LRK-1/LRRK2 and AP-3 regulate trafficking of synaptic vesicle precursors through active zone protein SYD-2/Liprin-α.

Synaptic vesicle proteins (SVps) are transported by the motor UNC-104/KIF1A. We show that SVps travel in heterogeneous carriers in C. elegans neuronal processes, with some SVp carriers co-transporting lysosomal proteins (SV-lysosomes). LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 play a critical role in the sorting of SVps and lysosomal proteins away from each other at the SV-lysosomal intermediate trafficking compartment. Both SVp carriers lacking lysosomal proteins and SV-lysosomes are dependent on the motor UNC-104/KIF1A for their transport. In lrk-1 mutants, both SVp carriers and SV-lysosomes can travel in axons in the absence of UNC-104, suggesting that LRK-1 plays an important role to enable UNC-104 dependent transport of synaptic vesicle proteins. Additionally, LRK-1 acts upstream of the AP-3 complex and regulates its membrane localization. In the absence of the AP-3 complex, the SV-lysosomes become more dependent on the UNC-104-SYD-2/Liprin-α complex for their transport. Therefore, SYD-2 acts to link upstream trafficking events with the transport of SVps likely through its interaction with the motor UNC-104. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. SYD-2 acts in concert with AP complexes to ensure polarized trafficking & transport of SVps.

Biallelic variants in the synaptic vesicle glycoprotein 2 A are associated with epileptic encephalopathy.

Synaptic Vesicle Glycoprotein 2 A (SV2A) is a membrane protein of synaptic vesicles and the binding site of antiepileptic drug levetiracetam. Biallelic Arg383Gln is reported in a family with intractable epilepsy earlier. Here, we report on the second family with early onset drug resistant epilepsy. We identified homozygous Arg289Ter variant by exome sequencing that segregated with the phenotype in the family. The affected children in these two families are normal at birth and developed recurrent seizures beginning in the second month of life and developed secondary failure of growth and development. Knock out mice models earlier had replicated the human phenotype observed in these two families. These findings support that biallelic loss of function variants in SV2A result in early onset intractable epilepsy in humans.

Insight into the regulation of axonal transport from the study of KIF1A-associated neurological disorder.

Neuronal function depends on axonal transport by kinesin superfamily proteins (KIFs). KIF1A is the molecular motor that transports synaptic vesicle precursors, synaptic vesicles, dense core vesicles and active zone precursors. KIF1A is regulated by an autoinhibitory mechanism; many studies, as well as the crystal structure of KIF1A paralogs, support a model whereby autoinhibited KIF1A is monomeric in solution, whereas activated KIF1A is dimeric on microtubules. KIF1A-associated neurological disorder (KAND) is a broad-spectrum neuropathy that is caused by mutations in KIF1A. More than 100 point mutations have been identified in KAND. In vitro assays show that most mutations are loss-of-function mutations that disrupt the motor activity of KIF1A, whereas some mutations disrupt its autoinhibition and abnormally hyperactivate KIF1A. Studies on disease model worms suggests that both loss-of-function and gain-of-function mutations cause KAND by affecting the axonal transport and localization of synaptic vesicles. In this Review, we discuss how the analysis of these mutations by molecular genetics, single-molecule assays and force measurements have helped to reveal the physiological significance of KIF1A function and regulation, and what physical parameters of KIF1A are fundamental to axonal transport.

De novo mutations in KIF1A-associated neuronal disorder (KAND) dominant-negatively inhibit motor activity and axonal transport of synaptic vesicle precursors.

