A suitable model to investigate acute neurological consequences of coronavirus infection.

OBJECTIVE AND DESIGN: The present study aimed to investigate the neurochemical and behavioral effects of the acute consequences after coronavirus infection through a murine model. MATERIAL: Wild-type C57BL/6 mice were infected intranasally (i.n) with the murine coronavirus 3 (MHV-3). METHODS: Mice underwent behavioral tests. Euthanasia was performed on the fifth day after infection (5 dpi), and the brain tissue was subjected to plaque assays for viral titration, ELISA, histopathological, immunohistochemical and synaptosome analysis. RESULTS: Increased viral titers and mild histological changes, including signs of neuronal degeneration, were observed in the cerebral cortex of infected mice. Importantly, MHV-3 infection induced an increase in cortical levels of glutamate and calcium, which is indicative of excitotoxicity, as well as increased levels of pro-inflammatory cytokines (IL-6, IFN-γ) and reduced levels of neuroprotective mediators (BDNF and CX3CL1) in the mice brain. Finally, behavioral analysis showed impaired motor, anhedonia-like and anxiety-like behaviors in animals infected with MHV-3. CONCLUSIONS: In conclusion, the data presented emulate many aspects of the acute neurological outcomes seen in patients with COVID-19. Therefore, this model may provide a preclinical platform to study acute neurological sequelae induced by coronavirus infection and test possible therapies.

A Biosafety Level 2 Mouse Model for Studying Betacoronavirus-Induced Acute Lung Damage and Systemic Manifestations.

The emergence of life-threatening zoonotic diseases caused by betacoronaviruses, including the ongoing coronavirus disease 19 (COVID-19) pandemic, has highlighted the need for developing preclinical models mirroring respiratory and systemic pathophysiological manifestations seen in infected humans. Here, we showed that C57BL/6J wild-type mice intranasally inoculated with the murine betacoronavirus murine hepatitis coronavirus 3 (MHV-3) develop a robust inflammatory response leading to acute lung injuries, including alveolar edema, hemorrhage, and fibrin thrombi. Although such histopathological changes seemed to resolve as the infection advanced, they efficiently impaired respiratory function, as the infected mice displayed restricted lung distention and increased respiratory frequency and ventilation. Following respiratory manifestation, the MHV-3 infection became systemic, and a high virus burden could be detected in multiple organs along with morphological changes. The systemic manifestation of MHV-3 infection was also marked by a sharp drop in the number of circulating platelets and lymphocytes, besides the augmented concentration of the proinflammatory cytokines interleukin 1 beta (IL-1ß), IL-6, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor (TNF), thereby mirroring some clinical features observed in moderate and severe cases of COVID-19. Importantly, both respiratory and systemic changes triggered by MHV-3 infection were greatly prevented by blocking TNF signaling, either via genetic or pharmacologic approaches. In line with this, TNF blockage also diminished the infection-mediated release of proinflammatory cytokines and virus replication of human epithelial lung cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Collectively, results show that MHV-3 respiratory infection leads to a large range of clinical manifestations in mice and may constitute an attractive, lower-cost, biosafety level 2 (BSL2) in vivo platform for evaluating the respiratory and multiorgan involvement of betacoronavirus infections. IMPORTANCE Mouse models have long been used as valuable in vivo platforms to investigate the pathogenesis of viral infections and effective countermeasures. The natural resistance of mice to the novel betacoronavirus SARS-CoV-2, the causative agent of COVID-19, has launched a race toward the characterization of SARS-CoV-2 infection in other animals (e.g., hamsters, cats, ferrets, bats, and monkeys), as well as adaptation of the mouse model, by modifying either the host or the virus. In the present study, we utilized a natural pathogen of mice, MHV, as a prototype to model betacoronavirus-induced acute lung injure and multiorgan involvement under biosafety level 2 conditions. We showed that C57BL/6J mice intranasally inoculated with MHV-3 develops severe disease, which includes acute lung damage and respiratory distress that precede systemic inflammation and death. Accordingly, the proposed animal model may provide a useful tool for studies regarding betacoronavirus respiratory infection and related diseases.

