Manu Sharma

α-Synuclein Promotes SNARE-Complex Assembly in Vivo and in Vitro

α-Synuclein and Aging Transgenic α-synuclein can reverse the otherwise lethal neurodegeneration of cysteine string protein-α knockout mice via changes in SNARE proteins, which mediate synaptic vesicle release. Using experiments with purified recombinant proteins, triple αβγ-synuclein knockout mice, and studies of mouse aging, Burré et al. (p. 1663, published online 26 August) now demonstrate that α-synuclein directly interacts with the SNARE protein synaptobrevin and functions as a catalyst for SNARE-complex assembly. The role of synucleins is fully dispensable in young animals, but becomes essential late in life, which suggests that α-synuclein maintains normal synaptic function during aging. A protein implicated in neurodegeneration promotes the assembly of membrane fusion complexes. Presynaptic nerve terminals release neurotransmitters repeatedly, often at high frequency, and in relative isolation from neuronal cell bodies. Repeated release requires cycles of soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)–complex assembly and disassembly, with continuous generation of reactive SNARE-protein intermediates. Although many forms of neurodegeneration initiate presynaptically, only few pathogenic mechanisms are known, and the functions of presynaptic proteins linked to neurodegeneration, such as α-synuclein, remain unclear. Here, we show that maintenance of continuous presynaptic SNARE-complex assembly required a nonclassical chaperone activity mediated by synucleins. Specifically, α-synuclein directly bound to the SNARE-protein synaptobrevin-2/vesicle-associated membrane protein 2 (VAMP2) and promoted SNARE-complex assembly. Moreover, triple-knockout mice lacking synucleins developed age-dependent neurological impairments, exhibited decreased SNARE-complex assembly, and died prematurely. Thus, synucleins may function to sustain normal SNARE-complex assembly in a presynaptic terminal during aging.

CASK Functions as a Mg2+-independent neurexin kinase

CASK is a unique MAGUK protein that contains an N-terminal CaM-kinase domain besides the typical MAGUK domains. The CASK CaM-kinase domain is presumed to be a catalytically inactive pseudokinase because it lacks the canonical DFG motif required for Mg2+ binding that is thought to be indispensable for kinase activity. Here we show, however, that CASK functions as an active protein kinase even without Mg2+ binding. High-resolution crystal structures reveal that the CASK CaM-kinase domain adopts a constitutively active conformation that binds ATP and catalyzes phosphotransfer without Mg2+. The CASK CaM-kinase domain phosphorylates itself and at least one physiological interactor, the synaptic protein neurexin-1, to which CASK is recruited via its PDZ domain. Thus, our data indicate that CASK combines the scaffolding activity of MAGUKs with an unusual kinase activity that phosphorylates substrates recuited by the scaffolding activity. Moreover, our study suggests that other pseudokinases (10% of the kinome) could also be catalytically active.

Autism-linked neuroligin-3 R451C mutation differentially alters hippocampal and cortical synaptic function

Multiple independent mutations in neuroligin genes were identified in patients with familial autism, including the R451C substitution in neuroligin-3 (NL3). Previous studies showed that NL3R451C knock-in mice exhibited modestly impaired social behaviors, enhanced water maze learning abilities, and increased synaptic inhibition in the somatosensory cortex, and they suggested that the behavioral changes in these mice may be caused by a general shift of synaptic transmission to inhibition. Here, we confirm that NL3R451C mutant mice behaviorally exhibit social interaction deficits and electrophysiologically display increased synaptic inhibition in the somatosensory cortex. Unexpectedly, however, we find that the NL3R451C mutation produced a strikingly different phenotype in the hippocampus. Specifically, in the hippocampal CA1 region, the NL3R451C mutation caused an ∼1.5-fold increase in AMPA receptor-mediated excitatory synaptic transmission, dramatically altered the kinetics of NMDA receptor-mediated synaptic responses, induced an approximately twofold up-regulation of NMDA receptors containing NR2B subunits, and enhanced long-term potentiation almost twofold. NL3 KO mice did not exhibit any of these changes. Quantitative light microscopy and EM revealed that the NL3R451C mutation increased dendritic branching and altered the structure of synapses in the stratum radiatum of the hippocampus. Thus, in NL3R451C mutant mice, a single point mutation in a synaptic cell adhesion molecule causes context-dependent changes in synaptic transmission; these changes are consistent with the broad impact of this mutation on murine and human behaviors, suggesting that NL3 controls excitatory and inhibitory synapse properties in a region- and circuit-specific manner.

