Presynaptic Protein Research Articles

Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling.

The activity-regulated cytoskeleton-associated (Arc) protein is essential for synaptic plasticity and memory formation. The Arc gene, which contains remnants of a structural GAG retrotransposon sequence, produces a protein that self-assembles into capsid-like structures harboring Arc mRNA. Arc capsids, released from neurons, have been proposed as a novel intercellular mechanism for mRNA transmission. Nevertheless, evidence for intercellular transport of Arc in the mammalian brain is still lacking. To enable the tracking of Arc molecules from individual neurons in vivo, we devised an adeno-associated virus (AAV) mediated approach to tag the N-terminal of the mouse Arc protein with a fluorescent reporter using CRISPR/Cas9 homologous independent targeted integration (HITI). We show that a sequence coding for mCherry can successfully be knocked in at the 5' end of the Arc open reading frame. While nine spCas9 gene editing sites surround the Arc start codon, the accuracy of the editing was highly sequence-dependent, with only a single target resulting in an in-frame reporter integration. When inducing long-term potentiation (LTP) in the hippocampus, we observed an increase of Arc protein highly correlated with an increase in fluorescent intensity and the number of mCherry-positive cells. By proximity ligation assay (PLA), we demonstrated that the mCherry-Arc fusion protein retains the Arc function by interacting with the transmembrane protein stargazin in postsynaptic spines. Finally, we recorded mCherry-Arc interaction with presynaptic protein Bassoon in mCherry-negative surrounding neurons at close proximity to mCherry-positive spines of edited neurons. This is the first study to provide support for inter-neuronal in vivo transfer of Arc in the mammalian brain.

Myosin Va, a Novel Interaction Partner of STXBP1, Is Required to Transport Syntaxin1A to the Plasma Membrane

Syntaxin-binding protein 1 (STXBP1, also known as Munc18-1) regulates exocytosis as a chaperone protein of Syntaxin1A. The haploinsufficiency of STXBP1 causes early infantile-onset developmental and epileptic encephalopathy, known as STXBP1 encephalopathy. Previously, we reported impaired cellular localization of Syntaxin1A in induced pluripotent stem cell-derived neurons from an STXBP1 encephalopathy patient harboring a nonsense mutation. However, the molecular mechanism of abnormal Syntaxin1A localization in the haploinsufficiency of STXBP1 remains unknown. This study aimed to identify the novel interacting partner of STXBP1 involved in transporting Syntaxin1A to the plasma membrane. Affinity purification coupled with mass spectrometry analysis identified a motor protein Myosin Va as a potential binding partner of STXBP1. Co-immunoprecipitation analysis of the synaptosomal fraction from the mouse and tag-fused recombinant proteins revealed that the STXBP1 short splice variant (STXBP1S) interacted with Myosin Va in addition to Syntaxin1A. These proteins colocalized at the tip of the growth cone and axons in primary cultured hippocampal neurons. Furthermore, RNAi-mediated gene silencing in Neuro2a cells showed that STXBP1 and Myosin Va were required for membrane trafficking of Syntaxin1A. In conclusion, this study proposes a potential role of STXBP1 in the trafficking of the presynaptic protein Syntaxin1A to the plasma membrane in conjunction with Myosin Va.

The mitochondrial uncoupling effects of nitazoxanide enhances cellular autophagy and promotes the clearance of α-synuclein: Potential role of AMPK-JNK pathway