Neuroprotective effect of CTK 01512-2 recombinant toxin at the spinal cord in a model of Huntington's disease.

NEW FINDINGS: What is the central question of this study? We investigated the effects of intrathecal administration of a novel toxin, CTK 01512-2, in a mouse model of Huntington's disease. We asked whether spinal cord neurons can represent a therapeutic target, given that the spinal cord seems to be involved in motor symptoms of Huntington's disease. Pharmacological approaches focusing on the spinal cord and skeletal muscles might represent a more feasible strategy than a high-risk brain intervention. What is the main finding and its importance? We provided evidence of a novel, local, neuroprotective effect of CTK 01512-2, paving a path for the development of approaches to treat motor symptoms of Huntington's disease beyond the brain. ABSTRACT: Phα1ß is a neurotoxin from the venom of the Phoneutria nigriventer spider, available as CTK 01512-2, a recombinant peptide. Owing to its antinociceptive and analgesic properties, CTK 01512-2 has been described to alleviate neuroinflammatory responses. Despite the diverse actions of CTK 01512-2 on the nervous system, little is known regarding its neuroprotective effect, especially in neurodegenerative conditions such as Huntington's disease (HD), a genetic movement disorder without cure. Here, we investigated whether CTK 01512-2 has a neuroprotective effect in a mouse model of HD. We hypothesized that spinal cord neurons might represent a therapeutic target, because the spinal cord seems to be involved in the motor symptoms of HD (BACHD) mice. We treated BACHD mice with CTK 01512-2 by intrathecal injection and performed in vivo motor behavioural and morphological analyses in the CNS (brain and spinal cord) and muscles. Our data showed that intrathecal injection of CTK 01512-2 significantly improved motor performance in the open field task. CTK 01512-2 protected neurons in the spinal cord (but not in the brain) from death, suggesting a local effect. CTK 01512-2 exerted its neuroprotective effect by inhibiting BACHD neuronal apoptosis, as revealed by a reduction in caspase-3 in the spinal cord. CTK 01512-2 was also able to revert BACHD muscle atrophy. In conclusion, our data suggest a novel role for CTK 01512-2 acting directly in the spinal cord to ameliorate morphofunctional aspects of spinal cord neurons and muscles and improve the performance of BACHD mice in motor behavioural tests. Given that HD shares similar symptoms with many neurodegenerative conditions, the findings presented herein might also be applicable to other disorders.

Renin-Angiotensin System in Huntington's Disease: Evidence from Animal Models and Human Patients.

The Renin-Angiotensin System (RAS) is expressed in the central nervous system and has important functions that go beyond blood pressure regulation. Clinical and experimental studies have suggested that alterations in the brain RAS contribute to the development and progression of neurodegenerative diseases. However, there is limited information regarding the involvement of RAS components in Huntington's disease (HD). Herein, we used the HD murine model, (BACHD), as well as samples from patients with HD to investigate the role of both the classical and alternative axes of RAS in HD pathophysiology. BACHD mice displayed worse motor performance in different behavioral tests alongside a decrease in the levels and activity of the components of the RAS alternative axis ACE2, Ang-(1-7), and Mas receptors in the striatum, prefrontal cortex, and hippocampus. BACHD mice also displayed a significant increase in mRNA expression of the AT1 receptor, a component of the RAS classical arm, in these key brain regions. Moreover, patients with manifest HD presented higher plasma levels of Ang-(1-7). No significant changes were found in the levels of ACE, ACE2, and Ang II. Our findings provided the first evidence that an imbalance in the RAS classical and counter-regulatory arms may play a role in HD pathophysiology.

Spatially Resolved Expression of Transposable Elements in Disease and Somatic Tissue with SpatialTE.

