Local Tetanus Begins with a Vesicle-Associated Membrane Protein Cleavage-Associated Neuromuscular Junction Paralysis around the Site of Tetanus Neurotoxin Release.

Local tetanus develops when limited amounts of tetanus neurotoxin (TeNT) are released by Clostridium tetani generated from spores inside a necrotic wound. Within days, a spastic paralysis restricted to the muscles of the affected anatomical area develops. This paralysis follows the retrograde transport of TeNT inside the axons of spinal cord motoneurons and its uptake by inhibitory interneurons with cleavage of a vesicle-associated membrane protein required for neurotransmitter release. Consequently, incontrollable excitation of motoneurons causes contractures of innervated muscles and leads to local spastic paralysis. Here, the initial events occurring close to the site of TeNT release were investigated in a mouse model of local tetanus. A peripheral flaccid paralysis was found to occur, before or overlapping, the spastic paralysis. At variance from the confined TeNT proteolytic activity at the periphery, central vesicle-associated membrane protein cleavage can be detected within inhibitory interneurons controlling motor neuron efferents innervating muscle groups distant from the site of TeNT release. These results indicate that TeNT does have peripheral activity in tetanus and explains why the spastic paralysis observed in local tetanus, although confined to single limbs, generally affects multiple muscles. The initial TeNT neuroparalytic activity can be detected by measuring the compound muscle action potential, providing a very early diagnosis and therapy, and thus preventing the ensuing life-threatening generalized tetanus.

Agonists of melatonin receptors strongly promote the functional recovery from the neuroparalysis induced by neurotoxic snakes.

Snake envenoming is a major, but neglected, tropical disease. Among venomous snakes, those inducing neurotoxicity such as kraits (Bungarus genus) cause a potentially lethal peripheral neuroparalysis with respiratory deficit in a large number of people each year. In order to prevent the development of a deadly respiratory paralysis, hospitalization with pulmonary ventilation and use of antivenoms are the primary therapies currently employed. However, hospitals are frequently out of reach for envenomated patients and there is a general consensus that additional, non-expensive treatments, deliverable even long after the snake bite, are needed. Traumatic or toxic degenerations of peripheral motor neurons cause a neuroparalysis that activates a pro-regenerative intercellular signaling program taking place at the neuromuscular junction (NMJ). We recently reported that the intercellular signaling axis melatonin-melatonin receptor 1 (MT1) plays a major role in the recovery of function of the NMJs after degeneration of motor axon terminals caused by massive Ca2+ influx. Here we show that the small chemical MT1 agonists: Ramelteon and Agomelatine, already licensed for the treatment of insomnia and depression, respectively, are strong promoters of the neuroregeneration after paralysis induced by krait venoms in mice, which is also Ca2+ mediated. The venom from a Bungarus species representative of the large class of neurotoxic snakes (including taipans, coral snakes, some Alpine vipers in addition to other kraits) was chosen. The functional recovery of the NMJ was demonstrated using electrophysiological, imaging and lung ventilation detection methods. According to the present results, we propose that Ramelteon and Agomelatine should be tested in human patients bitten by neurotoxic snakes acting presynaptically to promote their recovery of health. Noticeably, these drugs are commercially available, safe, non-expensive, have a long bench life and can be administered long after a snakebite even in places far away from health facilities.

Facial neuromuscular junctions and brainstem nuclei are the target of tetanus neurotoxin in cephalic tetanus.

Cephalic tetanus (CT) is a severe form of tetanus that follows head wounds and the intoxication of cranial nerves by tetanus neurotoxin (TeNT). Hallmarks of CT are cerebral palsy, which anticipates the spastic paralysis of tetanus, and rapid evolution of cardiorespiratory deficit even without generalized tetanus. How TeNT causes this unexpected flaccid paralysis, and how the canonical spasticity then rapidly evolves into cardiorespiratory defects, remain unresolved aspects of CT pathophysiology. Using electrophysiology and immunohistochemistry, we demonstrate that TeNT cleaves its substrate vesicle-associated membrane protein within facial neuromuscular junctions and causes a botulism-like paralysis overshadowing tetanus spasticity. Meanwhile, TeNT spreads among brainstem neuronal nuclei and, as shown by an assay measuring the ventilation ability of CT mice, harms essential functions like respiration. A partial axotomy of the facial nerve revealed a potentially new ability of TeNT to undergo intra-brainstem diffusion, which allows the toxin to spread to brainstem nuclei devoid of direct peripheral efferents. This mechanism is likely to be involved in the transition from local to generalized tetanus. Overall, the present findings suggest that patients with idiopathic facial nerve palsy should be immediately considered for CT and treated with antisera to block the potential progression to a life-threatening form of tetanus.