Upregulation and aggregation of the pre-synaptic protein, α-synuclein plays a key role in Parkinson's disease (PD) and mitochondrial dysfunction was surmised to be an upstream event in the disease pathogenesis. Emerging reports identified the role of nitazoxanide (NTZ), an anti-helminth drug, in enhancing mitochondrial oxygen consumption rate (OCR) and autophagy. In the present study, we have examined the mitochondrial effects of NTZ in mediating cellular autophagy and subsequent clearance of both endogenous and pre-formed aggregates of α-synuclein in cellular model of PD. Our results demonstrate that the mitochondrial uncoupling effects of NTZ results in the activation of AMPK and JNK, which in-turn leads to the enhancement of cellular autophagy. Also,1-methyl-4-phenylpyridinium (MPP+) mediated decrease in autophagic flux with a concomitant increase in the α-synuclein levels were ameliorated in cells treated with NTZ. However, in cells lacking functional mitochondria (ρ0 cells), NTZ did not mitigate MPP+ mediated alterations in the autophagic clearance of α-synuclein, indicating that the mitochondrial effects of NTZ play a crucial role in the clearance of α-synuclein by autophagy. Also, the ability of AMPK inhibitor, compound C, in abrogating NTZ mediated enhancement in the autophagic flux and α-synuclein clearance highlight the pivotal role of AMPK in NTZ mediated autophagy. Further, NTZ per se enhanced the clearance of preformed α-synuclein aggregates that were exogenously added to the cells. Overall, the results of our present study suggest that NTZ activates macroautophagy in cells due to its uncoupling effects on mitochondrial respiration via activation of AMPK-JNK pathway resulting in the clearance of both endogenous and pre-formed α-synuclein aggregates. As NTZ happens to possess good bioavailability and safety profile, considering this drug for PD based on its mitochondrial uncoupling and autophagy enhancing properties for mitigating mitochondrial reactive oxygen species (ROS) and α-synuclein toxicity appears to be a promising therapeutic option.

SNAP25 ameliorates postoperative cognitive dysfunction by facilitating PINK1-dependent mitophagy and impeding caspase-3/GSDME-dependent pyroptosis

Insufficient PTEN-induced kinase 1 (PINK1)-mediated mitophagy and activation of caspase-3/gasdermin E (GSDME)-dependent pyroptosis constitute the potential etiology of postoperative cognitive dysfunction (POCD), a severe neurological complication characterized by learning and memory deficits. Synaptosomal-Associated Protein 25 (SNAP25), a well-defined presynaptic protein that mediates the fusion between synaptic vesicles and plasma membrane, is crucial in autophagy and the trafficking of extracellular proteins to the mitochondria. We investigated whether SNAP25 regulates POCD via mitophagy and pyroptosis. SNAP25 downregulation was observed in the hippocampi of rats undergoing isoflurane anesthesia and laparotomy. SNAP25 silencing restrained PINK1-mediated mitophagy and promoted reactive oxygen species (ROS) production and caspase-3/GSDME-dependent pyroptosis in isoflurane (Iso) + lipopolysaccharide (LPS)-primed SH-SY5Y cells. SNAP25 depletion also destabilized PINK1 on the outer membrane of the mitochondria and blocked Parkin translocation to the mitochondria. In contrast, SNAP25 overexpression alleviated POCD and Iso + LPS-induced defective mitophagy and pyroptosis, which was reversed by PINK1 knockdown. These findings suggest that SNAP25 exerts neuroprotective effects against POCD by boosting PINK1-dependent mitophagy and hindering caspase-3/GSDME-dependent pyroptosis, providing a novel option for the management of POCD.

Tau expression and phosphorylation in enteroendocrine cells.

