Aquaporin 5 is degraded by autophagy in diabetic submandibular gland

Autophagy is a catabolic process which is involved in the development of many diseases including diabetes mellitus and its complications. Hyposalivation is a common complication of diabetes mellitus, whereas its mechanism remains unclear. Here, we observed that the stimulated salivary flow rate of SMG was significantly decreased in db/db mice, a diabetic mice model. The expressions of aquaporin 5(AQP5), a water channel protein, were decreased, whereas the m RNA level of AQP5 was increased in SMGs of both diabetic patients and mice. Under transmission electron microcope, more autophagosomes were detected in diabetic SMGs. Expressions of autophagy related proteins LC3 II, Beclin-1 and ATG5 were increased, meanwhile autophagy substrate p62 was decreased in SMGs of diabetic patients and mice, indicating that autophagy was activated in diabetic SMG.Double immunofluorescence staining showed that the colocalization of AQP5 and LC3 was increased in SMGs of diabetic mice.In cultured SMG-C6 cells, high glucose(HG), but not high osmotic pressure, reduced AQP5 protein expression and induced autophagy. Moreover, inhibition of autophagy by 3-methyladenin, an autophagy inhibitor, or by autophagy-related gene 5 siRNA, decreased HG-induced AQP5 reduction in SMG-C6 cells. Additionally, the expression of p-p85, p-Akt and p-mTOR were decreased in HG-treated SMG-C6 cells. Pretreatment with 740 Y-P, a PI3 K agonist, significantly suppressed HG-induced autophagy and AQP5 degradation. Taken together, these results indicate that autophagy plays a crucial role in AQP5 degradation in diabetic SMG via PI3 K/Akt/mTOR signaling pathway, which contributes to the dysfunction of diabetic SMG. Our study provides a novel mechanism of diabetic hyposalivation.     

MALAT1/miR-185-5p mediated high glucose-induced oxidative stress,mitochondrial injury and cardiomyocyte apoptosis via the RhoA/ROCK pathway

To explore the underlying mechanism of lncRNA MALAT1 in the pathogenesis of diabetic cardiomyopathy (DCM). DCM models were confirmed in db/db mice. MiRNAs in myocardium were detected by miRNA sequencing. The interactions of miR-185-5p with MALAT1 and RhoA were validated by dual-luciferase reporter assays. Primary neonatal cardiomyocytes were cultured with 5.5 or 30 mmol/L D-glucose (HG) in the presence or absence of MALAT1-shRNA and fasudil, a ROCK inhibitor. MALAT1 and miR-185-5p expression were determined by real-time quantitative PCR. The apoptotic cardiomyocytes were evaluated using flow cytometry and TUNEL staining. SOD activity and MDA contents were measured. The ROCK activity, phosphorylation of Drp1^S616, mitofusin 2 and apoptosis-related proteins were analysed by Western blotting. Mitochondrial membrane potential was examined by JC-1. MALAT1 was significantly up-regulated while miR-185-5p was down-regulated in myocardium of db/db mice and HG-induced cardiomyocytes. MALAT1 regulated RhoA/ROCK pathway via sponging miR-185-5p in cardiomyocytes in HG. Knockdown of MALAT1 and fasudil all inhibited HG-induced oxidative stress, and alleviated imbalance of mitochondrial dynamics and mitochondrial dysfunction, accompanied by reduced cardiomyocyte apoptosis. MALAT1 activated the RhoA/ROCK pathway via sponging miR-185-5p and mediated HG-induced oxidative stress, mitochondrial damage and apoptosis of cardiomyocytes in mice.     

