DRUG REPURPOSING OF RAPAMYCIN MITIGATES AMYLOID BETA ASSOCIATED PROTEOTOXICITY IN CAENORHABDITIS ELEGANS

Abstract

Alzheimer's disease (AD) is the leading cause of dementia worldwide, affecting an estimated 55 million people and projected to exceed 130 million by 2050. Defined neuropathologically by the extracellular accumulation of amyloid-beta (Aβ) peptides and intracellular neurofibrillary tangles of hyperphosphorylated tau, AD currently lacks disease-modifying therapies capable of halting or reversing neurodegeneration. The proteotoxic accumulation of Aβ is a central pathogenic event, and strategies that enhance endogenous clearance mechanisms represent a compelling therapeutic area.Rapamycin (sirolimus), a macrolide allosteric inhibitor of the mechanistic target of rapamycin complex 1 (mTORC1), is one of the most robustly validated pharmacological extenders of lifespan across model organisms and has demonstrated neuroprotective potential in mammalian AD models. Its capacity to provide mTORC1-mediated suppression of autophagy and to reduce neuroinflammation positions it as an attractive drug-repurposing candidate for AD. However, the extent to which rapamycin can mitigate Aβ proteotoxicity in Caenorhabditis elegans remains unexplored, and leveraging this model organism offers a powerful opportunity to study the mechanism by which rapamycin-mediated mTOR inhibition interacts with amyloid-β aggregation, clearance pathways, and proteostasis in the context of Alzheimer's-related neurodegeneration. In this study, we used two well-established transgenic Caenorhabditis elegans models of Aβ proteotoxicity: the muscle-specific temperature-inducible strain CL4176 (Pmyo-3::Aβ₁₋₄₂) and the pan-neuronal strain CL2355 (Psnb-1::Aβ₁₋₄₂). Following an empirical dose-finding strategy across four sequential titration experiments, 20 μM rapamycin was selected as the working concentration. Rapamycin treatment (20 μM) significantly delayed Aβ-induced paralysis in CL4176 animals, extending the time that 50% of the population was paralyzed (PT50) by approximately 1.8 hours (~23% relative to vehicle; log-rank p = 0.0017). Automated locomotion analysis further demonstrated that rapamycin improved peristaltic speed, center-point speed, and wavelength while markedly reducing mobility idle time (p < 0.05 to p < 0.0001). Swimming parameters as wave initiation rate, swimming speed, dynamic amplitude, and wave attenuation were similarly improved, collectively indicating a broad restoration of neuromuscular function. In the pan-neuronal CL2355 strain, rapamycin significantly rescued the Aβ-induced chemotaxis deficit, restoring the chemotaxis index toward control strain levels (one-way ANOVA, p = 0.015) without altering behavior in the non-transgenic CL2122 control. Kaplan–Meier lifespan analysis revealed that rapamycin significantly extended survival in CL2355 animals (log-rank, Bonferroni-adjusted p < 0.0001, with the most pronounced effect observed in the Aβ-expressing strain. Combined survival–locomotion analysis further demonstrated that rapamycin preserved neuromuscular healthspan, delaying the onset of age-related motor decline relative to vehicle-treated Aβ animals. At the molecular level, Western blot analysis using anti-Aβ immunoblotting revealed that rapamycin selectively reduced the accumulation of higher-molecular-weight Aβ oligomeric species (25 kDa and 60 kDa; p < 0.05 each), while monomeric and lower oligomeric Aβ levels were not significantly altered. Taken together, these findings demonstrate that rapamycin confers consistent, broad-spectrum neuroprotection across orthogonal measures of Aβ proteotoxicity in C. elegans, extending from acute paralysis and locomotion to chemosensory behavior, longevity, and healthspan. The selectivity of rescue in Aβ-expressing animals supports a mechanism engaged with proteotoxic pathology. These results establish C. elegans as a tractable platform for evaluating mTOR-directed therapies and strengthen the rationale for further preclinical investigation of rapamycin as a disease-modifying strategy in Alzheimer's disease.