There is mounting evidence to suggest that the gut-brain axis is involved in the development of Parkinson's disease (PD). In this regard, the enteroendocrine cells (EEC), which faces the gut lumen and are connected with both enteric neurons and glial cells have received growing attention. The recent observation showing that these cells express alpha-synuclein, a presynaptic neuronal protein genetically and neuropathologically linked to PD came to reinforce the assumption that EEC might be a key component of the neural circuit between the gut lumen and the brain for the bottom-up propagation of PD pathology. Besides alpha-synuclein, tau is another key protein involved in neurodegeneration and converging evidences indicate that there is an interplay between these two proteins at both molecular and pathological levels. There are no existing studies on tau in EEC and therefore we set out to examine the isoform profile and phosphorylation state of tau in these cells. Surgical specimens of human colon from control subjects were analyzed by immunohistochemistry using a panel of anti-tau antibodies together with chromogranin A and Glucagon-like peptide-1 (two EEC markers) antibodies. To investigate tau expression further, two EEC lines, namely GLUTag and NCI-H716 were analyzed by Western blot with pan-tau and tau isoform specific antibodies and by RT-PCR. Lambda phosphatase treatment was used to study tau phosphorylation in both cell lines. Eventually, GLUTag were treated with propionate and butyrate, two short chain fatty acids known to sense EEC, and analyzed at different time points by Western blot with an antibody specific for tau phosphorylated at Thr205. We found that tau is expressed and phosphorylated in EEC in adult human colon and that both EEC lines mainly express two tau isoforms that are phosphorylated under basal condition. Both propionate and butyrate regulated tau phosphorylation state by decreasing its phosphorylation at Thr205. Our study is the first to characterize tau in human EEC and in EEC lines. As a whole, our findings provide a basis to unravel the functions of tau in EEC and to further investigate the possibility of pathological changes in tauopathies and synucleinopathies.

Combination of Shengmai San and Radix puerariae ameliorates depression-like symptoms in diabetic rats at the nexus of PI3K/BDNF/SYN protein expression.

Hyperglycemia is a characteristic feature of diabetes that often results in neuropsychological complications such as depression. Diabetic individuals are more vulnerable to experience depression compared to the normal population. Thus, novel treatment approaches are required to reduce depressive symptoms among diabetic individuals. Traditional Chinese medicines (TCMs) such as Shengmai San (SMS) and Radix puerariae (R) are usually widely used to treat ailments such as neurological complications since ancient time. In this study, SMS was combined with R to prepare an R-SMS formulation and screened for their antidepressant activity in diabetic rats. The antidepressant potential of the prepared combination was evaluated behaviorally using open field test, novelty-induced hypophagia, and forced swim test in diabetic rats with biochemical and protein expression (PI3K, BDNF [brain-derived neurotrophic factor], and SYN [presynaptic vesicle protein]) analysis. Diabetic rats (streptozotocin, 45 mg/kg) showed elevated fasting blood glucose (FBG) >12 mM with depressive symptoms throughout the study. Treatment with R-SMS (0.5, 1.5, and 4.5 g/kg) significantly reverted depressive symptoms in diabetic rats as evinced by significantly (p < 0.05) reduced immobility time with an increased tendency to eat food in a novel environment. Treatment with R-SMS also significantly increased the protein expression of PI3K, BDNF, and SYN protein, which play a crucial role in depression. This study showed that R-SMS formulation antagonized depressive symptoms in diabetic rats; thus, this formulation might be studied further to develop as an antidepressant.

High ß‐synuclein plasma levels are coupled with amyloid PET positivity in memory clinic patients

