Abstract
Purpose :
Age-related macular degeneration (AMD) is a major cause of visual loss in the elderly. Studies indicate that AMD patients often have elevated homocysteine (Hcy) levels, leading to insufficient glucose reaching photoreceptor cells. This results in their death, as evidenced by our lab's findings of increased glycolysis in retinal pigmented epithelium (RPE) cells, causing increased glucose consumption and oxygen deprivation for photoreceptor cells. The metabolic shift's causes remain unknown, but we propose Hcy-induced mitochondrial dysfunction as a potential trigger. Investigating mitochondrial dynamics and functions could uncover the reasons behind RPE cell metabolic changes, enhancing our understanding of AMD and aiding in the development of targeted treatments.
Methods :
Two RPE cell lines were used to examine HHcy-induced toxicity on RPE mitochondria: 1) Human RPE cell line (ARPE-19) treated with/without 20, 50, and 100μM Hcy representing mild, moderate, and severe HHcy. 2) Primary RPE isolated from wild-type and CBS mice retina (mice with HHcy). Mitochondrial cytotoxicity assessment was performed using the HCS Mitochondrial Health Kit, which quantitatively measures two critical cell health parameters concurrently: mitotoxicity and cytotoxicity. The MitoHealth stain, reflecting changes in mitochondrial membrane potential, accumulated in active mitochondria. The green stain indicated cytotoxicity, penetrating only compromised plasma membranes. Additionally, autophagic degradation of defective mitochondria (Mitophagy) was evaluated by Mitophagy Detection Kit.
Results :
Elevating Hcy concentrations has demonstrated a significant influence on mitochondrial integrity and health, indicating minimal mitochondrial depolarization and mitophagy in ARPE control, 20μM Hcy, and wild-type RPE. Conversely, a notable escalation in mitochondrial depolarization, coupled with cellular toxicity and mitophagy, is observed in ARPE-19 cells subjected to higher Hcy concentrations (50 and 100μM Hcy) and CBS RPE. These results underscore a distinct correlation between HHcy and pronounced mitochondrial dysfunction.
Conclusions :
Hcy-induced mitochondrial dysfunction causes depolarization, reduced membrane potential, and ATP production. Addressing this dysfunction can offer insights into AMD mechanisms and therapeutic strategies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.