Synaptic Vesicle Dynamics Research Articles

Unveiling mitochondria as central components driving cognitive decline in alzheimer's disease through cross-transcriptomic analysis of hippocampus and entorhinal cortex microarray datasets

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder characterized by symptoms such as memory loss and impaired learning. This study conducted a cross-transcriptomic analysis of AD using existing microarray datasets from the hippocampus (HC) and entorhinal cortex (EC), comparing them with age-matched non-AD controls. Both of these brain regions are critical for learning and memory processing and are vulnerable areas that exhibit abnormalities in early AD. The cross-transcriptomic analysis identified 564 significantly differentially expressed genes in HC and 479 in EC. Among these, 151 genes were significantly differentially expressed in both tissues, with functions related to synaptic vesicle clustering, synaptic vesicle exocytosis/endocytosis, mitochondrial ATP synthesis, hydrogen ion transmembrane transport, and structural constituent of cytoskeleton, suggesting a potential association between cognitive decline in AD, synaptic vesicle dynamics, dysregulation of cytoskeleton organization, and mitochondrial dysfunction. Further gene ontology analysis specific to the HC revealed the gene ontology enrichment in aerobic respiration, synaptic vesicle cycle, and oxidative phosphorylation. The enrichment analysis in CA1 of HC revealed differentiation in gene expression related to mitochondrial membrane functions involved in bioenergetics, mitochondrial electron transport, and biological processes associated with microtubule-based process, while analysis in the EC region showed enrichment in synaptic vesicle dynamics which is associated with neurotransmitter release and the regulation of postsynaptic membrane potential and synaptic transmission of GABAergic and glutamatergic synapse. Protein-protein interaction analysis highlighted central hub proteins predominantly expressed in mitochondria, involved in regulation of oxidative stress and ATP synthesis. These hub proteins interact not only within the mitochondria but also with proteins in the vesicular membrane and neuronal cytoskeleton, indicating a central role of mitochondria. This finding underscores the association between clinical symptoms and mitochondrial dysregulation of synaptic vesicle dynamics, cytoskeleton organization, and mitochondrial processes in both the HC and EC of AD. Therefore, targeting these dysregulated pathways could provide promising therapeutic targets aimed at cognitive decline and memory impairment in early AD stages.

Electrochemical Monitoring of Real‐Time Vesicle Dynamics Induced by Tau in a Confined Nanopipette

AbstractThe microtubule‐associated protein tau participates in neurotransmission regulation via its interaction with synaptic vesicles (SVs). The precise nature and mechanics of tau‘s engagement with SVs, especially regarding alterations in vesicle dynamics, remain a matter of discussion. We report an electrochemical method using a synapse‐mimicking nanopipette to monitor vesicle dynamics induced by tau. A model vesicle of ~30 nm is confined within a lipid‐modified nanopipette orifice with a comparable diameter to mimic the synaptic lipid environment. Both tau and phosphorylated tau (p‐tau) present two‐state dynamic behavior in this biomimetic system, showing typical ionic current oscillation, induced by lipid‐tau interaction. The results indicate that p‐tau has a stronger affinity to the lipid vesicles in the confined environment, blocking the vesicle movement to a higher degree. Taken together, this method bridges a gap for sensing synaptic vesicle dynamics in a confined lipid environment, mimicking vesicle movement near the synaptic membrane. These findings contribute to understanding how different types of tau protein regulate synaptic vesicle motility and to underlying its functional and pathological behaviours in disease.

Electrochemical Monitoring of Real-Time Vesicle Dynamics Induced by Tau in a Confined Nanopipette.

The microtubule-associated protein tau participates in neurotransmission regulation via its interaction with synaptic vesicles (SVs). The precise nature and mechanics of tau's engagement with SVs, especially regarding alterations in vesicle dynamics, remain a matter of discussion. We report an electrochemical method using a synapse-mimicking nanopipette to monitor vesicle dynamics induced by tau. A model vesicle of ~30 nm is confined within a lipid-modified nanopipette orifice with a comparable diameter to mimic the synaptic lipid environment. Both tau and phosphorylated tau (p-tau) present two-state dynamic behavior in this biomimetic system, showing typical ionic current oscillation, induced by lipid-tau interaction. The results indicate that p-tau has a stronger affinity to the lipid vesicles in the confined environment, blocking the vesicle movement to a higher degree. Taken together, this method bridges a gap for sensing synaptic vesicle dynamics in a confined lipid environment, mimicking vesicle movement near the synaptic membrane. These findings contribute to understanding how different types of tau protein regulate synaptic vesicle motility and to underlying its functional and pathological behaviours in disease.

