc-13 and unc-18 phenotypes is paralleled by the coincidence of the binding sites for Munc13-1 and Munc18 in syntaxin. It is possible that the phenotype of unc-13 and unc-18 mutations is caused by the inability of the respective mutated gene products to bind to syntaxin.

    The Journal of biological chemistry 1997;272;4;2520-6

  • Insulin-responsive tissues contain the core complex protein SNAP-25 (synaptosomal-associated protein 25) A and B isoforms in addition to syntaxin 4 and synaptobrevins 1 and 2.

    Jagadish MN, Fernandez CS, Hewish DR, Macaulay SL, Gough KH, Grusovin J, Verkuylen A, Cosgrove L, Alafaci A, Frenkel MJ and Ward CW

    CSIRO, Division of Biomolecular Engineering, Parkville, Victoria, Australia.

    SNAP-25 (synaptosomal-associated protein 25), syntaxin and synaptobrevin are the three SNARE [soluble NSF attachment protein receptor (where NSF = N-ethylmaleimide-sensitive fusion protein)] proteins that form the core complex involved in synaptic vesicle docking and subsequent fusion with the target membrane. The present study is aimed at understanding the mechanisms of fusion of vesicles carrying glucose transporter proteins with the plasma membrane in human insulin-responsive tissues. It describes the isolation and characterization of cDNA molecules encoding SNAP-25 A and B isoforms, syntaxin 4 and synaptobrevins (also known as vehicle-associated membrane proteins) from two major human insulin-responsive tissues, skeletal muscle and fat. The DNA and deduced amino acid sequences of SNAP-25 revealed perfect identity with the previously reported human neural SNAP-25 A and B isoforms. Our results indicate the presence of both isoforms both in insulin-responsive tissues and in in vitro cultured 3T3-L1 cells, but suggest a differential pattern of gene expression: isoform A is the major species in adipose tissue, and isoform B is the major species in skeletal muscle. The presence of SNAP-25 protein in 3T3-L1 cells was demonstrated by immunofluorescence microscopy using an anti-SNAP-25 monoclonal antibody. Immunoprecipitation experiments using the same monoclonal antibody also revealed the presence of SNAP-25 protein in plasma membrane fractions from rat epididymal fat pads. The syntaxin 4-encoding region from skeletal muscle contains five nucleotide differences from the previously reported placental cDNA sequence, two of which result in amino acid changes: Asp-174 to Glu and Val-269 to Ala. The synaptobrevin 1 cDNA from skeletal muscle contains two nucleotide differences when compared with the corresponding clone from neural tissues, one of which is silent and the other resulting in the amino acid change Thr-102 to Ala. The cDNA sequence of the protein from fat is identical with that of human synaptobrevin 1 from neural tissues. Furthermore, we have confirmed the presence of syntaxin 4 in fat and of synaptobrevin 2 in skeletal muscle by PCR amplification and Southern hybridization analysis. Using the yeast two-hybrid system, an interaction was observed between the full-length cytoplasmic domains of syntaxin 4 and synaptobrevin 2, a vesicle membrane SNARE previously shown by others to be associated with vesicles carrying the GLUT4 glucose transporter protein, but no interaction was seen with synaptobrevin 1. Flow cytometry of low-density microsomes isolated from fat cells was used to demonstrate the binding of syntaxin 4 to a subset of vesicles carrying GLUT4 protein; whereas SNAP-25 on its own bound poorly to these vesicles, the syntaxin 4-SNAP-25 complex gave a strong interaction.

    The Biochemical journal 1996;317 ( Pt 3);945-54

  • Isoform-specific interaction of the alpha1A subunits of brain Ca2+ channels with the presynaptic proteins syntaxin and SNAP-25.

    Rettig J, Sheng ZH, Kim DK, Hodson CD, Snutch TP and Catterall WA

    Department of Pharmacology, University of Washington, Seattle 98195-7280, USA.

    Presynaptic Ca2+ channels are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. Here we report isoform-specific, stoichiometric interaction of the BI and rbA isoforms of the alpha1A subunit of P/Q-type Ca2+ channels with the presynaptic membrane proteins syntaxin and SNAP-25 in vitro and in rat brain membranes. The BI isoform binds to both proteins, while only interaction with SNAP-25 can be detected in vitro for the rbA isoform. The synaptic protein interaction ("synprint") site involves two adjacent segments of the intracellular loop connecting domains II and III between amino acid residues 722 and 1036 of the BI sequence. This interaction is competitively blocked by the corresponding region of the N-type Ca2+ channel, indicating that these two channels bind to overlapping regions of syntaxin and SNAP-25. Our results provide a molecular basis for a physical link between Ca2+ influx into nerve terminals and subsequent exocytosis of neurotransmitters at synapses that have presynaptic Ca2+ channels containing alpha1A subunits.

    Funded by: NIMH NIH HHS: 1F32 MH10775-01; NINDS NIH HHS: NS22625

    Proceedings of the National Academy of Sciences of the United States of America 1996;93;14;7363-8

  • Identification of a novel syntaxin- and synaptobrevin/VAMP-binding protein, SNAP-23, expressed in non-neuronal tissues.