AbstractBackgroundSynaptic dysfunction plays a key role in cognitive decline during progression of Alzheimer’s disease (AD). Higher levels of several synaptic proteins including the presynaptic protein ß‐synuclein have been measured in cerebrospinal fluid (CSF) of individuals with AD. Recently a quantitative assay using mass spectrometry allowed detection of ß‐synuclein in plasma. The aim of this study was to investigate the relationship between plasma ß‐synuclein levels and brain amyloid‐β (Aβ) plaque load in a cohort of memory clinic patients who underwent [18F]flutemetamol PET.MethodA clinical cohort of 118 patients (age = 65.6±8.3, 65F/53M), who despite extensive clinical assessment (including neuropsychological testing, CT/MRI, CSF biomarker analysis) at the Karolinska University Hospital, Sweden had an uncertain diagnosis and were referred to [18F]flutemetamol PET, was included in this work. The diagnostic groups were as follows: mild cognitive impairment (MCI, n = 16 Aβ+, and n = 24 Aβ‐), AD (n = 53), non‐AD dementia (n = 20),no dementia (n = 5). Plasma and CSF ß‐synuclein were measured using targeted mass spectrometry and ELISA.ResultWe found a significant correlation between β‐synuclein values in plasma and CSF (p&lt;0.001), with higher plasma β‐synuclein levels in AD (11.53±3.25pg/mL,p&lt;0.001) compared to MCI Aβ‐ (7.74±2.63pg/mL) and non‐AD (8.18±3.00 pg/mL), and also between MCI Aβ+ (10.85±2.75pg/mL,p&lt;0.01) in comparison to MCI Aβ‐. No significant difference was observed in plasma/CSF β‐synuclein levels between AD and MCI Aβ+. 18F‐Flutemetamol uptake positively correlated with plasma ß‐synuclein levels in the whole sample (R2 = 0.29,p&lt;0.001) and in the MCI group (R2 = 0.26,p&lt;0.01) (after controlling for sex and age). Individuals with comparable [18F]flutemetamol uptake values showed great variability in plasma ß‐synuclein levels. No correlation was observed between plasma ß‐synuclein and MMSE, brain atrophy, or white matter changes.ConclusionIn a clinical cohort of patients with [18F]flutemetamol‐PET, significantly elevated ß‐synuclein in plasma mirrored the higher levels of ß‐synuclein in CSF of Aß+ individuals. In the AD continuum, plasma ß‐synuclein seemed to capture pathological processes which were more closely associated with amyloid‐pathology rather than neuronal loss or clinical stage of the disease. The variability in plasma ß‐synuclein levels of Aß+ subjects warrants further exploration of factors that might contribute to increased plasma ß‐synuclein.

Early Alterations in Structural and Functional Properties in the Neuromuscular Junctions of Mutant FUS Mice.

Amyotrophic lateral sclerosis (ALS) is manifested as skeletal muscle denervation, loss of motor neurons and finally severe respiratory failure. Mutations of RNA-binding protein FUS are one of the common genetic reasons of ALS accompanied by a 'dying back' type of degeneration. Using fluorescent approaches and microelectrode recordings, the early structural and functional alterations in diaphragm neuromuscular junctions (NMJs) were studied in mutant FUS mice at the pre-onset stage. Lipid peroxidation and decreased staining with a lipid raft marker were found in the mutant mice. Despite the preservation of the end-plate structure, immunolabeling revealed an increase in levels of presynaptic proteins, SNAP-25 and synapsin 1. The latter can restrain Ca2+-dependent synaptic vesicle mobilization. Indeed, neurotransmitter release upon intense nerve stimulation and its recovery after tetanus and compensatory synaptic vesicle endocytosis were markedly depressed in FUS mice. There was a trend to attenuation of axonal [Ca2+]in increase upon nerve stimulation at 20 Hz. However, no changes in neurotransmitter release and the intraterminal Ca2+ transient in response to low frequency stimulation or in quantal content and the synchrony of neurotransmitter release at low levels of external Ca2+ were detected. At a later stage, shrinking and fragmentation of end plates together with a decrease in presynaptic protein expression and disturbance of the neurotransmitter release timing occurred. Overall, suppression of synaptic vesicle exo-endocytosis upon intense activity probably due to alterations in membrane properties, synapsin 1 levels and Ca2+ kinetics could be an early sign of nascent NMJ pathology, which leads to neuromuscular contact disorganization.

Vitamin D: A potent regulator of dopaminergic neuron differentiation and function.

Vitamin D has been identified as a key factor in dopaminergic neurogenesis and differentiation. Consequently, developmental vitamin D (DVD) deficiency has been linked to disorders of abnormal dopamine signalling with a neurodevelopmental basis such as schizophrenia. Here we provide further evidence of vitamin D's role as a mediator of dopaminergic development by showing that it increases neurite outgrowth, neurite branching, presynaptic protein re-distribution, dopamine production and functional release in various in vitro models of developing dopaminergic cells including SH-SY5Y cells, primary mesencephalic cultures and mesencephalic/striatal explant co-cultures. This study continues to establish vitamin D as an important differentiation agent for developing dopamine neurons, and now for the first time shows chronic exposure to the active vitamin D hormone increases the capacity of developing neurons to release dopamine. This study also has implications for understanding mechanisms behind the link between DVD deficiency and schizophrenia.

