An acute and highly reproducible motor axon terminal degeneration followed by complete regeneration is induced by some animal presynaptic neurotoxins, representing an appropriate and controlled system to dissect the molecular mechanisms underlying degeneration and regeneration of peripheral nerve terminals. We have previously shown that nerve terminals exposed to spider or snake presynaptic neurotoxins degenerate as a result of calcium overload and mitochondrial failure. Here we show that toxin-treated primary neurons release signaling molecules derived from mitochondria: hydrogen peroxide, mitochondrial DNA, and cytochrome c. These molecules activate isolated primary Schwann cells, Schwann cells cocultured with neurons and at neuromuscular junction in vivo through the MAPK pathway. We propose that this inter-and intracellular signaling is involved in triggering the regeneration of peripheral nerve terminals affected by other forms of neurodegenerative diseases.motor axon degeneration | presynaptic neurotoxins | mitochondrial alarmins | Schwann cells

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.

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.

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.

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.