Clathrin-mediated Synaptic Vesicle Endocytosis Research Articles

Acute introduction of monomeric or multimeric α-synuclein induces distinct impacts on synaptic vesicle trafficking at lamprey giant synapses.

Synaptic aggregation of α-synuclein often occurs in Parkinson's disease (PD), dementia with Lewy bodies (DLB) and other synucleinopathies and is associated with cognitive deficits and dementia. Thus, it is important to understand how accumulation of α-synuclein affects synapse structure and function. Native, physiological α-synuclein comprises a mixture of tetramers and related physiological oligomers (60-100kDa) in equilibrium with monomeric α-synuclein. We previously demonstrated that acutely increasing the levels of physiologicalα-synuclein impaired intracellular synaptic vesicle trafficking and produced a pleiotropic phenotype, raising questions about which aspects of the synaptic phenotype were due to multimeric versus monomeric α-synuclein. Here, we address this by taking advantage of the unique features of the lamprey giant reticulospinal (RS) synapse, a vertebrate synapse that is amenable to acute perturbations of presynaptic processes via microinjection of purified proteins. α-Synuclein monomers and multimers were purified from HEK cells and separately introduced to lamprey synapses. Ultrastructural analysis revealed that both multimeric and monomeric α-synuclein impaired intracellular vesicle trafficking, leading to a loss of synaptic vesicles and buildup of endosomes. However, while monomeric α-synuclein additionally induced atypical fusion/fission at the active zone and impaired clathrin-mediated endocytosis, multimeric α-synuclein did not. Conversely, multimeric α-synuclein led to a decrease in synaptic vesicle docking, which was not observed with monomeric α-synuclein. These data provide further evidence that different molecular species of α-synuclein produce distinct and complex impacts on synaptic vesicle trafficking and reveal important insights into the cell biological processes that are affected in PD and DLB. KEY POINTS: α-Synuclein accumulation at synapses is associated with cognitive decline and dementia in Parkinson's disease and other synucleinopathies. We previously showed that acute introduction of excess human brain-derived α-synuclein to lamprey giant synapses caused pleiotropic phenotypes on synaptic vesicle trafficking, probably due to the mixture of molecular species of α-synuclein. Here, we dissected which aspects of the synaptic phenotypes were caused by monomeric (14kDa) or multimeric (60-100kDa) α-synuclein by purifying each molecular species and introducing each one separately to synapses via axonal microinjection. While monomeric α-synuclein inhibited clathrin-mediated synaptic vesicle endocytosis, multimeric α-synuclein primarily impaired endosomal trafficking. These findings reveal that different molecular species of α-synuclein have distinct impacts on synapses, suggesting different cellular and molecular targets.

The SAC1 phosphatase domain of synaptojanin-1 is activated by interacting with polyunsaturated fatty acid-containing phosphatidic acids.

Although there are many phosphatidic acid (PA) molecular species based on its fatty acyl compositions, their interacting partners have been poorly investigated. Here, we identified synaptojanin-1 (SYNJ1), Parkinson's disease-related protein that is essential for regulating clathrin-mediated synaptic vesicle endocytosis via dually dephosphorylating D5 and D4 position phosphates from phosphatidylinositol (PI) (4,5)-bisphosphate, as a 1-stearoyl-2-docosahexaenoyl (18:0/22:6)-PA-binding protein. SYNJ1 failed to substantially associate with other acidic phospholipids. Although SYNJ1 interacted with 18:0/20:4-PA in addition to 18:0/22:6-PA, the association of the enzyme with 16:0/16:0-, 16:0/18:1-, 18:0/18:0-, or 18:1/18:1-PA was not considerable. 18:0/20:4- and 18:0/22:6-PAs bound to SYNJ1 via its SAC1 domain, which preferentially hydrolyses D4 position phosphate. Moreover, 18:0/20:4- and 18:0/22:6-PA selectively enhanced the D4-phosphatase activity, but not the D5-phosphatase activity, of SYNJ1.

Effects of Excess Brain-Derived Human α-Synuclein on Synaptic Vesicle Trafficking.

