Mutations in the Neuronal Vesicular SNARE VAMP2 Affect Synaptic Membrane Fusion and Impair Human Neurodevelopment

VAMP2 encodes the vesicular SNARE protein VAMP2 (also called synaptobrevin-2). Together with its partners syntaxin-1A and synaptosomal-associated protein 25 (SNAP25), VAMP2 mediates fusion of synaptic vesicles to release neurotransmitters. VAMP2 is essential for vesicular exocytosis and activity-dependent neurotransmitter release. Here, we report five heterozygous de novo mutations in VAMP2 in unrelated individuals presenting with a neurodevelopmental disorder characterized by axial hypotonia (which had been present since birth), intellectual disability, and autistic features. In total, we identified two single-amino-acid deletions and three non-synonymous variants affecting conserved residues within the C terminus of the VAMP2 SNARE motif. Affected individuals carrying de novo non-synonymous variants involving the C-terminal region presented a more severe phenotype with additional neurological features, including central visual impairment, hyperkinetic movement disorder, and epilepsy or electroencephalography abnormalities. Reconstituted fusion involving a lipid-mixing assay indicated impairment in vesicle fusion as one of the possible associated disease mechanisms. The genetic synaptopathy caused by VAMP2 de novo mutations highlights the key roles of this gene in human brain development and function. VAMP2 encodes the vesicular SNARE protein VAMP2 (also called synaptobrevin-2). Together with its partners syntaxin-1A and synaptosomal-associated protein 25 (SNAP25), VAMP2 mediates fusion of synaptic vesicles to release neurotransmitters. VAMP2 is essential for vesicular exocytosis and activity-dependent neurotransmitter release. Here, we report five heterozygous de novo mutations in VAMP2 in unrelated individuals presenting with a neurodevelopmental disorder characterized by axial hypotonia (which had been present since birth), intellectual disability, and autistic features. In total, we identified two single-amino-acid deletions and three non-synonymous variants affecting conserved residues within the C terminus of the VAMP2 SNARE motif. Affected individuals carrying de novo non-synonymous variants involving the C-terminal region presented a more severe phenotype with additional neurological features, including central visual impairment, hyperkinetic movement disorder, and epilepsy or electroencephalography abnormalities. Reconstituted fusion involving a lipid-mixing assay indicated impairment in vesicle fusion as one of the possible associated disease mechanisms. The genetic synaptopathy caused by VAMP2 de novo mutations highlights the key roles of this gene in human brain development and function. Chemical synaptic transmission relies on precisely coordinated, activity-dependent neurotransmitter release. 1Jahn R. Fasshauer D. Molecular machines governing exocytosis of synaptic vesicles. Nature. 2012; 490: 201-207Crossref PubMed Scopus (674) Google Scholar A fundamental step in this pathway is the fusion of synaptic vesicles with the presynaptic plasma membrane. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate membrane fusion and are essential for the fusion of synaptic vesicles. 1Jahn R. Fasshauer D. Molecular machines governing exocytosis of synaptic vesicles. Nature. 2012; 490: 201-207Crossref PubMed Scopus (674) Google Scholar, 2Hu C. Ahmed M. Melia T. J. Söllner T. H. Mayer T. Rothman J. E. Fusion of cells by flipped SNAREs. Science. 2003; 300: 1745-1749Crossref PubMed Scopus (183) Google Scholar At mammalian central nervous system (CNS) synapses, neuronal SNAREs consist of vesicle-associated membrane protein 2 (VAMP2, also called synaptobrevin-2) on the vesicle membrane (v-SNARE) and the binary complex of syntaxin1A (STX1A) and synaptosomal-associated protein 25 Kd (SNAP25) on the plasma membrane (target or t-SNARE). 3Chen Y. A. Scales S. J. Patel S. M. Doung Y. C. Scheller R. H. SNARE complex formation is triggered by Ca2+ and drives membrane fusion. Cell. 1999; 97: 165-174Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar The v- and t-SNARE proteins assemble in a polarized manner starting from the N termini distal from the membranes and proceeding towards the C termini and are held together by discrete interacting residues (numbered -7 to +8), including 15 hydrophobic contacts and central ionic residues. 