The mechanisms through which synaptic vesicle membranes are reinternalized after exocytosis remain a matter of debate. Because several vesicular transport steps require GTP hydrolysis, GTP-gamma S may help identify intermediates in synaptic vesicle recycling. In GTP-gamma S-treated nerve terminals, we observed tubular invaginations of the plasmalemma that were often, but not always, capped by a clathrin-coated bud. Strikingly, the walls of these tubules were decorated by transverse electron-dense rings that were morphologically similar to structures formed by dynamin around tubular templates. Dynamin is a GTPase implicated in synaptic vesicle endocytosis and here we show that the walls of these membranous tubules, but not their distal ends, were positive for dynamin immunoreactivity. These findings demonstrate that dynamin and clathrin act at different sites in the formation of endocytic vesicles. They strongly support a role for dynamin in the fission reaction and suggest that stabilization of the GTP-bound conformation of dynamin leads to tubule formation by progressive elongation of the vesicle stalk.

Graphical Abstract Highlights d SynGO is a public knowledge base and online analysis platform for synapse research d SynGO has annotated 1,112 genes with synaptic localization and/or function d SynGO genes are exceptionally large, well conserved, and intolerant to mutations d SynGO genes are strongly enriched among genes associated with brain disorders Correspondence guus.smit@cncr.vu.nl (A.B.S.), matthijs@cncr.vu.nl (M.V.) In BriefThe SynGO consortium presents a framework to annotate synaptic protein locations and functions and annotations for 1,112 synaptic genes based on published experimental evidence. SynGO reports exceptional features and disease associations for synaptic genes and provides an online data analysis platform. SUMMARYSynapses are fundamental information-processing units of the brain, and synaptic dysregulation is central to many brain disorders (''synaptopathies''). However, systematic annotation of synaptic genes and ontology of synaptic processes are currently lacking. We established SynGO, an interactive knowledge base that accumulates available research about synapse biology using Gene Ontology (GO) annotations to novel ontology terms: 87 synaptic locations and 179 synaptic processes. SynGO annotations are exclusively based on published, expert-curated evidence. Using 2,922 annotations for 1,112 genes, we show that synaptic genes are exceptionally well conserved and less tolerant to mutations than other genes. Many SynGO terms are significantly overrepresented among gene variations associated with intelligence, educational attainment, ADHD, autism, and bipolar disorder and among de novo variants associated with neurodevelopmental disorders, including schizophrenia. SynGO is a public, universal reference for synapse research and an online analysis platform for interpretation of large-scale -omics data (https://syngoportal.org and

Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Heritability and polygenic predictionIn the EUR sample, the SNP-based heritability (h 2 SNP ) (that is, the proportion of variance in liability attributable to all measured SNPs)

Synaptojanin is a nerve terminal protein of relative molecular mass 145,000 which appears to participate with dynamin in synaptic vesicle recycling. The central region of synaptojanin defines it as a member of the inositol-5-phosphatase family, which includes the product of the gene that is defective in the oculocerebrorenal syndrome of Lowe. Synaptojanin has 5-phosphatase activity and its amino-terminal domain is homologous with the yeast protein Sac1 (Rsd1), which is genetically implicated in phospholipid metabolism and in the function of the actin cytoskeleton. The carboxy terminus, which is of different lengths in adult and developing neurons owing to the alternative use of two termination sites, is proline-rich, consistent with the reported interaction of synaptojanin with the SH3 domains of Grb2 (refs 1, 2). Synaptojanin is the only other major brain protein besides dynamin that binds the SH3 domain of amphiphysin, a presynaptic protein with a putative function in endocytosis. Our results suggest a link between phosphoinositide metabolism and synaptic vesicle recycling.

Phosphorylated products of phosphatidylinositol play critical roles in the regulation of membrane traffic, in addition to their classical roles as second messengers in signal transduction at the cell surface. Growing evidence suggests that phosphorylation-dephosphorylation of the polar heads of phosphoinositides (polyphosphorylated inositol lipids) in specific intracellular locations signals either the recruitment or the activation of proteins essential for vesicular transport. Cross talk between phosphatidylinositol metabolites and guanosine triphosphatases is an important feature of these regulatory mechanisms.

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.

