Abstract
This study was conducted to elucidate the involvement of the PINK1-Parkin pathway in ethanol-induced mitophagy among Sertoli cells (SCs). In the research, adult rats were given intraperitoneal injections of ethanol (5 gm/kg) and sacrificed at various time periods within 24 h. Transmission electron microscopy was applied to reveal enhanced mitochondrial damage in SCs of the ethanol-treated rats (ETRs) in association with a significant increase in numbers of mitophagic vacuoles (mitophagosomes and autolysosomes) in contrast to very low levels in a control group treated with phosphate-buffered saline (PBS). This enhancement was ultra-structurally verified via observation of trapped mitochondria within LC3-labeled membranes, upregulation of LC3 protein levels, colocalization of LC3 and cytochrome c, and reduced expression of mitochondrial proteins. Importantly, Parkin expression was found to be upregulated in ETR SCs, specifically in mitochondria and mitophagosomes in addition to colocalization with PINK1 and pan-cathepsin, indicating augmented mitophagy. Transcription factor EB (TFEB, a transcription factor for autophagy and mitophagy proteins) was also found to be upregulated in nuclei of ETR SCs and associated with enhanced expression of iNOS. Enhanced Parkin-related mitophagy in ETR SCs may be a protective mechanism with therapeutic implications. To the authors’ knowledge, this is the first report demonstrating the ultrastructural characteristics and molecular mechanisms of Parkin-related mitophagy in ETR SCs.
Highlights
Autophagy is a catabolic pathway for lysosomal degradation of most cellular components under basal conditions and upon exposure to various stressors such as starvation, oxidative/nitrosative stress, mitochondrial damage, and lipogenic challenge [1,2,3].Selective autophagic removal of damaged mitochondria, or mitophagy, is an antiapoptotic mechanism induced and upregulated as a response to various damaging agents such as protonophore carbonyl cyanide m-chlorophenyl hydrazine and ethanol in animal models [4,5,6]
Multilamellar bodies (Figure 1k) were frequently observed, indicating enhanced mitochondrial damage [6,16,26]. This juxtanuclear accumulation of Mitophagic Vacuoles (MVs) in ethanol-treated rats (ETRs) Sertoli cells (SCs) is shown with low-power magnification (Figure S1)
Based on transmission electron microscopy (TEM) and Transferase dUTP-Mediated Nick-End Labeling (TUNEL) (Figure S2), germ cell apoptosis was frequently observed in ETR testes, but SCs nuclei appeared normal
Summary
Autophagy (or macroautophagy) is a catabolic pathway for lysosomal degradation of most cellular components under basal conditions and upon exposure to various stressors such as starvation, oxidative/nitrosative stress, mitochondrial damage, and lipogenic challenge [1,2,3].Selective autophagic removal of damaged mitochondria, or mitophagy, is an antiapoptotic mechanism induced and upregulated as a response to various damaging agents such as protonophore carbonyl cyanide m-chlorophenyl hydrazine (or CCCP; used for in vitro studies) and ethanol in animal models [4,5,6]. Cells 2019, 8, 283 ethanol) were recently reported by the authors’ laboratory These include the engulfment of damaged mitochondria by microtubule-associated protein 1 light chain (LC3)-mediated autophagosomal membranes forming mitophagosomes that fuse with lysosomes, creating autolysosomes with perinuclear localization. The specific molecular mechanisms of ethanol-induced hepatic mitophagy were recently reported to be related to the PINK1-Parkin pathway [6,7,10,11,12,13]. In these studies, ethanol-induced mitochondrial damage via mechanisms related to mitochondrial DNA (mt DNA) damage, oxidative stress, and other factors caused the stabilization of PINK1 (a sensor of mitochondrial damage) on damaged mitochondria
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