α-Synuclein is a presynaptic protein that regulates synaptic vesicle trafficking under physiological conditions. However, in several neurodegenerative diseases, including Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy, α-synuclein accumulates throughout the neuron, including at synapses, leading to altered synaptic function, neurotoxicity, and motor, cognitive, and autonomic dysfunction. Neurons typically contain both monomeric and multimeric forms of α-synuclein, and it is generally accepted that disrupting the balance between them promotes aggregation and neurotoxicity. However, it remains unclear how distinct molecular species of α-synuclein affect synapses where α-synuclein is normally expressed. Using the lamprey reticulospinal synapse model, we previously showed that acute introduction of excess recombinant monomeric or dimeric α-synuclein impaired distinct stages of clathrin-mediated synaptic vesicle endocytosis, leading to a loss of synaptic vesicles. Here, we expand this knowledge by investigating the effects of native, physiological α-synuclein isolated from the brain of a neuropathologically normal human subject, which comprised predominantly helically folded multimeric α-synuclein with a minor component of monomeric α-synuclein. After acute introduction of excess brain-derived human α-synuclein, there was a moderate reduction in the synaptic vesicle cluster and an increase in the number of large, atypical vesicles called “cisternae.” In addition, brain-derived α-synuclein increased synaptic vesicle and cisternae sizes and induced atypical fusion/fission events at the active zone. In contrast to monomeric or dimeric α-synuclein, the brain-derived multimeric α-synuclein did not appear to alter clathrin-mediated synaptic vesicle endocytosis. Taken together, these data suggest that excess brain-derived human α-synuclein impairs intracellular vesicle trafficking and further corroborate the idea that different molecular species of α-synuclein produce distinct trafficking defects at synapses. These findings provide insights into the mechanisms by which excess α-synuclein contributes to synaptic deficits and disease phenotypes.

DNAJC6 Mutations Disrupt Dopamine Homeostasis in Juvenile Parkinsonism-Dystonia.

BackgroundJuvenile forms of parkinsonism are rare conditions with onset of bradykinesia, tremor and rigidity before the age of 21 years. These atypical presentations commonly have a genetic aetiology, highlighting important insights into underlying pathophysiology. Genetic defects may affect key proteins of the endocytic pathway and clathrin‐mediated endocytosis (CME), as in DNAJC6‐related juvenile parkinsonism.ObjectiveTo report on a new patient cohort with juvenile‐onset DNAJC6 parkinsonism‐dystonia and determine the functional consequences on auxilin and dopamine homeostasis.MethodsTwenty‐five children with juvenile parkinsonism were identified from a research cohort of patients with undiagnosed pediatric movement disorders. Molecular genetic investigations included autozygosity mapping studies and whole‐exome sequencing. Patient fibroblasts and CSF were analyzed for auxilin, cyclin G–associated kinase and synaptic proteins.ResultsWe identified 6 patients harboring previously unreported, homozygous nonsense DNAJC6 mutations. All presented with neurodevelopmental delay in infancy, progressive parkinsonism, and neurological regression in childhood. 123I‐FP‐CIT SPECT (DaTScan) was performed in 3 patients and demonstrated reduced or absent tracer uptake in the basal ganglia. CSF neurotransmitter analysis revealed an isolated reduction of homovanillic acid. Auxilin levels were significantly reduced in both patient fibroblasts and CSF. Cyclin G–associated kinase levels in CSF were significantly increased, whereas a number of presynaptic dopaminergic proteins were reduced.ConclusionsDNAJC6 is an emerging cause of recessive juvenile parkinsonism‐dystonia. DNAJC6 encodes the cochaperone protein auxilin, involved in CME of synaptic vesicles. The observed dopamine dyshomeostasis in patients is likely to be multifactorial, secondary to auxilin deficiency and/or neurodegeneration. Increased patient CSF cyclin G–associated kinase, in tandem with reduced auxilin levels, suggests a possible compensatory role of cyclin G–associated kinase, as observed in the auxilin knockout mouse. DNAJC6 parkinsonism‐dystonia should be considered as a differential diagnosis for pediatric neurotransmitter disorders associated with low homovanillic acid levels. Future research in elucidating disease pathogenesis will aid the development of better treatments for this pharmacoresistant disorder. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

α-Synuclein-112 Impairs Synaptic Vesicle Recycling Consistent With Its Enhanced Membrane Binding Properties.

