Albiflorin Decreases Glutamate Release from Rat Cerebral Cortex Nerve Terminals (Synaptosomes) through Depressing P/Q-Type Calcium Channels and Protein Kinase A Activity.

The purpose of this study was to investigate whether and how albiflorin, a natural monoterpene glycoside, affects the release of glutamate, one of the most important neurotransmitters involved in neurotoxicity, from cerebrocortical nerve terminals (synaptosomes) in rats. The results showed that albiflorin reduced 4-aminopyridine (4-AP)-elicited glutamate release from synaptosomes, which was abrogated in the absence of extracellular Ca2+ or in the presence of the vesicular glutamate transporter inhibitor or a P/Q-type Ca2+ channel inhibitor, indicating a mechanism of action involving Ca2+-dependent depression of vesicular exocytotic glutamate release. Albiflorin failed to alter the increase in the fluorescence intensity of 3,3-diethylthiacarbocyanine iodide (DiSC3(5)), a membrane-potential-sensitive dye. In addition, the suppression of protein kinase A (PKA) abolished the effect of albiflorin on glutamate release. Albiflorin also reduced the phosphorylation of PKA and synaptosomal-associated protein of 25 kDa (SNAP-25) and synapsin I at PKA-specific residues, which correlated with decreased available synaptic vesicles. The results of transmission electron microscopy (TEM) also observed that albiflorin reduces the release competence of synaptic vesicles evoked by 4-AP in synaptosomes. In conclusion, by studying synaptosomally released glutamate, we suggested that albiflorin reduces vesicular exocytotic glutamate release by decreasing extracellular Ca2+ entry via P/Q-type Ca2+ channels and reducing PKA-mediated synapsin I and SNAP-25 phosphorylation.

[A case of myasthenia gravis with coexistence of anti-acetylcholine receptor antibodies and anti-P/Q-type VGCC antibodies].

A 79-year-old woman who presented ptosis and dysphagia were admitted to our hospital. Anti-acetylcholine receptor antibodies and anti-P/Q-type VGCC antibodies were both positive. Electrophysiological examination showed postsynaptic pattern which supported myasthenia gravis. She did not meet the diagnostic criteria for Lambert-Eaton myasthenic syndrome (LEMS). In cases which these antibodies coexist, careful electrophysiological evaluation is required for the diagnosis. In addition, although anti-P/Q-type VGCC antibodies have been specific to LEMS, patients with these antibodies represent various symptoms other than LEMS. Low and middle titer of the antibodies may be not specific to LEMS.

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.

[LEMS: Clinical Significance of Pathogenic Autoantibodies].

Approximately 90% of patients with Lambert-Eaton myasthenic syndrome (LEMS) are positive for P/Q-type voltage-gated calcium channels (VGCCs) antibodies, and can be broadly divided into two groups: paraneoplastic, especially with small cell lung carcinoma and, non-paraneoplastic, without cancer. Under the Japanese LEMS diagnostic criteria 2022, abnormal electrophysiological results is mandatory for diagnosis in addition to muscle weakness. Contrastingly, autoantibodies are useful for diagnosing the etiology and influence treatment strategies. We comprehensively reviewed the MG/LEMS 2022 practice guidelines. Moreover, we presented a case of PCD without LEMS that was positive for P/Q-type VGCCs antibodies_and discussed the clinical significance of the autoantibodies.

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.

Paraneoplastic Cerebellar Degeneration With P/Q-VGCC vs Yo Autoantibodies.