Co-occurrence of PRKN and SYNJ1 variants in Early-Onset Parkinson's disease.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease globally, with a fast-growing prevalence. The etiology of PD exhibits a multifactorial complex nature and remains challenging. Herein, we described clinical, molecular, and integrative bioinformatics findings from a Brazilian female affected by Early-Onset PD (EOPD) harboring a recurrent homozygous pathogenic deletion in the parkin RBR E3 ubiquitin protein ligase gene (PRKN; NM_004562.3:c.155delA; p.Asn52Metfs*29; rs754809877), along with a novel heterozygous variant in the synaptojanin 1 gene (SYNJ1; NM_003895.3:c.62G > T; p.Cys21Phe; rs1486511197) found by Whole Exome Sequencing. Uncommon or unreported PRKN-related clinical features in the patient include cognitive decline, auditory and visual hallucinations, REM sleep disorder, and depression, previously observed in SYNJ1-related conditions. Moreover, PRKN interacts with endophilin A1, which is a major binding partner of SYNJ1. This protein plays a pivotal role in regulating the dynamics of synaptic vesicles, particularly in the context of endocytosis and recycling processes. Altogether, our comprehensive analyses underscore a potential synergistic effect between the PRKN and SYNJ1 variants over the pathogenesis of EOPD.

A stem cell-based assay platform demonstrates alpha-synuclein dependent synaptic dysfunction in patient-derived cortical neurons

Alpha-synuclein (αS)-rich Lewy bodies and neurites in the cerebral cortex correlate with the presence of dementia in Parkinson disease (PD) and Dementia with Lewy bodies (DLB), but whether αS influences synaptic vesicle dynamics in human cortical neurons is unknown. Using a new iPSC-based assay platform for measuring synaptic vesicle cycling, we found that in human cortical glutamatergic neurons, increased αS from either transgenic expression or triplication of the endogenous locus in patient-derived neurons reduced synaptic vesicle cycling under both stimulated and spontaneous conditions. Thus, using a robust, easily adopted assay platform, we show for the first time αS-induced synaptic dysfunction in human cortical neurons, a key cellular substrate for PD dementia and DLB.

Excess phosphoserine-129 α-synuclein induces synaptic vesicle trafficking and declustering defects at a vertebrate synapse.

α-Synuclein is a presynaptic protein that regulates synaptic vesicle (SV) trafficking. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), α-synuclein aberrantly accumulates throughout neurons, including at synapses. During neuronal activity, α-synuclein is reversibly phosphorylated at serine 129 (pS129). While pS129 comprises ∼4% of total α-synuclein under physiological conditions, it dramatically increases in PD and DLB brains. The impacts of excess pS129 on synaptic function are currently unknown. We show here that compared with wild-type (WT) α-synuclein, pS129 exhibits increased binding and oligomerization on synaptic membranes and enhanced vesicle "microclustering" in vitro. Moreover, when acutely injected into lamprey reticulospinal axons, excess pS129 α-synuclein robustly localized to synapses and disrupted SV trafficking in an activity-dependent manner, as assessed by ultrastructural analysis. Specifically, pS129 caused a declustering and dispersion of SVs away from the synaptic vicinity, leading to a significant loss of total synaptic membrane. Live imaging further revealed altered SV cycling, as well as microclusters of recently endocytosed SVs moving away from synapses. Thus, excess pS129 caused an activity-dependent inhibition of SV trafficking via altered vesicle clustering/reclustering. This work suggests that accumulation of pS129 at synapses in diseases like PD and DLB could have profound effects on SV dynamics.