    Ravichandran V, Chawla A and Roche PA

    Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.

    The specificity of vesicular transport is regulated, in part, by the interaction of a vesicle-associated membrane protein termed synaptobrevin/VAMP with a target compartment membrane protein termed syntaxin. These proteins, together with SNAP-25 (synaptosome-associated protein of 25 kDa), form a complex which serves as a binding site for the general membrane fusion machinery. Synaptobrevin/VAMP and syntaxin are ubiquitously expressed proteins and are believed to be involved in vesicular transport in most (if not all) cells. However, SNAP-25 is present almost exclusively in the brain, suggesting that a ubiquitously expressed homolog of SNAP-25 exists to facilitate transport vesicle/target membrane fusion in other tissues. Using the yeast two-hybrid system, we have identified a 23-kDa protein from human B lymphocytes (termed SNAP-23) that binds tightly to multiple syntaxins and synaptobrevins/VAMPs in vitro. SNAP-23 is 59% identical with SNAP-25. Unlike SNAP-25, SNAP-23 was expressed in all tissues examined. These findings suggest that SNAP-23 is an essential component of the high affinity receptor for the general membrane fusion machinery and an important regulator of transport vesicle docking and fusion in all mammalian cells.

    The Journal of biological chemistry 1996;271;23;13300-3

  • Radiation hybrid mapping of SNAP, PCSK2, and THBD (human chromosome 20p).

    Maglott DR, Feldblyum TV, Durkin AS and Nierman WC

    American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852-1776, USA.

    Mammalian genome : official journal of the International Mammalian Genome Society 1996;7;5;400-1

  • Complexins: cytosolic proteins that regulate SNAP receptor function.

    McMahon HT, Missler M, Li C and Südhof TC

    Howard Hughes Medical Institute, University of Texas Southwestern Medical School, Dallas 75235, USA.

    A family of proteins called complexins was discovered that compete with alpha-SNAP, but not synaptotagmin, for SNAP receptor binding. Complexins I and II are highly homologous hydrophilic proteins that are tightly conserved, with 100% identity among mouse, rat, and human complexin II. They are enriched in neurons where they colocalize with syntaxin and SNAP-25; in addition, complexin II is expressed ubiquitously at low levels. Complexins bind weakly to syntaxin alone and not at all to synaptobrevin and SNAP-25, but strongly to the SNAP receptor-core complex composed of these three molecules. They compete with alpha-SNAP for binding to the core complex but not with other interacting molecules, including synaptotagmin I, suggesting that the complexins regulate the sequential interactions of alpha-SNAP and synaptotagmins with the SNAP receptor during exocytosis.

    Cell 1995;83;1;111-9

  • The N-ethylmaleimide-sensitive fusion protein and alpha-SNAP induce a conformational change in syntaxin.

    Hanson PI, Otto H, Barton N and Jahn R

    Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

    The N-ethylmaleimide-sensitive fusion protein (NSF) plays an essential role in intracellular membrane fusion events and has been implicated in the exocytosis of synaptic vesicles. NSF binds through soluble NSF attachment proteins (SNAPs) to a complex of neuronal membrane proteins comprised of synaptobrevin, syntaxin, and SNAP-25. Disassembly of this complex by NSF is thought to be a critical step in the molecular events which lead to vesicle fusion with the plasma membrane. Here we have studied the interaction of alpha-SNAP and NSF with individual components of this complex and have identified syntaxin as a primary substrate for NSF/alpha-SNAP. We find that alpha-SNAP binds directly to syntaxin 1A as well as weakly to SNAP-25, while it does not bind to synaptobrevin II. NSF binds to syntaxin through alpha-SNAP and in the presence of ATP catalyzes a conformational rearrangement which abolishes binding of itself and alpha-SNAP. This reaction leads to the previously described disassembly of the fusion complex, since synaptobrevin binding to syntaxin is also reduced. alpha-SNAP binds to a carboxyl-terminal syntaxin fragment (residues 194-288) that also binds synaptobrevin and SNAP-25. However, NSF action on this syntaxin fragment has no effect on the binding of alpha-SNAP or synaptobrevin. This suggests that the conformational change normally induced by NSF in syntaxin depends on an interaction between carboxyl- and amino-terminal domains of syntaxin.

    The Journal of biological chemistry 1995;270;28;16955-61

  • A novel ubiquitous form of Munc-18 interacts with multiple syntaxins. Use of the yeast two-hybrid system to study interactions between proteins involved in membrane traffic.

    Hata Y and Südhof TC

    Howard Hughes Medical Institute, University of Texas Southwestern Medical School, Dallas 75235, USA.