ß3AR-Dependent Brain-Derived Neurotrophic Factor (BDNF) Generation Limits Chronic Postischemic Heart Failure.

BACKGROUND: Loss of brain-derived neurotrophic factor (BDNF)/TrkB (tropomyosin kinase receptor B) signaling accounts for brain and cardiac disorders. In neurons, ß-adrenergic receptor stimulation enhances local BDNF expression. It is unclear if this occurs in a pathophysiological relevant manner in the heart, especially in the ß-adrenergic receptor-desensitized postischemic myocardium. Nor is it fully understood whether and how TrkB agonists counter chronic postischemic left ventricle (LV) decompensation, a significant unmet clinical milestone. METHODS: We conducted in vitro studies using neonatal rat and adult murine cardiomyocytes, SH-SY5Y neuronal cells, and umbilical vein endothelial cells. We assessed myocardial ischemia (MI) impact in wild type, ß3AR knockout, or myocyte-selective BDNF knockout (myoBDNF KO) mice in vivo (via coronary ligation [MI]) or in isolated hearts with global ischemia-reperfusion (I/R). RESULTS: In wild type hearts, BDNF levels rose early after MI (<24 hours), plummeting at 4 weeks when LV dysfunction, adrenergic denervation, and impaired angiogenesis ensued. The TrkB agonist, LM22A-4, countered all these adverse effects. Compared with wild type, isolated myoBDNF KO hearts displayed worse infarct size/LV dysfunction after I/R injury and modest benefits from LM22A-4. In vitro, LM22A-4 promoted neurite outgrowth and neovascularization, boosting myocyte function, effects reproduced by 7,8-dihydroxyflavone, a chemically unrelated TrkB agonist. Superfusing myocytes with the ß3AR-agonist, BRL-37344, increased myocyte BDNF content, while ß3AR signaling underscored BDNF generation/protection in post-MI hearts. Accordingly, the ß1AR blocker, metoprolol, via upregulated ß3ARs, improved chronic post-MI LV dysfunction, enriching the myocardium with BDNF. Last, BRL-37344-imparted benefits were nearly abolished in isolated I/R injured myoBDNF KO hearts. CONCLUSIONS: BDNF loss underscores chronic postischemic heart failure. TrkB agonists can improve ischemic LV dysfunction via replenished myocardial BDNF content. Direct cardiac ß3AR stimulation, or ß-blockers (via upregulated ß3AR), is another BDNF-based means to fend off chronic postischemic heart failure.

Defective excitation-contraction coupling and mitochondrial respiration precede mitochondrial Ca2+ accumulation in spinobulbar muscular atrophy skeletal muscle.

Polyglutamine expansion in the androgen receptor (AR) causes spinobulbar muscular atrophy (SBMA). Skeletal muscle is a primary site of toxicity; however, the current understanding of the early pathological processes that occur and how they unfold during disease progression remains limited. Using transgenic and knock-in mice and patient-derived muscle biopsies, we show that SBMA mice in the presymptomatic stage develop a respiratory defect matching defective expression of genes involved in excitation-contraction coupling (ECC), altered contraction dynamics, and increased fatigue. These processes are followed by stimulus-dependent accumulation of calcium into mitochondria and structural disorganization of the muscle triads. Deregulation of expression of ECC genes is concomitant with sexual maturity and androgen raise in the serum. Consistent with the androgen-dependent nature of these alterations, surgical castration and AR silencing alleviate the early and late pathological processes. These observations show that ECC deregulation and defective mitochondrial respiration are early but reversible events followed by altered muscle force, calcium dyshomeostasis, and dismantling of triad structure.

