3.6 The role of DACA in mitochondrial function and autophagy
Mitochondria, a highly adaptable and dynamic organelle, play a crucial role in neuronal apoptosis. We investigated the potential protective effect of DACA on mitochondria. JC-1 staining was employed to assess mitochondrial membrane potential (MMP) in SH-SY5Y cells and primary neurons, mitochondrial superoxide localization was used to evaluate mitochondrial redox potential. Our findings demonstrate that MPP+ significantly increased the number of cells with mitochondrial depolarization (green), which was effectively reversed by DACA treatment (Fig. 6A). Furthermore, DACA treatment successfully restored the imbalance in mitochondrial REDOX potential caused by MPP+ exposure (Fig. 6B). MPP+induces neuronal cell death through regulation of pro-apoptotic members within the Bcl-2 protein family. To investigate this further, we performed Western blot analysis to examine changes in Bcl-2 and Bax expression following DACA treatment. The results revealed that DACA counteracted the decrease in Bcl-2 levels and attenuated the increase in Bax induced by MPP+, thereby enhancing the ratio of Bcl-2/Bax. These findings suggest that Nrf2 activation mediated by DACA not only preserves mitochondrial health but also prevents cellular apoptosis (Fig. 6E-H).
Mitophagy is a selective cellular process that eliminates senescent, damaged, or redundant mitochondria through autophagy, serving as a crucial pathway for maintaining mitochondrial quality control. Previous studies have demonstrated the regulatory role of Nrf2 activation in autophagy, potentially mediated by p62-Nrf2 interaction. To investigate the impact of DACA on autophagy, we assessed the expression levels of p62, PINK1, and LC3 proteins. Our results (Fig. 6E-H) revealed decreased levels of p62 and PINK1 proteins along with an increased LC3Ⅱ/LC3Ⅰ ratio in MPP+-treated cells. As hypothesized, DACA activated Nrf2 signaling pathway and subsequently upregulated p62 and PINK1 expression (Fig. 6E-H). Consistently observed in both SH-SY5Y cells and primary neurons, these findings suggest that DACA plays a regulatory role in modulating normal autophagic flux to safeguard cellular homeostasis.