The neuromuscular junction (NMJ) is the site of transmission of the electrical impulses from the motor axon terminal to the muscle; the anatomical organization of this highly dynamic system also includes the perisynaptic Schwann cells (PSCs), and therefore the NMJ has to be considered structurally and functionally as a tripartite system. These non-myelinating SCs are intimately associated with the nerve muscle contact and act as dynamic partners at the synapse: they are involved in many physiological functions including the embryonic development and the maintenance of adult NMJs. Moreover, they are able to detect and reciprocally modulate synaptic activity, through the activation of muscarinin and purinergic receptors present on their surface.
In addition, non-traditional roles for PSCs in the recovery after nerve injury are being recognized. Following denervation or reduced synaptic activity, PSCs de-differentiate to an earlier developmental stage, becoming “reactive” PSCs, and start proliferating. These reactive PSCs actively participate in the process of nerve degeneration and regeneration: they undergo changes in their gene expression and acquire macrophagic-like activities, thus contributing to the removal of nerve debris as well as to the recruitment of macrophages, by releasing cytokines and chemokines. Moreover, following nerve terminals degeneration, PSCs at denervated end-plates extend long processes that induce and guide nerve regrowth.
Given the increasing incidence of non cell-autonomous and dying-back axonopathies - such as amyotrophic lateral sclerosis (ALS) and autoimmune neuropathies - which affect predominantly motor axons terminals, it becomes very important to characterize the crosstalk between degenerating nerve terminals and adjacent PSCs at the NMJ; in particular, the identification of molecular mediators involved in PSCs activation and in nerve terminals regeneration would be crucial for the improvement of therapeutic strategies.
This is the general aim of the present thesis and with this purpose in mind, we have adopted an innovative experimental approach, alternative to the traditional cut/crush surgical model employed till now. To confine the nerve damage to the sole motor axon terminal, thus avoiding the involvement of many cell types and inflammatory mediators, we exploited our knowledge on the mechanism of action of two classes of animal presynaptic neurotoxins: α-Ltx, a pore forming toxin of the venom of black widow spiders, and some snake neurotoxins endowed with phospholipase A2 activity called SPANs. Both kinds of neurotoxins induce an acute and highly reproducible motor axon terminal degeneration, which is followed in few days by complete regeneration: thus, this model represents an appropriate and controlled system to dissect the molecular mechanisms underlying de- and re-generation of peripheral nerve terminals, and to define how PSCs contribute to such processes.
We have previously shown that nerve terminals exposed to spider or snake neurotoxins degenerate owing to calcium overload and mitochondrial failure. Here, we found that toxin-treated cultured neurons increase their mitochondrial production of hydrogen peroxide (H2O2), which can easily diffuse across membranes, thus acting as a paracrine signal on neighbouring cellS. Indeed, exposure of cultured SCs to H2O2 leads to ERK phosphorylation and to the activation of downstream pathways. The ERK signalling pathway plays a central role in controlling SCs plasticity during nerve repair in-vivo, but so far the molecular mediators responsible for its activation were unknown: neurons-derived H2O2 represents an ideal candidate for this role.
In support of this hypothesis, we observed that ERK phosphorylation is reduced in intoxicated neurons-SCs co-cultures pre-incubated with catalase - which converts H2O2 to oxygen and water -, indicating that H2O2 produced inside neurons diffuses to reach nearby SCs, contributing to ERK activation in their cytosol. ERK phosphorylation takes place also in PSCs at intoxicated NMJs in-vivo. To confirm the involvement of H2O2 in promoting nerve regeneration, we performed electrophysiological recordings and immunohistochemistry on intoxicated muscles, and we found that co-injection of catalase together with neurotoxins delays nerve regeneration, confirming the prominent role of H2O2 in promoting NMJ recovery. Injection of the MAP kinase inhibitor PD98059 also impairs nerve repair in a way similar to that observed with catalase, supporting the finding that H2O2 enhances nerve terminals regeneration through the activation of ERK pathway in PSC