Spatial transcriptomics (ST) is transforming the way we can study gene expression and its regulation through position-specific resolution within tissues. However, as in bulk RNA-Seq, transposable elements (TEs) are not being studied due to their highly repetitive nature. In recent years, TEs have been recognized as important regulators of gene expression, and thus, TE expression analysis in a spatially resolved manner could further help to understand their role in gene regulation within tissues. We present SpatialTE, a tool to analyze TE expression from ST datasets and show its application in somatic and diseased tissues. The results indicate that TEs have spatially regulated expression patterns and that their expression profiles are spatially altered in ALS disease, indicating that TEs might perform differential regulatory functions within tissue organs. We have made SpatialTE publicly available as open-source software under an MIT license.

Evaluation of the neuromuscular junction in a middle-aged mouse model of congenital myasthenic syndrome.

INTRODUCTION: Reduced expression of the vesicular acetylcholine transporter (VAChT) leads to changes in the distribution and shape of synaptic vesicles (SVs) at neuromuscular junctions (NMJs), suggesting vesicular acetylcholine (ACh) as a key component of synaptic structure and function. It is poorly understood how long-term changes in cholinergic transmission contribute to age- and disease-related degeneration in the motor system. METHODS: In this study we performed confocal imaging, electrophysiology, electron microscopy, and analyses of respiratory mechanics of the diaphragm NMJ components in 12-month-old wild-type (WT) and VAChTKDHOM mice. RESULTS: Diaphragms of NMJs of the VAChTKDHOM mice were similar to those in WT mice in number, colocalization, and fragmentation of pre-/postsynaptic components. However, they had increased spontaneous SV exocytosis, miniature endplate potential frequency, and diminished MEPP amplitude. No impairment in respiratory mechanics at rest was observed, probably due to the large neurotransmission safety factor of the diaphragm. DISCUSSION: The present findings help us to understand the consequences of reduced ACh release at the NMJs during aging.

Muscle atrophy is associated with cervical spinal motoneuron loss in BACHD mouse model for Huntington's disease.

Involuntary choreiform movements are clinical hallmark of Huntington's disease, an autosomal dominant neurodegenerative disorder caused by an increased number of CAG trinucleotide repeats in the huntingtin gene. Involuntary movements start with an impairment of facial muscles and then affect trunk and limbs muscles. Huntington's disease symptoms are caused by changes in cortex and striatum neurons induced by mutated huntingtin protein. However, little is known about the impact of this abnormal protein in spinal cord motoneurons that control movement. Therefore, in this study we evaluated abnormalities in the motor unit (spinal cervical motoneurons, motor axons, neuromuscular junctions and muscle) in a mouse model for Huntington's disease (BACHD). Using light, fluorescence, confocal, and electron microscopy, we showed significant changes such as muscle fibers atrophy, fragmentation of neuromuscular junctions, axonal alterations, and motoneurons death in BACHD mice. Noteworthy, the surviving motoneurons from BACHD spinal cords were smaller than WT. We suggest that this loss of larger putative motoneurons is accompanied by a decrease in the expression of fast glycolytic muscle fibers in this model for Huntington's disease. These observations show spinal cord motoneurons loss in BACHD that might help to understand neuromuscular changes in Huntington's disease.

Comparative presynaptic effects of the volatile anesthetics sevoflurane and isoflurane at the mouse neuromuscular junction.

INTRODUCTION: Sevoflurane and isoflurane are anesthetics that cause muscle relaxation and potentiate the effects of neuromuscular blocking agents. Their presynaptic mechanisms of action are not understood completely, especially at the motor nerve terminal. METHODS: We compared the presynaptic effects of these anesthetics on the exocytosis of synaptic vesicles labeled with the dye FM1-43 at the mouse neuromuscular junction. RESULTS: Neither anesthetic evoked spontaneous exocytosis of synaptic vesicles, but both significantly inhibited the depolarization evoked by 4-aminopyridine and veratridine, suggesting a putative action on sodium channels. Exocytosis evoked by veratridine was inhibited by tetrodotoxin alone or in conjunction with sevoflurane or isoflurane, indicating that both agents may target voltage-gated sodium channels. CONCLUSIONS: We suggest that sevoflurane and isoflurane inhibit exocytosis evoked by sodium-dependent depolarization and might act on tetrodotoxin-sensitive sodium channels. These findings contribute to a better understanding of some clinical neuromuscular effects induced by these anesthetics.

