The Role of Proton-Mediated Signaling at the Neuromuscular Junction in ALS Models - PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is marked by progressive loss of motor neurons in the central and peripheral nervous system, leading to a devastating loss of motor function and eventually death. There is no cure. Degeneration of the motor nerve terminal proceeds depending on the muscle of interest, some being resistant and some being susceptible to denervation. Previous work suggests that dysregulation of neuromuscular communication may be an early and important component of ALS. The mechanism behind this phenomenon is not understood and is the focus of this proposal. Extracellular proton (H+) concentration, measured as pH, is characteristic of ALS and has been shown to increase at several tissues in ALS models, suggesting pH dysregulation in ALS pathology. Extracellular protons, being potent neuromodulators, can influence the movement of ions on both the pre- and postsynaptic sides of the synapse at the neuromuscular junction (NMJ). In ALS models, experimentally reducing acidosis and inhibiting matrix metalloproteases was found to improve survival in ALS mouse models, whereas reducing pH buffering capacity was detrimental to survival. We have described synaptic cleft pH transients associated with activity at the healthy mouse NMJ. Our data suggest the activity-dependent pH transients at muscle and neuron are segregated in the cleft due to the basal lamina, a specialized extracellular matrix, dividing the NMJ. These cleft pH transients remain unknown in muscles susceptible to ALS. To address this gap in knowledge, we will investigate synaptic cleft pH in two transgenic mouse lines that have been used to model familial and sporadic ALS, SOD1-G93A and TDP-43-Q331K, respectively. Our central hypothesis is that dysregulation of proton concentrations at NMJs in susceptible muscles contributes to the progression of ALS in model mice. This hypothesis will be tested by measuring basal- and activity-dependent pH transients in the NMJ cleft of ALS mutant and healthy mice using the genetically-encoded pH-sensitive fluorescent probe pHusion-Ex. We will measure these changes in resistant and susceptible muscle groups: the soleus (a resistant muscle) and the extensor digitorum longus (EDL, susceptible to ALS). Since we suggest presynaptic and postsynaptic pH transients are segregated, both presynaptic and postsynaptic transients at the NMJ will be measured separately. Electrophysiology recordings in muscle will be used to correlate neurotransmission with pH changes and disease progression. Immunohistochemistry will be used to identify changes in basal lamina composition and structure. To assess the effects of prolonged pH changes, pH will be stabilized at the mouse soleus or EDL, induced via acidic and alkaline saline delivered by a surgically embedded osmotic pump. The effect of induced acidification or alkalization will be monitored through immunohistochemistry, measuring NMJ atrophy and denervation. These experiments will provide insight into the role of NMJ pH changes in ALS to help us understand why some motor neurons and muscle groups remain resistant to disease progression.
