DISCUSSION AND CONCLUSIONS
Our investigation centered on modulating RyR activity, specifically through a novel class of triazole molecules known as MP compounds that act as FKBP12 ligands . The primary focus of our study was on MP-010, a RyR stabilizer with favorable pharmacokinetics and CNS permeability. Our results in SOD1G93A mice, a widely accepted ALS model, demonstrated significant benefits following MP-010 administration. Notably, increased survival, protection against MN loss, muscle denervation, and enhanced neuromuscular function were observed, highlighting the potential of MP-010 as a therapeutic intervention for ALS. The robustness of our findings is emphasized by their replication in two distinct colonies of SOD1G93A mice with a mixed hybrid B6SJL genetic background, across two independent laboratories. This replication is particularly noteworthy considering the potential genetic drift that hybrid ALS SOD1 colonies may undergo among different research labs, leading to variability in disease onset or symptom severity . This potential confounding factor makes the replication of our findings in two independent laboratories even more significant. Remarkably, despite differing ages of motor symptom onset between the two colonies (14 weeks in one laboratory and 17 weeks in the other), MP-010 demonstrated beneficial effects in both settings at the dose of 61 mg/kg. The compound exhibited remarkable tolerability and appeared devoid of apparent toxicity even at high doses. Doubling the dose did not induce a change in body weight, a recognized clinical symptom of pharmacological toxicity. Nevertheless, the compound’s therapeutic efficacy was compromised at this higher dose. This unexpected loss of efficacy could be attributed to off-target effects. These unintended interactions with non-target proteins could be interfering with the compound’s ability to reach its intended target and exert its therapeutic effects. Further investigations are warranted to identify and characterize specific off-target mechanisms, enabling the development of strategies to mitigate them and optimize the compound’s efficacy.
As previously introduced, cytosolic Ca2+ overload assumes particular importance in the vulnerability of spinal MNs subjected to ALS toxicity , and deficient Ca2+handling from ER and mitochondrial stores has emerged as a key player in the pathogenesis of ALS. This sets the stage for a positive feedback loop that amplifies pathological Ca2+ overload, intensifying the neurodegenerative processes associated with the disease . Mutations in genes such as SIGMA1R and VAPB , identified in ALS patients, underscore the significance of ER and mitochondrial proteins in Ca2+ homeostasis . In the context of the present study, the rationale for targeting RyR stemmed from its central role in Ca2+-induced Ca2+ release from the ER, aiming to prevent excessive cytosolic Ca2+ accumulation, especially in the most vulnerable MNs associated with ALS. The outcomes of the current study, demonstrating the therapeutic benefits of a drug promoting the interaction between RyR and its endogenous ligand FKBP12 in the SOD1G93A mouse model of ALS, are in accordance with earlier research that has emphasized reductions in FKBP12 in the context of ALS . Also in the SOD1G93A mouse, various alternative strategies to reduce the Ca2+ burden on MNs have been successfully implemented. These strategies include the application of the AMPA receptor antagonist talampanel and SIGMA1R agonists , both exerting their effects through the modulation of extracellular Ca2+ influx to cytosol.
The dysregulation of RyR signaling emerges as a fundamental contributor to the pathogenesis of various neurodegenerative disorders, notably Alzheimer’s disease (AD). Specifically, RyRs have been demonstrated to interact with amyloid-beta (Aβ) peptide, a protein known to play a pivotal role in AD development. The binding of Aβ to RyRs can modulate their function, resulting in heightened Ca2+ release from the ER, subsequently leading to excitotoxicity, neuronal death, and cognitive deficits . Although our investigation cannot definitively ascertain the existence of Ca2+ leakage through RyR in ALS MNs or whether this pathological phenomenon is replicated in the SOD1G93A mouse model, the administration of the compound MP-010 emphasizes the potential therapeutic significance of targeting FKBP12 to safeguard the RyR-dependent proper release of Ca2+ and alleviate the downstream effects of disrupted Ca2+ homeostasis. This approach offers a targeted intervention for ALS. However, while it is important to note that an aberrant increase in RyR-mediated Ca2+ release can contribute to disrupting neuronal homeostasis, triggering excitotoxicity, conversely, diminished RyR function may compromise the crucial Ca2+ signaling required for proper neuronal operation. This impairment might lead to a breakdown in synaptic transmission and plasticity, potentially contributing to the manifestation of motor deficits in individuals with ALS. Consequently, inhibiting RyR under this premise may not be a plausible strategy for treating neurons susceptible to ALS. Indeed, prior research utilizing dantrolene, an RyR1 inhibitor clinically used to treat malignant hyperthermia, failed to demonstrate significant improvements when administered to the SOD1G93A mouse model , confirming the inadequacy of RyR inhibition as a therapeutic avenue. In contrast, compounds exhibiting RyR-stabilizing activity, previously recognized under the nomenclature ”rycals” , adopt a distinct approach by modulating RyR channel opening. These compounds achieve this modulation by facilitating the interaction between the topoisomerase FKBP12 and RyR, particularly in pathological scenarios characterized by persistent Ca2+ leakage . Thus, the strategic targeting of RyR modulation through rycal compounds offers a more nuanced and potentially effective therapeutic strategy compared to direct inhibition.
Comparative analysis with MP-001, another compound characterized by limited CNS availability, provides additional insights. The lack of MP-001 effect underscore the significance of targeting Ca2+ homeostasis at the CNS level to achieve therapeutic efficacy. Thus, it dismisses the possibility that the therapeutic effects could be mediated by the modulation of Ca2+ dynamics in cells of the innate immune system. It is crucial to consider this aspect, especially given that immune cells heavily rely on Ca2+-dependent processes and tightly regulated Ca2+ homeostasis , and that the innate immune response appears to play an active role in the pathophysiology of ALS and the SOD1G93A mouse model .
While our study presents promising results, several avenues for future research emerge. Elucidating the molecular mechanisms underlying the observed effects of MP-010, particularly its impact on Ca2+ dynamics and neuronal survival, is critical for a comprehensive understanding of ALS pathology. Additionally, considerations of toxicity, dosage optimization, and potential side effects are paramount before translating these findings into clinical trials. In conclusion, our study contributes to the evolving landscape of ALS research by highlighting the therapeutic potential of targeting intracellular Ca2+ dynamics through the modulation of RyR activity. MP-010, with its favorable preclinical outcomes, offers a promising avenue for further exploration and potential clinical development.