KIF1A is a kinesin superfamily motor protein that transports synaptic vesicle precursors in axons. Cargo binding stimulates the dimerization of KIF1A molecules to induce processive movement along microtubules. Mutations in human Kif1a lead to a group of neurodegenerative diseases called KIF1A-associated neuronal disorder (KAND). KAND mutations are mostly de novo and autosomal dominant; however, it is unknown if the function of wild-type KIF1A motors is inhibited by heterodimerization with mutated KIF1A. Here, we have established Caenorhabditis elegans models for KAND using CRISPR-Cas9 technology and analyzed the effects of human KIF1A mutation on axonal transport. In our C. elegans models, both heterozygotes and homozygotes exhibited reduced axonal transport. Suppressor screening using the disease model identified a mutation that recovers the motor activity of mutated human KIF1A. In addition, we developed in vitro assays to analyze the motility of heterodimeric motors composed of wild-type and mutant KIF1A. We find that mutant KIF1A significantly impaired the motility of heterodimeric motors. Our data provide insight into the molecular mechanism underlying the dominant nature of de novo KAND mutations.

Motor domain-mediated autoinhibition dictates axonal transport by the kinesin UNC-104/KIF1A.

The UNC-104/KIF1A motor is crucial for axonal transport of synaptic vesicles, but how the UNC-104/KIF1A motor is activated in vivo is not fully understood. Here, we identified point mutations located in the motor domain or the inhibitory CC1 domain, which resulted in gain-of-function alleles of unc-104 that exhibit hyperactive axonal transport and abnormal accumulation of synaptic vesicles. In contrast to the cell body localization of wild type motor, the mutant motors accumulate on neuronal processes. Once on the neuronal process, the mutant motors display dynamic movement similarly to wild type motors. The gain-of-function mutation on the motor domain leads to an active dimeric conformation, releasing the inhibitory CC1 region from the motor domain. Genetically engineered mutations in the motor domain or CC1 of UNC-104, which disrupt the autoinhibitory interface, also led to the gain of function and hyperactivation of axonal transport. Thus, the CC1/motor domain-mediated autoinhibition is crucial for UNC-104/KIF1A-mediated axonal transport in vivo.

TRPM7 is critical for short-term synaptic depression by regulating synaptic vesicle endocytosis.

Transient receptor potential melastatin 7 (TRPM7) contributes to a variety of physiological and pathological processes in many tissues and cells. With a widespread distribution in the nervous system, TRPM7 is involved in animal behaviors and neuronal death induced by ischemia. However, the physiological role of TRPM7 in central nervous system (CNS) neuron remains unclear. Here, we identify endocytic defects in neuroendocrine cells and neurons from TRPM7 knockout (KO) mice, indicating a role of TRPM7 in synaptic vesicle endocytosis. Our experiments further pinpoint the importance of TRPM7 as an ion channel in synaptic vesicle endocytosis. Ca2+ imaging detects a defect in presynaptic Ca2+ dynamics in TRPM7 KO neuron, suggesting an importance of Ca2+ influx via TRPM7 in synaptic vesicle endocytosis. Moreover, the short-term depression is enhanced in both excitatory and inhibitory synaptic transmissions from TRPM7 KO mice. Taken together, our data suggests that Ca2+ influx via TRPM7 may be critical for short-term plasticity of synaptic strength by regulating synaptic vesicle endocytosis in neurons.

Reexamination of N-terminal domains of syntaxin-1 in vesicle fusion from central murine synapses.

Syntaxin-1 (STX1) and Munc18-1 are two requisite components of synaptic vesicular release machinery, so much so synaptic transmission cannot proceed in their absence. They form a tight complex through two major binding modes: through STX1's N-peptide and through STX1's closed conformation driven by its Habc- domain. However, physiological roles of these two reportedly different binding modes in synapses are still controversial. Here we characterized the roles of STX1's N-peptide, Habc-domain, and open conformation with and without N-peptide deletion using our STX1-null mouse model system and exogenous reintroduction of STX1A mutants. We show, on the contrary to the general view, that the Habc-domain is absolutely required and N-peptide is dispensable for synaptic transmission. However, STX1A's N-peptide plays a regulatory role, particularly in the Ca2+-sensitivity and the short-term plasticity of vesicular release, whereas STX1's open conformation governs the vesicle fusogenicity. Strikingly, we also show neurotransmitter release still proceeds when the two interaction modes between STX1A and Munc18-1 are presumably intervened, necessitating a refinement of the conceptualization of STX1A-Munc18-1 interaction.

Reciprocal regulation of spontaneous synaptic vesicle fusion by Fragile X mental retardation protein and group I metabotropic glutamate receptors.

