INTRODUCTION
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that targets both upper and lower motor neurons within the brain and spinal cord. Tragically, ALS is characterized by a brief survival span of 2-3 years, with only 10-20% of patients surpassing a decade of life post-diagnosis. Familial cases, attributable to genetic mutations, account for 5-10% of all ALS cases, with the rest classified as sporadic . Despite intense ongoing research, current medications such as Riluzole, Edaravone, Relyvrio, and Qalsody provide only a modest delay in disease progression. The pathogenesis of ALS is a complex interplay of multiple processes occurring within motor neurons (MNs), encompassing amongst other proposed mechanism: excitotoxicity, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, oxidative stress, RNA processing aberrations, protein misfolding, endosomal trafficking disruptions, impaired axonal transport or neuroinflammation . Computational models have underscored the significance of disturbances in calcium (Ca2+) homeostasis and energy imbalances as pivotal factors predicting neuronal dysfunction and degeneration in ALS .
One distinguishing feature in early ALS-affected MNs is the excessive influx of Ca2+ ions due to high-frequency rhythmic activity and an increased expression of Ca2+-permeable AMPA receptors . These MNs exhibit low expression of Ca2+ buffering proteins like parvalbumin and calbindin . Overexpression of these proteins has been shown to confer resistance to ALS-induced toxicity in MNs . Conversely, MNs that exhibit resistance to ALS degeneration, such as those of oculomotor and Onuf’s nuclei, possess higher levels of Ca2+ buffering proteins . Notably, ALS-associated mutations, including those in VAPB ,SIGMAR1 and SOD1 can potentiate Ca2+deregulation and heighten vulnerability to the effects of Ca2+ influx . These findings emphasize the inherent susceptibility of MNs to intracellular Ca2+ overload as a significant risk factor for degeneration .
Mitochondria play a pivotal role in regulating intracellular Ca2+ levels through the mitochondrial uniporter, particularly crucial in MNs with low intrinsic cytosolic Ca2+ buffering capacity. In these vulnerable MNs, mitochondria are responsible for absorbing over 50% of intracellular Ca2+ increases, setting them apart from other cell types. Consequently, the pronounced demand placed on specialized Ca2+ storage organelles makes MNs even more susceptible to Ca2+ imbalances , especially in ALS where mitochondria are frequently damaged . Indeed, impaired mitochondrial Ca2+ buffering in MNs derived from patient-induced pluripotent stem cells carrying mutations inTARDBP or C9orf72 has been reported .
Apart from excessive Ca2+ influx and diminished Ca2+ buffering capacity, augmented Ca2+ leakage from ER stores in MNs can further contribute to cytosolic Ca2+ overload. Ryanodine receptors (RyR) are tetrameric channels that play a crucial role in mediating Ca2+-induced Ca2+ release from the ER, play a crucial role in this process . RyR channels are activated by Ca2+ and can also be triggered by the oxidation of redox-sensing thiols by reactive oxygen species (ROS), creating a positive feedback loop exacerbating pathological cytoplasmic Ca2+ overload . The endogenous ligand of RyR, FKBP12 (calstabin), normally stabilizes RyR, preventing Ca2+leakage from the ER. Reduced FKBP12 levels in MNs of ALS patients underscore the importance of maintaining equilibrium between FKBP12 and RyR in neurodegeneration .
Overall, this cumulative evidence suggests the prospect of RyRs as a potential therapeutic targets for ameliorating intracellular Ca2+ dysregulation and forestalling Ca2+-induced neurodegeneration within susceptible neurons and neuromuscular units in ALS. In this context, we introduce a novel class of triazole molecules, empirically validated as FKBP12 ligands to facilitate FKBP12-RyR binding. This binding capability serves to stabilize the Ca2+ flux mediated by RyR, as demonstrated in our previous works . In this study, we introduce MP compounds, novel FKBP12 ligands that exhibit cytosolic calcium modulating activity, with the potential to be used for the treatment of ALS. This proposal is based on the encouraging results observed in transgenic SOD1G93A mice, a mouse model that recapitulates the key clinical, electrophysiological, and histopathological features of the disease. .