Search Results (34)

Aging is characterized by progressive degeneration of neuromuscular junctions (NMJs), which significantly contributes to muscle weakness and the development of sarcopenia. Photobiomodulation (PBM), a non-invasive therapeutic method based on the use of low-intensity light, has shown promising results in mitigating muscle degeneration in both experimental and clinical studies. The aim of this study was to evaluate the ultrastructural effects of photobiomodulation on neuromuscular junctions and skeletal muscle fibers in the m. vastus lateralis muscle of aged rats using light and transmission electron microscopy. Male Wistar rats (18 months old, body weight 650–800 g, n = 10) were subjected to photobiomodulation of the right m. vastus lateralis muscle (650 nm, 6 J/cm², four consecutive daily sessions of 3 min each). The contralateral left limb served as an untreated control. Muscle samples were analyzed by light and transmission electron microscopy. Histological examination revealed typical age-related changes in control muscles, including variability in muscle fiber diameter, centrally located nuclei, and an increased volume of connective tissue. Ultrastructural analysis confirmed signs of skeletal muscle aging, such as myofibril fragmentation, sarcomere disorganization, lipofuscin accumulation, and tubular aggregate formation. Morphometric analysis of neuromuscular junctions after photobiomodulation showed an increase in the number of active zones on the presynaptic membrane, elongation of the postsynaptic membrane, and a reduction in the width of the synaptic cleft. In addition, mitochondrial hyperplasia was observed in presynaptic terminals, while the total number of synaptic vesicles decreased. These findings indicate a compensatory reorganization of neuromuscular junctions and suggest that photobiomodulation can enhance their functional activity in aged skeletal muscle. Full article

Neuronal synapses are the functional units of communication in the central nervous system. This review describes the molecular mechanisms regulating synaptic transmission, plasticity, and circuit refinement. At the presynaptic active zone, scaffolding proteins including bassoon, piccolo, RIMs, and munc13 organize vesicle priming and the localization of voltage-gated calcium channels. Neurotransmitter release is mediated by the SNARE complex, comprising syntaxin-1, SNAP25, and synaptobrevin, and triggered by the calcium sensor synaptotagmin-1. Following exocytosis, synaptic vesicles are recovered through clathrin-mediated, ultrafast, bulk, or kiss-and-run endocytic pathways. Postsynaptically, the postsynaptic density (PSD) serves as a protein hub where scaffolds such as PSD-95, shank, homer, and gephyrin anchor excitatory (AMPA, NMDA) and inhibitory (GABA-A, Glycine) receptors are observed. Synaptic strength is modified during long-term potentiation (LTP) and depression (LTD) through signaling cascades involving kinases like CaMKII, PKA, and PKC, or phosphatases such as PP1 and calcineurin. These pathways regulate receptor trafficking, Arc-mediated endocytosis, and actin-dependent remodeling of dendritic spines. Additionally, synapse formation and elimination are guided by cell adhesion molecules, including neurexins and neuroligins, and by microglial pruning via the complement cascade (C1q, C3) and “don’t eat me” signals like CD47. Molecular diversity is further expanded by alternative splicing and post-translational modifications. A unified model of synaptic homeostasis is required to understand the basis of neuropsychiatric and neurological disorders. Full article

Evidence increasingly indicates that synaptic vesicle dysfunction emerges early in Parkinson’s disease (PD), preceding overt dopaminergic neuron loss rather than arising solely as a downstream consequence of neurodegeneration. α-Synuclein (αSyn), a presynaptic protein that regulates vesicle clustering, trafficking, and neurotransmitter release under physiological conditions, exhibits dose-, conformation-, and context-dependent actions that distinguish its normal regulatory roles from pathological effects observed in disease models. This narrative review synthesizes findings from a structured search of PubMed and Scopus, with emphasis on α-syn-knockout (αSynKO) and BAC transgenic (αSynBAC) mouse models, which do not recapitulate the full human PD trajectory but provide complementary insights into αSyn physiological function and dosage-sensitive vulnerability. Priority was given to studies integrating ultrastructural approaches—such as cryo-electron tomography, high-pressure freezing/freeze-substitution TEM, and super-resolution microscopy—with proteomic and lipidomic analyses. Across these methodologies, several convergent presynaptic alterations are consistently observed. In vivo and ex vivo studies associate αSyn perturbation with impaired vesicle acidification, consistent with altered expression or composition of vacuolar-type H⁺-ATPase subunits. Lipidomic analyses reveal age- and genotype-dependent remodeling of vesicle membrane lipids, particularly curvature- and charge-sensitive phospholipids, which may destabilize αSyn–membrane interactions. Complementary biochemical and cell-based studies support disruption of SNARE complex assembly and nanoscale release-site organization, while ultrastructural analyses demonstrate reduced vesicle docking, altered active zone geometry, and vesicle pool disorganization, collectively indicating compromised presynaptic efficiency. These findings support a synapse-centered framework in which presynaptic dysfunction represents an early and mechanistically relevant feature of PD. Rather than advocating αSyn elimination, emerging therapeutic concepts emphasize preservation of physiological vesicle function—through modulation of vesicle acidification, SNARE interactions, or membrane lipid homeostasis. Although such strategies remain exploratory, they identify the presynaptic terminal as a potential window for early intervention aimed at maintaining synaptic resilience and delaying functional decline in PD. Full article

