Ropinirole hydrochloride mitigates oxidative stress and neuroinflammation via the PI3K–mTOR pathway in TDP-43 hiPSC-derived microglial-like cells

Abstract Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron loss accompanied by neuroinflammation, oxidative stress, and impaired proteostasis, with pathological aggregation of TDP-43 as its defining hallmark. Microglia are recognized as central contributors to ALS pathogenesis, yet the mechanisms underlying their intrinsic dysfunction in the context of TDP-43 remain insufficiently characterized. Methods We characterized microglia-like cells (iMGLs) differentiated from isogenic TDP-43 M337V human induced pluripotent stem cell lines. We performed integrated functional assays, transcriptomic profiling and drug repurposing analysis to systematically compare mutant and control iMGLs. To assess therapeutic potential, we evaluated the effects of ROPI, a dopamine D2 receptor agonist previously advanced to clinical trials for ALS, on disease-relevant phenotypes in TDP-43 iMGLs. Results TDP-43 M337V/M337V iMGLs presented ALS-associated abnormalities, including cytoplasmic TDP-43 accumulation with impaired phagocytosis and elevated oxidative stress, impaired autophagy and mitophagy, altered cytokine profiles, and reduced ferritin levels. Building on our previous identification of ropinirole hydrochloride as neuroprotective for ALS motor neurons, we now show that it confers therapeutic benefits in mitigating microglial pathology. ROPI treatment significantly reduced oxidative stress and caspase-3/7 activity and partially restored cytokine homeostasis in TDP-43 M337V/M337V iMGLs, independent of autophagy/mitophagy modulation. Transcriptomic profiling revealed that ropinirole modulated disease-associated gene expression signatures involving protein folding, extracellular matrix organization, and oxidative stress responses. Furthermore, connectivity map–based analysis prioritized PI3K-Akt-mTOR inhibitors as candidates for reversing TDP-43 iMGL signatures, and ropinirole was found to modulate mTOR signaling. These converging lines of evidence support a mechanistic role for ropinirole in restoring microglial homeostasis via PI3K-mTOR pathway regulation. Taken together, our findings position ropinirole as a promising candidate dual action therapeutic candidate capable of targeting both neuronal and microglial dysfunction in ALS, suggesting broader applicability of ropinirole in modulating neuroinflammatory cascades across other neurodegenerative conditions. Conclusion Ropinirole broadens ALS therapy from motor neurons to microglia, underscoring its promise for integrated and clinically meaningful treatment strategies.