Model reduction for ‐induced vesicle fusion dynamics

AbstractIn this work, we adapt an established model for the ‐induced fusion dynamics of synaptic vesicles and employ a lumping method to reduce its complexity. In the reduced system, sequential ‐binding steps are merged to a single releasable state, while keeping the important dependence of the reaction rates on the local concentration. We examine the feasibility of this model reduction for a representative stimulus train over the physiologically relevant site‐channel distances. Our findings show that the approximation error is generally small and exhibits an interesting nonlinear and non‐monotonic behavior where it vanishes for very low distances and is insignificant at intermediary distances. Furthermore, we give expressions for the reduced model's reaction rates and suggest that our approach may be used to directly compute effective fusion rates for assessing the validity of a fusion model, thereby circumventing expensive simulations.

Benzo[a]pyrene represses synaptic vesicle exocytosis by inhibiting P/Q-type calcium channels in hippocampal neurons

Humans are exposed to the common carcinogen benzo[a]pyrene (BaP) by ingesting contaminated foods and water or inhaling polluted air. Given the enriched lipids and reduced antioxidative properties in the brain and the accumulation of BaP in the brain due to its high lipophilicity, the brain is susceptible to BaP-induced toxicity. Exposure to BaP leads to impairments in learning and memory, increased anxiety behavior, and neuronal death. It induces protein dysfunctions in neuronal compartments that play essential roles in neuronal activity or physiology. However, the neurotoxicity of BaP on presynaptic terminals, which is crucial to neurotransmission by releasing synaptic vesicles that contain neurotransmitters, has not yet been investigated. In the present study, we investigated the toxicity of BaP at presynaptic terminals in living hippocampal neurons. These neurons were sourced from transgenic mice pups (postnatal 1-day, a total of 12 pups, equal numbers for each sex) that endogenously express synaptic vesicle-fused pHluorin, which is a green fluorescent protein that enables monitoring of synaptic vesicle dynamics. We observed that BaP suppressed synaptic vesicle exocytosis by inhibiting presynaptic Ca2+ entry via P/Q-type Ca2+ channels. Together with molecular docking simulation, we speculate that BaP and metabolites may bind to the P/Q Ca2+ channels. These results suggest the toxic mechanism of BaP exposure-induced abnormal behavior that provides a basis to evaluate the risk assessment of BaP-induced neurotoxicity.

Effects of alpha-synuclein and synaptic vesicles on the material properties of synapsin condensates.

Biomolecular condensates can recruit and segregate client molecules and are involved in various physiological processes. The material properties of a biomolecular condensate, as a mesoscale manifestation of intermolecular interactions, are crucial to its biological and pathological functions. Despite emerging reports about the changes in material properties of condensates in disease-related proteins, quantitative studies of the material properties of condensates are still rare. Here, we apply a calibration-free micropipette aspiration (MPA) assay to quantify the surface tension and viscosity of synapsin condensates. Furthermore, we investigated the effect of alpha-synuclein and synaptic vesicles (SVs) on the material properties of synapsin condensate. The results suggest that the recruitment of alpha-synuclein will increase the viscosity of synapsin condensate, and indicate the potential effects of SVs in mediating the material properties of synapsin condensates. Synapsin condensates are known to be essential for the formation of SV clusters. Therefore, our findings will help understand the regulation of SV dynamics during neurotransmission.

Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses.

Ribbon synapses of cochlear inner hair cells (IHCs) are specialized to indefatigably transmit sound information at high rates. To understand the underlying mechanisms, structure-function analysis of the active zone (AZ) of these synapses is essential. Previous electron microscopy studies of synaptic vesicle (SV) dynamics at the IHC AZ used potassium stimulation, which limited the temporal resolution to minutes. Here, we established optogenetic IHC stimulation followed by quick freezing within milliseconds and electron tomography to study the ultrastructure of functional synapse states with good temporal resolution in mice. We characterized optogenetic IHC stimulation by patch-clamp recordings from IHCs and postsynaptic boutons revealing robust IHC depolarization and neurotransmitter release. Ultrastructurally, the number of docked SVs increased upon short (17-25 ms) and long (48-76 ms) light stimulation paradigms. We did not observe enlarged SVs or other morphological correlates of homotypic fusion events. Our results indicate a rapid recruitment of SVs to the docked state upon stimulation and suggest that univesicular release prevails as the quantal mechanism of exocytosis at IHC ribbon synapses.

Identification of the common neurobiological process disturbed in genetic and non-genetic models for autism spectrum disorders.