4Li F. Kümmel D. Coleman J. Reinisch K. M. Rothman J. E. Pincet F. A half-zippered SNARE complex represents a functional intermediate in membrane fusion. J. Am. Chem. Soc. 2014; 136: 3456-3464Crossref PubMed Scopus (48) Google Scholar This “zippering” process pulls the membranes together and provides the energy to fuse the lipid bilayers. 5Gao Y. Zorman S. Gundersen G. Xi Z. Ma L. Sirinakis G. Rothman J. E. Zhang Y. Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science. 2012; 337: 1340-1343Crossref PubMed Scopus (282) Google Scholar, 6Rothman J. E. Söllner T. H. Throttles and dampers: controlling the engine of membrane fusion. Science. 1997; 276: 1212-1213Crossref PubMed Scopus (93) Google Scholar The SNAREs alone are sufficient to drive fusion of synaptic vesicles, but this process is tightly regulated by a number of synaptic proteins to enable Ca2+-regulated neurotransmitter release. 7Weber T. Zemelman B. V. McNew J. A. Westermann B. Gmachl M. Parlati F. Söllner T. H. Rothman J. E. SNAREpins: minimal machinery for membrane fusion. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2010) Google Scholar The key regulatory elements at excitatory CNS synapses include chaperones (Munc18 and Munc13), the primary Ca2+ sensor synaptotagmin-1, and the auxiliary protein complexin. 7Weber T. Zemelman B. V. McNew J. A. Westermann B. Gmachl M. Parlati F. Söllner T. H. Rothman J. E. SNAREpins: minimal machinery for membrane fusion. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2010) Google Scholar, 8Melia T. J. Weber T. McNew J. A. Fisher L. E. Johnston R. J. Parlati F. Mahal L. K. Sollner T. H. Rothman J. E. Regulation of membrane fusion by the membrane-proximal coil of the t-SNARE during zippering of SNAREpins. J. Cell Biol. 2002; 158: 929-940Crossref PubMed Scopus (170) Google Scholar, 9Brunger A. T. Structure and function of SNARE and SNARE-interacting proteins. Q. Rev. Biophys. 2005; 38: 1-47Crossref PubMed Scopus (208) Google Scholar, 10Rizo J. Rosenmund C. Synaptic vesicle fusion. Nat. Struct. Mol. Biol. 2008; 15: 665-674Crossref PubMed Scopus (401) Google Scholar VAMP2 (MIM: 185881) encodes a neuronal v-SNARE essential for the fusion of synaptic vesicles at mammalian central nerve terminals. 5Gao Y. Zorman S. Gundersen G. Xi Z. Ma L. Sirinakis G. Rothman J. E. Zhang Y. Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science. 2012; 337: 1340-1343Crossref PubMed Scopus (282) Google Scholar, 6Rothman J. E. Söllner T. H. Throttles and dampers: controlling the engine of membrane fusion. Science. 1997; 276: 1212-1213Crossref PubMed Scopus (93) Google Scholar, 7Weber T. Zemelman B. V. McNew J. A. Westermann B. Gmachl M. Parlati F. Söllner T. H. Rothman J. E. SNAREpins: minimal machinery for membrane fusion. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2010) Google Scholar Introduction of specific engineered mutations affecting its SNARE motif has been reported to alter vesicle fusion in vitro by impairing either formation of the SNARE complex or the interaction of VAMP2 with other (auxiliary) presynaptic proteins. 11Hernandez J. M. Stein A. Behrmann E. Riedel D. Cypionka A. Farsi Z. Walla P. J. Raunser S. Jahn R. Membrane fusion intermediates via directional and full assembly of the SNARE complex. Science. 2012; 336: 1581-1584Crossref PubMed Scopus (166) Google Scholar, 12Shen C. Rathore S. S. Yu H. Gulbranson D. R. Hua R. Zhang C. Schoppa N. E. Shen J. The trans-SNARE-regulating function of Munc18-1 is essential to synaptic exocytosis. Nat. Commun. 2015; 6: 8852Crossref PubMed Scopus (41) Google Scholar Vamp2−/− mice present severely decreased rates of both spontaneous and Ca2+-triggered synaptic-vesicle fusion, and these mice die immediately after birth. 13Schoch S. Deák F. Königstorfer A. Mozhayeva M. Sara Y. Südhof T. C. Kavalali E. T. SNARE function analyzed in synaptobrevin/VAMP knockout mice. Science. 2001; 294: 1117-1122Crossref PubMed Scopus (510) Google Scholar Also, synapses from VAMP2-deficient mice display changes in synaptic-vesicle morphology and size—and delayed stimulus-dependent endocytosis. 