Synucleinopathies are neurological disorders associated with α-synuclein overexpression and aggregation. While it is well-established that overexpression of wild type α-synuclein (α-syn-140) leads to cellular toxicity and neurodegeneration, much less is known about other naturally occurring α-synuclein splice isoforms. In this study we provide the first detailed examination of the synaptic effects caused by one of these splice isoforms, α-synuclein-112 (α-syn-112). α-Syn-112 is produced by an in-frame excision of exon 5, resulting in deletion of amino acids 103–130 in the C-terminal region. α-Syn-112 is upregulated in the substantia nigra, frontal cortex, and cerebellum of parkinsonian brains and higher expression levels are correlated with susceptibility to Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA). We report here that α-syn-112 binds strongly to anionic phospholipids when presented in highly curved liposomes, similar to α-syn-140. However, α-syn-112 bound significantly stronger to all phospholipids tested, including the phosphoinositides. α-Syn-112 also dimerized and trimerized on isolated synaptic membranes, while α-syn-140 remained largely monomeric. When introduced acutely to lamprey synapses, α-syn-112 robustly inhibited synaptic vesicle recycling. Interestingly, α-syn-112 produced effects on the plasma membrane and clathrin-mediated synaptic vesicle endocytosis that were phenotypically intermediate between those caused by monomeric and dimeric α-syn-140. These findings indicate that α-syn-112 exhibits enhanced phospholipid binding and oligomerization in vitro and consequently interferes with synaptic vesicle recycling in vivo in ways that are consistent with its biochemical properties. This study provides additional evidence suggesting that impaired vesicle endocytosis is a cellular target of excess α-synuclein and advances our understanding of potential mechanisms underlying disease pathogenesis in the synucleinopathies.

An integrated transcriptomics and proteomics analysis reveals functional endocytic dysregulation caused by mutations in LRRK2

BackgroundMutations in LRRK2 are the most common cause of autosomal dominant Parkinson's disease, and the relevance of LRRK2 to the sporadic form of the disease is becoming ever more apparent. It is therefore essential that studies are conducted to improve our understanding of the cellular role of this protein. Here we use multiple models and techniques to identify the pathways through which LRRK2 mutations may lead to the development of Parkinson's disease. MethodsA novel integrated transcriptomics and proteomics approach was used to identify pathways that were significantly altered in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blotting, immunostaining and functional assays including FM1-43 analysis of synaptic vesicle endocytosis were performed to confirm these findings in iPSC-derived dopaminergic neuronal cultures carrying either the LRRK2-G2019S or the LRRK2-R1441C mutation, and LRRK2 BAC transgenic rats, and post-mortem human brain tissue from LRRK2-G2019S patients. ResultsOur integrated -omics analysis revealed highly significant dysregulation of the endocytic pathway in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blot analysis confirmed that key endocytic proteins including endophilin I-III, dynamin-1, and various RAB proteins were downregulated in these cultures and in cultures carrying the LRRK2-R1441C mutation, compared with controls. We also found changes in expression of 25 RAB proteins. Changes in endocytic protein expression led to a functional impairment in clathrin-mediated synaptic vesicle endocytosis. Further to this, we found that the endocytic pathway was also perturbed in striatal tissue of aged LRRK2 BAC transgenic rats overexpressing either the LRRK2 wildtype, LRRK2-R1441C or LRRK2-G2019S transgenes. Finally, we found that clathrin heavy chain and endophilin I-III levels are increased in human post-mortem tissue from LRRK2-G2019S patients compared with controls. ConclusionsOur study demonstrates extensive alterations across the endocytic pathway associated with LRRK2 mutations in iPSC-derived dopaminergic neurons and BAC transgenic rats, as well as in post-mortem brain tissue from PD patients carrying a LRRK2 mutation. In particular, we find evidence of disrupted clathrin-mediated endocytosis and suggest that LRRK2-mediated PD pathogenesis may arise through dysregulation of this process.

