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.