In addition, overexpressed TMEM166 suppresses AMPK activation, while TMEM166 knockdown enhances AMPK activation. Overexpression of TMEM166 is sufficient to induce autophagy and mitophagy and promotes autophagy and mitophagy under MPP + treatment, while knockdown of TMEM166 inhibits basal autophagic degradation. Overexpression of TMEM166 induces mitochondria fragmentation and dysfunction and exacerbates MPP +-induced oxidative stress and cell viability reduction. We found that TMEM166 is up-regulated in transcript levels, but up-regulated first and then down-regulated by autophagic degradation in protein levels upon MPP +-treatment. The colocalization of mitochondria with LC3, the colocalization of Tom20 with LAMP1 and Tom20 expression were analyzed to evaluate mitophagy. The effect on autophagy induction was evaluated by analyzing p62 and LC3-II/I expression and by observing the LC3 puncta and the colocalization of LC3 with LAMP1/ LAMP2. Reactive oxygen species (ROS) production and mitochondrial membrane potential were checked to assess the oxidative stress induced by MPP + and the cellular ATP generated was determined to evaluate mitochondrial function. This work aims to investigate the involvement of TMEM166, an endoplasmic-reticulum-localized autophagy-regulating protein, in the process of PD-associated oxidative stress through the classic cellular PD model of neuroblastoma SH-SY5Y cells exposed to 1-methyl-4-phenylpyridinium (MPP +). Growing evidence shows that autophagy confers neuroprotection in oxidative-stress-associated PD. Neuronal oxidative stress caused by mitochondrial dysfunction plays a crucial role in the development of Parkinson’s disease (PD).

What is cccp treated cells