The effects of volatile anesthetics on the extracellular accumulation of [(3)H]GABA in rat brain cortical slices.

GABA is an inhibitory neurotransmitter that appears to be associated with the action of volatile anesthetics. These anesthetics potentiate GABA-induced postsynaptic currents by synaptic GABAA receptors, although recent evidence suggests that these agents also significantly affect extrasynaptic GABA receptors. However, the effect of volatile anesthetics on the extracellular concentration of GABA in the central nervous system has not been fully established. In the present study, rat brain cortical slices loaded with [(3)H]GABA were used to investigate the effect of halothane and sevoflurane on the extracellular accumulation of this neurotransmitter. The accumulation of [(3)H]GABA was significantly increased by sevoflurane (0.058, 0.11, 0.23, 0.46, and 0.93 mM) and halothane (0.006, 0.012, 0.024, 0.048, 0072, and 0.096 mM) with an EC50 of 0.26 mM and 35 µM, respectively. TTX (blocker of voltage-dependent Na(+) channels), EGTA (an extracellular Ca(2+) chelator) and BAPTA-AM (an intracellular Ca(2+) chelator) did not interfere with the accumulation of [(3)H]GABA induced by 0.23 mM sevoflurane and 0.048 mM halothane. SKF 89976A, a GABA transporter type 1 (GAT-1) inhibitor, reduced the sevoflurane- and halothane-induced increase in the accumulation of GABA by 57 and 63 %, respectively. Incubation of brain cortical slices at low temperature (17 °C), a condition that inhibits GAT function and reduces GABA release through reverse transport, reduced the sevoflurane- and halothane-induced increase in the accumulation of [(3)H]GABA by 82 and 75 %, respectively, relative to that at normal temperature (37 °C). Ouabain, a Na(+)/K(+) ATPase pump inhibitor, which is known to induce GABA release through reverse transport, abolished the sevoflurane and halothane effects on the accumulation of [(3)H]GABA. The effect of sevoflurane and halothane did not involve glial transporters because ß-alanine, a blocker of GAT-2 and GAT-3, did not inhibit the effect of the anesthetics. In conclusion, the present study suggests that sevoflurane and halothane increase the accumulation of GABA by inducing the reverse transport of this neurotransmitter. Therefore, volatile anesthetics could interfere with neuronal excitability by increasing the action of GABA on synaptic and extrasynaptic GABA receptors.

Influence of ß2-adrenergic selective agonist formoterol on the motor unit of a mouse model of a congenital myasthenic syndrome with complete VAChT deletion.

Congenital Myasthenic Syndromes (CMS) are a set of genetic diseases that affect the neuromuscular transmission causing muscular weakness. The standard pharmacological treatment aims at ameliorating the myasthenic symptom by acetylcholinesterase inhibitors. Most patients respond well in the short and medium term, however, over time the beneficial effects rapidly fade, and the efficacy of the treatment diminishes. Increasing evidence shows that ß2-adrenergic agonists can be a suitable choice for the treatment of neuromuscular disorders, including CMS, as they promote beneficial effects in the neuromuscular system. The exact mechanism on which they rely is not completely understood, although patients and animal models respond well to the treatment, especially over extended periods. Here, we report the use of the long-lasting specific ß2-adrenergic agonist formoterol in a myasthenic mouse model (mnVAChT-KD), featuring deletion of VAChT (Vesicular Acetylcholine Transporter) specifically in the α-motoneurons. Our findings demonstrate that formoterol treatment (300 µg/kg/day; sc) for 30 days increased the neuromuscular junction area, induced skeletal muscle hypertrophy and altered fibre type composition in myasthenic mice. Interestingly, ß2-adrenergic agonists have shown efficacy even in the absence of ACh (acetylcholine). Our data provide important evidence supporting the potential of ß2-adrenergic agonists in treating neuromuscular disorders of pre-synaptic origin and characterized by disruptions in nerve-muscle communication, through a direct and beneficial action within the motor unit.

Membrane cholesterol regulates different modes of synaptic vesicle release and retrieval at the frog neuromuscular junction.