BACKGROUND AND OBJECTIVES: Paraneoplastic cerebellar degeneration (PCD) is characterized by a widespread loss of Purkinje cells (PCs) and may be associated with autoantibodies against intracellular antigens such as Yo or cell surface neuronal antigens such as the P/Q-type voltage-gated calcium channel (P/Q-VGCC). Although the intracellular location of the target antigen in anti-Yo-PCD supports a T cell-mediated pathology, the immune mechanisms in anti-P/Q-VGCC-PCD remain unclear. In this study, we compare neuropathologic characteristics of PCD with anti-P/Q-VGCC and anti-Yo autoantibodies in an archival autopsy cohort. METHODS: We performed neuropathology, immunohistochemistry, and multiplex immunofluorescence on formalin-fixed and paraffin-embedded brain tissue of 1 anti-P/Q-VGCC, 2 anti-Yo-PCD autopsy cases and controls. RESULTS: Anti-Yo-PCD revealed a diffuse and widespread PC loss together with microglial nodules with pSTAT1+ and CD8+granzymeB+ T cells and neuronal upregulation of major histocompatibility complex (MHC) Class I molecules. Some neurons showed a cytoplasmic immunoglobulin G (IgG) staining. In contrast, PC loss in anti-P/Q-VGCC-PCD was focal and predominantly affected the upper vermis, whereas caudal regions and lateral hemispheres were spared. Inflammation was characterized by scattered CD8+ T cells, single CD20+/CD79a+ B/plasma cells, and an IgG staining of the neuropil in the molecular layer of the cerebellar cortex and neuronal cytoplasms. No complement deposition or MHC-I upregulation was detected. Moreover, synaptophysin was reduced, and neuronal P/Q-VGCC was downregulated. In affected areas, axonal spheroids and the accumulation of amyloid precursor protein and glucose-regulated protein 78 in PCs indicate endoplasmatic reticulum stress and impairment of axonal transport. In both PCD types, calbindin expression was reduced or lost in the remaining PCs. DISCUSSION: Anti-Yo-PCD showed characteristic features of a T cell-mediated pathology, whereas this was not observed in 1 case of anti-P/Q-VGCC-PCD. Our findings support a pathogenic role of anti-P/Q-VGCC autoantibodies in causing neuronal dysfunction, probably due to altered synaptic transmission resulting in calcium dysregulation and subsequent PC death. Because disease progression may lead to irreversible PC loss, anti-P/Q-VGCC-PCD patients could benefit from early oncologic and immunologic therapies.

Cerebellar contribution to threat probability in a SCA6 mouse model.

Fear and anxiety have proven to be essential during the evolutionary process. However, the mechanisms involved in recognizing and categorizing threat probability (i.e. low to high) to elicit the appropriate defensive behavior are yet to be determined. In this study, we investigated the cerebellar contribution in evoking appropriate defensive escape behavior using a purely cerebellar, neurodegenerative mouse model for spinocerebellar ataxia type 6 which is caused by an expanded CAG repeat in exon 47 of the P/Q type calcium channel α1A subunit. These mice overexpress the carboxy terminus (CT) of the P/Q type calcium channel containing an expanded 27 CAG repeat specifically in cerebellar Purkinje cells (CT-longQ27PC). We found that our CT-longQ27PC mice exhibit anxiolytic behavior in the open field, elevated plus maze and light/dark place preference tests, which could be recovered with more threatening conditions such as brighter lighting, meowing sounds and an ultrasound repellent. Their innate fear to find safety in the Barnes maze and visual cliff tests was also diminished with subsequent trials, which could be partially recovered with an ultrasound repellent in the Barnes maze. However, under higher threat conditions such as in the light/dark place preference with ultrasound repellent and in the looming tests, CT-longQ27PC mice responded with higher defensive escape behaviors as controls. Moreover, CT-longQ27PC mice displayed increased levels of CT-labeled aggregates compared with controls. Together these data suggest that cerebellar degeneration by overexpression of CT-longQ27PC is sufficient to impair defensive escape responses in those mice.

Clinical and molecular spectrum of P/Q type calcium channel Cav2.1 in epileptic patients.

