NADPH oxidase promotes Parkinsonian phenotypes by impairing autophagic flux in an mTORC1-independent fashion in a cellular model of Parkinson’s disease

Abstract

Oxidative stress and aberrant accumulation of misfolded proteins in the cytosol are key pathological features associated with Parkinson's disease (PD). NADPH oxidase (Nox2) is upregulated in the pathogenesis of PD; however, the underlying mechanism(s) of Nox2-mediated oxidative stress in PD pathogenesis are still unknown. Using a rotenone-inducible cellular model of PD, we observed that a short exposure to rotenone (0.5 μM) resulted in impaired autophagic flux through activation of a Nox2 dependent Src/PI3K/Akt axis, with a consequent disruption of a Beclin1-VPS34 interaction that was independent of mTORC1 activity. Sustained exposure to rotenone at a higher dose (10 μM) decreased mTORC1 activity; however, autophagic flux was still impaired due to dysregulation of lysosomal activity with subsequent induction of the apoptotic machinery. Cumulatively, our results highlight a complex pathogenic mechanism for PD where short-and long-term oxidative stress alters different signaling pathways, ultimately resulting in anomalous autophagic activity and disease phenotype. Inhibition of Nox2-dependent oxidative stress attenuated the impaired autophagy and cell death, highlighting the importance and therapeutic potential of these pathways for treating patients with PD.Reactive oxygen species (ROS) play pivotal roles in regulating signaling molecules, but when in excess they induce oxidative stress; which has been implicated as a key pathological factor in "sporadic" forms of Parkinson's disease (PD) and other neurodegenerative diseases. Environmental toxins, such as rotenone, have been well-established as causal agents of sporadic form of PD due to its ability to generate reactive oxygen and nitrogen species (ROS/ RNS) 1 . Previous work has shown that oxidative stress impairs autophagic flux and decreases lysosomal biogenesis and function in animal models of PD 2,3 . Up-regulation of NADPH oxidase (Nox2), a major superoxide-producing enzyme complex 4 , has recently been associated with PD pathogenesis in human patients and animal models 5 . Shacka and colleagues suggest that rotenone-induced oxidative stress impairs autophagic flux and promotes accumulation of protein aggregates in an in vitro model of PD 6 . We and others have recently demonstrated the ability of rotenone to induce oxidative stress via activation of the Nox2 complex [7][8][9] . We have also shown that enhanced Nox2-dependent ROS production drives impaired autophagic flux and lysosomal dysfunction by activation of Src/PI3K/mTORC1 pathway in a mouse model of muscle degeneration 10 . The classical paradigm of autophagy in mammalian cells involves ULK1, a key pro-autophagy adapter kinase essential to the nucleation of the autophagophore membrane. Activation of the serine/threonine kinase mTORC1 inhibits autophagy through phosphorylation of ULK1 at S757, thus subsequent inhibition of ULK1 activity 11 . The energy sensitive AMP activated protein kinase (AMPK) promotes autophagy, in an mTORC1-independent manner, by directly activating ULK1 through

Mitochondria, lysosomes, and dysfunction: their meaning in neurodegeneration

“…In addition, NADPH oxidase (NOX2) promotes parkinsonian phenotypes by altering autophagic flux in an mTORC1‐independent fashion in a cellular model of PD (Pal et al . ). Specifically, rotenone treatment of SHSY‐5Y cells for a short time increased NOX2‐ROS generation and impaired autophagic flux in an mTORC1/AMPK‐independent fashion by up‐regulating Rous sarcoma oncogene (SRC)/phosphatidylinositol‐4,5‐bisphosphate 3‐kinase/AKT‐mediated phosphorylation of Beclin1 at S295 and concomitant disruption of Beclin1‐phosphatidylinositol 3‐kinase catalytic subunit type 3 complex.…”

Disorders of lysosomal acidification—The emerging role of v-ATPase in aging and neurodegenerative disease

“…As a weak base, methamphetamine collapses the pH gradient across acidic organelles, including lysosomes (Funakoshi-Hirose et al, 2013). Similarly, rotenone, another environmental risk factor for PD (Betarbet et al, 2000), impairs lysosomal acidification and lysosomal activity in an NADPH oxidase-dependent manner (Pal et al, 2016). MPP + , a neurotoxin which promotes Parkinsonian symptoms, also causes de-acidification of lysosomes in cultured cells (Bourdenx et al, 2016).…”

The inhibition of apoptosis through myocardial postconditioning by affecting Fas/FasIg signaling through miR139‐3p and miR181a‐1

“…Autophagy inhibitor 3‐methyladenine inhibited the induction of Beclin and LC3, as well as the effect of PostC that reduces the volume of infarction 17 . Furthermore, it was demonstrated that the oxidative stress caused by the mild activation of Cybb disrupted the autophagic flux by disrupting the Becn1‐VPS34 interaction—a complex required for the maturation of autophagosome 37 . In another study, autophagy was induced by rapamycin application, and the protective effect was demonstrated in Fas/FasL signaling and apoptosis activation.…”

“…17 Furthermore, it was demonstrated that the oxidative stress caused by the mild activation of Cybb disrupted the autophagic flux by disrupting the Becn1-VPS34 interaction-a complex required for the maturation of autophagosome. 37 In another study, autophagy was induced by rapamycin application, and the protective effect was demonstrated in Fas/FasL signaling and apoptosis activation. The study further revealed that autophagy inhibitor 3-methyladenine could block the antiapoptotic effect of autophagy and that Becn1 interacted with members of the Fas/FasL signaling apoptosis pathway in a primary rat proximal tubule cell culture.…”