We investigated the effects of cholesterol removal on spontaneous and KCl-evoked synaptic vesicle recycling at the frog neuromuscular junction. Cholesterol removal by methyl-ß-cyclodextrin (MßCD) induced an increase in the frequency of miniature end-plate potentials (MEPPs) and spontaneous destaining of synaptic vesicles labeled with the styryl dye FM1-43. Treatment with MßCD also increased the size of MEPPs without causing significant changes in nicotinic receptor clustering. At the ultrastructural level, synaptic vesicles from nerve terminals treated with MßCD were larger than those from control. In addition, treatment with MßCD reduced the fusion of synaptic vesicles that are mobilized during KCl-evoked stimulation, but induced recycling of those vesicles that fuse spontaneously. We therefore suggest that MßCD might favor the release of vesicles that belong to a pool that is different from that involved in the KCl-evoked release. These results reveal fundamental differences in the synaptic vesicle cycle for spontaneous and evoked release, and suggest that deregulation of cholesterol affects synaptic vesicle biogenesis and increases transmitter packing.

Neuromuscular defects after infection with a beta coronavirus in mice.

COVID-19 affects primarily the lung. However, several other systemic alterations, including muscle weakness, fatigue and myalgia have been reported and may contribute to the disease outcome. We hypothesize that changes in the neuromuscular system may contribute to the latter symptoms observed in COVID-19 patients. Here, we showed that C57BL/6J mice inoculated intranasally with the murine betacoronavirus hepatitis coronavirus 3 (MHV-3), a model for studying COVID-19 in BSL-2 conditions that emulates severe COVID-19, developed robust motor alterations in muscle strength and locomotor activity. The latter changes were accompanied by degeneration and loss of motoneurons that were associated with the presence of virus-like particles inside the motoneuron. At the neuromuscular junction level, there were signs of atrophy and fragmentation in synaptic elements of MHV-3-infected mice. Furthermore, there was muscle atrophy and fiber type switch with alteration in myokines levels in muscles of MHV-3-infected mice. Collectively, our results show that acute infection with a betacoronavirus leads to robust motor impairment accompanied by neuromuscular system alteration.

Weight-drop model as a valuable tool to study potential neurobiological processes underlying behavioral and cognitive changes secondary to mild traumatic brain injury.

The pathophysiology of post-traumatic brain injury (TBI) behavioral and cognitive changes is not fully understood, especially in its mild presentation. We designed a weight drop TBI model in mice to investigate the role of neuroinflammation in behavioral and cognitive sequelae following mild TBI. C57BL/6 mice displayed depressive-like behavior at 72 h after mild TBI compared with controls, as indicated by a decrease in the latency to first immobility and climbing time in the forced swim test. Additionally, anxiety-like behavior and hippocampal-associated spatial learning and memory impairment were found in the elevated plus maze and in the Barnes maze, respectively. Levels of a set of inflammatory mediators and neurotrophic factors were analyzed at 6 h, 24 h, 72 h, and 30 days after injury in ipsilateral and contralateral hemispheres of the prefrontal cortex and hippocampus. Principal components analysis revealed two principal components (PC), which represented 59.1% of data variability. PC1 (cytokines and chemokines) expression varied between both hemispheres, while PC2 (neurotrophic factors) expression varied only across the investigated brain areas. Our model reproduces mild TBI-associated clinical signs and pathological features and might be a valuable tool to broaden the knowledge regarding mild TBI pathophysiology as well as to test potential therapeutic targets.

Non-neuronal cholinergic machinery present in cardiomyocytes offsets hypertrophic signals.