Fragile X mental retardation protein (FMRP) is a neuronal protein mediating multiple functions, with its absence resulting in one of the most common monogenic causes of autism, Fragile X syndrome (FXS). Analyses of FXS pathophysiology have identified a range of aberrations in synaptic signaling pathways and plasticity associated with group I metabotropic glutamate (mGlu) receptors. These studies, however, have mostly focused on the post-synaptic functions of FMRP and mGlu receptor activation, and relatively little is known about their presynaptic effects. Neurotransmitter release is mediated via multiple forms of synaptic vesicle (SV) fusion, each of which contributes to specific neuronal functions. The impacts of mGlu receptor activation and loss of FMRP on these SV fusion events remain unexplored. Here we combined electrophysiological and fluorescence imaging analyses on primary hippocampal cultures prepared from an Fmr1 knockout (KO) rat model. Compared to wild-type (WT) hippocampal neurons, KO neurons displayed an increase in the frequency of spontaneous excitatory post-synaptic currents (sEPSCs), as well as spontaneous SV fusion events. Pharmacological activation of mGlu receptors in WT neurons caused a similar increase in spontaneous SV fusion and sEPSC frequency. Notably, this increase in SV fusion was not observed when spontaneous activity was blocked using the sodium channel antagonist tetrodotoxin. Importantly, the effect of mGlu receptor activation on spontaneous SV fusion was occluded in Fmr1 KO neurons. Together, our results reveal that FMRP represses spontaneous presynaptic SV fusion, whereas mGlu receptor activation increases this event. This reciprocal control appears to be mediated via their regulation of intrinsic neuronal excitability.

Synapsin Is Required for Dense Core Vesicle Capture and cAMP-Dependent Neuropeptide Release.

Release of neuropeptides from dense core vesicles (DCVs) is essential for neuromodulation. Compared with the release of small neurotransmitters, much less is known about the mechanisms and proteins contributing to neuropeptide release. By optogenetics, behavioral analysis, electrophysiology, electron microscopy, and live imaging, we show that synapsin SNN-1 is required for cAMP-dependent neuropeptide release in Caenorhabditis elegans hermaphrodite cholinergic motor neurons. In synapsin mutants, behaviors induced by the photoactivated adenylyl cyclase bPAC, which we previously showed to depend on ACh and neuropeptides (Steuer Costa et al., 2017), are altered as in animals with reduced cAMP. Synapsin mutants have slight alterations in synaptic vesicle (SV) distribution; however, a defect in SV mobilization was apparent after channelrhodopsin-based photostimulation. DCVs were largely affected in snn-1 mutants: DCVs were ∼30% reduced in synaptic terminals, and their contents not released following bPAC stimulation. Imaging axonal DCV trafficking, also in genome-engineered mutants in the serine-9 protein kinase A phosphorylation site, showed that synapsin captures DCVs at synapses, making them available for release. SNN-1 colocalized with immobile, captured DCVs. In synapsin deletion mutants, DCVs were more mobile and less likely to be caught at release sites, and in nonphosphorylatable SNN-1B(S9A) mutants, DCVs traffic less and accumulate, likely by enhanced SNN-1 dependent tethering. Our work establishes synapsin as a key mediator of neuropeptide release.SIGNIFICANCE STATEMENT Little is known about mechanisms that regulate how neuropeptide-containing dense core vesicles (DCVs) traffic along the axon, how neuropeptide release is orchestrated, and where it occurs. We found that one of the longest known synaptic proteins, required for the regulation of synaptic vesicles and their storage in nerve terminals, synapsin, is also essential for neuropeptide release. By electrophysiology, imaging, and electron microscopy in Caenorhabditis elegans, we show that synapsin regulates this process by tethering the DCVs to the cytoskeleton in axonal regions where neuropeptides are to be released. Without synapsin, DCVs cannot be captured at the release sites and, consequently, cannot fuse with the membrane, and neuropeptides are not released. We suggest that synapsin fulfills this role also in vertebrates, including humans.