14Deák F. Schoch S. Liu X. Südhof T. C. Kavalali E. T. Synaptobrevin is essential for fast synaptic-vesicle endocytosis. Nat. Cell Biol. 2004; 6: 1102-1108Crossref PubMed Scopus (189) Google Scholar Thus, VAMP2 exerts a complex influence on synaptic transmission; it plays fundamental roles in vesicle fusion, neurotransmitter release, and vesicle endocytosis. Despite the critical role of VAMP2 in presynaptic molecular events, little is known of the consequences of disrupted VAMP2 function in human neurodevelopment. Here, we describe five unrelated individuals who had shown hypotonia since birth and who had intellectual disability (ID) with autistic features, including variable motor stereotypies resembling Rett syndrome (RTT), and, in some children, also central visual impairment, hyperkinetic movements, and epilepsy and/or electroencephalography (EEG) abnormalities. Table 1 summarizes the detailed phenotypes of the individuals (1–5), aged between 3 and 14 years. Table 1Clinical Features of Individuals with De Novo VAMP2 MutationsIndividual Number Gender AgeVariantGrowth/OFCHypotonia/DDIDEpileptic SeizuresEEGASDRTT-Like FeaturesMovement DisorderCentral Visual DefectsSpeech ImpairmentBrain ImagingAdditional Features1F3 yrc. 223T>C, p. Ser75Pronormalyesseverenohigh-voltage delta activity, sharp wave-slow wave complexesyesstereotyped hand movements, absent purposeful hand movementschoreic movement, flapping, dystonic posturesyesabsent speechthin corpus callosum, delayed myelinationinability to walk2M10 yrc. 233A>C, p. Glu78Alanormalyesseverefocal seizures, GTCSfast rhythmic activity, sharp wave-slow wave complexesyesbody rocking, head banging, screaming, absent purposeful hand movementsgeneralizedchoreayesabsent speechunremarkableabnormal behavior, self-injury, inability to walk3M13yrc. 230T>C, p. Phe77Sernormalyessevereinfantile spasms, convulsive status epilepticusdisorganized EEG paroxysmsyesstereotyped hand movements, absent purposeful hand movementschoreic movement, myoclonic jerksyesabsent speechunremarkableabnormal behavior, inability to walk, severe constipation4M14yrc. 128₁30delTGG, p. Val43delnormalyesmoderatefocal seizuresgeneralized and multifocal abnormalitiesyesstereotyped hand movements (wringing), absent purposeful hand movementsnonoonly 5–10 spoken wordsunremarkableclumsiness, abnormal behavior5F3 yrc. 135₁37delCAT, p. Ile45delnormalyesmoderatenodisorganized EEG paroxysmsyesstereotyped hand movements (washing) nonoonly 5 spoken wordsunremarkableabnormal behaviorAbbreviations are as follows: ASD = autism spectrum disorder; DD = developmental delay; EEG = electroencephalography; FC = focal seizures; GTCS = generalized tonic-clonic seizures; ID = intellectual disability; and OFC = occipital-frontal circumference. Variants are named according to the GenBank: NM₀14232 reference transcript. Open table in a new tab Abbreviations are as follows: ASD = autism spectrum disorder; DD = developmental delay; EEG = electroencephalography; FC = focal seizures; GTCS = generalized tonic-clonic seizures; ID = intellectual disability; and OFC = occipital-frontal circumference. Variants are named according to the GenBank: NM₀14232 reference transcript. In all affected children, family histories, pregnancies, and birth histories were unremarkable, and neurodevelopmental impairment occurred within the first year of life. The earliest sign of neurological involvement was axial hypotonia at birth. Poor visual fixation (with only brief and occasional visual contact, lasting up to a few seconds) had been evident since the first months of life in three affected individuals (1–3) ; these individuals were later diagnosed with central visual impairment (Table 1). Three children (individuals 1–3) exhibited a hyperkinetic movement disorder starting in the first year of life (Videos S1, S2, S3, and S4). Abnormal movements ranged from dystonic posturing (mainly involving the trunk, neck, and lower limbs) and moderate chorea (individuals 1 and 3) to a mixed-movement disorder with severe chorea and dystonic posturing (individual 2) or myoclonic jerks (individual 3). All children showed autistic features, typically including flapping or flailing of the arms, as well as hand wringing or clapping. Additional repetitive behavior patterns included body rocking and head banging. Self-injurious behaviors were evident in individual 2. A virtual absence of purposeful hand movements was present in all cases (Table 1, Videos S1, S2, S3, S4, and S5). Motor development in individuals 1–3 was severely impaired, and these children had not attained the ability to walk. Severe language impairment was present in the three more severely affected children (individuals 1–3), none of whom had attained meaningful speech production, but individuals 4 and 5 were capable of saying 5–10 words (Table 1). with 1 at the of and 1, with the de novo presented with hypotonia at the of 2 at the of 5 showed a hyperkinetic movement disorder, hand and