Neurobeachin controls the asymmetric subcellular distribution of electrical synapse proteins

The subcellular positioning of synapses and their specialized molecular compositions form the fundamental basis of neural circuits. Like chemical synapses, electrical synapses are constructed from an assortment of adhesion, scaffolding, and regulatory molecules, yet little is known about how these molecules localize to specific neuronal compartments. Here, we investigate the relationship between the autism- and epilepsy-associated gene Neurobeachin, the neuronal gap junction channel-forming Connexins, and the electrical synapse scaffold ZO1. Using the zebrafish Mauthner circuit, we find Neurobeachin localizes to the electrical synapse independently of ZO1 and Connexins. By contrast, we show Neurobeachin is required postsynaptically for the robust localization of ZO1 and Connexins. We demonstrate that Neurobeachin binds ZO1 but not Connexins. Finally, we find Neurobeachin is required to restrict electrical postsynaptic proteins to dendrites, but not electrical presynaptic proteins to axons. Together, the results reveal an expanded understanding of electrical synapse molecular complexity and the hierarchical interactions required to build neuronal gap junctions. Further, these findings provide novel insight into the mechanisms by which neurons compartmentalize the localization of electrical synapse proteins and provide a cell biological mechanism for the subcellular specificity of electrical synapse formation and function.

Blue Native PAGE-Antibody Shift in Conjunction with Mass Spectrometry to Reveal Protein Subcomplexes: Detection of a Cerebellar α1/α6-Subunits Containing γ-Aminobutyric Acid Type A Receptor Subtype.

The pentameric γ-Aminobutyric acid type A receptors (GABAARs) are ligand-gated ion channels that mediate the majority of inhibitory neurotransmission in the brain. In the cerebellum, the two main receptor subtypes are the 2α1/2β/γ and 2α6/2β/δ subunits. In the present study, an interaction proteomics workflow was used to reveal additional subtypes that contain both α1 and α6 subunits. Immunoprecipitation of the α6 subunit from mouse brain cerebellar extract co-purified the α1 subunit. In line with this, pre-incubation of the cerebellar extract with anti-α6 antibodies and analysis by blue native gel electrophoresis mass-shifted part of the α1 complexes, indicative of the existence of an α1α6-containing receptor. Subsequent mass spectrometry of the blue native gel showed the α1α6-containing receptor subtype to exist in two main forms, i.e., with or without Neuroligin-2. Immunocytochemistry on a cerebellar granule cell culture revealed co-localization of α6 and α1 in post-synaptic puncta that apposed the presynaptic marker protein Vesicular GABA transporter, indicative of the presence of this synaptic GABAAR subtype.

Mice deficient for G-protein-coupled receptor 75 display altered presynaptic structural protein expression and disrupted fear conditioning recall.

There are a number of G-protein-coupled receptors (GPCRs) that are considered "orphan receptors" because the information on their known ligands is incomplete. Yet, these receptors are important targets to characterize, as the discovery of their ligands may lead to potential new therapies. GPR75 was recently deorphanized because at least two ligands appear to bind to it, the chemokine CCL5 and the eicosanoid 20-Hydroxyeicosatetraenoic acid. Recent reports suggest that GPR75 may play a role in regulating insulin secretion and obesity. However, little is known about the function of this receptor in the brain. To study the function of GPR75, we have generated a knockout (KO) mouse model of this receptor and we evaluated the role that this receptor plays in the adult hippocampus by an array of histological, proteomic, and behavioral endpoints. Using RNAscope® technology, we identified GPR75 puncta in several Rbfox3-/NeuN-positive cells in the hippocampus, suggesting that this receptor has a neuronal expression. Proteomic analysis of the hippocampus in 3-month-old GPR75 KO animals revealed that several markers of synapses, including synapsin I and II are downregulated compared with wild type (WT). To examine the functional consequence of this down-regulation, WT and GPR75 KO mice were tested on a hippocampal-dependent behavioral task. Both contextual memory and anxiety-like behaviors were significantly altered in GPR75 KO, suggesting that GPR75 plays a role in hippocampal activity.