    Munc-18-1 is a 67-kDa neuronal protein that binds tightly to syntaxin 1 and functions in synaptic vesicle exocytosis (Hata, Y., Slaughter, C.A., and Südhof, T.C. (1993a) Nature 366, 347-351). We have now characterized a new Munc-18 isoform, Munc-18-2, that exhibits 63% amino acid sequence identity with Munc-18-1. Munc-18-2 is expressed in most tissues, whereas Munc-18-1 is primarily expressed in brain. Using recombinant Munc-18-1 and Munc-18-2 produced in COS cells, we show that both forms of Munc-18 bind tightly to syntaxins 1A, 2, and 3 but not to syntaxin 4. In an independent approach to study the binding specificities of Munc-18-1 and Munc-18-2, we used the yeast two-hybrid system. This assay system depends on protein-protein interactions in the cell nucleus. We validated its utility for studying membrane trafficking proteins by testing well characterized interactions between cytosolic proteins that are known to be physiologically important in exocytosis. Strong interactions, such as the binding of syntaxins 1-4 with SNAP-25, were effectively detected by the yeast two-hybrid assay, but weak binding, such as the binding of syntaxins to synaptotagmin or of synaptotagmin to neurexins, was not. Studies on full-length and truncated forms of Munc-18s by the yeast two-hybrid system confirmed their interactions with syntaxins. Both the N and the C terminus of Munc-18 were essential for binding. Munc-18-1 and Munc-18-2 bind only to syntaxins 1A, 2, and 3 but not 4 and 5 by yeast-two hybrid system assays. Our studies demonstrate that neural and non-neural tissues have distinct forms of Munc-18, which may function in different types of exocytosis. The lack of specificity of the interactions between syntaxins and Munc-18s indicates that specificity of membrane trafficking reactions is not dependent on this interaction.

    The Journal of biological chemistry 1995;270;22;13022-8

  • SNAP-25, a t-SNARE which binds to both syntaxin and synaptobrevin via domains that may form coiled coils.

    Chapman ER, An S, Barton N and Jahn R

    Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510.

    The membrane proteins SNAP-25, syntaxin, and synaptobrevin (vesicle-associated membrane protein) have recently been implicated as central elements of an exocytotic membrane fusion complex in neurons. Here we report that SNAP-25 binds directly to both syntaxin and synaptobrevin. The SNAP-25-binding domain of syntaxin lies between residues 199 and 243, within the region previously shown to mediate synaptobrevin binding (Calakos, N., Bennett, M. K., Peterson, K. E., and Scheller, R. H. (1994) Science 263, 1146-1149). The syntaxin-binding domain of SNAP-25 encompasses most of the amino-terminal half of SNAP-25, including its putative palmitoylation sites. Truncation of the carboxyl-terminal 9 residues of SNAP-25, which yields a fragment corresponding to that generated by botulinum neurotoxin A, diminishes the interaction of SNAP-25 with synaptobrevin, but not with syntaxin. Sequence analysis revealed that the regions that mediate the interaction between SNAP-25 and syntaxin contain heptad repeats characteristic of certain classes of alpha-helices. Similar repeats are also present at the carboxyl terminus of SNAP-25 and in synaptobrevin. These domains have a moderate to high probability of forming coiled coils. We conclude that SNAP-25 can interact with both syntaxin and synaptobrevin and that binding may be mediated by alpha-helical domains that form intermolecular coiled-coil structures.

    The Journal of biological chemistry 1994;269;44;27427-32

  • Cloning and sequence analysis of the human SNAP25 cDNA.

    Zhao N, Hashida H, Takahashi N and Sakaki Y

    Human Genome Center, University of Tokyo, Japan.

    During the screening of human brain-specific cDNAs by a modified differential hybridization analysis, we found a clone which was highly and specifically expressed in the (adult) brain. This clone was subjected to nucleotide sequence analysis and proven to be a nearly full-length cDNA of a human homologue of the previously reported mouse synaptosomal-associated protein 25 (mSNAP25). The human SNAP25 (hSNAP25) and mSNAP25 showed perfect amino-acid sequence conservation.

    Gene 1994;145;2;313-4

  • Human cDNA clones encoding two different isoforms of the nerve terminal protein SNAP-25.

    Bark IC and Wilson MC

    Scripps Research Institute, Department of Neuropharmacology, La Jolla, CA 92037.

    Two distinct cDNA sequences, corresponding to alternative isoforms of the human nerve terminal protein SNAP-25 (synaptosomal associated protein of 25 kDa), were cloned and characterized. Sequence analysis demonstrated that the two isoforms are generated by alternative splicing between two distinct but homologous exons 5, a and b each encoding 39 amino acids (aa). Although the two isoforms, SNAP-25a and SNAP-25b, differ by only 9 aa, this domain encodes the portion of the protein that is a substrate for post-translational fatty acylation, and therefore might be important for regulating subcellular localization and membrane targeting.

    Funded by: NIMH NIH HHS: MH48989

    Gene 1994;139;2;291-2

  • Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.

    Maruyama K and Sugano S

    Institute of Medical Science, University of Tokyo, Japan.

    We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

    Gene 1994;138;1-2;171-4

Gene lists (10)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000039 G2C Homo sapiens Pocklington H8 Human orthologues of cluster 8 (mouse) from Pocklington et al (2006) 3
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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