BACKGROUND: Epilepsy is a neurological disorder characterized by the potential to induce seizure and accompanied by cognitive, psychological, and social consequences. CACNA1A gene is a voltage-gated P/Q-type Cav2.1 channel that is broadly expressed in the central nervous system, and the pathogenic variants within this gene may be associated with the epileptic phenotype. In the present study, we collected clinical and molecular data related to epileptic patients with CACNA1A pathogenic variants and investigated possible meaningful relationship between age at onset, neurodevelopmental disorders, type of seizures, brain imaging abnormalities, genotype, and protein domains. RESULTS: In our retrospective literature studies, from among 890 articles reviewed, a total of 90 individuals were related to epilepsy phenotype. Our findings showed that about 90 percent of patients have shown the first symptoms in childhood and teenage years and different types of neurodevelopmental disorders, such as intellectual disability, developmental arrest, and behavioral disorders, have been common findings for these patients. Further, a wide range of abnormalities have been observed in their brain imaging, and generalized seizures have been the most type of seizures in these patients. However, our data showed no specific genotype-phenotype correlation in epileptic patients with CACNA1A pathogenic alterations. CONCLUSIONS: Our study focused on epileptic phenotype in patients with CACNA1A pathogenic variants and showed a wide range of clinical and molecular heterogeneity with no specific genotype-phenotype correlation. It seems that incomplete penetrance, de-novo variants, and modifier genes are obstacles in predicting the clinical outcome.

Chlorogenic Acid Decreases Glutamate Release from Rat Cortical Nerve Terminals by P/Q-Type Ca2+ Channel Suppression: A Possible Neuroprotective Mechanism.

The glutamatergic neurotransmitter system has received substantial attention in research on the pathophysiology and treatment of neurological disorders. The study investigated the effect of the polyphenolic compound chlorogenic acid (CGA) on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). CGA inhibited 4-aminopyridine (4-AP)-induced glutamate release from synaptosomes. This inhibition was prevented in the absence of extracellular Ca2+ and was associated with the inhibition of 4-AP-induced elevation of Ca2+ but was not attributed to changes in synaptosomal membrane potential. In line with evidence observed through molecular docking, CGA did not inhibit glutamate release in the presence of P/Q-type Ca2+ channel inhibitors; therefore, CGA-induced inhibition of glutamate release may be mediated by P/Q-type Ca2+ channels. CGA-induced inhibition of glutamate release was also diminished by the calmodulin and Ca2+/calmodilin-dependent kinase II (CaMKII) inhibitors, and CGA reduced the phosphorylation of CaMKII and its substrate, synapsin I. Furthermore, pretreatment with intraperitoneal CGA injection attenuated the glutamate increment and neuronal damage in the rat cortex that were induced by kainic acid administration. These results indicate that CGA inhibits glutamate release from cortical synaptosomes by suppressing P/Q-type Ca2+ channels and CaMKII/synapsin I pathways, thereby preventing excitotoxic damage to cortical neurons.

Natural Product Isoliquiritigenin Activates GABAB Receptors to Decrease Voltage-Gate Ca2+ Channels and Glutamate Release in Rat Cerebrocortical Nerve Terminals.

Reduction in glutamate release is a key mechanism for neuroprotection and we investigated the effect of isoliquiritigenin (ISL), an active ingredient of Glycyrrhiza with neuroprotective activities, on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). ISL produced a concentration-dependent inhibition of glutamate release and reduced the intraterminal [Ca2+] increase. The inhibition of glutamate release by ISL was prevented after removing extracellular Ca2+ or blocking P/Q-type Ca2+ channels. This inhibition was mediated through the γ-aminobutyric acid type B (GABAB) receptors because ISL was unable to inhibit glutamate release in the presence of baclofen (an GABAB agonist) or CGP3548 (an GABAB antagonist) and docking data revealed that ISL interacted with GABAB receptors. Furthermore, the ISL inhibition of glutamate release was abolished through the inhibition of Gi/o-mediated responses or Gßγ subunits, but not by 8-bromoadenosine 3',5'-cyclic monophosphate or adenylate cyclase inhibition. The ISL inhibition of glutamate release was also abolished through the inhibition of protein kinase C (PKC), and ISL decreased the phosphorylation of PKC. Thus, we inferred that ISL, through GABAB receptor activation and Gßγ-coupled inhibition of P/Q-type Ca2+ channels, suppressed the PKC phosphorylation to cause a decrease in evoked glutamate release at rat cerebrocortical nerve terminals.

Primary and secondary motoneurons use different calcium channel types to control escape and swimming behaviors in zebrafish.