DJ-1 promotes energy balance by regulating both mitochondrial and autophagic homeostasis.

The protein DJ-1 is mutated in rare familial forms of recessive Parkinson's disease and in parkinsonism accompanied by amyotrophic lateral sclerosis symptoms and dementia. DJ-1 is considered a multitasking protein able to confer protection under various conditions of stress. However, the precise cellular function still remains elusive. In the present work, we evaluated fruit flies lacking the expression of the DJ-1 homolog dj-1ß as compared to control aged-matched individuals. Behavioral evaluations included lifespan, locomotion in an open field arena, sensitivity to oxidative insults, and resistance to starvation. Molecular analyses were carried out by analyzing the mitochondrial morphology and functionality, and the autophagic response. We demonstrated that dj-1ß null mutant flies are hypoactive and display higher sensitivity to oxidative insults and food deprivation. Analysis of mitochondrial homeostasis revealed that loss of dj-1ß leads to larger and more circular mitochondria, characterized by impaired complex-I-linked respiration while preserving ATP production capacity. Additionally, dj-1ß null mutant flies present an impaired autophagic response, which is suppressed by treatment with the antioxidant molecule N-Acetyl-L-Cysteine. Overall, our data point to a mechanism whereby DJ-1 plays a critical role in the maintenance of energy homeostasis, by sustaining mitochondrial homeostasis and affecting the autophagic flux through the maintenance of the cellular redox state. In light of the involvement of DJ-1 in neurodegenerative diseases and considering that neurons are highly energy-demanding cells, particularly sensitive to redox stress, our study sheds light on a key role of DJ-1 in the maintenance of cellular homeostasis.

Towards a unified vision on animal navigation.

The study of animal navigation is a complex and fertile field of research: Several questions regarding how animals relate to external stimuli, integrating them to perform their everyday movement routine, have been or are being addressed in different organisms and taxa, both from the behavioural and the neuronal activity point of view. Several invertebrate model organisms are the object of studies aimed at unravelling how they navigate and their ability to precisely return to a starting point and also how navigational information is communicated to conspecifics when precise social structures are present. Also, vertebrates are studied because of the interest in their orientation abilities while migrating, homing over impressive distances and studying exploration, orientation and space recognition. Last, research on the navigation capabilities of humans pursues a better understanding of the neural architecture involved in these processes in the remarkable effort to find answers and possible solutions to impairments, lesions and diseases. However, an 'all-inclusive' vision of navigation still appears to be in its embryonic state: A better perspective could (and should) shift from a paradigm where single research teams are centred on studying navigation in a single genus or species towards a more comprehensive evolutionary-centred view, searching systematically for behavioural analogies, and possibly for homologies in neural architecture between different taxa. In this review, we introduce examples of relevant topics in animal navigation from distinct animal groups, highlighting the similar approaches of those studies, and why, in our opinion, this research field could profit from a 'new' perspective.

Hydrogen peroxide induced by nerve injury promotes axon regeneration via connective tissue growth factor.

Regeneration of the neuromuscular junction (NMJ) leverages on extensive exchange of factors released from motor axon terminals (MATs), muscle fibers and perisynaptic Schwann cells (PSCs), among which hydrogen peroxide (H2O2) is a major pro-regenerative signal. To identify critical determinants of NMJ remodeling in response to injury, we performed temporal transcriptional profiling of NMJs from 2 month-old mice during MAT degeneration/regeneration, and cross-referenced the differentially expressed genes with those elicited by H2O2 in SCs. We identified an enrichment in extracellular matrix (ECM) transcripts, including Connective Tissue Growth Factor (Ctgf), which is usually expressed during development. We discovered that Ctgf levels are increased in a Yes-associated protein (YAP)-dependent fashion in response to rapid, local H2O2 signaling generated by stressed mitochondria in the injured sciatic nerve, a finding highlighting the importance of signals triggered by mechanical force to motor nerve repair. Through sequestration of Ctgf or inactivation of H2O2, we delayed the recovery of neuromuscular function by impairing SC migration and, in turn, axon-oriented re-growth. These data indicate that H2O2 and its downstream effector Ctgf are pro-regenerative factors that enable axonal growth, and reveal a striking ECM remodeling process during nerve regeneration upon local H2O2 signaling. Our study identifies key transcriptomic changes at the regenerating NMJ, providing a rich source of pro-regenerative factors with potential for alleviating the consequences of peripheral nerve injuries.