to at the of the individual is in the the virtual absence of purposeful hand with 1 at the of 15 1, with the de novo presented with developmental inability to visual contact, hand stereotypies movement disorder, dystonic and to with 2 at the of with the de novo at the of presented with developmental of purposeful hand movements, to and dystonic posturing of the by severe generalized with 3 at the of with the de novo presented with developmental visual contact, a virtual absence of purposeful hand movements, stereotypies and generalized movements with myoclonic with 4 at the of 14 with the de novo at the of 14 presented some motor and hand stereotypies or abnormal EEG occurred in affected 1 not present with seizures, but EEG at the of 15 months showed delta with complexes the central and brain 2 from focal these after birth and were characterized on EEG by fast rhythmic by complexes At individual 3 presented with were associated with EEG 4 with at 5 of and had a of status at the of including and been in individuals of were in individual who has been since the of and has had 2 a for at the of months and had a affecting the and at the of had a was in all children in individual 1, for whom and a corpus was at the of 2 1). 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A. for disease gene and a for with in the 2015; PubMed Scopus Google Scholar, F. D. A. A for with in the 2015; PubMed Scopus Google Scholar Three de novo non-synonymous variants in VAMP2 were identified in three affected individuals and at as of analyzed the genetic from the of and from individuals affected with neurological children with neurodevelopmental impairment and for variants in VAMP2 and identified a (individual carrying a de novo at and F. C. D. A. for disease gene and a for with in the 2015; PubMed Scopus Google Scholar, F. D. A. 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J. of by Full Text Full Text PDF PubMed Scopus Google Scholar were to the and and showed these variants were and with S5). all to the were in vitro fusion to the two non-synonymous variants and shown in the VAMP2 the and of fusion to with VAMP2 the had little to and The in the fusion associated with was to in the the of a at this with the assembly of the SNARE proteins and VAMP2 fusion the fusion associated with the was from of the shown chaperones SNARE assembly via with the VAMP2 C-terminal C. Rathore S. S. Yu H. Gulbranson D. R. Hua R. Zhang C. Schoppa N. E. Shen J. The trans-SNARE-regulating function of Munc18-1 is essential to synaptic exocytosis. Nat. Commun. 2015; 6: 8852Crossref PubMed Scopus (41) Google Scholar, T. C. Rothman J. E. Membrane with SNARE and PubMed Scopus Google Scholar the of the disease variants of Munc18-1 in the and of fusion VAMP2 was not the fusion by the VAMP2 we a phenotype with the these In was to the fusion by VAMP2 this not the SNARE assembly process or its the of the individuals carrying heterozygous de novo VAMP2 we also the of the of VAMP2 with the disease variants S4). in the of the fusion for the was to the fusion for the only the proteins S4). This with and this the phenotype with this genetic and functional de novo mutations in VAMP2 neurodevelopmental impairment associated with variable features. Individuals carrying de novo non-synonymous variants affecting the C terminus of the VAMP2 SNARE motif and presented a severe neurological phenotype with motor impairment inability to central visual hyperkinetic movements, and, in two of epilepsy starting in Individuals 4 and carrying de novo single-amino-acid deletions involving residues at and presented a severe neurological the ability to and were to a few showed mutations in the C terminus of this region within the SNARE The in vitro lipid-mixing assay a in vesicle fusion as a of the but had functional The phenotype for the to with regulatory proteins were not included in the in vitro the assembly of the C-terminal region of the SNARE proteins is critical to membrane Y. Zorman S. Gundersen G. Xi Z. Ma L. Sirinakis G. Rothman J. E. Zhang Y. Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science. 2012; 337: 1340-1343Crossref PubMed Scopus (282) Google Scholar, Y. and pathway of SNARE and assembly by PubMed Scopus Google Scholar and synaptic regulatory proteins vesicle fusion by the C-terminal of the SNARE C. Rathore S. S. Yu H. Gulbranson D. R. Hua R. Zhang C. Schoppa N. E. Shen J. The trans-SNARE-regulating function of Munc18-1 is essential to synaptic exocytosis. Nat. Commun. 