Autism spectrum disorders (ASD) are neurodevelopmental disorders. Genetic factors, along with non-genetic triggers, have been shown to play a causative role. Despite the various causes, a triad of common symptoms defines individuals with ASD; pervasive social impairments, impaired social communication, and repeated sensory-motor behaviors. Therefore, it can be hypothesized that different genetic and environmental factors converge on a single hypothetical neurobiological process that determines these behaviors. However, the cellular and subcellular signature of this process is, so far, not well understood. Here, we performed a comparative study using "omics" approaches to identify altered proteins and, thereby, biological processes affected in ASD. In this study, we mined publicly available repositories for genetic mouse model data sets, identifying six that were suitable, and compared them with in-house derived proteomics data from prenatal zinc (Zn)-deficient mice, a non-genetic mouse model with ASD-like behavior. Findings derived from these comparisons were further validated using in vitro neuronal cell culture models for ASD. We could show that a protein network, centered on VAMP2, STX1A, RAB3A, CPLX2, and AKAP5, is a key convergence point mediating synaptic vesicle release and recycling, a process affected across all analyzed models. Moreover, we demonstrated that Zn availability has predictable functional effects on synaptic vesicle release in line with the alteration of proteins in this network. In addition, drugs that target kinases, reported to regulate key proteins in this network, similarly impacted the proteins' levels and distribution. We conclude that altered synaptic stability and plasticity through abnormal synaptic vesicle dynamics and function may be the common neurobiological denominator of the shared behavioral abnormalities in ASD and, therefore, a prime drug target for developing therapeutic strategies.

A novel SYNJ1 homozygous variant causing developmental and epileptic encephalopathy in an Afro-Caribbean individual.

SYNJ1 encodes Synaptojanin-1, a dual-function poly-phosphoinositide phosphatase that is expressed in the brain to regulate neuronal synaptic vesicle dynamics. Biallelic SYNJ1 variants cause a spectrum of clinical manifestations, from early onset parkinsonism to developmental and epileptic encephalopathy. Proband-only exome sequencing was used to identify a homozygous SYNJ1 pathogenic variant in an individual with epileptic encephalopathy. Sanger sequencing was used to confirm the variant. We present an Afro-Caribbean female who developed uncontrollable seizures shortly after birth, accompanied by developmental delay and severe generalized dystonia. She had homozygosity for a novel c.242-2A > G variant in SYNJ1 with both parents being heterozygous carriers. An older sister was reported to have had a similar presentation but was not examined. Both siblings died at an approximate age of 16 years. We report a novel pathogenic variant in SYNJ1 present in homozygosity leading to developmental and epileptic encephalopathy. Currently, there are only 4 reports describing 10 individuals with SYNJ1-related developmental and epileptic encephalopathy. This case expands the clinical knowledge and the allelic heterogeneity associated with SYNJ1 variants.

Crosstalk of Synapsin1 palmitoylation and phosphorylation controls the dynamicity of synaptic vesicles in neurons

The dynamics of synaptic vesicles (SVs) within presynaptic domains are tightly controlled by synapsin1 phosphorylation; however, the mechanism underlying the anchoring of synapsin1 with F-actin or SVs is not yet fully understood. Here, we found that Syn1 is modified with protein palmitoylation, and examining the roles of Syn1 palmitoylation in neurons led us to uncover that Syn1 palmitoylation is negatively regulated by its phosphorylation; together, they manipulate the clustering and redistribution of SVs. Using the combined approaches of electron microscopy and genetics, we revealed that Syn1 palmitoylation is vital for its binding with F-actin but not SVs. Inhibition of Syn1 palmitoylation causes defects in SVs clustering and a reduced number of total SVs in vivo. We propose a model in which SVs redistribution is triggered by upregulated Syn1 phosphorylation and downregulated Syn1 palmitoylation, and they reversibly promote SVs clustering. The crosstalk of Syn1 palmitoylation and phosphorylation thereby bidirectionally manipulates SVs dynamics in neurons.