CSPα promotes SNARE-complex assembly by chaperoning SNAP-25 during synaptic activity

A neuron forms thousands of presynaptic nerve terminals on its axons, far removed from the cell body. The protein CSPα resides in presynaptic terminals, where it forms a chaperone complex with Hsc70 and SGT. Deletion of CSPα results in massive neurodegeneration that impairs survival in mice and flies. In CSPα-knockout mice, levels of presynaptic SNARE complexes and the SNARE protein SNAP-25 are reduced, suggesting that CSPα may chaperone SNARE proteins, which catalyse synaptic vesicle fusion. Here, we show that the CSPα–Hsc70–SGT complex binds directly to monomeric SNAP-25 to prevent its aggregation, enabling SNARE-complex formation. Deletion of CSPα produces an abnormal SNAP-25 conformer that inhibits SNARE-complex formation, and is subject to ubiquitylation and proteasomal degradation. Even in wild-type mouse terminals, SNAP-25 degradation is regulated by synaptic activity; this degradation is decreased by CSPα overexpression, and enhanced by CSPα deletion. Thus, SNAP-25 function is maintained during rapid SNARE cycles by equilibrium between CSPα-dependent chaperoning and ubiquitin-dependent degradation, revealing unique protein quality-control machinery within the presynaptic compartment.

Native α-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2

α-Synuclein is a presynaptic protein that is implicated in Parkinsons and other neurodegenerative diseases. Physiologically, native α-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native α-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinsons disease-related point mutants of α-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. α-Synuclein had little effect on Ca2+-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a ‘buffer’ of synaptic vesicles, without affecting neurotransmitter release itself. DOI: http://dx.doi.org/10.7554/eLife.00592.001

Properties of native brain α-synuclein

Arising from T. Bartels, J. G. Choi & D. J. Selkoe. 477, 107–110 (2011).10.1038/nature10324α-Synuclein is an abundant presynaptic protein that binds to negatively charged phospholipids, functions as a SNARE-complex chaperone and contributes to Parkinson’s disease pathogenesis. Recombinant α-synuclein in solution is largely unfolded and devoid of tertiary structure, but Bartels et al. have proposed that native α-synuclein purified from human erythrocytes forms a stably folded, soluble tetramer that resists aggregation. By contrast, we show here that native α-synuclein purified from mouse brain consists of a largely unstructured monomer, exhibits no stable tetramer formation, and is prone to aggregation. The native state of α-synuclein is important for understanding its pathological effects as a stably folded protein would be much less prone to aggregation than a conformationally labile protein. There is a Reply to this Brief Communication Arising by Bartels, T. & Selkoe, D. J. Nature 498, http://dx.doi.org/10.1038/nature12126 (2013).

An autism-associated point mutation in the neuroligin cytoplasmic tail selectively impairs AMPA receptor-mediated synaptic transmission in hippocampus

Neuroligins are evolutionarily conserved postsynaptic cell‐adhesion molecules that function, at least in part, by forming trans‐synaptic complexes with presynaptic neurexins. Different neuroligin isoforms perform diverse functions and exhibit distinct intracellular localizations, but contain similar cytoplasmic sequences whose role remains largely unknown. Here, we analysed the effect of a single amino‐acid substitution (R704C) that targets a conserved arginine residue in the cytoplasmic sequence of all neuroligins, and that was associated with autism in neuroligin‐4. We introduced the R704C mutation into mouse neuroligin‐3 by homologous recombination, and examined its effect on synapses in vitro and in vivo. Electrophysiological and morphological studies revealed that the neuroligin‐3 R704C mutation did not significantly alter synapse formation, but dramatically impaired synapse function. Specifically, the R704C mutation caused a major and selective decrease in AMPA receptor‐mediated synaptic transmission in pyramidal neurons of the hippocampus, without similarly changing NMDA or GABA receptor‐mediated synaptic transmission, and without detectably altering presynaptic neurotransmitter release. Our results suggest that the cytoplasmic tail of neuroligin‐3 has a central role in synaptic transmission by modulating the recruitment of AMPA receptors to postsynaptic sites at excitatory synapses.