Simulations of active zone structure and function at mammalian NMJs predict that loss of calcium channels alone is not sufficient to replicate LEMS effects.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune-mediated neuromuscular disease thought to be caused by autoantibodies against P/Q-type voltage-gated calcium channels (VGCCs), which attack and reduce the number of VGCCs within transmitter release sites (active zones; AZs) at the neuromuscular junction (NMJ), resulting in neuromuscular weakness. However, patients with LEMS also have antibodies to other neuronal proteins, and about 15% of patients with LEMS are seronegative for antibodies against VGCCs. We hypothesized that a reduction in the number of P/Q-type VGCCs alone is not sufficient to explain LEMS effects on transmitter release. Here, we used a computational model to study a variety of LEMS-mediated effects on AZ organization and transmitter release constrained by electron microscopic, pharmacological, immunohistochemical, voltage imaging, and electrophysiological observations. We show that models of healthy AZs can be modified to predict the transmitter release and short-term facilitation characteristics of LEMS and that in addition to a decrease in the number of AZ VGCCs, disruption in the organization of AZ proteins, a reduction in AZ number, a reduction in the amount of synaptotagmin, and the compensatory expression of L-type channels outside the remaining AZs are important contributors to LEMS-mediated effects on transmitter release. Furthermore, our models predict that antibody-mediated removal of synaptotagmin in combination with disruption in AZ organization alone could mimic LEMS effects without the removal of VGCCs (a seronegative model). Overall, our results suggest that LEMS pathophysiology may be caused by a collection of pathological alterations to AZs at the NMJ, rather than by a simple loss of VGCCs.NEW & NOTEWORTHY We used a computational model of the active zone (AZ) in the mammalian neuromuscular junction to investigate Lambert-Eaton myasthenic syndrome (LEMS) pathophysiology. This model suggests that disruptions in presynaptic active zone organization and protein content (particularly synaptotagmin), beyond the simple removal of presynaptic calcium channels, play an important role in LEMS pathophysiology.

Critical Role of the Presynaptic Protein CAST in Maintaining the Photoreceptor Ribbon Synapse Triad.

The cytomatrix at the active zone-associated structural protein (CAST) and its homologue, named ELKS, being rich in glutamate (E), leucine (L), lysine (K), and serine (S), belong to a family of proteins that organize presynaptic active zones at nerve terminals. These proteins interact with other active zone proteins, including RIMs, Munc13s, Bassoon, and the β subunit of Ca2+ channels, and have various roles in neurotransmitter release. A previous study showed that depletion of CAST/ELKS in the retina causes morphological changes and functional impairment of this structure. In this study, we investigated the roles of CAST and ELKS in ectopic synapse localization. We found that the involvement of these proteins in ribbon synapse distribution is complex. Unexpectedly, CAST and ELKS, in photoreceptors or in horizontal cells, did not play a major role in ribbon synapse ectopic localization. However, depletion of CAST and ELKS in the mature retina resulted in degeneration of the photoreceptors. These findings suggest that CAST and ELKS play critical roles in maintaining neural signal transduction in the retina, but the regulation of photoreceptor triad synapse distribution is not solely dependent on their actions within photoreceptors and horizontal cells.

Revisiting Alpha-Synuclein Pathways to Inflammation.