Recent data in invertebrates demonstrated that huntingtin (htt) is essential for fast axonal trafficking. Here, we provide direct and functional evidence that htt is involved in fast axonal trafficking in mammals. Moreover, expression of full-length mutant htt (mhtt) impairs vesicular and mitochondrial trafficking in mammalian neurons in vitro and in whole animals in vivo. Particularly, mitochondria become progressively immobilized and stop more frequently in neurons from transgenic animals. These defects occurred early in development prior to the onset of measurable neurological or mitochondrial abnormalities. Consistent with a progressive loss of function, wild-type htt, trafficking motors, and mitochondrial components were selectively sequestered by mhtt in human Huntington's disease-affected brain. Data provide a model for how loss of htt function causes toxicity; mhtt-mediated aggregation sequesters htt and components of trafficking machinery leading to loss of mitochondrial motility and eventual mitochondrial dysfunction.Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion mutation in the coding region of a novel gene. The mechanism of HD is unknown. However, most data suggest that polyglutamine-mediated aggregation contributes to the pathology (32). Studies of human brain (14), mouse models (48), and cells (8, 28) demonstrate that mutant huntingtin (mhtt) binds and sequesters its normal counterpart as well as many cellular proteins (41). But whether pathophysiology results from a loss of normal function or a gain of a new function in adult neurons is not well understood.A major gap in our understanding of the disease mechanism is the absence of a known function for normal huntingtin (htt). Emerging evidence suggests that htt is likely to be a multifunctional protein that can mediate transactions in both the nucleus and the cytoplasm. Transcriptional dysfunction caused by mhtt has been proposed to lead to toxicity. The mutation in full-length htt prevents its normal ability to bind and sequester a repressor of brain-derived neurotrophic factor expression, reducing the availability of brain-derived neurotrophic factor to striatal neurons (54). The N-terminal, truncated form of mhtt can bind to and interfere with nuclear factors such as CREB (51), CREB binding protein (30, 39), corepressor (22), and transcriptional activator Sp1 (12,23).Cytoplasmic dysfunction has also been implicated as a toxic mechanism. Recently, novel data obtained with Drosophila (17) and isolated squid axoplasm (42) have provided direct evidence that htt is an essential protein involved in fast axonal trafficking. Additionally, these data demonstrate that the mutation in htt causes trafficking abnormalities. Reduction of htt expression in Drosophila caused axonal transport defects in larval nerves and the same neurodegenerative phenotype in adult eyes as expression of mutant dynein or p150 Glued (17). In invertebrate models for HD, expression of truncated proteins with an expanded gluta...

Amphiphysin, a major autoantigen in paraneoplastic Stiff-Man syndrome, is an SH3 domain-containing neuronal protein, concentrated in nerve terminals. Here, we demonstrate a specific, SH3 domain-mediated, interaction between amphiphysin and dynamin by gel overlay and affinity chromatography. In addition, we show that the two proteins are colocalized in nerve terminals and are coprecipitated from brain extracts consistent with their interactions in situ. We also report that a region of amphiphysin distinct from its SH3 domain mediates its binding to the a, subunit of AP2 adaptin, which is also concentrated in nerve terminals. These findings support a role of amphiphysin in synaptic vesicle endocytosis.Strong evidence implicates the GTPase dynamin (1, 2) in the internalization of synaptic vesicle membranes after exocytosis and, more generally, in internalization of clathrin-coated vesicles. Temperature-sensitive mutations of the dynamin gene (shibire) in Drosophila cause a selective arrest of the synaptic vesicle cycle at the stage of invaginated plasmalemmal pits (3)(4)(5)(6), and transfection of dominant negative dynamin mutants in fibroblastic cells blocks clathrin-mediated endocytosis (7,8). Recent studies have shown that dynamin forms rings at the neck of invaginated clathrin-coated vesicles and suggested that a conformational change of the rings which correlates with GTP hydrolysis leads to vesicle fission (9, 10). The identification of dynamin's physiological binding partner will be an important next step toward a full elucidation of endocytotic mechanisms.Dynamin has a proline-rich C-terminal region that binds to a subset of SH3 domains. It was found to bind most effectively to the SH3 domains of Grb2, phospholipase Cy,, and the p85 subunit of phosphatidylinositol 3-kinase (11-14). However, none of these proteins was shown to be concentrated in nerve terminals and the significance of these interactions for synaptic vesicle recycling remains unclear. In this study we have explored the possibility that amphiphysin, a neuronal SH3 domain-containing protein selectively concentrated in axon endings (15-17), may represent a physiological partner for dynamin. Amphiphysin is a hydrophilic, highly acidic protein, which is found in soluble and particulate fractions of brain homogenates including synaptic vesicle membranes but is not enriched in purified synaptic vesicles (15)(16)(17) MATERIALS AND METHODS Antibodies. Polyclonal antibodies (CD5 and CD6) directed against full-length glutathione S-transferase (GST)-amphiphysin were raised in rabbits and affinity purified on polyhistidinetagged amphiphysin (His-amph) fusion proteins. Polyclonal antibodies directed against dynamin were obtained by injecting rabbits with gel slices containing rat brain dynamin purified on a Grb2 column. A polyclonal anti-synapsin antibody (G246) was previously described (20). The T7 tag antibody which recognizes an 11-amino acid (aa) sequence in the pTrcHis constructs was from Novagen. The following antibodies were generous gifts: ...