Systematic Mutagenesis of α-Synuclein Reveals Distinct Sequence Requirements for Physiological and Pathological Activities

α-Synuclein is an abundant presynaptic protein that binds to phospholipids and synaptic vesicles. Physiologically, α-synuclein functions as a SNARE-protein chaperone that promotes SNARE-complex assembly for neurotransmitter release. Pathologically, α-synuclein mutations and α-synuclein overexpression cause Parkinsons disease, and aggregates of α-synuclein are found as Lewy bodies in multiple neurodegenerative disorders (“synucleinopathies”). The relation of the physiological functions to the pathological effects of α-synuclein remains unclear. As an initial avenue of addressing this question, we here systematically examined the effect of α-synuclein mutations on its physiological and pathological activities. We generated 26 α-synuclein mutants spanning the entire molecule, and analyzed them compared with wild-type α-synuclein in seven assays that range from biochemical studies with purified α-synuclein, to analyses of α-synuclein expression in cultured neurons, to examinations of the effects of virally expressed α-synuclein introduced into the mouse substantia nigra by stereotactic injections. We found that both the N-terminal and C-terminal sequences of α-synuclein were required for its physiological function as SNARE-complex chaperone, but that these sequences were not essential for its neuropathological effects. In contrast, point mutations in the central region of α-synuclein, referred to as nonamyloid β component (residues 61–95), as well as point mutations linked to Parkinsons disease (A30P, E46K, and A53T) increased the neurotoxicity of α-synuclein but did not affect its physiological function in SNARE-complex assembly. Thus, our data show that the physiological function of α-synuclein, although protective of neurodegeneration in some contexts, is fundamentally distinct from its neuropathological effects, thereby dissociating the two activities of α-synuclein.

CSPα knockout causes neurodegeneration by impairing SNAP-25 function.

At a synapse, the synaptic vesicle protein cysteine‐string protein‐α (CSPα) functions as a co‐chaperone for the SNARE protein SNAP‐25. Knockout (KO) of CSPα causes fulminant neurodegeneration that is rescued by α‐synuclein overexpression. The CSPα KO decreases SNAP‐25 levels and impairs SNARE‐complex assembly; only the latter but not the former is reversed by α‐synuclein. Thus, the question arises whether the CSPα KO phenotype is due to decreased SNAP‐25 function that then causes neurodegeneration, or due to the dysfunction of multiple as‐yet uncharacterized CSPα targets. Here, we demonstrate that decreasing SNAP‐25 levels in CSPα KO mice by either KO or knockdown of SNAP‐25 aggravated their phenotype. Conversely, increasing SNAP‐25 levels by overexpression rescued their phenotype. Inactive SNAP‐25 mutants were unable to rescue, showing that the rescue was specific. Under all conditions, the neurodegenerative phenotype precisely correlated with SNARE‐complex assembly, indicating that impaired SNARE‐complex assembly due to decreased SNAP‐25 levels is the ultimate correlate of neurodegeneration. Our findings suggest that the neurodegeneration in CSPα KO mice is primarily produced by defective SNAP‐25 function, which causes neurodegeneration by impairing SNARE‐complex assembly.

Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ

Significance Synaptic vesicle fusion is catalyzed by multiprotein complexes that bring two lipid bilayers into close opposition. Several assembly mechanisms have been proposed for the synaptic vesicle fusion machinery, but exactly how these proteins interact in vivo remains unclear. We developed two-color fluorescence nanoscopy to directly visualize molecular interactions in situ and discovered that syntaxin-1, SNAP-25, and Munc18-1 (mammalian uncoordinated-18), three essential components for neurotransmission, closely colocalize on the plasma membrane, suggesting possible pathways for SNARE-mediated membrane fusion. Our superresolution method provides a framework for delineating the molecular underpinnings of the synaptic vesicle fusion machinery. Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ.