The neuromuscular junction (NMJ) is a ‘tripartite’ synapse, composed of the presynaptic motor axon terminal (MAT), the muscle fiber and perisynaptic Schwann cells (PSCs). NMJ functionality is essential for the execution of body movements and it is anatomically exposed, becoming an easy target of bacterial and animal neurotoxins, toxic chemicals, mechanical trauma, and autoimmune diseases.
In the Miller Fisher Syndrome (MFS) autoantibodies against specific gangliosides (>90% GQ1b) bind to MAT and in turn activate the complement system cascade at its surface, leading to nerve degeneration. Such damage is reversible, as the motor neuron is able to fully regenerate and restore neurotransmission.
PSCs are main supporters of NMJ regeneration: to date, however, the current understanding of PSCs role in this autoimmune neuropathy is mostly phenomenological, and molecular studies are needed. It was recently reported that the degenerating MAT release alarm signals (alarmins) able to activate the pro-regenerating phenotype of PSCs. Therefore, MAT can be considered an active player of its own regeneration. In addition, many other signals are thought to be generated by all the three main components of the NMJ, thus generating a complex inter-cellular communication network, which has been only partially identified.
In order to better elucidate the molecular and cellular events driving PSCs response to MAT damage in MFS, we recently developed a novel in vivo MFS model. The combination of FS3, a monoclonal antibody against gangliosides related to MFS, and normal human serum (NHS) as a source of complement, administered subcutaneously in LAL muscles and intramuscularly in soleus muscle in mice, causes the degeneration of MAT. Soon after MAT destruction, neuronal debris are engulfed and digested by PSCs. Within few days after injection MAT regrowth is morphologically and functionally complete, as assessed by immunofluorescence analysis and electrophysiological recordings. The effect is antibody- and complement-dependent, as no MAT degeneration takes place in the absence of FS3, nor when NHS is heat-inactivated.
To identify the neuronal alarmins responsible for PSCs activation and the signaling pathways engaged, we have parallely set up an in vitro MFS model consisting of administration of FS3 plus NHS to primary cerebellar neurons and spinal cord motor neurons, which causes a complement-dependent massive Ca2+ overload in neurite, together with the formation of neurite enlargements, named bulges. Bulges are sites of accumulation of swollen and dysfunctional mitochondria, and of localized hydrogen peroxide (H2O2) production. Hydrogen peroxide is an alarm signal for SCs. Indeed, in FS3 plus NHS attacked neurons-SCs co-cultures, neuron-derived H2O2 induces activation of the ERK1/2 pathway in SCs, a known crucial player of the switch toward a pro-regenerative phenotype of SCs.
In addition, we identified adenosine triphosphate (ATP) as an additional alarmin involved in SCs activation. Indeed, primary neurons exposed to FS3 plus NHS release ATP in the extracellular medium, which in turn evokes intracellular calcium spikes within SCs in co-cultures with neurons. These spikes were significantly abolished in the presence of the ATP-inactivating enzyme apyrase in the incubation medium.
Furthermore, experiments with a FRET-based cyclic AMP (cAMP) sensor show that, upon FS3 plus NHS addition in neurons-SCs co-cultures, cAMP levels rise in SCs, and this event eventually results in an ATP-dependent increased phosphorylation the transcription factor CREB (cAMP response element-binding protein).
In conclusion, the work performed during this PhD project has led to the development of novel in vitro and in vivo models of MFS, in order to study the molecular communication between MAT and PSCs. Hydrogen peroxide and ATP were found to be important neuronal alarmins, able to activate pro-regenerative pathways within SCs.
We believe these results throw light on the molecular and cellular events taking place in MFS, and may well be extended to other MAT affecting pathologies.
