Characterization of mitochondrial dysfunction induced by BAX trigger site activator 1 in the ARPE-19 retinal pigment epithelial cell model

Mitochondrial dysfunction in the retinal pigment epithelium (RPE) is a key pathological feature of age-related macular degeneration (AMD). However, mechanistically defined experimental models that recapitulate stress-mediated mitochondrial injury remain limited. Bcl-2-associated X (Bax), a key pro-apoptotic effector, serves as a critical upstream regulator of mitochondrial outer membrane permeabilization. In this study, we systematically characterized mitochondrial dysfunction induced by Bax trigger site activator 1 (BTSA1), a selective small-molecule Bax Activator, in ARPE-19 cells. Treatment with BTSA1 (3-60 μM) for 24 and 48 h induced a concentration- and time-dependent reduction in cell viability, accompanied by Caspase-3 activation. Mitochondrial membrane potential, assessed via tetramethylrhodamine ethyl ester staining, was markedly reduced in a BAX-dependent manner and associated with increased Reactive Oxygen Species production following prolonged exposure or at high concentrations. BTSA1 profoundly altered mitochondrial dynamics by promoting DRP1-mediated fission while suppressing fusion through MFN2 downregulation and stress-associated OPA1 processing, resulting in pronounced mitochondrial fragmentation. Furthermore, Bax activation elicited a biphasic response in mitochondrial quality control pathways: mild stress induced impaired autophagic flux and compensatory mitochondrial biogenesis, whereas severe stress triggered Mitophagy accompanied by failure of biogenic compensation. These coordinated alterations closely mirror mitochondrial pathologies observed in the degenerating RPE in AMD. Collectively, our findings demonstrate that Bax activation by BTSA1 is sufficient to induce a comprehensive cascade of mitochondrial dysfunction. This system represents a mechanistically defined experimental model for dissecting BAX-mediated mitochondrial pathology and evaluating therapeutic strategies to preserve mitochondrial integrity in AMD.

Age-related macular degeneration (AMD); BAX activation; Mitochondrial dynamics; Mitochondrial dysfunction; Mitochondrial quality control; Retinal pigment epithelium (RPE).