The escape response and rhythmic swimming in zebrafish are distinct behaviors mediated by two functionally distinct motoneuron (Mn) types. The primary (1°Mn) type depresses and has a large quantal content (Qc) and a high release probability (Pr). Conversely, the secondary (2°Mn) type facilitates and has low and variable Qc and Pr. This functional duality matches well the distinct associated behaviors, with the 1°Mn providing the strong, singular C bend initiating escape and the 2°Mn conferring weaker, rhythmic contractions. Contributing to these functional distinctions is our identification of P/Q-type calcium channels mediating transmitter release in 1°Mns and N-type channels in 2°Mns. Remarkably, despite these functional and behavioral distinctions, all ∼15 individual synapses on each muscle cell are shared by a 1°Mn bouton and at least one 2°Mn bouton. This blueprint of synaptic sharing provides an efficient way of controlling two different behaviors at the level of a single postsynaptic cell.

[1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol, an indole-3-carbinol derivative, inhibits glutamate release in rat cerebrocortical nerve terminals by suppressing the P/Q-type Ca2+ channels and Ca2+/calmodulin/protein kinase A pathway.

Indole-3-carbinol (I3C), found in cruciferous vegetables, has been proposed to exhibit neuroprotective effects. This study aimed to investigate the effect of the I3C derivative [1(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol (CIM), which has superior pharmacokinetic properties to I3C, on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). We observed that CIM dose-dependently inhibited glutamate release evoked by the potassium channel blocker 4-aminopyridine (4-AP). CIM-mediated inhibition of glutamate release was attributed to reduced exocytosis, as it correlated with the removal of extracellular calcium and blocking of the vesicular glutamate transporter but not the glutamate transporter. In addition, CIM decreased 4-AP-evoked intrasynaptosomal Ca2+ elevation; however, it did not alter the synaptosomal membrane potential. The inhibition of P/Q-typeCa2+ channels abolished the effect of CIM on 4-AP-evoked glutamate release, and the effect was not prevented by intracellular Ca2+ release inhibitors. Moreover, the molecular docking study showed that CIM exhibited the highest binding affinity with the P/Q-type Ca2+channels. Finally, the CIM-mediated inhibition of glutamate release was sensitive to calmodulin, adenylate cyclase (AC), and protein kinase A (PKA) inhibitors. Based on these results, we propose that CIM, through the direct suppression of P/Q-type Ca2+ channels, decreases Ca2+ influx and the activation of Ca2+/calmodulin/AC/PKA signaling, thereby inhibiting glutamate release. This finding is crucial for understanding the role of CIM in the central nervous system and for exploiting its potential in therapeutic interventions.

Synapsin I Synchronizes GABA Release in Distinct Interneuron Subpopulations.

Neurotransmitters can be released either synchronously or asynchronously with respect to action potential timing. Synapsins (Syns) are a family of synaptic vesicle (SV) phosphoproteins that assist gamma-aminobutyric acid (GABA) release and allow a physiological excitation/inhibition balance. Consistently, deletion of either or both Syn1 and Syn2 genes is epileptogenic. In this work, we have characterized the effect of SynI knockout (KO) in the regulation of GABA release dynamics. Using patch-clamp recordings in hippocampal slices, we demonstrate that the lack of SynI impairs synchronous GABA release via a reduction of the readily releasable SVs and, in parallel, increases asynchronous GABA release. The effects of SynI deletion on synchronous GABA release were occluded by ω-AgatoxinIVA, indicating the involvement of P/Q-type Ca2+channel-expressing neurons. Using in situ hybridization, we show that SynI is more expressed in parvalbumin (PV) interneurons, characterized by synchronous release, than in cholecystokinin or SOM interneurons, characterized by a more asynchronous release. Optogenetic activation of PV and SOM interneurons revealed a specific reduction of synchronous release in PV/SynIKO interneurons associated with an increased asynchronous release in SOM/SynIKO interneurons. The results demonstrate that SynI is differentially expressed in interneuron subpopulations, where it boosts synchronous and limits asynchronous GABA release.

Ca2+ -independent and voltage-dependent exocytosis in mouse chromaffin cells.

