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Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors
Published on: July 28, 2023
Characterization of mitochondrial dysfunction induced by BAX trigger site activator 1 in the ARPE-19 retinal pigment
Toshihide Kashihara1, Yuka Akiyama1, Akane Morita1
1Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, 108-8641, Japan.
Abstract:
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.
Insights
This study shows that activating BAX with BTSA1 causes mitochondrial dysfunction in retinal cells, mimicking AMD pathology. This provides a new model for studying and treating mitochondrial damage in AMD.
Area of Science:
- Cell Biology
- Ophthalmology
- Mitochondrial Biology
Background:
- Mitochondrial dysfunction in retinal pigment epithelium (RPE) is central to age-related macular degeneration (AMD).
- Mechanistically defined models for stress-induced mitochondrial injury in RPE are scarce.
- Bcl-2-associated X (BAX) protein is a key regulator of mitochondrial outer membrane permeabilization.
Purpose of the Study:
- To systematically characterize mitochondrial dysfunction induced by BTSA1, a selective BAX activator, in ARPE-19 cells.
- To establish a mechanistically defined model for BAX-mediated mitochondrial pathology relevant to AMD.
Main Methods:
- ARPE-19 cells were treated with varying concentrations and durations of BTSA1.
- Assessed cell viability, caspase-3 activation, mitochondrial membrane potential, and reactive oxygen species (ROS) production.
- Analyzed mitochondrial dynamics (fission/fusion proteins) and quality control pathways (autophagy, biogenesis, mitophagy).
Main Results:
- BTSA1 induced dose- and time-dependent decreases in cell viability and increased caspase-3 activation.
- BAX activation reduced mitochondrial membrane potential and increased ROS production.
- BTSA1 promoted BAX- and DRP1-mediated mitochondrial fission, suppressed fusion, and caused fragmentation.
- Observed biphasic mitochondrial quality control responses: impaired autophagy/compensatory biogenesis under mild stress, and mitophagy failure under severe stress.
Conclusions:
- BAX activation by BTSA1 is sufficient to induce a comprehensive cascade of mitochondrial dysfunction in RPE cells.
- This BTSA1-induced model recapitulates key mitochondrial pathologies seen in AMD.
- This system offers a valuable tool for dissecting BAX-mediated mitochondrial pathology and evaluating AMD therapeutics.