Retinal ribbon synapses are continuously active chemical synapses. The eponymous synaptic ribbon is anchored to the active zone neurotransmitter release sites of ribbon synapses, recruits synaptic vesicles and guides ribbon-associated synaptic vesicles to the release sites. RIBEYE is the major protein component of synaptic ribbons. But likely, additional proteins contribute to ribbon synapse function. The synaptic ribbon of photoreceptor synapses is embedded into a highly polarized microtubule cytoskeleton. Interestingly, proteins of the photoreceptor primary cilium, such as NPHP4 and other ciliary proteins, including KIF3A, were shown to be localized to photoreceptor synaptic ribbons. Previous studies demonstrated that the microtubule motor protein KIF13B catalyzes secretory vesicle transport to the plus ends of microtubules and identified an interaction of KIF13B with NPHP4 at primary cilia. However, the localization of KIF13B, a kinesin-3 family motor protein, in the retina is still unknown. In the present study, we used two different antibodies against KIF13B and high-resolution confocal microscopy, super-resolution structured illumination microscopy (SR-SIM), and post-embedding immunogold electron microscopy to determine the localization of KIF13B in retinal photoreceptors. Apart from its localization at the primary photoreceptor cilium, we found a strong enrichment of KIF13B at photoreceptor synaptic ribbons. The synaptic ribbon is needed for the synaptic enrichment of KIF13B as shown by analyses of synaptic ribbon-deficient RIBEYE knockout mice. These findings suggest that KIF13B performs vesicle trafficking functions at the photoreceptor synaptic ribbon complex at the interface between the synaptic ribbon and the presynaptic microtubule transport system. Full article

Super-resolution single-molecule localization microscopy (SMLM) of presynaptic active zones (AZs) and postsynaptic densities contributed to the observation of protein nanoclusters that are involved in defining functional characteristics and in plasticity of synaptic connections. Among SMLM techniques, direct stochastic optical reconstruction microscopy (dSTORM) depends on organic fluorophores that exert high brightness and reliable photoswitching. While multicolor imaging is highly desirable, the requirements necessary for high-quality dSTORM make it challenging to identify combinations of equally performing, spectrally separated dyes. Red-excited carbocyanine dyes, e.g., Alexa Fluor 647 (AF647) or Cy5, are currently regarded as “gold standard” fluorophores for dSTORM imaging. However, a recent study introduced a set of chemically modified rhodamine dyes, including CF583R, that promise to display similar performance in dSTORM. In this study, we defined CF583R’s performance compared to AF647 and CF568 based on a nanoscopic analysis of Bruchpilot (Brp), a nanotopologically well-characterized scaffold protein at Drosophila melanogaster AZs. We demonstrate equal suitability of AF647, CF568 and CF583R for basal AZ morphometry, while in Brp subcluster analysis CF583R outperforms CF568 and is on par with AF647. Thus, the AF647/CF583R combination will be useful in future dSTORM-based analyses of AZs and other subcellularly located marker molecules and their role in physiological and pathophysiological contexts. Full article