2015; 6: 8852Crossref PubMed Scopus (41) Google Scholar, J. F. Rothman J. E. Melia T. J. of by Full Text Full Text PDF PubMed Scopus Google Scholar, T. C. Rothman J. E. Membrane with SNARE and PubMed Scopus Google Scholar Thus, mutations affecting this region the SNARE complex assembly by of SNARE proteins and/or its with regulatory elements as Munc18-1 or In the this as the of Ca2+-triggered neurotransmitter release. a of the fusion in vitro to a on the release of release at the neuronal This the severe neurodevelopmental impairment in the VAMP2 variants affecting the been shown to the Munc18-1 by impairing its ability to C. Rathore S. S. Yu H. Gulbranson D. R. Hua R. Zhang C. Schoppa N. E. Shen J. The trans-SNARE-regulating function of Munc18-1 is essential to synaptic exocytosis. Nat. Commun. 2015; 6: 8852Crossref PubMed Scopus (41) Google Scholar, J. F. Rothman J. E. Melia T. J. of by Full Text Full Text PDF PubMed Scopus Google Scholar and variants involving also Ca2+-regulated neurotransmitter T. T. E. J. The SNARE protein is to fast of 2002; PubMed Scopus Google Scholar The present to the neurodevelopmental a genetic and a of including developmental and autism spectrum J. S. L. H. Liu Z. knockout mice neurological of J. Full Text Full Text PDF PubMed Scopus Google Scholar, M. de A. A. A. B. de Novo and variants in and of a distinct neurodevelopmental J. Full Text Full Text PDF PubMed Scopus Google Scholar impairment, and movement also A. The genetic of the of and 15: Full Text Full Text PDF PubMed Scopus Google Scholar, M. N. E. complex hyperkinetic PubMed Scopus Google Scholar variants in a number of presynaptic proteins in Ca2+-regulated neurotransmitter release been identified in individuals affected by a spectrum of neurological include the variants in (MIM: and these variants been identified in with seizures, and D. J. and intellectual 2014; PubMed Scopus Google Scholar, H. E. A. S. S. T. A. in two affected from family and PubMed Scopus Google variants in (MIM: encodes the for these variants are in individuals with and hyperkinetic H. F. T. J. D. M. for neurodevelopmental A PubMed Scopus Google Scholar, D. M. M. E. T. of a human synaptic vesicle 2015; Google variants in the or the and these variants been identified in of and (MIM: M. S. B. L. A. J. H. J. of in 1 in in 2 2005; PubMed Scopus Google Scholar, Y. J. Zhang mutations in PubMed Scopus Google variants in (MIM: the synaptic these variants been to with G. R. protein and movement PubMed Scopus Google variants in (MIM: these variants with epilepsy and autistic H. M. T. H. J. S. A. novo mutations in the gene 2008; PubMed Scopus Google Scholar The phenotypes associated with the VAMP2 synaptopathy reported are of the reported in some individuals who variants in or in and who present with a of neurodevelopmental impairment, hyperkinetic movements chorea and and EEG or of variable H. F. T. J. D. M. for neurodevelopmental A PubMed Scopus Google Scholar, H. M. M. H. M. A. A neurodevelopmental disorder including PubMed Scopus (189) Google Scholar a heterozygous in a encodes a protein in vesicle fusion at Y. Y. The role of in Ca2+-triggered neurotransmitter release at the PubMed Scopus Google Scholar has been to in from M. in J. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar mutations in been identified in with a phenotype of hypotonia and and in three of these of presynaptic transmission impairment was and to a of presynaptic A. M. Liu Y. S. A. S. A. mutations in a presynaptic PubMed Scopus Google Scholar, R. H. P. J. J. D. PubMed Scopus Google Scholar, D. M. M. Z. M. H. M. A. Z. of genetic in on the first and 136: PubMed Scopus Google Scholar In we identified a neurodevelopmental disease is associated with additional neurological features, including epilepsy and hyperkinetic movements, and is caused by de novo mutations in spectrum of human caused by variants in SNAREs and essential regulatory of the synaptic The of these is presynaptic at nerve this in a of features, including neurodevelopmental impairment, seizures, and abnormal The genetic synaptopathy caused by VAMP2 mutations highlights the key roles of this gene in human brain development and function. in the of VAMP2 in vitro the presynaptic of the affected children, and this highlights a of The all the for in this This was by the the and the to the and the de and the to are also by the for the and the The for the reported in this are in the Open with and Table S1, and and of the and in