Event-triggered STED imaging

Monitoring the proteins and lipids that mediate all cellular processes requires imaging methods with increased spatial and temporal resolution. STED (stimulated emission depletion) nanoscopy enables fast imaging of nanoscale structures in living cells but is limited by photobleaching. Here, we present event-triggered STED, an automated multiscale method capable of rapidly initiating two-dimensional (2D) and 3D STED imaging after detecting cellular events such as protein recruitment, vesicle trafficking and second messengers activity using biosensors. STED is applied in the vicinity of detected events to maximize the temporal resolution. We imaged synaptic vesicle dynamics at up to 24 Hz, 40 ms after local calcium activity; endocytosis and exocytosis events at up to 11 Hz, 40 ms after local protein recruitment or pH changes; and the interaction between endosomal vesicles at up to 3 Hz, 70 ms after approaching one another. Event-triggered STED extends the capabilities of live nanoscale imaging, enabling novel biological observations in real time.

Detecting Fear-Memory-Related Genes from Neuronal scRNA-seq Data by Diverse Distributions and Bhattacharyya Distance.

The detection of differentially expressed genes (DEGs) is one of most important computational challenges in the analysis of single-cell RNA sequencing (scRNA-seq) data. However, due to the high heterogeneity and dropout noise inherent in scRNAseq data, challenges in detecting DEGs exist when using a single distribution of gene expression levels, leaving much room to improve the precision and robustness of current DEG detection methods. Here, we propose the use of a new method, DEGman, which utilizes several possible diverse distributions in combination with Bhattacharyya distance. DEGman can automatically select the best-fitting distributions of gene expression levels, and then detect DEGs by permutation testing of Bhattacharyya distances of the selected distributions from two cell groups. Compared with several popular DEG analysis tools on both large-scale simulation data and real scRNA-seq data, DEGman shows an overall improvement in the balance of sensitivity and precision. We applied DEGman to scRNA-seq data of TRAP; Ai14 mouse neurons to detect fear-memory-related genes that are significantly differentially expressed in neurons with and without fear memory. DEGman detected well-known fear-memory-related genes and many novel candidates. Interestingly, we found 25 DEGs in common in five neuron clusters that are functionally enriched for synaptic vesicles, indicating that the coupled dynamics of synaptic vesicles across in neurons plays a critical role in remote memory formation. The proposed method leverages the advantage of the use of diverse distributions in DEG analysis, exhibiting better performance in analyzing composite scRNA-seq datasets in real applications.

Postsynaptic Neuroligin-1 Mediates Presynaptic Endocytosis During Neuronal Activity.

Fast, high-fidelity neurotransmission and synaptic efficacy requires tightly regulated coordination of pre- and postsynaptic compartments and alignment of presynaptic release sites with postsynaptic receptor nanodomains. Neuroligin-1 (Nlgn1) is a postsynaptic cell-adhesion protein exclusively localised to excitatory synapses that is crucial for coordinating the transsynaptic alignment of presynaptic release sites with postsynaptic AMPA receptors as well as postsynaptic transmission and plasticity. However, little is understood about whether the postsynaptic machinery can mediate the molecular architecture and activity of the presynaptic nerve terminal, and thus it remains unclear whether there are presynaptic contributions to Nlgn1-dependent control of signalling and plasticity. Here, we employed a presynaptic reporter of neurotransmitter release and synaptic vesicle dynamics, synaptophysin-pHluorin (sypHy), to directly assess the presynaptic impact of loss of Nlgn1. We show that lack of Nlgn1 had no effect on the size of the readily releasable or entire recycling pool of synaptic vesicles, nor did it impact exocytosis. However, we observed significant changes in the retrieval of synaptic vesicles by compensatory endocytosis, specifically during activity. Our data extends growing evidence that synaptic adhesion molecules critical for forming transsynaptic scaffolds are also important for regulating activity-induced endocytosis at the presynapse.

Tianeptine modulates synaptic vesicle dynamics and favors synaptic mitochondria processes in socially isolated rats