Alpha-synuclein (α-Syn) is a short presynaptic protein with an active role on synaptic vesicle traffic and the neurotransmitter release and reuptake cycle. The α-Syn pathology intertwines with the formation of Lewy Bodies (multiprotein intraneuronal aggregations), which, combined with inflammatory events, define various α-synucleinopathies, such as Parkinson's Disease (PD). In this review, we summarize the current knowledge on α-Syn mechanistic pathways to inflammation, as well as the eventual role of microbial dysbiosis on α-Syn. Furthermore, we explore the possible influence of inflammatory mitigation on α-Syn. In conclusion, and given the rising burden of neurodegenerative disorders, it is pressing to clarify the pathophysiological processes underlying α-synucleinopathies, in order to consider the mitigation of existing low-grade chronic inflammatory states as a potential pathway toward the management and prevention of such conditions, with the aim of starting to search for concrete clinical recommendations in this particular population.

The activation of mGluR4 rescues parallel fiber synaptic transmission and LTP, motor learning and social behavior in a mouse model of Fragile X Syndrome

BackgroundFragile X syndrome (FXS), the most common inherited intellectual disability, is caused by the loss of expression of the Fragile X Messenger Ribonucleoprotein (FMRP). FMRP is an RNA-binding protein that negatively regulates the expression of many postsynaptic as well as presynaptic proteins involved in action potential properties, calcium homeostasis and neurotransmitter release. FXS patients and mice lacking FMRP suffer from multiple behavioral alterations, including deficits in motor learning for which there is currently no specific treatment.MethodsWe performed electron microscopy, whole-cell patch-clamp electrophysiology and behavioral experiments to characterise the synaptic mechanisms underlying the motor learning deficits observed in Fmr1KO mice and the therapeutic potential of positive allosteric modulator of mGluR4.ResultsWe found that enhanced synaptic vesicle docking of cerebellar parallel fiber to Purkinje cell Fmr1KO synapses was associated with enhanced asynchronous release, which not only prevents further potentiation, but it also compromises presynaptic parallel fiber long-term potentiation (PF-LTP) mediated by β adrenergic receptors. A reduction in extracellular Ca2+ concentration restored the readily releasable pool (RRP) size, basal synaptic transmission, β adrenergic receptor-mediated potentiation, and PF-LTP. Interestingly, VU 0155041, a selective positive allosteric modulator of mGluR4, also restored both the RRP size and PF-LTP in mice of either sex. Moreover, when injected into Fmr1KO male mice, VU 0155041 improved motor learning in skilled reaching, classical eyeblink conditioning and vestibuloocular reflex (VOR) tests, as well as the social behavior alterations of these mice.LimitationsWe cannot rule out that the activation of mGluR4s via systemic administration of VU0155041 can also affect other brain regions. Further studies are needed to stablish the effect of a specific activation of mGluR4 in cerebellar granule cells.ConclusionsOur study shows that an increase in synaptic vesicles, SV, docking may cause the loss of PF-LTP and motor learning and social deficits of Fmr1KO mice and that the reversal of these changes by pharmacological activation of mGluR4 may offer therapeutic relief for motor learning and social deficits in FXS.

In silico investigation of the structural stability as the origin of the pathogenicity of α-synuclein protofibrils

α-Synuclein is a presynaptic neuronal protein. The fibril form of α-synuclein is a major constituent of the intraneuronal inclusion called Lewy body, a characteristic hallmark of Parkinson’s disease. Recent ssNMR and cryo-EM experiments of wild-type α-synuclein fibrils have shown polymorphism and observed two major polymorphs, rod and twister. To associate the cytotoxicity of α-synuclein fibrils with their structural features, it is essential to understand the origins of their structural stability. In this study, we performed molecular dynamics simulations of the two major polymorphs of wild-type α-synuclein fibrils. The predominance of specific fibril polymorphs was rationalized in terms of relative structural stability in aqueous environments, which was attributed to the cooperative contributions of various stabilizing features. The results of the simulations indicated that highly stable structures in aqueous environments could be maintained by the cooperation of compact sidechain packing in the hydrophobic core, backbone geometry of the maximal β-sheet content wrapping the hydrophobic core, and solvent-exposed sidechains with large fluctuations maximizing the solvation entropy. The paired structure of the two protofilaments provides additional stability, especially at the interface region, by forming steric zipper interactions and hiding the hydrophobic residues from exposure to water. The sidechain interaction analyses and pulling simulations showed that the rod polymorph has stronger sidechain interactions and exhibits higher dissociation energy than the twister polymorph. It is expected that our study will provide a basis for understanding the pathogenic behaviors of diverse amyloid strains in terms of their structural properties.Communicated by Ramaswamy H. Sarma