Identification of putative second genetic hits in schizophrenia carriers of high-risk copy number variants and resequencing in additional samples

Copy number variants (CNVs) conferring risk of schizophrenia present incomplete penetrance, suggesting the existence of second genetic hits. Identification of second hits may help to find genes with rare variants of susceptibility to schizophrenia. The aim of this work was to search for second hits of moderate/high risk in schizophrenia carriers of risk CNVs and resequencing of the relevant genes in additional samples. To this end, ten patients with risk CNVs at cytobands 15q11.2, 15q11.2-13.1, 16p11.2, or 16p13.11, were subjected to whole-exome sequencing. Rare single nucleotide variants, defined as those absent from main public databases, were classified according to bioinformatic prediction of pathogenicity by CADD scores. The average number of rare predicted pathogenic variants per sample was 13.6 (SD 2.01). Two genes, BFAR and SYNJ1, presented rare predicted pathogenic variants in more than one sample. Follow-up resequencing of these genes in 432 additional cases and 432 controls identified a significant excess of rare predicted pathogenic variants in case samples at SYNJ1. Taking into account its function in clathrin-mediated synaptic vesicle endocytosis at presynaptic terminals, our results suggest an impairment of this process in schizophrenia.

Endophilin A1 regulates dendritic spine morphogenesis and stability through interaction with p140Cap.

Dendritic spines are actin-rich membrane protrusions that are the major sites of excitatory synaptic input in the mammalian brain, and their morphological plasticity provides structural basis for learning and memory. Here we report that endophilin A1, with a well-established role in clathrin-mediated synaptic vesicle endocytosis at the presynaptic terminal, also localizes to dendritic spines and is required for spine morphogenesis, synapse formation and synaptic function. We identify p140Cap, a regulator of cytoskeleton reorganization, as a downstream effector of endophilin A1 and demonstrate that disruption of their interaction impairs spine formation and maturation. Moreover, we demonstrate that knockdown of endophilin A1 or p140Cap impairs spine stabilization and synaptic function. We further show that endophilin A1 regulates the distribution of p140Cap and its downstream effector, the F-actin-binding protein cortactin as well as F-actin enrichment in dendritic spines. Together, these results reveal a novel function of postsynaptic endophilin A1 in spine morphogenesis, stabilization and synaptic function through the regulation of p140Cap.

LRRK2 functions in synaptic vesicle endocytosis through a kinase-dependent mechanism.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson’s disease, but the precise physiological function of the protein remains ill-defined. Recently, our group proposed a model in which LRRK2 kinase activity is part of an EndoA phosphorylation cycle that facilitates efficient vesicle formation at synapses in the Drosophila melanogaster neuromuscular junctions.Flies harbor only one Lrrk gene, which might encompass the functions of both mammalian LRRK1 and LRRK2. We therefore studied the role of LRRK2 in mammalian synaptic function and provide evidence that knockout or pharmacological inhibition of LRRK2 results in defects in synaptic vesicle endocytosis, altered synaptic morphology and impairments in neurotransmission. In addition, our data indicate that mammalian endophilin A1 (EndoA1,also known as SH3GL2) is phosphorylated by LRRK2 in vitro at T73 and S75, two residues in the BAR domain. Hence, our results indicate that LRRK2 kinase activity has an important role in the regulation of clathrin-mediated endocytosis of synaptic vesicles and subsequent neurotransmission at the synapse.