Previous studies have suggested a role for selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (Prozac^®) in the treatment of dizziness and inner ear vestibular dysfunction. The potential mechanism of action within the vestibular system remains unclear; however, fluoxetine has been reported to block certain types of K⁺ channel in other systems. Here, we investigated the direct actions of fluoxetine on membrane currents in presynaptic hair cells and postsynaptic calyx afferents of the gerbil peripheral vestibular system using whole cell patch clamp recordings in crista slices. We explored differences in K⁺ currents in peripheral zone (PZ) and central zone (CZ) calyces of the crista and their response to fluoxetine application. Outward K⁺ currents in PZ calyces showed greater inactivation at depolarized membrane potentials compared to CZ calyces. The application of 100 μM fluoxetine notably reduced K⁺ currents in calyx terminals within both zones of the crista, and the remaining currents exhibited distinct traits. In PZ cells, fluoxetine inhibited a non-inactivating K⁺ current and revealed a rapidly activating and inactivating K⁺ current, which was sensitive to blocking by 4-aminopyridine. This was in contrast to CZ calyces, where low-voltage-activated and non-inactivating K⁺ currents persisted following application of 100 μM fluoxetine. Additionally, marked inhibition of transient inward Na⁺ currents by fluoxetine was observed in calyces from both crista zones. Different concentrations of fluoxetine were tested, and the EC₅₀ values were found to be 40 µM and 32 µM for K⁺ and Na⁺ currents, respectively. In contrast, 100 μM fluoxetine had no impact on voltage-dependent K⁺ currents in mechanosensory type I and type II vestibular hair cells. In summary, micromolar concentrations of fluoxetine are expected to strongly reduce both Na⁺ and K⁺ conductance in afferent neurons of the peripheral vestibular system in vivo. This would lead to inhibition of action potential firing in vestibular sensory neurons and has therapeutic implications for disorders of balance. Full article

The faithful formation and, consequently, function of a synapse requires continuous and tightly controlled delivery of synaptic material. At the presynapse, a variety of proteins with unequal molecular properties are indispensable to compose and control the molecular machinery concerting neurotransmitter release through synaptic vesicle fusion with the presynaptic membrane. As presynaptic proteins are produced mainly in the neuronal soma, they are obliged to traffic along microtubules through the axon to reach the consuming presynapse. This anterograde transport is performed by highly specialised and diverse presynaptic precursor vesicles, membranous organelles able to transport as different proteins such as synaptic vesicle membrane and membrane-associated proteins, cytosolic active zone proteins, ion-channels, and presynaptic membrane proteins, coordinating synaptic vesicle exo- and endocytosis. This review aims to summarise and categorise the diverse and numerous findings describing presynaptic precursor cargo, mode of trafficking, kinesin-based axonal transport and the molecular mechanisms of presynaptic precursor vesicles biogenesis in both vertebrate and invertebrate model systems. Full article

Ribbon synapses reliably transmit synaptic signals over a broad signalling range. Rod photoreceptor ribbon synapses are capable of transmitting signals generated by the absorption of single photons. The high precision of ribbon synapses emphasizes the need for particularly efficient signalling mechanisms. Synaptic ribbons are presynaptic specializations of ribbon synapses and are anchored to the active zone. Synaptic ribbons bind many synaptic vesicles that are delivered to the active zone for continuous and faithful signalling. In the present study we demonstrate with independent antibodies at the light- and electron microscopic level that rabconnectin-3α (RC3α)—alternative name Dmx-like 2 (DMXL2)—is localized to the synaptic ribbons of rod photoreceptor synapses in the mouse retina. In the brain, RC3α-containing complexes are known to interact with important components of synaptic vesicles, including Rab3-activating/inactivating enzymes, priming proteins and the vesicular H⁺-ATPase that acidifies the synaptic vesicle lumen to promote full neurotransmitter loading. The association of RC3α/DMXL2 with rod synaptic ribbons of the mouse retina could enable these structures to deliver only fully signalling-competent synaptic vesicles to the active zone thus contributing to reliable synaptic communication. Full article

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. Full article

Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations. Full article

Alpha-synuclein is a presynaptic protein linked to Parkinson’s disease with a poorly characterized physiological role in regulating the synaptic vesicle cycle. Using RBL-2H3 cells as a model system, we earlier reported that wild-type alpha-synuclein can act as both an inhibitor and a potentiator of stimulated exocytosis in a concentration-dependent manner. The inhibitory function is constitutive and depends on membrane binding by the helix-2 region of the lipid-binding domain, while potentiation becomes apparent only at high concentrations. Using structural and functional characterization of conformationally selective mutants via a combination of spectroscopic and cellular assays, we show here that binding affinity for isolated vesicles similar in size to synaptic vesicles is a primary determinant of alpha-synuclein-mediated potentiation of vesicle release. Inhibition of release is sensitive to changes in the region linking the helix-1 and helix-2 regions of the N-terminal lipid-binding domain and may require some degree of coupling between these regions. Potentiation of release likely occurs as a result of alpha-synuclein interactions with undocked vesicles isolated away from the active zone in internal pools. Consistent with this, we observe that alpha-synuclein can disperse vesicles from in vitro clusters organized by condensates of the presynaptic protein synapsin-1. Full article

