New method to assess mitophagy flux by flow cytometry

SUMMARY Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disease caused by TSC1 or TSC2 mutations and subsequent activation of the mTORC1 kinase. Upon mTORC1 activation, anabolic metabolism, which requires mitochondria, is induced, yet at the same time the principal pathway for mitochondrial turnover, autophagy, is compromised. How mTORC1 activation impacts mitochondrial turnover in neurons remains unknown. Here we demonstrate impaired mitochondrial homeostasis in neuronal in vitro and in vivo models of TSC. We find that Tsc1/2-deficient neurons accumulate mitochondria in cell bodies but are depleted of axonal mitochondria, including those supporting presynaptic sites. Axonal and global mitophagy of damaged mitochondria is impaired, suggesting that decreased turnover may act upstream of impaired mitochondrial metabolism. Importantly, blocking mTORC1 or inducing mTOR-independent autophagy restores mitochondrial homeostasis. Our study clarifies the complex relationship between the TSC-mTORC1 pathway, autophagy and mitophagy, and defines mitochondrial homeostasis as a therapeutic target for TSC and related diseases.

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“…To address this, we employed a flow cytometry-based assay (Mauro-Lizcano et al, 2015) to quantitate mitochondrial content (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

Impaired Mitochondrial Dynamics and Mitophagy in Neuronal Models of Tuberous Sclerosis Complex

Ebrahimi‐Fakhari¹,

Saffari²,

Wahlster³

et al. 2016

Cell Reports

128

108

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“…67 However, Phen is unable to exert similar effects in neuronal-like SH-SY5Y cells, suggesting that its mitophagic activity might be cell type dependent. 68 In addition, the structurally similar siderophore 2'2-bipyridyl was shown to extend lifespan in C. elegans by inducing a mitophagic response that was dependent on PINK1 as well as PBR-1 and DCT-1, the C. elegans homologues of Parkin and NIX1/BNIP3 respectively ( Table 1 and Figure 5b). …”

Section: Iron Chelatorsmentioning

confidence: 99%

The pharmacological regulation of cellular mitophagy

2017

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Small molecules are pharmacological tools of considerable value in dissecting complex biological processes and identifying potential therapeutic interventions.Recently, the cellular quality control process of mitophagy attracted considerable research interest, however, the limited availability of suitable chemical probes restricted our understanding of the molecular mechanisms involved.Current approaches to initiate mitophagy include acute dissipation of the mitochondrial membrane potential (ΔΨm) by mitochondrial uncouplers (e.g. FCCP/CCCP), or the use of Antimycin A/Oligomycin to impair respiration. Both approaches impair mitochondrial homeostasis, and therefore limit the scope for dissection of subtle, bio-energy related regulatory phenomena.Recently, novel mitophagy activators acting independently of the respiration collapse have been reported, offering new opportunities to understand the process and potential for therapeutic exploitation.We have therefore summarized the current status of mitophagy modulators and analyzed the available chemical tools, commenting on their advantages, limitations and current applications.

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“…3-methyladenine and LY294002) (25,31) and inhibiting the fusion of autophagosomes with lysosomes or the degradation capacity of autophagolysosomes (e.g. chloroquine, bafilomycin A1, leupeptin and liensinine) (25,31,32).…”

Section: Inhibition Of Mitophagy Enhances Drug Sensitivitymentioning

confidence: 99%

“…autophagyrelated 5 (ATG5), Parkin, PINK1, BNIP3 and BNIP3L) (5,25,31), blocking the formation of autophagosomes by phosphoinositide 3-kinase (PI3K) inhibitors (e.g. 3-methyladenine and LY294002) (25,31) and inhibiting the fusion of autophagosomes with lysosomes or the degradation capacity of autophagolysosomes (e.g.…”

Section: Inhibition Of Mitophagy Enhances Drug Sensitivitymentioning

confidence: 99%

Dual Role of Mitophagy in Cancer Drug Resistance

Yan

2018

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New method to assess mitophagy flux by flow cytometry

Cited by 118 publications

References 59 publications

Impaired Mitochondrial Dynamics and Mitophagy in Neuronal Models of Tuberous Sclerosis Complex

Impaired Mitochondrial Dynamics and Mitophagy in Neuronal Models of Tuberous Sclerosis Complex

The pharmacological regulation of cellular mitophagy

Dual Role of Mitophagy in Cancer Drug Resistance

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