Recent work has provided compelling evidence that increased levels of acetylcholine (ACh) can be protective in heart failure, whereas reduced levels of ACh secretion can cause heart malfunction. Previous data show that cardiomyocytes themselves can actively secrete ACh, raising the question of whether this cardiomyocyte derived ACh may contribute to the protective effects of ACh in the heart. To address the functionality of this non-neuronal ACh machinery, we used cholinesterase inhibitors and a siRNA targeted to AChE (acetylcholinesterase) as a way to increase the availability of ACh secreted by cardiac cells. By using nitric oxide (NO) formation as a biological sensor for released ACh, we showed that cholinesterase inhibition increased NO levels in freshly isolated ventricular myocytes and that this effect was prevented by atropine, a muscarinic receptor antagonist, and by inhibition of ACh synthesis or vesicular storage. Functionally, cholinesterase inhibition prevented the hypertrophic effect as well as molecular changes and calcium transient alterations induced by adrenergic overstimulation in cardiomyocytes. Moreover, inhibition of ACh storage or atropine blunted the anti-hypertrophic action of cholinesterase inhibition. Altogether, our results show that cardiomyocytes possess functional cholinergic machinery that offsets deleterious effects of hyperadrenergic stimulation. In addition, we show that adrenergic stimulation upregulates expression levels of cholinergic components. We propose that this cardiomyocyte cholinergic signaling could amplify the protective effects of the parasympathetic nervous system in the heart and may counteract or partially neutralize hypertrophic adrenergic effects.

Inflammasome activation and assembly in Huntington's disease.

Huntington's disease (HD) is a rare neurodegenerative disease characterized by motor, cognitive, and psychiatric symptoms. Inflammasomes are multiprotein complexes capable of sensing pathogen-associated and damage-associated molecular patterns, triggering innate immune pathways. Activation of inflammasomes results in a pro-inflammatory cascade involving, among other molecules, caspases and interleukins. NLRP3 (nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing 3) is the most studied inflammasome complex, and its activation results in caspase-1 mediated cleavage of the pro-interleukins IL-1ß and IL-18 into their mature forms, also inducing a gasdermin D mediated form of pro-inflammatory cell death, i.e. pyroptosis. Accumulating evidence has implicated NLRP3 inflammasome complex in neurodegenerative diseases. The evidence in HD is still scant and mostly derived from pre-clinical studies. This review aims to present the available evidence on NLRP3 inflammasome activation in HD and to discuss whether targeting this innate immune system complex might be a promising therapeutic strategy to alleviate its symptoms.

Effect of intravenous anesthetic propofol on synaptic vesicle exocytosis at the frog neuromuscular junction.

AIM: To investigate the presynaptic effects of propofol, a short-acting intravenous anesthetic, in the frog neuromuscular junction. METHODS: Frog cutaneous pectoris nerve muscle preparations were prepared. A fluorescent tool (FM1-43) was used to visualize the effect of propofol on synaptic vesicle exocytosos in the frog neuromuscular junction. RESULTS: Low concentrations of propofol, ranging from 10 to 25 µmol/L, enhanced spontaneous vesicle exocytosis monitored by FM1-43 in a Ca(2+)-dependent and Na(+)-independent fashion. Higher concentrations of propofol (50, 100, and 200 µmol/L) had no effect on spontaneous exocytosis. By contrast, higher concentrations of propofol inhibited the Na(+)-dependent exocytosis evoked by 4-aminopyridine but did not affect the Na(+)-independent exocytosis evoked by KCl. This action was similar and non-additive with that observed by tetrodotoxin, a Na(+) channel blocker. CONCLUSION: Our data suggest that propofol has a dose-dependent presynaptic effect at the neuromuscular transmission which may help to understand some of the clinical effects of this agent on neuromuscular function.

Motoneuron-specific loss of VAChT mimics neuromuscular defects seen in congenital myasthenic syndrome.