Calcineurin Selectively Docks with the Dynamin Ixb Splice Variant to Regulate Activity-dependent Bulk Endocytosis

Depolarization of nerve terminals stimulates rapid dephosphorylation of two isoforms of dynamin I (dynI), mediated by the calcium-dependent phosphatase calcineurin (CaN). Dephosphorylation at the major phosphorylation sites Ser-774/778 promotes a dynI-syndapin I interaction for a specific mode of synaptic vesicle endocytosis called activity-dependent bulk endocytosis (ADBE). DynI has two main splice variants at its extreme C terminus, long or short (dynIxa and dynIxb) varying only by 20 (xa) or 7 (xb) residues. Recombinant GST fusion proteins of dynIxa and dynIxb proline-rich domains (PRDs) were used to pull down interacting proteins from rat brain nerve terminals. Both bound equally to syndapin, but dynIxb PRD exclusively bound to the catalytic subunit of CaNA, which recruited CaNB. Binding of CaN was increased in the presence of calcium and was accompanied by further recruitment of calmodulin. Point mutations showed that the entire C terminus of dynIxb is a CaN docking site related to a conserved CaN docking motif (PXIXI(T/S)). This sequence is unique to dynIxb among all other dynamin variants or genes. Peptide mimetics of the dynIxb tail blocked CaN binding in vitro and selectively inhibited depolarization-evoked dynI dephosphorylation in nerve terminals but not of other dephosphins. Therefore, docking to dynIxb is required for the regulation of both dynI splice variants, yet it does not regulate the phosphorylation cycle of other dephosphins. The peptide blocked ADBE, but not clathrin-mediated endocytosis of synaptic vesicles. Our results indicate that Ca(2+) influx regulates assembly of a fully active CaN-calmodulin complex selectively on the tail of dynIxb and that the complex is recruited to sites of ADBE in nerve terminals.

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

Members of the DISABLED (DAB) family of proteins are known to play a conserved role in endocytic trafficking of cell surface receptors by functioning as monomeric CLATHRIN-associated sorting proteins that recruit cargo proteins into endocytic vesicles. Here, we report a Drosophila disabled mutant revealing a novel role for DAB proteins in chemical synaptic transmission. This mutant exhibits impaired synaptic function, including a rapid activity-dependent reduction in neurotransmitter release and disruption of synaptic vesicle endocytosis. In presynaptic boutons, Drosophila DAB and CLATHRIN were highly colocalized within two distinct classes of puncta, including relatively dim puncta that were located at active zones and may reflect endocytic mechanisms operating at neurotransmitter release sites. Finally, broader analysis of endocytic proteins, including DYNAMIN, supported a general role for CLATHRIN-mediated endocytic mechanisms in rapid clearance of neurotransmitter release sites for subsequent vesicle priming and refilling of the release-ready vesicle pool.

Endocytosis and clathrin-uncoating defects at synapses of auxilin knockout mice

Neuronally expressed auxilin and ubiquitously expressed cyclin-G-dependent kinase (GAK) are homologous proteins that act as cochaperones to support the Hsc70-dependent clathrin uncoating of clathrin-coated vesicles. GAK was previously shown to be essential in mouse during embryonic development and in the adult. We have now engineered an auxilin knockout mouse. Mutant mice had a high rate of early postnatal mortality and surviving pups generally had a lower body weight than wild-type pups, although they had a normal life span. GAK was up-regulated as much as 3-fold in the brains of both surviving neonates and adult mutant mice. An increased number of clathrin-coated vesicles and empty cages were present at knockout synapses both in situ and in primary neuronal cultures. Additionally, clathrin-mediated endocytosis of synaptic vesicles in knockout hippocampal neurons was impaired, most likely due to sequestration of coat components in assembled coats and cages. Collectively, our results demonstrate the specialized role of auxilin in the recycling of synaptic vesicles at synapses, but also show that its function can be partially compensated for by up-regulation of GAK.

Molecular Basis for Association of PIPKIγ-p90 with Clathrin Adaptor AP-2

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the I gamma-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P(2) metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKI gamma-p90 associates with both the mu and beta2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKI gamma-p90 tail binds to a cognate recognition site on the sandwich subdomain of the beta2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2mu, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKI gamma-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKI gamma tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2 beta and AP-2mu. Our data also suggest that interactions between AP-2 and the tail domain of PIPKI gamma-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKI gamma-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P(2) synthesis during clathrin-mediated SV endocytosis.

