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The Degradation of TMEM166 by Autophagy Promotes AMPK Activation to Protect SH-SY5Y Cells Exposed to MPP(+)

Neuronal oxidative stress caused by mitochondrial dysfunction plays a crucial role in the development of Parkinson’s disease (PD). Growing evidence shows that autophagy confers neuroprotection in oxidative-stress-associated PD. This work aims to investigate the involvement of TMEM166, an endoplasmic...

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Autores principales: Liao, Zhaozhong, Gong, Zunshuang, Wang, Zhe, Yang, Weiyan, Liu, Wenjing, Hou, Lin, Liu, Xiaokun, Hua, Junnan, Wang, Bin, Li, Ning
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9454683/
https://www.ncbi.nlm.nih.gov/pubmed/36078115
http://dx.doi.org/10.3390/cells11172706
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author Liao, Zhaozhong
Gong, Zunshuang
Wang, Zhe
Yang, Weiyan
Liu, Wenjing
Hou, Lin
Liu, Xiaokun
Hua, Junnan
Wang, Bin
Li, Ning
author_facet Liao, Zhaozhong
Gong, Zunshuang
Wang, Zhe
Yang, Weiyan
Liu, Wenjing
Hou, Lin
Liu, Xiaokun
Hua, Junnan
Wang, Bin
Li, Ning
author_sort Liao, Zhaozhong
collection PubMed
description Neuronal oxidative stress caused by mitochondrial dysfunction plays a crucial role in the development of Parkinson’s disease (PD). Growing evidence shows that autophagy confers neuroprotection in oxidative-stress-associated PD. 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(+)). 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. 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. The colocalization of mitochondria with LC3, the colocalization of Tom20 with LAMP1 and Tom20 expression were analyzed to evaluate mitophagy. 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. Overexpression of TMEM166 induces mitochondria fragmentation and dysfunction and exacerbates MPP(+)-induced oxidative stress and cell viability reduction. 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. In addition, overexpressed TMEM166 suppresses AMPK activation, while TMEM166 knockdown enhances AMPK activation. Pharmacological activation of AMPK alleviates the exacerbation of oxidative stress induced by TMEM166 overexpression and increases cell viability, while pharmacological inhibition mitophagy aggravates the oxidative stress induced by MPP(+) treatment combined with TMEM166 overexpression. Finally, we find that overexpressed TMEM166 partially localizes to mitochondria and, simultaneously, the active AMPK in mitochondria is decreased. Collectively, these findings suggest that TMEM166 can translocate from ER to mitochondria and inhibit AMPK activation and, in response to mitochondrial oxidative stress, neuronal cells choose to up-regulate TMEM166 to promote autophagy/mitophagy; then, the enhancing autophagy/mitophagy degrades the TMEM166 to activate AMPK, by the two means to maintain cell survival. The continuous synthesis and degradation of TMEM166 in autophagy/mitochondria flux suggest that TMEM166 may act as an autophagy/mitochondria adaptor.
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spelling pubmed-94546832022-09-09 The Degradation of TMEM166 by Autophagy Promotes AMPK Activation to Protect SH-SY5Y Cells Exposed to MPP(+) Liao, Zhaozhong Gong, Zunshuang Wang, Zhe Yang, Weiyan Liu, Wenjing Hou, Lin Liu, Xiaokun Hua, Junnan Wang, Bin Li, Ning Cells Article Neuronal oxidative stress caused by mitochondrial dysfunction plays a crucial role in the development of Parkinson’s disease (PD). Growing evidence shows that autophagy confers neuroprotection in oxidative-stress-associated PD. 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(+)). 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. 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. The colocalization of mitochondria with LC3, the colocalization of Tom20 with LAMP1 and Tom20 expression were analyzed to evaluate mitophagy. 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. Overexpression of TMEM166 induces mitochondria fragmentation and dysfunction and exacerbates MPP(+)-induced oxidative stress and cell viability reduction. 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. In addition, overexpressed TMEM166 suppresses AMPK activation, while TMEM166 knockdown enhances AMPK activation. Pharmacological activation of AMPK alleviates the exacerbation of oxidative stress induced by TMEM166 overexpression and increases cell viability, while pharmacological inhibition mitophagy aggravates the oxidative stress induced by MPP(+) treatment combined with TMEM166 overexpression. Finally, we find that overexpressed TMEM166 partially localizes to mitochondria and, simultaneously, the active AMPK in mitochondria is decreased. Collectively, these findings suggest that TMEM166 can translocate from ER to mitochondria and inhibit AMPK activation and, in response to mitochondrial oxidative stress, neuronal cells choose to up-regulate TMEM166 to promote autophagy/mitophagy; then, the enhancing autophagy/mitophagy degrades the TMEM166 to activate AMPK, by the two means to maintain cell survival. The continuous synthesis and degradation of TMEM166 in autophagy/mitochondria flux suggest that TMEM166 may act as an autophagy/mitochondria adaptor. MDPI 2022-08-30 /pmc/articles/PMC9454683/ /pubmed/36078115 http://dx.doi.org/10.3390/cells11172706 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Liao, Zhaozhong
Gong, Zunshuang
Wang, Zhe
Yang, Weiyan
Liu, Wenjing
Hou, Lin
Liu, Xiaokun
Hua, Junnan
Wang, Bin
Li, Ning
The Degradation of TMEM166 by Autophagy Promotes AMPK Activation to Protect SH-SY5Y Cells Exposed to MPP(+)
title The Degradation of TMEM166 by Autophagy Promotes AMPK Activation to Protect SH-SY5Y Cells Exposed to MPP(+)
title_full The Degradation of TMEM166 by Autophagy Promotes AMPK Activation to Protect SH-SY5Y Cells Exposed to MPP(+)
title_fullStr The Degradation of TMEM166 by Autophagy Promotes AMPK Activation to Protect SH-SY5Y Cells Exposed to MPP(+)
title_full_unstemmed The Degradation of TMEM166 by Autophagy Promotes AMPK Activation to Protect SH-SY5Y Cells Exposed to MPP(+)
title_short The Degradation of TMEM166 by Autophagy Promotes AMPK Activation to Protect SH-SY5Y Cells Exposed to MPP(+)
title_sort degradation of tmem166 by autophagy promotes ampk activation to protect sh-sy5y cells exposed to mpp(+)
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9454683/
https://www.ncbi.nlm.nih.gov/pubmed/36078115
http://dx.doi.org/10.3390/cells11172706
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