Neurotransmission Recovery by Melatonin Measured by CMAP.

Compound muscle action potential (CMAP) recordings provide a sensitive electromyographic approach to measure nerve conduction and assess neuromuscular junction functionality in humans and rodents. In humans, it represents a diagnostic tool for neuromuscular disorders. In rodents, this approach is widely employed to dissect the molecular mechanisms driving peripheral nerve degeneration/regeneration, as well as to evaluate the effect of candidate pro-regenerative compounds. The method described here allows recording CMAP from the gastrocnemius muscle of mice after sciatic nerve stimulation. We report some representative traces of CMAP recorded from adult, healthy mice, after sciatic nerve compression and during neurotransmission recovery stimulated by melatonin administration.

Nerve-dependent distribution of subsynaptic type 1 inositol 1,4,5-trisphosphate receptor at the neuromuscular junction.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are enriched at postsynaptic membrane compartments of the neuromuscular junction (NMJ), surrounding the subsynaptic nuclei and close to nicotinic acetylcholine receptors (nAChRs) of the motor endplate. At the endplate level, it has been proposed that nerve-dependent electrical activity might trigger IP3-associated, local Ca2+ signals not only involved in excitation-transcription (ET) coupling but also crucial to the development and stabilization of the NMJ itself. The present study was undertaken to examine whether denervation affects the subsynaptic IP3R distribution in skeletal muscles and which are the underlying mechanisms. Fluorescence microscopy, carried out on in vivo denervated muscles (following sciatectomy) and in vitro denervated skeletal muscle fibers from flexor digitorum brevis (FDB), indicates that denervation causes a reduction in the subsynaptic IP3R1-stained region, and such a decrease appears to be determined by the lack of muscle electrical activity, as judged by partial reversal upon field electrical stimulation of in vitro denervated skeletal muscle fibers.

Recovery from the Neuroparalysis Caused by the Micrurus nigrocinctus Venom Is Accelerated by an Agonist of the CXCR4 Receptor.

Snake envenoming is a major but neglected human disease in tropical and subtropical regions. Among venomous snakes in the Americas, coral snakes of the genus Micrurus are particularly dangerous because they cause a peripheral neuroparalysis that can persist for many days or, in severe cases, progress to death. Ventilatory support and the use of snake species-specific antivenoms may prevent death from respiratory paralysis in most cases. However, there is a general consensus that additional and non-expensive treatments that can be delivered even long after the snake bite are needed. Neurotoxic degeneration of peripheral motor neurons activates pro-regenerative intercellular signaling programs, the greatest of which consist of the chemokine CXCL12α, produced by perisynaptic Schwann cells, which act on the CXCR4 receptor expressed on damaged neuronal axons. We recently found that the CXCR4 agonist NUCC-390 promotes axonal growth. Here, we show that the venom of the highly neurotoxic snake Micrurus nigrocinctus causes a complete degeneration of motor axon terminals of the soleus muscle, followed by functional regeneration whose time course is greatly accelerated by NUCC-390. These results suggest that NUCC-390 is a potential candidate for treating human patients envenomed by Micrurus nigrocinctus as well as other neurotoxic Micrurus spp. in order to improve the recovery of normal neuromuscular physiology, thus reducing the mortality and hospital costs of envenoming.

Lifespan and ROS levels in different Drosophila melanogaster strains after 24†h hypoxia exposure.