Amphiphysin I and regulation of synaptic vesicle endocytosis.

Amphiphysin I, known as a major dynamin-binding partner localized on the collars of nascent vesicles, plays a key role in clathrin-mediated endocytosis (CME) of synaptic vesicles. Amphiphysin I mediates the invagination and fission steps of synaptic vesicles by sensing or facilitating membrane curvature and stimulating the GTPase activity of dynamin. Amphiphysin I may form a homodimer by itself or a heterodimer with amphiphysin II in vivo. Both amphiphysin I and II function as multilinker proteins in the clathrin-coated complex. Under normal physiological conditions, the functions of amphiphysin I and some other endocytic proteins are known to be regulated by phosphorylation and dephosphorylation. During hyperexcited conditions, the most recent data showed that amphiphysin I is truncated by the ca2-dependent protease calpain. Overexpression of the truncated form of amphiphysin I inhibited transferrin uptake and synaptic vesicle endocytosis (SVE). This suggests that amphiphysin I may be an important regulator for SVE when massive amounts of Ca2 flow into presynaptic terminals, a phenomenon observed in neurodegenerative disorders such as ischemia/anoxia, epilepsy, stroke, trauma and Alzheimer's disease. This review describes current knowledge regarding the general properties and functions of amphiphysin I as well as the functional regulations such as phosphorylation and proteolysis in nerve terminals.

The Drosophila Epsin 1 is Required for Ubiquitin‐Dependent Synaptic Growth and Function but Not for Synaptic Vesicle Recycling

The ubiquitin-proteasome system plays an important role in synaptic development and function. However, many components of this system, and how they act to affect synapses, are still not well understood. In this study, we use the Drosophila neuromuscular junction to study the in vivo function of Liquid facets (Lqf), a homolog of mammalian epsin 1. Our data show that Lqf plays a novel role in synapse development and function. Contrary to prior models, Lqf is not required for clathrin-mediated endocytosis of synaptic vesicles. Lqf is required to maintain bouton size and shape and to sustain synapse growth by acting as a specific substrate of the deubiquitinating enzyme Fat facets. However, Lqf is not a substrate of the Highwire (Hiw) E3 ubiquitin ligase; neither is it required for synapse overgrowth in hiw mutants. Interestingly, Lqf converges on the Hiw pathway by negatively regulating transmitter release in the hiw mutant. These observations demonstrate that Lqf plays distinct roles in two ubiquitin pathways to regulate structural and functional plasticity of the synapse.

Role of epsin 1 in synaptic vesicle endocytosis

Epsin has been suggested to act as an alternate adaptor in several endocytic pathways. Its role in synaptic vesicle recycling remains, however, unclear. Here, we examined the role of epsin in this process by using the lamprey reticulospinal synapse as a model system. We characterized a lamprey ortholog of epsin 1 and showed that it is accumulated at release sites at rest and also at clathrin-coated pits in the periactive zone during synaptic activity. Disruption of epsin interactions, by presynaptic microinjection of antibodies to either the epsin-N-terminal homology domain (ENTH) or the clathrin/AP2 binding region (CLAP), caused profound loss of vesicles in stimulated synapses. CLAP antibody-injected synapses displayed a massive accumulation of distorted coated structures, including coated vacuoles, whereas in synapses perturbed with ENTH antibodies, very few coated structures were found. In both cases coated pits on the plasma membrane showed a shift to early intermediates (shallow coated pits) and an increase in size. Moreover, in CLAP antibody-injected synapses flat clathrin-coated patches occurred on the plasma membrane. We conclude that epsin is involved in clathrin-mediated synaptic vesicle endocytosis. Our results support a model, based on in vitro studies, suggesting that epsin coordinates curvature generation with coat assembly and further indicating that epsin limits clathrin coat assembly to the size of newly formed vesicles. We propose that these functions of epsin 1 provide an additional mechanism for generation of uniformly sized synaptic vesicles.

