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.

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.

Sulfation at Glycopolymer Side Chains Switches Activity at the Macrophage Mannose Receptor (CD206) In Vitro and In Vivo.

The mannose receptor (CD206) is an endocytic receptor expressed by selected innate immune cells and nonvascular endothelium, which plays a critical role in both homeostasis and pathogen recognition. Although its involvement in the development of several diseases and viral infections is well established, molecular tools able to both provide insight on the chemistry of CD206-ligand interactions and, importantly, effectively modulate its activity are currently lacking. Using novel SO4-3-Gal-glycopolymers targeting its cysteine-rich lectin ectodomain, this study uncovers and elucidates a previously unknown mechanism of CD206 blockade involving the formation of stable intracellular SO4-3-Gal-glycopolymer-CD206 complexes that prevents receptor recycling to the cell membrane. Further, we show that SO4-3-Gal glycopolymers inhibit CD206 both in vitro and in vivo, revealing hitherto unknown receptor function and demonstrating their potential as CD206 modulators within future immunotherapies.

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.

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.

Models and methods to study Schwann cells.

Schwann cells (SCs) are fundamental components of the peripheral nervous system (PNS) of all vertebrates and play essential roles in development, maintenance, function, and regeneration of peripheral nerves. There are distinct populations of SCs including: (1) myelinating SCs that ensheath axons by a specialized plasma membrane, called myelin, which enhances the conduction of electric impulses; (2) non-myelinating SCs, including Remak SCs, which wrap bundles of multiple axons of small caliber, and perysinaptic SCs (PSCs), associated with motor axon terminals at the neuromuscular junction (NMJ). All types of SCs contribute to PNS regeneration through striking morphological and functional changes in response to nerve injury, are affected in peripheral neuropathies and show abnormalities and a diminished plasticity during aging. Therefore, methodological approaches to study and manipulate SCs in physiological and pathophysiological conditions are crucial to expand the present knowledge on SC biology and to devise new therapeutic strategies to counteract neurodegenerative conditions and age-derived denervation. We present here an updated overview of traditional and emerging methodologies for the study of SCs for scientists approaching this research field.

Polymer Coated Oncolytic Adenovirus to Selectively Target Hepatocellular Carcinoma Cells.

Despite significant advances in chemotherapy, the overall prognosis of hepatocellular carcinoma (HCC) remains extremely poor. HCC targeting strategies were combined with the tumor cell cytotoxicity of oncolytic viruses (OVs) to develop a more efficient and selective therapeutic system. OVs were coated with a polygalactosyl-b-agmatyl diblock copolymer (Gal32-b-Agm29), with high affinity for the asialoglycoprotein receptor (ASGPR) expressed on the liver cell surface, exploiting the electrostatic interaction of the positively charged agmatine block with the negatively charged adenoviral capsid surface. The polymer coating altered the viral particle diameter (from 192 to 287 nm) and zeta-potential (from -24.7 to 23.3 mV) while hiding the peculiar icosahedral symmetrical OV structure, as observed by TEM. Coated OVs showed high potential therapeutic value on the human hepatoma cell line HepG2 (cytotoxicity of 72.4% ± 4.96), expressing a high level of ASGPRs, while a lower effect was attained with ASPGR-negative A549 cell line (cytotoxicity of 54.4% ± 1.59). Conversely, naked OVs showed very similar effects in both tested cell lines. Gal32-b-Agm29 OV coating enhanced the infectivity and immunogenic cell death program in HepG2 cells as compared to the naked OV. This strategy provides a rationale for future studies utilizing oncolytic viruses complexed with polymers toward effective treatment of hepatocellular carcinoma.

Melatonin promotes regeneration of injured motor axons via MT1 receptors.

Melatonin is an ancient multi-tasking molecule produced by the pineal gland and by several extrapineal tissues. A variety of activities has been ascribed to this hormone in different physiological and pathological contexts, but little is known about its role in peripheral neuroregeneration. Here, we have exploited two different types of injury to test the capability of melatonin to stimulate regeneration of motor axons: (a) the acute and reversible presynaptic degeneration induced by the spider neurotoxin α-Latrotoxin and (b) the compression/transection of the sciatic nerve. We found that in both cases melatonin administration accelerates the process of nerve repair. This pro-regenerative action is MT1 -mediated, and at least in part due to a sustained activation of the ERK1/2 pathway. These findings reveal a receptor-mediated, pro-regenerative action of melatonin in vivo that holds important clinical implications, as it posits melatonin as a safe candidate molecule for the treatment of a number of peripheral neurodegenerative conditions.

An agonist of the CXCR4 receptor accelerates the recovery from the peripheral neuroparalysis induced by Taipan snake envenomation.