Motoneurons (MNs) control muscle activity by releasing the neurotransmitter acetylcholine (ACh) at the level of neuromuscular junctions. ACh is packaged into synaptic vesicles by the vesicular ACh transporter (VAChT), and disruptions in its release can impair muscle contraction, as seen in congenital myasthenic syndromes (CMS). Recently, VAChT gene mutations were identified in humans displaying varying degrees of myasthenia. Moreover, mice with a global deficiency in VAChT expression display several characteristics of CMS. Despite these findings, little is known about how a long-term decrease in VAChT expression in vivo affects MNs structure and function. Using Cre-loxP technology, we generated a mouse model where VAChT is deleted in select groups of MNs (mnVAChT-KD). Molecular analysis revealed that the VAChT deletion was specific to MNs and affected approximately 50% of its population in the brainstem and spinal cord, with alpha-MNs primarily targeted (70% in spinal cord). Within each animal, the cell body area of VAChT-deleted MNs was significantly smaller compared to MNs with VAChT preserved. Likewise, muscles innervated by VAChT-deleted MNs showed atrophy while muscles innervated by VAChT-containing neurons appeared normal. In addition, mnVAChT KD mice had decreased muscle strength, were hypoactive, leaner and exhibited kyphosis. This neuromuscular dysfunction was evident at 2 months of age and became progressively worse by 6 months. Treatment of mutants with a cholinesterase inhibitor was able to improve some of the motor deficits. As these observations mimic what is seen in CMS, this new line could be valuable for assessing the efficacy of potential CMS drugs.

Increased Cholinergic Tone Causes Pre-synaptic Neuromuscular Degeneration and is Associated with Impaired Diaphragm Function.

In vertebrates, muscle activity is dependent on acetylcholine (ACh) released from neuromuscular junctions (NMJs), and changes in cholinergic neurotransmission are linked to a variety of neuromuscular diseases, including congenital myasthenic syndromes (CMS). The storage and release of ACh depends on the activity of the Vesicular Acetylcholine Transporter (VAChT), a rate-limiting step for cholinergic neurotransmission whose loss of function mutations was shown to cause human congenital myasthenia. However, we know much less about increased VAChT activity, due to copy number variations, for example. Therefore, here we investigated the impact of increased VAChT expression and consequently ACh levels at the synaptic cleft of the diaphragm NMJs. We analyzed structure and function of nerve and muscles from a mouse model of cholinergic hyperfunction (ChAT-ChR2-EYFP) with increased expression of VAChT. Our results showed a significant increase of ACh released under evoked stimuli. However, we observed deleterious changes in synaptic vesicles cycle (impaired endocytosis and decrease in vesicles number), together with structural alterations of NMJs. Interestingly, ultrastructure analyses showed that synaptic vesicles from ChAT-ChR2-EYFP mice NMJs were larger, which might be related to increased ACh load. We also observed that these larger synaptic vesicles were less rounded in comparison with control. Finally, we showed that ChAT-ChR2-EYFP mice NMJs have compromised safety factor, possible due to the structural alterations we described. These findings reveal that physiological cholinergic activity is important to maintain the structure and function of the neuromuscular system and help to understand some of the neuromuscular adverse effects experienced by chronically increased NMJ neurotransmission, such as individuals treated with cholinesterase inhibitors.

Protective effect of a spider recombinant toxin in a murine model of Huntington's disease.

Abnormal calcium influx and glutamatergic excitotoxicity have been extensively associated with neuronal death in Huntington's disease (HD), a genetic movement disorder. Currently, there is no effective treatment for this fatal condition. The neurotoxin Phα1ß has demonstrated therapeutic effects as a calcium channel blocker, for example during pain control. However, little is known about its neuroprotective effect in HD. Herein, we investigated if Phα1ß is effective in inhibiting neuronal cell death in the BACHD mouse model for HD. We performed intrastriatal injection of Phα1ß in WT and BACHD mice. No side effects or unusual behaviors were observed upon Phα1ß administration. Using three different motor behavior tests, we observed that injection of the toxin in BACHD mice greatly improved the animals' motor-force as seen in the Wire-hang test, and also the locomotor performance, according to the Open field test. NeuN labeling for mature neuron detection revealed that Phα1ß toxin promoted neuronal preservation in the striatum and cortex, when injected locally. Intrastriatal injection of Phα1ß was not able to preserve neurons from the spinal cord and also not revert muscle atrophy in BACHD mice. Finally, we observed that Phα1ß might, at least in part, exert its protective effect by decreasing L-glutamate, measured in cerebrospinal fluid. Our data provide evidence of a novel neuroprotector effect of Phα1ß, paving a path for the development of new approaches to treat HD motor symptoms.