Sorting Nexin 9 Interacts with Dynamin 1 and N-WASP and Coordinates Synaptic Vesicle Endocytosis

Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins, each of which contains a characteristic Phox homology domain. SNX9 is widely expressed and plays a role in clathrin-mediated endocytosis, but it is not known if it is present in neuronal cells. We report that SNX9 is expressed in the presynaptic compartment of cultured hippocampal neurons, where it binds to dynamin-1 and N-WASP. Overexpression of full-length SNX9 or a C-terminal truncated version caused severe defects in synaptic vesicle endocytosis during, as well as after, stimulation. Knockdown of SNX9 with short interfering RNA also reduced synaptic vesicle endocytosis, and the W39A mutation of SNX9 abolished the inhibitory effect of SNX9 on endocytosis. Rescue experiments showed that most of the effect of SNX9 on endocytosis results from its interaction with dynamin 1, although its interaction with N-WASP contributes in some degree. We further showed that SNX9 dimerizes through its C-terminal domain, suggesting that it may interact simultaneously with dynamin 1 and N-WASP. We propose that SNX9 interacts with dynamin-1 and N-WASP in presynaptic terminals, where it links actin dynamics and synaptic vesicle endocytosis.

Disturbance of synaptic vesicle recycling resulting from deletion of a mammalian actin-binding protein, mAbp1

Physiological and ultrastructural studies of synapses between hippocampal neurons of animals with knock-out of a mammalian actin-binding protein, mAbp1, demonstrated that recycling of synaptic vesicles undergoes, in this case, significant modifications. Thus, mAbp1 is rather important from this aspect, which can be related to the noticeable role of actin in clathrin-mediated endocytosis of synaptic vesicles.

Truncations of amphiphysin I by calpain inhibit vesicle endocytosis during neural hyperexcitation

Under normal physiological conditions, synaptic vesicle endocytosis is regulated by phosphorylation and Ca(2+)-dependent dephosphorylation of endocytic proteins such as amphiphysin and dynamin. To investigate the regulatory mechanisms that may occur under the conditions of excessive presynaptic Ca(2+) influx observed preceding neural hyperexcitation, we examined hippocampal slices following high-potassium or high-frequency electrical stimulation (HFS). In both cases, three truncated forms of amphiphysin I resulted from cleavage by the protease calpain. In vitro, the binding of truncated amphiphysin I to dynamin I and copolymerization into rings with dynamin I were inhibited, but its interaction with liposomes was not affected. Moreover, overexpression of the truncated form of amphiphysin I inhibited endocytosis of transferrin and synaptic vesicles. Inhibiting calpain prevented HFS-induced depression of presynaptic transmission. Finally, calpain-dependent amphiphysin I cleavage attenuated kainate-induced seizures. These results suggest that calpain-dependent cleavage of amphiphysin I inhibits synaptic vesicle endocytosis during neural hyperexcitation and demonstrate a novel post-translational regulation of endocytosis.

SH3P7/mAbp1 deficiency leads to tissue and behavioral abnormalities and impaired vesicle transport

The intracellular adaptor protein SH3P7 is the mammalian ortholog of yeast actin-binding protein 1 and thus alternatively named as mAbp1 (or HIP55). Structural properties, biochemical analysis of its interaction partners and siRNA studies implicated mAbp1 as an accessory protein in clathrin-mediated endocytosis (CME). Here, we describe the generation and characterization of mice deficient for SH3P7/mAbp1 owing to targeted gene disruption in embryonic stem cells. Mutant animals are viable and fertile without obvious deficits during the first weeks of life. Abnormal structure and function of organs including the spleen, heart, and lung is observed at about 3 months of age in both heterozygous and homozygous mouse mutants. A moderate reduction of both receptor-mediated and synaptic endocytosis is observed in embryonic fibroblasts and in synapses of hippocampal neurons, respectively. Recycling of synaptic vesicles in hippocampal boutons is severely impaired and delayed four-fold. The presynaptic defect of SH3P7/mAbp1 mouse mutants is associated with their constricted physical capabilities and disturbed neuromotoric behaviour. Our data reveal a nonredundant role of SH3P7/mAbp1 in CME and places its function downstream of vesicle fission.