Envenomation by snakes is a major neglected human disease. Hospitalization and use of animal-derived antivenom are the primary therapeutic supports currently available. There is consensus that additional, not expensive, treatments that can be delivered even long after the snake bite are needed. We recently showed that the drug dubbed NUCC-390 shortens the time of recovery from the neuroparalysis caused by traumatic or toxic degeneration of peripheral motor neurons. These syndromes are characterized by the activation of a pro-regenerative molecular axis, consisting of the CXCR4 receptor expressed at the damaged site in neuronal axons and by the release of its ligand CXCL12α, produced by surrounding Schwann cells. This intercellular signaling axis promotes axonal growth and functional recovery from paralysis. NUCC-390 is an agonist of CXCR4 acting similarly to CXCL12α. Here, we have tested its efficacy in a murine model of neuroparalytic envenoming by a Papuan Taipan (Oxyuranus scutellatus) where a degeneration of the motor axon terminals caused by the presynaptic PLA2 toxin Taipoxin, contained in the venom, occurs. Using imaging of the neuromuscular junction and electrophysiological analysis, we found that NUCC-390 administration after injection of either the purified neuroparalytic Taipoxin or the whole Taipan venom, significantly accelerates the recovery from paralysis. These results indicate that NUCC-390, which is non-toxic in mice, should be considered for trials in humans to test its efficacy in accelerating the recovery from the peripheral neuroparalysis induced by Taipans. NUCC-390 should be tested as well in the envenomation by other snakes that cause neuroparalytic syndromes in humans. NUCC-390 could become an additional treatment, common to many snake envenomings, that can be delivered after the bite to reduce death by respiratory deficits and to shorten and improve functional recovery.

Signals Orchestrating Peripheral Nerve Repair.

The peripheral nervous system has retained through evolution the capacity to repair and regenerate after assault from a variety of physical, chemical, or biological pathogens. Regeneration relies on the intrinsic abilities of peripheral neurons and on a permissive environment, and it is driven by an intense interplay among neurons, the glia, muscles, the basal lamina, and the immune system. Indeed, extrinsic signals from the milieu of the injury site superimpose on genetic and epigenetic mechanisms to modulate cell intrinsic programs. Here, we will review the main intrinsic and extrinsic mechanisms allowing severed peripheral axons to re-grow, and discuss some alarm mediators and pro-regenerative molecules and pathways involved in the process, highlighting the role of Schwann cells as central hubs coordinating multiple signals. A particular focus will be provided on regeneration at the neuromuscular junction, an ideal model system whose manipulation can contribute to the identification of crucial mediators of nerve re-growth. A brief overview on regeneration at sensory terminals is also included.

A CXCR4 receptor agonist strongly stimulates axonal regeneration after damage.

OBJECTIVE: To test whether the signaling axis CXCL12α-CXCR4 is activated upon crush/cut of the sciatic nerve and to test the activity of NUCC-390, a new CXCR4 agonist, in promoting nerve recovery from damage. METHODS: The sciatic nerve was either crushed or cut. Expression and localization of CXCL12α and CXCR4 were evaluated by imaging with specific antibodies. Their functional involvement in nerve regeneration was determined by antibody-neutralization of CXCL12α, and by the CXCR4 specific antagonist AMD3100, using as quantitative read-out the compound muscle action potential (CMAP). NUCC-390 activity on nerve regeneration was determined by imaging and CMAP recordings. RESULTS: CXCR4 is expressed at the injury site within the axonal compartment, whilst its ligand CXCL12α is expressed in Schwann cells. The CXCL12α-CXCR4 axis is involved in the recovery of neurotransmission of the injured nerve. More importantly, the small molecule NUCC-390 is a strong promoter of the functional and anatomical recovery of the nerve, by acting very similarly to CXCL12α. This pharmacological action is due to the capability of NUCC-390 to foster elongation of motor neuron axons both in vitro and in vivo. INTERPRETATION: Imaging and electrophysiological data provide novel and compelling evidence that the CXCL12α-CXCR4 axis is involved in sciatic nerve repair after crush/cut. This makes NUCC-390 a strong candidate molecule to stimulate nerve repair by promoting axonal elongation. We propose this molecule to be tested in other models of neuronal damage, to lay the basis for clinical trials on the efficacy of NUCC-390 in peripheral nerve repair in humans.

An Agonist of the CXCR4 Receptor Strongly Promotes Regeneration of Degenerated Motor Axon Terminals.