Synaptophysin controls synaptobrevin-II retrieval via a cryptic C-terminal interaction site.

The accurate retrieval of synaptic vesicle (SV) proteins during endocytosis is essential for the maintenance of neurotransmission. Synaptophysin (Syp) and synaptobrevin-II (SybII) are the most abundant proteins on SVs. Neurons lacking Syp display defects in the activity-dependent retrieval of SybII and a general slowing of SV endocytosis. To determine the role of the cytoplasmic C terminus of Syp in the control of these two events, we performed molecular replacement studies in primary cultures of Syp knockout neurons using genetically encoded reporters of SV cargo trafficking at physiological temperatures. Under these conditions, we discovered, 1) no slowing in SV endocytosis in Syp knockout neurons, and 2) a continued defect in SybII retrieval in knockout neurons expressing a form of Syp lacking its C terminus. Sequential truncations of the Syp C-terminus revealed a cryptic interaction site for the SNARE motif of SybII that was concealed in the full-length form. This suggests that a conformational change within the Syp C terminus is key to permitting SybII binding and thus its accurate retrieval. Furthermore, this study reveals that the sole presynaptic role of Syp is the control of SybII retrieval, since no defect in SV endocytosis kinetics was observed at physiological temperatures.

Dysfunction of the SNARE complex in neurological and psychiatric disorders.

The communication between neurons constitutes the basis of all neural activities, and synaptic vesicle exocytosis is the fundamental biological event that mediates most communication between neurons in the central nervous system. The SNARE complex is the core component of the protein machinery that facilitates the fusion of synaptic vesicles with presynaptic terminals and thereby the release of neurotransmitters. In synapses, each release event is dependent on the assembly of the SNARE complex. In recent years, basic research on the SNARE complex has provided a clearer understanding of the mechanism underlying the formation of the SNARE complex and its role in vesicle formation. Emerging evidence indicates that abnormal expression or dysfunction of the SNARE complex in synapse physiology might contribute to abnormal neurotransmission and ultimately to synaptic dysfunction. Clinical research using postmortem tissues suggests that SNARE complex dysfunction is correlated with various neurological diseases, and some basic research has also confirmed the important role of the SNARE complex in the pathology of these diseases. Genetic and pharmacogenetic studies suggest that the SNARE complex and individual proteins might represent important molecular targets in neurological disease. In this review, we summarize the recent progress toward understanding the SNARE complex in regulating membrane fusion events and provide an update of the recent discoveries from clinical and basic research on the SNARE complex in neurodegenerative, neuropsychiatric, and neurodevelopmental diseases.

The docking of synaptic vesicles on the presynaptic membrane induced by α-synuclein is modulated by lipid composition.

α-Synuclein (αS) is a presynaptic disordered protein whose aberrant aggregation is associated with Parkinson's disease. The functional role of αS is still debated, although it has been involved in the regulation of neurotransmitter release via the interaction with synaptic vesicles (SVs). We report here a detailed characterisation of the conformational properties of αS bound to the inner and outer leaflets of the presynaptic plasma membrane (PM), using small unilamellar vesicles. Our results suggest that αS preferentially binds the inner PM leaflet. On the basis of these studies we characterise in vitro a mechanism by which αS stabilises, in a concentration-dependent manner, the docking of SVs on the PM by establishing a dynamic link between the two membranes. The study then provides evidence that changes in the lipid composition of the PM, typically associated with neurodegenerative diseases, alter the modes of binding of αS, specifically in a segment of the sequence overlapping with the non-amyloid component region. Taken together, these results reveal how lipid composition modulates the interaction of αS with the PM and underlie its functional and pathological behaviours in vitro.

Regulation of a subset of release-ready vesicles by the presynaptic protein Mover.