Neurotransmitter release from presynaptic terminals is primarily regulated by rapid Ca²⁺ influx through membrane-resident voltage-gated Ca²⁺ channels (VGCCs). Moreover, accumulating evidence indicates that the endoplasmic reticulum (ER) is extensively present in axonal terminals of neurons and plays a modulatory role in synaptic transmission by regulating Ca²⁺ levels. Familial Alzheimer’s disease (FAD) is marked by enhanced Ca²⁺ release from the ER and downregulation of Ca²⁺ buffering proteins. However, the precise consequence of impaired Ca²⁺ signaling within the vicinity of VGCCs (active zone (AZ)) on exocytosis is poorly understood. Here, we perform in silico experiments of intracellular Ca²⁺ signaling and exocytosis in a detailed biophysical model of hippocampal synapses to investigate the effect of aberrant Ca²⁺ signaling on neurotransmitter release in FAD. Our model predicts that enhanced Ca²⁺ release from the ER increases the probability of neurotransmitter release in FAD. Moreover, over very short timescales (30–60 ms), the model exhibits activity-dependent and enhanced short-term plasticity in FAD, indicating neuronal hyperactivity—a hallmark of the disease. Similar to previous observations in AD animal models, our model reveals that during prolonged stimulation (~450 ms), pathological Ca²⁺ signaling increases depression and desynchronization with stimulus, causing affected synapses to operate unreliably. Overall, our work provides direct evidence in support of a crucial role played by altered Ca²⁺ homeostasis mediated by intracellular stores in FAD. Full article

Within 1 millisecond of action potential arrival at presynaptic terminals voltage–gated Ca²⁺ channels open. The Ca²⁺ channels are linked to synaptic vesicles which are tethered by active zone proteins. Ca²⁺ entrance into the active zone triggers: (1) the fusion of the vesicle and exocytosis, (2) the replenishment of the active zone with vesicles for incoming exocytosis, and (3) various types of endocytosis for vesicle reuse, dependent on the pattern of firing. These time-dependent vesicle dynamics are controlled by presynaptic Ca²⁺ sensor proteins, regulating active zone scaffold proteins, fusion machinery proteins, motor proteins, endocytic proteins, several enzymes, and even Ca²⁺ channels, following the decay of Ca²⁺ concentration after the action potential. Here, I summarize the Ca²⁺-dependent protein controls of synchronous and asynchronous vesicle release, rapid replenishment of the active zone, endocytosis, and short-term plasticity within 100 msec after the action potential. Furthermore, I discuss the contribution of active zone proteins to presynaptic plasticity and to homeostatic readjustment during and after intense activity, in addition to activity-dependent endocytosis. Full article

Synapses serve as the interface for the transmission of information between neurons in the central nervous system. The structural and functional characteristics of synapses are highly dynamic, exhibiting extensive plasticity that is shaped by neural activity and regulated primarily by trans-synaptic cell-adhesion molecules (CAMs). Prototypical trans-synaptic CAMs, such as neurexins (Nrxs) and neuroligins (Nlgs), directly regulate the assembly of presynaptic and postsynaptic molecules, including synaptic vesicles, active zone proteins, and receptors. Therefore, the trans-synaptic adhesion mechanisms mediated by Nrx–Nlg interaction can contribute to a range of synaptopathies in the context of pathological pain and other neurological disorders. The present review provides an overview of the current understanding of the roles of Nrx–Nlg interaction in the regulation of trans-synaptic connections, with a specific focus on Nrx and Nlg structures, the dynamic shaping of synaptic function, and the dysregulation of Nrx–Nlg in pathological pain. Additionally, we discuss a range of proteins capable of modulating Nrx–Nlg interactions at the synaptic cleft, with the objective of providing a foundation to guide the future development of novel therapeutic agents for managing pathological pain. Full article

The mouse neuromuscular junction (NMJ) has long been used as a model synapse for the study of neurotransmission in both healthy and disease states of the NMJ. Neurotransmission from these neuromuscular nerve terminals occurs at highly organized structures called active zones (AZs). Within AZs, the relationships between the voltage-gated calcium channels and docked synaptic vesicles govern the probability of acetylcholine release during single action potentials, and the short-term plasticity characteristics during short, high frequency trains of action potentials. Understanding these relationships is important not only for healthy synapses, but also to better understand the pathophysiology of neuromuscular diseases. In particular, we are interested in Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disorder in which neurotransmitter release from the NMJ decreases, leading to severe muscle weakness. In LEMS, the reduced neurotransmission is traditionally thought to be caused by the antibody-mediated removal of presynaptic voltage-gated calcium channels. However, recent experimental data and AZ computer simulations have predicted that a disruption in the normally highly organized active zone structure, and perhaps autoantibodies to other presynaptic proteins, contribute significantly to pathological effects in the active zone and the characteristics of chemical transmitters. Full article