The activation of the G-protein coupled receptor CXCR4 by its ligand CXCL12α is involved in a large variety of physiological and pathological processes, including the growth of B cells precursors and of motor axons, autoimmune diseases, stem cell migration, inflammation, and several neurodegenerative conditions. Recently, we demonstrated that CXCL12α potently stimulates the functional recovery of damaged neuromuscular junctions via interaction with CXCR4. This result prompted us to test the neuroregeneration activity of small molecules acting as CXCR4 agonists, endowed with better pharmacokinetics with respect to the natural ligand. We focused on NUCC-390, recently shown to activate CXCR4 in a cellular system. We designed a novel and convenient chemical synthesis of NUCC-390, which is reported here. NUCC-390 was tested for its capability to induce the regeneration of motor axon terminals completely degenerated by the presynaptic neurotoxin α-Latrotoxin. NUCC-390 was found to strongly promote the functional recovery of the neuromuscular junction, as assayed by electrophysiology and imaging. This action is CXCR4 dependent, as it is completely prevented by AMD3100, a well-characterized CXCR4 antagonist. These data make NUCC-390 a strong candidate to be tested in human therapy to promote nerve recovery of function after different forms of neurodegeneration.

Hydrogen peroxide is a neuronal alarmin that triggers specific RNAs, local translation of Annexin A2, and cytoskeletal remodeling in Schwann cells.

Schwann cells are key players in neuro-regeneration: They sense "alarm" signals released by degenerating nerve terminals and differentiate toward a proregenerative phenotype, with phagocytosis of nerve debris and nerve guidance. At the murine neuromuscular junction, hydrogen peroxide (H2O2) is a key signal of Schwann cells' activation in response to a variety of nerve injuries. Here we report that Schwann cells exposed to low doses of H2O2 rewire the expression of several RNAs at both transcriptional and translational levels. Among the genes positively regulated at both levels, we identified an enriched cluster involved in cytoskeleton remodeling and cell migration, with the Annexin (Anxa) proteins being the most represented family. We show that both Annexin A2 (Anxa2) transcript and protein accumulate at the tips of long pseudopods that Schwann cells extend upon H2O2 exposure. Interestingly, Schwann cells reply to this signal and to nerve injury by locally translating Anxa2 in pseudopods, and undergo an extensive cytoskeleton remodeling. Our results show that, similarly to neurons, Schwann cells take advantage of local protein synthesis to change shape and move toward damaged axonal terminals to facilitate axonal regeneration.

Mouse Phrenic Nerve Hemidiaphragm Assay (MPN).

The neuromuscular junction (NMJ) is the specialized synapse by which peripheral motor neurons innervate muscle fibers and control skeletal muscle contraction. The NMJ is the target of several xenobiotics, including chemicals, plant, animal and bacterial toxins, as well as of autoantibodies raised against NMJ antigens. Depending on their biochemical nature, the site they target (either the nerve or the muscle) and their mechanism of action, substances affecting NMJ produce very specific alterations of neuromuscular functionality. Here we provide a detailed protocol to isolate the diaphragmatic muscle from mice and to set up two autonomously innervated hemidiaphragms. This preparation can be used to study bioactive substances like toxins, venoms and neuroactive molecules of various origin, or to measure the force of skeletal muscle contraction. The 'mouse phrenic nerve hemidiaphragm assay' (MPN) is an established model of ex vivo NMJ and recapitulates the complexity of neuromuscular transmission in a system easy to control and to manipulate, thus representing a valuable tool to study both NMJ physiology and the mechanism of action of toxins and other molecules acting at this synapse.

Electrophysiological Recordings of Evoked End-Plate Potential on Murine Neuro-muscular Synapse Preparations.

Neuromuscular junction (NMJ) is the specialized chemical synapse that mediates the transmission of the electrical impulse running along motor neuron axons to skeletal muscle fibers. NMJ is the best characterized chemical synapse and its study along many years of research has provided most of the general knowledge of synapse development, structure and functionality. Electrophysiology is the most accurate experimental procedure to study NMJ physiology and it largely contributed to the elucidation of synaptic transmission basic principles. Many electrophysiological techniques have been developed to study NMJ physiology and physiopathology. In this paper, we describe an ex vivo tissue preparation for electrophysiology that can be applied to investigate nerve-muscle transmission functionality in mice. It is routinely used in our laboratory to study presynaptic neurotoxins, antitoxins, and to monitor NMJ degeneration and regeneration. This is a broadly applicable technique which can also be adopted to investigate alterations of NMJ activity in mouse models of neuromuscular diseases, including peripheral neuropathies, motor neuron disorders and myasthenic syndromes.

Botulinum neurotoxin C mutants reveal different effects of syntaxin or SNAP-25 proteolysis on neuromuscular transmission.