During recent decades, model organisms such as Drosophila melanogaster have made it possible to study the effects of different environmental oxygen conditions on lifespan and oxidative stress. However, many studies have often yielded controversial results usually assigned to variations in Drosophila genetic background and differences in study design. In this study, we compared longevity and ROS levels in young, unmated males of three laboratory wild-type lines (Canton-S, Oregon-R and Berlin-K) and one mutant line (Sod1n1) as a positive control of redox imbalance, under both normoxic and hypoxic (2% oxygen for 24 h) conditions. Lifespan was used to detect the effects of hypoxic treatment and differences were analysed by means of Kaplan-Meier survival curves and log-rank tests. Electron paramagnetic resonance spectroscopy was used to measure ROS levels and analysis of variance was used to estimate the effects of hypoxic treatment and to assess ROS differences between strains. We observed that the genetic background is a relevant factor involved in D. melanogaster longevity and ROS levels. Indeed, as expected, in normoxia Sod1n1 are the shortest-lived, while the wild-type strains, despite a longer lifespan, show some differences, with the Canton-S line displaying the lowest mortality rate. After hypoxic stress these variances are amplified, with Berlin-K flies showing the highest mortality rate and most evident reduction of lifespan. Moreover, our analysis highlighted differential effects of hypoxia on redox balance/unbalance. Canton-S flies had the lowest increase of ROS level compared to all the other strains, confirming it to be the less sensitive to hypoxic stress. Sod1n1 flies displayed the highest ROS levels in normoxia and after hypoxia. These results should be used to further standardize future Drosophila research models designed to investigate genes and pathways that may be involved in lifespan and/or ROS, as well as comparative studies on specific mutant strains.

Dopamine Modulation of Drosophila Ellipsoid Body Neurons, a Nod to the Mammalian Basal Ganglia.

The central complex (CX) is a neural structure located on the midline of the insect brain that has been widely studied in the last few years. Its role in navigation and goal-oriented behaviors resembles those played by the basal ganglia in mammals. However, the neural mechanisms and the neurotransmitters involved in these processes remain unclear. Here, we exploited an in vivo bioluminescence Ca2+ imaging technique to record the activity in targeted neurons of the ellipsoid body (EB). We used different drugs to evoke excitatory Ca2+-responses, depending on the putative neurotransmitter released by their presynaptic inputs, while concomitant dopamine administration was employed to modulate those excitations. By using a genetic approach to knockdown the dopamine 1-like receptors, we showed that different dopamine modulatory effects are likely due to specific receptors expressed by the targeted population of neurons. Altogether, these results provide new data concerning how dopamine modulates and shapes the response of the ellipsoid body neurons. Moreover, they provide important insights regarding the similitude with mammals as far as the role played by dopamine in increasing and stabilizing the response of goal-related information.

Latrotoxin-Induced Neuromuscular Junction Degeneration Reveals Urocortin 2 as a Critical Contributor to Motor Axon Terminal Regeneration.

We used α-Latrotoxin (α-LTx), the main neurotoxic component of the black widow spider venom, which causes degeneration of the neuromuscular junction (NMJ) followed by a rapid and complete regeneration, as a molecular tool to identify by RNA transcriptomics factors contributing to the structural and functional recovery of the NMJ. We found that Urocortin 2 (UCN2), a neuropeptide involved in the stress response, is rapidly expressed at the NMJ after acute damage and that inhibition of CRHR2, the specific receptor of UCN2, delays neuromuscular transmission rescue. Experiments in neuronal cultures show that CRHR2 localises at the axonal tips of growing spinal motor neurons and that its expression inversely correlates with synaptic maturation. Moreover, exogenous UCN2 enhances the growth of axonal sprouts in cultured neurons in a CRHR2-dependent manner, pointing to a role of the UCN2-CRHR2 axis in the regulation of axonal growth and synaptogenesis. Consistently, exogenous administration of UCN2 strongly accelerates the regrowth of motor axon terminals degenerated by α-LTx, thereby contributing to the functional recovery of neuromuscular transmission after damage. Taken together, our results posit a novel role for UCN2 and CRHR2 as a signalling axis involved in NMJ regeneration.

A heuristic underlies the search for relief in Drosophila melanogaster.

Humans rely on multiple types of sensory information to make decisions, and strategies that shorten decision-making time by taking into account fewer but essential elements of information are preferred to strategies that require complex analyses. Such shortcuts to decision making are known as heuristics. The identification of heuristic principles in species phylogenetically distant to humans would shed light on the evolutionary origin of speed-accuracy trade-offs and offer the possibility for investigating the brain representations of such trade-offs, urgency and uncertainty. By performing experiments on spatial learning in the invertebrate Drosophila melanogaster, we show that the fly's search strategies conform to a spatial heuristic-the nearest neighbor rule-to avoid bitter taste (a negative stimulation). That is, Drosophila visits a salient location closest to its current position to stop the negative stimulation; only if this strategy proves unsuccessful does the fly use other learned associations to avoid bitter taste. Characterizing a heuristic in D. melanogaster supports the view that invertebrates can, when making choices, operate on economic principles, as well as the conclusion that heuristic decision making dates to at least 600 million years ago.