Deregulation of synaptic function and neurotransmission has been linked with the development of major depression disorder (MDD). Tianeptine (Tian) has been used as antidepressant with anxiolytic properties and recently as a nootropic to improve cognitive performance, but its mechanism of action is unknown. We conducted a proteomic study on the hippocampal synaptosomal fractions of adult male Wistar rats exposed to chronic social isolation (CSIS, 6 weeks), an animal model of depression and after chronic Tian treatment in controls (nootropic effect) and CSIS-exposed rats (lasting 3 weeks of 6-week CSIS) (therapeutic effect). Increased expression of Syn1 and Camk2-related neurotransmission, vesicle transport and energy processes in Tian-treated controls were found. CSIS led to upregulation of proteins associated with actin cytoskeleton, signaling transduction and glucose metabolism. In CSIS rats, Tian up-regulated proteins involved in mitochondrial energy production, mitochondrial transport and dynamics, antioxidative defense and glutamate clearance, while attenuating the CSIS-increased glycolytic pathway and cytoskeleton organization proteins expression and decreased the expression of proteins involved in V-ATPase and vesicle endocytosis. Our overall findings revealed that synaptic vesicle dynamics, specifically exocytosis, and mitochondria-related energy processes might be key biological pathways modulated by the effective nootropic and antidepressant treatment with Tian and be a potential target for therapeutic efficacy of the stress-related mood disorders.

Secreted C-type lectin regulation of neuromuscular junction synaptic vesicle dynamics modulates coordinated movement.

The synaptic cleft manifests enriched glycosylation, with structured glycans coordinating signaling between presynaptic and postsynaptic cells. Glycosylated signaling ligands orchestrating communication are tightly regulated by secreted glycan-binding lectins. Using the Drosophila neuromuscular junction (NMJ) as a model glutamatergic synapse, we identify a new Ca2+-binding (C-type) lectin, Lectin-galC1 (LGC1), which modulates presynaptic function and neurotransmission strength. We find that LGC1 is enriched in motoneuron presynaptic boutons and secreted into the NMJ extracellular synaptomatrix. We show that LGC1 limits locomotor peristalsis and coordinated movement speed, with a specific requirement for synaptic function, but not NMJ architecture. LGC1 controls neurotransmission strength by limiting presynaptic active zone (AZ) and postsynaptic glutamate receptor (GluR) aligned synapse number, reducing both spontaneous and stimulation-evoked synaptic vesicle (SV) release, and capping SV cycling rate. During high-frequency stimulation (HFS), mutants have faster synaptic depression and impaired recovery while replenishing depleted SV pools. Although LGC1 removal increases the number of glutamatergic synapses, we find that LGC1-null mutants exhibit decreased SV density within presynaptic boutons, particularly SV pools at presynaptic active zones. Thus, LGC1 regulates NMJ neurotransmission to modulate coordinated movement.

Caveolin-1 deficiency impairs synaptic transmission in hippocampal neurons

In addition to providing structural support, caveolin-1 (Cav1), a component of lipid rafts, including caveolae, in the plasma membrane, is involved in various cellular mechanisms, including signal transduction. Although pre-synaptic membrane dynamics and trafficking are essential cellular processes during synaptic vesicle exocytosis/synaptic transmission and synaptic vesicle endocytosis/synaptic retrieval, little is known about the involvement of Cav1 in synaptic vesicle dynamics. Here we demonstrate that synaptic vesicle exocytosis is significantly impaired in Cav1–knockdown (Cav1–KD) neurons. Specifically, the size of the synaptic recycled vesicle pool is modestly decreased in Cav1–KD synapses and the kinetics of synaptic vesicle endocytosis are somewhat slowed. Notably, neurons rescued by triple mutants of Cav1 lacking palmitoylation sites mutants show impairments in both synaptic transmission and retrieval. Collectively, our findings implicate Cav1 in activity-driven synaptic vesicle dynamics—both exocytosis and endocytosis—and demonstrate that palmitoylation of Cav1 is important for this activity.

Neurotransmitter Release Site Replenishment and Presynaptic Plasticity.

An action potential (AP) triggers neurotransmitter release from synaptic vesicles (SVs) docking to a specialized release site of presynaptic plasma membrane, the active zone (AZ). The AP simultaneously controls the release site replenishment with SV for sustainable synaptic transmission in response to incoming neuronal signals. Although many studies have suggested that the replenishment time is relatively slow, recent studies exploring high speed resolution have revealed SV dynamics with milliseconds timescale after an AP. Accurate regulation is conferred by proteins sensing Ca2+ entering through voltage-gated Ca2+ channels opened by an AP. This review summarizes how millisecond Ca2+ dynamics activate multiple protein cascades for control of the release site replenishment with release-ready SVs that underlie presynaptic short-term plasticity.