AIM: It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca2+ entry through voltage-dependent Ca2+ channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca2+ current and intracellular Ca2+ increase. In this work we investigated if such a mechanism contributes to secretory vesicle exocytosis in mouse chromaffin cells. METHODS: Exocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic Ca2+ was estimated using epifluorescence microscopy and fluo-8 (salt form). RESULTS: Cells stimulated by brief depolatizations in absence of extracellular Ca+2 show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca+2 release from intracellular stores. This exocytosis is tightly dependent on membrane potential, is inhibited by neurotoxin Bont-B (cleaves the v-SNARE synaptobrevin), is very fast (saturates with time constant <10 ms), it is followed by a fast endocytosis sensitive to the application of an anti-dynamin monoclonal antibody, and recovers after depletion in <5 s. Finally, this exocytosis was inhibited by: (i) ω-agatoxin IVA (blocks P/Q-type Ca2+ channel gating), (ii) in cells from knock-out P/Q-type Ca2+ channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel-exocytic proteins association). CONCLUSION: We demonstrated that Ca2+ -independent and voltage-dependent exocytosis is present in chromaffin cells. This process is tightly coupled to membrane depolarization, and is able to support secretion during action potentials at low basal rates. P/Q-type Ca2+ channels can operate as voltage sensors of this process.

RIMB-1/RIM-Binding Protein and UNC-10/RIM Redundantly Regulate Presynaptic Localization of the Voltage-Gated Calcium Channel in Caenorhabditis elegans.

Presynaptic active zones (AZs) contain many molecules essential for neurotransmitter release and are assembled in a highly organized manner. A network of adaptor proteins known as cytomatrix at the AZ (CAZ) is important for shaping the structural characteristics of AZ. Rab3-interacting molecule (RIM)-binding protein (RBP) family are binding partners of the CAZ protein RIM and also bind the voltage-gated calcium channels (VGCCs) in mice and flies. Here, we investigated the physiological roles of RIMB-1, the homolog of RBPs in the nematode Caenorhabditis elegans RIMB-1 is expressed broadly in neurons and predominantly localized at presynaptic sites. Loss-of-function animals of rimb-1 displayed slight defects in motility and response to pharmacological inhibition of synaptic transmission, suggesting a modest involvement of rimb-1 in synapse function. We analyzed genetic interactions of rimb-1 by testing candidate genes and by an unbiased forward genetic screen for rimb-1 enhancer. Both analyses identified the RIM homolog UNC-10 that acts together with RIMB-1 to regulate presynaptic localization of the P/Q-type VGCC UNC-2/Cav2. We also find that the precise localization of RIMB-1 to presynaptic sites requires presynaptic UNC-2/Cav2. RIMB-1 has multiple FN3 and SH3 domains. Our transgenic rescue analysis with RIMB-1 deletion constructs revealed a functional requirement of a C-terminal SH3 in regulating UNC-2/Cav2 localization. Together, these findings suggest a redundant role of RIMB-1/RBP and UNC-10/RIM to regulate the abundance of UNC-2/Cav2 at the presynaptic AZ in C. elegans, depending on the bidirectional interplay between CAZ adaptor and channel proteins.SIGNIFICANCE STATEMENT Presynaptic active zones (AZs) are highly organized structures for synaptic transmission with characteristic networks of adaptor proteins called cytomatrix at the AZ (CAZ). In this study, we characterized a CAZ protein RIMB-1, named for RIM-binding protein (RBP), in the nematode Caenorhabditis elegans Through systematic analyses of genetic interactions and an unbiased genetic enhancer screen of rimb-1, we revealed a redundant role of two CAZ proteins RIMB-1/RBP and UNC-10/RIM in regulating presynaptic localization of UNC-2/Cav2, a voltage-gated calcium channel (VGCC) critical for proper neurotransmitter release. Additionally, the precise localization of RIMB-1/RBP requires presynaptic UNC-2/Cav2. These findings provide new mechanistic insight about how the interplay among multiple CAZ adaptor proteins and VGCC contributes to the organization of presynaptic AZ.