Neurotransmitter release occurs by regulated exocytosis from synaptic vesicles (SVs). Evolutionarily conserved proteins mediate the essential aspects of this process, including the membrane fusion step and priming steps that make SVs release-competent. Unlike the proteins constituting the core fusion machinery, the SV protein Mover does not occur in all species and all synapses. Its restricted expression suggests that Mover may modulate basic aspects of transmitter release and short-term plasticity. To test this hypothesis, we analyzed synaptic transmission electrophysiologically at the mouse calyx of Held synapse in slices obtained from wild-type mice and mice lacking Mover. Spontaneous transmission was unaffected, indicating that the basic release machinery works in the absence of Mover. Evoked release and vesicular release probability were slightly reduced, and the paired pulse ratio was increased in Mover knockout mice. To explore whether Mover's role is restricted to certain subpools of SVs, we analyzed our data in terms of two models of priming. A model assuming two SV pools in parallel showed a reduced release probability of so-called "superprimed vesicles" while "normally primed" ones were unaffected. For the second model, which holds that vesicles transit sequentially from a loosely docked state to a tightly docked state before exocytosis, we found that knocking out Mover selectively decreased the release probability of tight state vesicles. These results indicate that Mover regulates a subclass of primed SVs in the mouse calyx of Held.

The BAD-BAX-Caspase-3 Cascade Modulates Synaptic Vesicle Pools via Autophagy.

The BAD-BAX-caspase-3 cascade is a canonical apoptosis pathway. Macroautophagy ("autophagy" hereinafter) is a process by which organelles and aggregated proteins are delivered to lysosomes for degradation. Here, we report a new function of the BAD-BAX-caspase-3 cascade and autophagy in the control of synaptic vesicle pools. We found that, in hippocampal neurons of male mice, the BAD-BAX-caspase-3 pathway regulates autophagy, which in turn limits the size of synaptic vesicle pools and influences the kinetics of activity-induced depletion and recovery of synaptic vesicle pools. Moreover, the caspase-autophagy pathway is engaged by fear conditioning to facilitate associative fear learning and memory. This work identifies a new mechanism for controlling synaptic vesicle pools, and a novel, nonapoptotic, presynaptic function of the BAD-BAX-caspase-3 cascade.SIGNIFICANCE STATEMENT Despite the importance of synaptic vesicles for neurons, little is known about how the size of synaptic vesicle pools is maintained under basal conditions and regulated by neural activity. This study identifies a new mechanism for the control of synaptic vesicle pools, and a new, nonapoptotic function of the BAD-BAX-caspase-3 pathway in presynaptic terminals. Additionally, it indicates that autophagy is not only a homeostatic mechanism to maintain the integrity of cells and tissues, but also a process engaged by neural activity to regulate synaptic vesicle pools for optimal synaptic responses, learning, and memory.

Combining Single Molecule Super-Resolution Imaging Techniques to Unravel the Nanoscale Organization of the Presynapse.

The fusion of synaptic vesicles with the plasma membrane underpins neurotransmission. A number of presynaptic proteins play a critical role in overcoming the energy barrier inherent to the fusion of the negatively charged vesicular and plasma membranes. Emerging concepts suggest that this process is hierarchical and dependent on rapid and transient reorganization of proteins in and out of small nanoclusters located in the active zones of nerve terminals. Examining the nanoscale organization of presynaptic molecules requires super-resolution microscopy to overcome the limits of conventional light microscopy. In this chapter, we describe three super-resolution techniques that allow for the examination of the nanoscale organization of proteins within live hippocampal nerve terminals. We used (1) single-particle tracking photoactivated localization microscopy (sptPALM) to resolve the mobility and clustering of syntaxin1A (STX1A), (2) universal Point Accumulation Imaging in Nanoscale Topography (uPAINT) to study the mobility of a pool of vesicular-associated membrane protein 2 (VAMP2) transiting on the plasma membrane, and (3) subdiffractional Tracking of Internalized Molecules (sdTIM) to track VAMP2-positive recycling synaptic vesicles in conjunction with Cholera Toxin subunit B (CTB), which has recently been shown to be trafficked retrogradely from the presynapse to the cell body via signaling endosomes.

Vesicle Tethering on the Surface of Phase-Separated Active Zone Condensates.