Botulinum neurotoxin serotype C (BoNT/C) is a neuroparalytic toxin associated with outbreaks of animal botulism, particularly in birds, and is the only BoNT known to cleave two different SNARE proteins, SNAP-25 and syntaxin. BoNT/C was shown to be a good substitute for BoNT/A1 in human dystonia therapy because of its long lasting effects and absence of neuromuscular damage. Two triple mutants of BoNT/C, namely BoNT/C S51T/R52N/N53P (BoNT/C α-51) and BoNT/C L200W/M221W/I226W (BoNT/C α-3W), were recently reported to selectively cleave syntaxin and have been used here to evaluate the individual contribution of SNAP-25 and syntaxin cleavage to the effect of BoNT/C in vivo. Although BoNT/C α-51 and BoNT/C α-3W toxins cleave syntaxin with similar efficiency, we unexpectedly found also cleavage of SNAP-25, although to a lesser extent than wild type BoNT/C. Interestingly, the BoNT/C mutants exhibit reduced lethality compared to wild type toxin, a result that correlated with their residual activity against SNAP-25. In spite of this, a local injection of BoNT/C α-51 persistently impairs neuromuscular junction activity. This is due to an initial phase in which SNAP-25 cleavage causes a complete blockade of neurotransmission, and to a second phase of incomplete impairment ascribable to syntaxin cleavage. Together, these results indicate that neuroparalysis of BoNT/C at the neuromuscular junction is due to SNAP-25 cleavage, while the proteolysis of syntaxin provides a substantial, but incomplete, neuromuscular impairment. In light of this evidence, we discuss a possible clinical use of BoNT/C α-51 as a botulinum neurotoxin endowed with a wide safety margin and a long lasting effect.

CXCL12α/SDF-1 from perisynaptic Schwann cells promotes regeneration of injured motor axon terminals.

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter-cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12α, also abbreviated as stromal-derived factor-1 (SDF-1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by α-latrotoxin. CXCL12α acts via binding to the neuronal CXCR4 receptor. A CXCL12α-neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration in vivo Recombinant CXCL12α in vivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons in vitro These findings indicate that the CXCL12α-CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage.

Schwann cells are activated by ATP released from neurons in an in vitro cellular model of Miller Fisher syndrome.

The neuromuscular junction is exposed to different types of insult, including mechanical trauma, toxins and autoimmune antibodies and, accordingly, has retained through evolution a remarkable ability to regenerate. Regeneration is driven by multiple signals that are exchanged among the cellular components of the junction. These signals are largely unknown. Miller Fisher syndrome is a variant of Guillain-Barré syndrome caused by autoimmune antibodies specific for epitopes of peripheral axon terminals. Using an animal model of Miller Fisher syndrome, we recently reported that a monoclonal anti-polysialoganglioside GQ1b antibody plus complement damages nerve terminals with production of mitochondrial hydrogen peroxide, which activates Schwann cells. Several additional signaling molecules are likely to be involved in the activation of the regeneration program in these cells. Using an in vitro cellular model consisting of co-cultured primary neurons and Schwann cells, we found that ATP is released by neurons injured by the anti-GQ1b antibody plus complement. Neuron-derived ATP acts as an alarm messenger for Schwann cells, where it induces the activation of intracellular pathways, including calcium signaling, cAMP and CREB, which, in turn, produce signals that promote nerve regeneration. These results contribute to defining the cross-talk taking place at the neuromuscular junction when it is attacked by anti-gangliosides autoantibodies plus complement, which is crucial for nerve regeneration and is also likely to be important in other peripheral neuropathies.

An animal model of Miller Fisher syndrome: Mitochondrial hydrogen peroxide is produced by the autoimmune attack of nerve terminals and activates Schwann cells.

The neuromuscular junction is a tripartite synapse composed of the presynaptic nerve terminal, the muscle and perisynaptic Schwann cells. Its functionality is essential for the execution of body movements and is compromised in a number of disorders, including Miller Fisher syndrome, a variant of Guillain-Barré syndrome: this autoimmune peripheral neuropathy is triggered by autoantibodies specific for the polysialogangliosides GQ1b and GT1a present in motor axon terminals, including those innervating ocular muscles, and in sensory neurons. Their binding to the presynaptic membrane activates the complement cascade, leading to a nerve degeneration that resembles that caused by some animal presynaptic neurotoxins. Here we have studied the intra- and inter-cellular signaling triggered by the binding and complement activation of a mouse monoclonal anti-GQ1b/GT1a antibody to primary cultures of spinal cord motor neurons and cerebellar granular neurons. We found that a membrane attack complex is rapidly assembled following antibody binding, leading to calcium accumulation, which affects mitochondrial functionality. Consequently, using fluorescent probes specific for mitochondrial hydrogen peroxide, we found that this reactive oxygen species is rapidly produced by mitochondria of damaged neurons, and that it triggers the activation of the MAP kinase pathway in Schwann cells. These results throw light on the molecular and cellular pathogenesis of Miller Fisher syndrome, and may well be relevant to other pathologies of the motor axon terminals, including some subtypes of the Guillain Barré syndrome.