Parkin,as a Regulator,Participates in Arsenic Trioxide-Triggered Mitophagy in HeLa Cells

Parkin is a well-established synergistic mediator of mitophagy in dysfunctional mitochondria. Mitochondria are the main target of arsenic trioxide (ATO) cytotoxicity, and the effect of mitophagy on ATO action remains unclear. In this study, we used stable Parkin-expressing (YFP-Parkin) and Parkin loss-of-function mutant (Parkin C431S) HeLa cell models to ascertain whether Parkin-mediated mitophagy participates in ATO-induced apoptosis/cell death. Our data showed that the overexpression of Parkin significantly sensitized HeLa cells to ATO-initiated proliferation inhibition and apoptosis; however, the mutation of Parkin C431S significantly weakened this Parkin-mediated responsiveness. Our further investigation found that ATO significantly downregulated two fusion proteins (Mfn1/2) and upregulated fission-related protein (Drp1). Autophagy was also activated as evidenced by the formation of autophagic vacuoles and mitophagosomes, increased expression of PINK1, and recruitment of Parkin to impaired mitochondria followed by their degradation, accompanied by the increased transformation of LC3-I to LC3-II, increased expression of Beclin1 and decreased expression of P62 in YFP-Parkin HeLa cells. Enhanced mitochondrial fragmentation and autophagy indicated that mitophagy was activated. Furthermore, during the process of mitophagy, the overproduction of ROS implied that ROS might represent a key factor that initiates mitophagy following Parkin recruitment to mitochondria. In conclusion, our findings indicate that Parkin is critically involved in ATO-triggered mitophagy and functions as a potential antiproliferative target in cancer cells.     

Hypoxic postconditioning promotes mitophagy against transient global cerebral ischemia via PINK1/Parkin-induced mitochondrial ubiquitination in adult rats

Mitophagy alleviates neuronal damage after cerebral ischemia by selectively removing dysfunctional mitochondria. Phosphatase and tensin homolog (PTEN) induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy is the most well-known type of mitophagy. However, little is known about the role of PINK1/Parkin-mediated mitophagy in ischemic tolerance induced by hypoxic postconditioning (HPC) with 8% O₂ against transient global cerebral ischemia (tGCI). Hence, we aimed to test the hypothesis that HPC-mediated PINK1/Parkin-induced mitochondrial ubiquitination and promotes mitophagy, thus exerting neuroprotection in the hippocampal CA1 subregion against tGCI. We found that mitochondrial clearance was disturbed at the late phase of reperfusion after tGCI, which was reversed by HPC, as evidenced by the reduction of the translocase of outer mitochondrial membrane 20 homologs (TOMM20), translocase of inner mitochondrial membrane 23 (TIMM23) and heat shock protein 60 (HSP60) in CA1 after HPC. In addition, HPC further increased the ratio of LC3II/I in mitochondrial fraction and promoted the formation of mitophagosomes in CA1 neurons after tGCI. The administration of lysosome inhibitor chloroquine (CQ) intraperitoneally or mitophagy inhibitor (Mdivi-1) intracerebroventricularly abrogated HPC-induced mitochondrial turnover and neuroprotection in CA1 after tGCI. We also found that HPC activated PINK1/Parkin pathway after tGCI, as shown by the augment of mitochondrial PINK1 and Parkin and the promotion of mitochondrial ubiquitination in CA1. In addition, PINK1 or Parkin knockdown with small-interfering RNA (siRNA) suppressed the activation of PINK1/Parkin pathway and hampered mitochondrial clearance and attenuated neuroprotection induced by HPC, whereas PINK1 overexpression promoted PINK1/Parkin-mediated mitophagy and ameliorated neuronal damage in CA1 after tGCI. Taken together, the new finding in this study is that HPC-induced neuroprotection against tGCI through promoting mitophagy mediated by PINK1/Parkin-dependent pathway.Subject terms: Cell death in the nervous system, Stroke     

Spatial parkin translocation and degradation of damaged mitochondria via mitophagy in live cortical neurons