Tethering of synaptic vesicles (SVs) to the active zone determines synaptic strength, although the molecular basis governing SV tethering is elusive. Here, we discover that small unilamellar vesicles (SUVs) and SVs from rat brains coat on the surface of condensed liquid droplets formed by active zone proteins RIM, RIM-BP, and ELKS via phase separation. Remarkably, SUV-coated RIM/RIM-BP condensates are encapsulated by synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools that exist in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca2+ channels anchored on membranes. Thus, we reconstitute a presynaptic bouton-like structure mimicking the SV-tethered active zone with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates. The distinct interaction modes between membraneless protein condensates and membrane-based organelles revealed here have general implications in cellular processes, including vesicular formation and trafficking, organelle biogenesis, and autophagy.

Multimodal analysis of gene expression from postmortem brains and blood identifies synaptic vesicle trafficking genes to be associated with Parkinson's disease.

OBJECTIVE: We aimed to identify key susceptibility gene targets in multiple datasets generated from postmortem brains and blood of Parkinson's disease (PD) patients and healthy controls (HC). METHODS: We performed a multitiered analysis to integrate the gene expression data using multiple-gene chips from 244 human postmortem tissues. We identified hub node genes in the highly PD-related consensus module by constructing protein-protein interaction (PPI) networks. Next, we validated the top four interacting genes in 238 subjects (90 sporadic PD, 125 HC and 23 Parkinson's Plus Syndrome (PPS)). Utilizing multinomial logistic regression analysis (MLRA) and receiver operating characteristic (ROC), we analyzed the risk factors and diagnostic power for discriminating PD from HC and PPS. RESULTS: We identified 1333 genes that were significantly different between PD and HCs based on seven microarray datasets. The identified MEturquoise module is related to synaptic vesicle trafficking (SVT) dysfunction in PD (P < 0.05), and PPI analysis revealed that SVT genes PPP2CA, SYNJ1, NSF and PPP3CB were the top four hub node genes in MEturquoise (P < 0.001). The levels of these four genes in PD postmortem brains were lower than those in HC brains. We found lower blood levels of PPP2CA, SYNJ1 and NSF in PD compared with HC, and lower SYNJ1 in PD compared with PPS (P < 0.05). SYNJ1, negatively correlated to PD severity, displayed an excellent power to discriminating PD from HC and PPS. CONCLUSIONS: This study highlights that SVT genes, especially SYNJ1, may be promising markers in discriminating PD from HCs and PPS.

Going Deep into Synaptic Vesicle Machinery Genes and Migraine Susceptibility - A Case-Control Association Study.

OBJECTIVE: A number of observations, including among our study population, have implicated variants in the syntaxin-1A, a component of the synaptic vesicles, in migraine susceptibility. Therefore, we hypothesize that variants in other components of the vesicle machinery are involved in migraine. BACKGROUND: Migraine is a common and complex neurologic disorder that affects approximately 15-18% of the general population. The exact cause of migraine is unknown; however, genetic studies have made possible substantial progress toward the identification of underlying molecular pathways. Neurotransmitters have been for long considered to have a key role in migraine pathophysiology; so we investigated common variants in genes involved in the synaptic vesicle machinery and their impact in migraine susceptibility. METHODS: We performed a case-control study comprising 188 unrelated patients with headache and 286 healthy controls in a population from the north of Portugal. Benefiting from the presence of linkage disequilibrium, we selected and genotyped 119 tagging single-nucleotide polymorphisms in 18 genes. RESULTS: We found significant associations between single-nucleotide variants and migraine in 7 genes, SYN1, SYN2, SNAP25, VAMP2, STXBP1, STXBP5, and UNC13A, either conferring an increased risk or protection of migraine. Due to SYN1 X-chromosomal location, we performed the statistical analysis separated by gender and, in the female group, the C allele of rs5906435 increased the risk for migraine susceptibility (P = .021; OR = 1.69; 95% CI: 1.21-2.34). In contrast, the TT genotype of the same variant emerged as a potential protective factor (P = .003; OR = 0.45; 95% CI: 0.27-0.74). The SYN2 analysis supported the rs3773364's G allele (P = .014) as a risk factor for migraine, and although not statistically significant after correction, the AG genotype (P = .006; OR = 1.86; 95% CI: 1.20-2.90) reinforced the allelic findings. Additionally, we found the SNAP25-rs363039's CT genotype (P = .001; OR = 2.14; 95% CI: 1.36-3.34), the STXBP5-rs1765028's T allele (P = .041; OR = 1.46; 95% CI: 1.13-1.90), and the UNC13B-rs7851161's TT genotype (P = .001; OR = 2.14; 95% CI: 1.36-3.34) as statistically significant risk factors for migraine liability. VAMP2-rs1150's G allele revealed a risk association to migraine, not statistically significant after correction (P = .068). Additionally, we found haplotypes in SYN1, SYN2, STXBP1, and UNC13B to be associated with migraine. CONCLUSIONS: Overall, this study provides a new insight into migraine liability, identifying possible starting points for functional studies.