Unraveling the Complex Interplay between Alpha-Synuclein and Epigenetic Modification

Alpha-synuclein (αS) is a small, presynaptic neuronal protein encoded by the SNCA gene. Point mutations and gene multiplication of SNCA cause rare familial forms of Parkinson's disease (PD). Misfolded αS is cytotoxic and is a component of Lewy bodies, which are a pathological hallmark of PD. Because SNCA multiplication is sufficient to cause full-blown PD, gene dosage likely has a strong impact on pathogenesis. In sporadic PD, increased SNCA expression resulting from a minor genetic background and various environmental factors may contribute to pathogenesis in a complementary manner. With respect to genetic background, several risk loci neighboring the SNCA gene have been identified, and epigenetic alterations, such as CpG methylation and regulatory histone marks, are considered important factors. These alterations synergistically upregulate αS expression and some post-translational modifications of αS facilitate its translocation to the nucleus. Nuclear αS interacts with DNA, histones, and their modifiers to alter epigenetic status; thereby, influencing the stability of neuronal function. Epigenetic changes do not affect the gene itself but can provide an appropriate transcriptional response for neuronal survival through DNA methylation or histone modifications. As a new approach, publicly available RNA sequencing datasets from human midbrain-like organoids may be used to compare transcriptional responses through epigenetic alterations. This informatic approach combined with the vast amount of transcriptomics data will lead to the discovery of novel pathways for the development of disease-modifying therapies for PD.

Genetic disorders of neurotransmitter release machinery.

Synaptic neurotransmitter release is an evolutionarily conserved process that mediates rapid information transfer between neurons as well as several peripheral tissues. Release of neurotransmitters are ensured by successive events such as synaptic vesicle docking and priming that prepare synaptic vesicles for rapid fusion. These events are orchestrated by interaction of different presynaptic proteins and are regulated by presynaptic calcium. Recent studies have identified various mutations in different components of neurotransmitter release machinery resulting in aberrant neurotransmitter release, which underlie a wide spectrum of psychiatric and neurological symptoms. Here, we review how these genetic alterations in different components of the core neurotransmitter release machinery affect the information transfer between neurons and how aberrant synaptic release affects nervous system function.

DOPAL initiates αSynuclein-dependent impaired proteostasis and degeneration of neuronal projections in Parkinson’s disease

Dopamine dyshomeostasis has been acknowledged among the determinants of nigrostriatal neuron degeneration in Parkinson’s disease (PD). Several studies in experimental models and postmortem PD patients underlined increasing levels of the dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is highly reactive towards proteins. DOPAL has been shown to covalently modify the presynaptic protein αSynuclein (αSyn), whose misfolding and aggregation represent a major trait of PD pathology, triggering αSyn oligomerization in dopaminergic neurons. Here, we demonstrated that DOPAL elicits αSyn accumulation and hampers αSyn clearance in primary neurons. DOPAL-induced αSyn buildup lessens neuronal resilience, compromises synaptic integrity, and overwhelms protein quality control pathways in neurites. The progressive decline of neuronal homeostasis further leads to dopaminergic neuron loss and motor impairment, as showed in in vivo models. Finally, we developed a specific antibody which detected increased DOPAL-modified αSyn in human striatal tissues from idiopathic PD patients, corroborating the translational relevance of αSyn-DOPAL interplay in PD neurodegeneration.