Abstract 12308: Presenilin-2 Regulates Mitophagy via Promoting Intracellular Translocation of Parkin to Mitochondria

Abstract

Introduction: Presenilin-2 (PS2) is a component of the γ-secretase complex, intracellular protease. Variant in PSEN2 , the causative genes of Alzheimer's disease, are also implicated in the development of dilated cardiomyopathy. Hypoxia increases PSEN2 expression 10-fold over normal, and PSEN2 knockout (PS2KO) cells exhibit significantly lower ATP production. However, the mechanism of the mitochondrial dysfunction has not been fully elucidated. Hypothesis: PS2 regulates mitochondrial selective autophagy (mitophagy) by promoting the translocation of Parkin to mitochondria. Methods: Expression of autophagy and mitophagy markers was examined using cultured rat cardiomyocytes (H9c2) silenced for PSEN2 and PSEN1 , exposed to 10 μM carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler used to induce mitophagy. Results: Mitochondrial respiration of PS2KO-H9c2 was showed reduced (maximal respiration capacity, 68.4 ± 27.0 pmol/min/cells) compared to PS1KO-H9c2 (147.6 ± 27.2 pmol/min/cells) or normal cells (276.6 ± 11.9 pmol/min/cells) (P<0.001). When mitophagy-dye modified with perylene-3,4-dicarboxylic anhydride was used, mitophagy detection rates were significantly increased by the addition of FCCP to normal H9c2 (34% increased, P=0.041) and PS1KO-H9c2 (46% increased, P=0.035), but there was no change with PS2KO-H9c2 (25% decreased, P=0.232). When FCCP was added to PS2KO-H9c2, Parkin was not only enhanced in whole cell lysate of PS2KO-H9c2, but also increased even at mRNA level; however, in the isolated mitochondrial fraction, Parkin was significantly reduced, and PINK1 and phosphorylated ubiquitin (serine 65) were also attenuated. In Immunofluorescent staining confirmed that Parkin was not co-localized with mitochondria in PS2KO-H9c2, implicating PS2 in promoting the transfer of Parkin from the cytoplasm to mitochondria. As mitochondrial depolarization causes PINK1 stabilization needed for Parkin recruitment, we interrogated mitochondrial membrane potential measurement using Rhodamine-123 and examined expression of OPA1 isoforms; no significant results were obtained. Conclusions: PS2 contributes to the intracellular transport of Parkin to regulate mitophagy.