Perturbed BMP signaling and denervation promote muscle wasting in cancer cachexia.

Most patients with advanced solid cancers exhibit features of cachexia, a debilitating syndrome characterized by progressive loss of skeletal muscle mass and strength. Because the underlying mechanisms of this multifactorial syndrome are incompletely defined, effective therapeutics have yet to be developed. Here, we show that diminished bone morphogenetic protein (BMP) signaling is observed early in the onset of skeletal muscle wasting associated with cancer cachexia in mouse models and in patients with cancer. Cancer-mediated factors including Activin A and IL-6 trigger the expression of the BMP inhibitor Noggin in muscle, which blocks the actions of BMPs on muscle fibers and motor nerves, subsequently causing disruption of the neuromuscular junction (NMJ), denervation, and muscle wasting. Increasing BMP signaling in the muscles of tumor-bearing mice by gene delivery or pharmacological means can prevent muscle wasting and preserve measures of NMJ function. The data identify perturbed BMP signaling and denervation of muscle fibers as important pathogenic mechanisms of muscle wasting associated with tumor growth. Collectively, these findings present interventions that promote BMP-mediated signaling as an attractive strategy to counteract the loss of functional musculature in patients with cancer.

Whole brain functional recordings at cellular resolution in zebrafish larvae with 3D scanning multiphoton microscopy.

Optical recordings of neuronal activity at cellular resolution represent an invaluable tool to investigate brain mechanisms. Zebrafish larvae is one of the few model organisms where, using fluorescence-based reporters of the cell activity, it is possible to optically reconstruct the neuronal dynamics across the whole brain. Typically, leveraging the reduced light scattering, methods like lightsheet, structured illumination, and light-field microscopy use spatially extended excitation profiles to detect in parallel activity signals from multiple cells. Here, we present an alternative design for whole brain imaging based on sequential 3D point-scanning excitation. Our approach relies on a multiphoton microscope integrating an electrically tunable lens. We first apply our approach, adopting the GCaMP6s activity reporter, to detect functional responses from retinal ganglion cells (RGC) arborization fields at different depths within the zebrafish larva midbrain. Then, in larvae expressing a nuclear localized GCaMP6s, we recorded whole brain activity with cellular resolution. Adopting a semi-automatic cell segmentation, this allowed reconstructing the activity from up to 52,000 individual neurons across the brain. In conclusion, this design can easily retrofit existing imaging systems and represents a compact, versatile and reliable tool to investigate neuronal activity across the larva brain at high resolution.

Immuno-electrophysiology on Neuromuscular Junctions of Drosophila Third Instar Larva.

Alterations in synaptic transmission are critical early events in neuromuscular disorders. However, reliable methodologies to analyze the functional organization of the neuromuscular synapses are still needed. This manuscript provides a detailed protocol to analyze the molecular assembly of the neuromuscular synapses through immune-electrophysiology in Drosophila melanogaster. This technique allows the quantification of the molecular behavior of the neuromuscular synapses by correlating the structural configuration of the synaptic boutons with their electrical activity.

Tetanus and tetanus neurotoxin: From peripheral uptake to central nervous tissue targets.

Tetanus is a deadly but preventable disease caused by a protein neurotoxin produced by Clostridium tetani. Spores of C. tetani may contaminate a necrotic wound and germinate into a vegetative bacterium that releases a toxin, termed tetanus neurotoxin (TeNT). TeNT enters the general circulation, binds to peripheral motor neurons and sensory neurons, and is transported retroaxonally to the spinal cord. It then enters inhibitory interneurons and blocks the release of glycine or GABA causing a spastic paralysis. This review attempts to correlate the metalloprotease activity of TeNT and its trafficking and localization into the vertebrate body to the nature and sequence of appearance of the symptoms of tetanus.