Passive Transfer of Sera from ALS Patients with Identified Mutations Evokes an Increased Synaptic Vesicle Number and Elevation of Calcium Levels in Motor Axon Terminals, Similar to Sera from Sporadic Patients.

Previously, we demonstrated increased calcium levels and synaptic vesicle densities in the motor axon terminals (MATs) of sporadic amyotrophic lateral sclerosis (ALS) patients. Such alterations could be conferred to mice with an intraperitoneal injection of sera from these patients or with purified immunoglobulin G. Later, we confirmed the presence of similar alterations in the superoxide dismutase 1 G93A transgenic mouse strain model of familial ALS. These consistent observations suggested that calcium plays a central role in the pathomechanism of ALS. This may be further reinforced by completing a similar analytical study of the MATs of ALS patients with identified mutations. However, due to the low yield of muscle biopsy samples containing MATs, and the low incidence of ALS patients with the identified mutations, these examinations are not technically feasible. Alternatively, a passive transfer of sera from ALS patients with known mutations was used, and the MATs of the inoculated mice were tested for alterations in their calcium homeostasis and synaptic activity. Patients with 11 different ALS-related mutations participated in the study. Intraperitoneal injection of sera from these patients on two consecutive days resulted in elevated intracellular calcium levels and increased vesicle densities in the MATs of mice, which is comparable to the effect of the passive transfer from sporadic patients. Our results support the idea that the pathomechanism underlying the identical manifestation of the disease with or without identified mutations is based on a common final pathway, in which increasing calcium levels play a central role.

Proteomic profiling of sclerotic hippocampus revealed dysregulated packaging of vesicular neurotransmitters in temporal lobe epilepsy.

PURPOSE: Temporal lobe epilepsy (TLE) is the most common type of epilepsy. Hippocampal sclerosis is the most distinctive pathological feature of TLE; however, its role in the pathogenesis of TLE remains to be clarified. We performed global protein expression analysis of hippocampus from TLE patients and controls, aiming to reveal the molecular signaling pathways related to TLE. METHOD: Proteomic and bioinformatic analyses of the hippocampus were performed on 4 TLE and 4 control samples. High-resolution liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), in combination with TMT-6plex quantification, was applied for global protein expression analysis. The proteomics results were validated by Western blot with 25 TLE and 25 control individuals and Immunohistochemistry analysis with 33 TLE and 10 control individuals. RESULTS: Bioinformatics analysis demonstrated differentially expressed proteins in the synaptic vesicle pathway, the prostaglandin synthesis and regulation pathway and endocannabinoids and retrograde modulation of synaptic transmission pathway. Among these, excitatory amino acid transporter 1 (EAAT1) and Vesicular glutamate transporter 1 (VGLUT1) are critical for TLE and dysregulated expression might be closely related to the uptake of extracellular glutamate and contribute to the pathophysiology of TLE. Ras-related protein Rab-3A (RAB3A) downregulation might indicate the TLE-induced compensatory deficit in glutamate release. CONCLUSION: Our study indicates that expression of some proteins involved in the packaging of vesicular neurotransmitters is altered in TLE. In addition, upregulated expression of annexin family proteins, which are also related to TLE, might play an important role in protection against TLE.