Mitochondria are essential for neuronal survival and function. Proper degradation of aged and damaged mitochondria through mitophagy is a key cellular pathway for mitochondrial quality control. Recent studies have indicated that PINK1/Parkin-mediated pathways ensure mitochondrial integrity and function. Translocation of Parkin to damaged mitochondria induces mitophagy in many nonneuronal cell types. However, evidence showing Parkin translocation in primary neurons is controversial, leaving unanswered questions as to how and where Parkin-mediated mitophagy occurs in neurons. Here, we report the unique process of dissipating mitochondrial Δψ(m)-induced and Parkin-mediated mitophagy in mature cortical neurons. Compared with nonneuronal cells, neuronal mitophagy is a much slower and compartmentally restricted process, coupled with reduced anterograde mitochondrial transport. Parkin-targeted mitochondria are accumulated in the somatodendritic regions where mature lysosomes are predominantly located. Time-lapse imaging shows dynamic formation and elimination of Parkin- and LC3-ring-like structures surrounding depolarized mitochondria through the autophagy-lysosomal pathway in the soma. Knocking down Parkin in neurons impairs the elimination of dysfunctional mitochondria. Thus, our study provides neuronal evidence for dynamic and spatial Parkin-mediated mitophagy, which will help us understand whether altered mitophagy contributes to pathogenesis of several major neurodegenerative diseases characterized by mitochondrial dysfunction and impaired transport.     

Inhibition of Peroxynitrite-Induced Mitophagy Activation Attenuates Cerebral Ischemia-Reperfusion Injury

Activated autophagy/mitophagy has been intensively observed in ischemic brain, but its roles remain controversial. Peroxynitrite (ONOO^?), as a representative of reactive nitrogen species, is considered as a critical neurotoxic factor in mediating cerebral ischemia-reperfusion (I/R) injury, but its roles in autophagy/mitophagy activation remain unclear. Herein, we hypothesized that ONOO^? could induce PINK1/Parkin-mediated mitophagy activation via triggering dynamin-related protein 1 (Drp1) recruitment to damaged mitochondria, contributing to cerebral I/R injury. Firstly, we found PINK1/Parkin-mediated mitophagy activation was predominant among general autophagy, leading to rat brain injury at the reperfusion phase after cerebral ischemia. Subsequently, increased nitrotyrosine was found in the plasma of ischemic stroke patients and ischemia-reperfused rat brains, indicating the generation of ONOO^? in ischemic stroke. Moreover, in vivo animal experiments illustrated that ONOO^? was dramatically increased, accompanied with mitochondrial recruitment of Drp1, PINK1/Parkin-mediated mitophagy activation, and progressive infarct size in rat ischemic brains at the reperfusion phase. FeTMPyP, a peroxynitrite decomposition catalyst, remarkably reversed mitochondrial recruitment of Drp1, mitophagy activation, and brain injury. Intriguingly, further study revealed that ONOO^? induced tyrosine nitration of Drp1 peptide, which might contribute to mitochondrial recruitment of Drp1 for mitophagy activation. In vitro cell experiments yielded consistent results with in vivo animal experiments. Taken together, all above findings support the hypothesis that ONOO^?-induced mitophagy activation aggravates cerebral I/R injury via recruiting Drp1 to damaged mitochondria.     

Klotho attenuates diabetic nephropathy in db/db mice and ameliorates high glucose-induced injury of human renal glomerular endothelial cells

Glomerular endothelial cell injury plays an important role in the development and progression of diabetic nephropathy (DN). The expression and function of klotho in glomerular endothelial cells remain unclear. Thus, this study aimed to investigate the expression and the functional role of klotho in DN progression in mice and in high glucose (HG)-induced cell injury of human renal glomerular endothelial cells (HRGECs) and the underlying mechanism. In this study, HRGECs were cultured with media containing HG to induce endothelial cell injury and db/db mice were used as DN model mice. Klotho was overexpressed or knocked down in HRECs to evaluate its role in HG-induced HRGECs injury. klotho-overexpressing adenovirus (rAAV-klotho) was injected into db/db mice via the tail vein to further validate the protective effect of klotho in DN. Decreased klotho expression was observed in DN patients, DN mice, and HG-exposed HRGECs. Furthermore, klotho overexpression significantly abolished the HG-induced HRGECs injury and activation of Wnt/β-catenin pathway and RAAS. In contrast, klotho knockdown exerted the opposite effects. Moreover, klotho attenuated diabetic nephropathy in db/db mice, which was also associated with inhibition of the Wnt/β-catenin pathway and RAAS. In conclusion, klotho attenuates DN in db/db mice and ameliorates HG-induced injury of HRGECs.     

Zinc prevents mitochondrial superoxide generation by inducing mitophagy in the setting of hypoxia/reoxygenation in cardiac cells

Zinc plays a role in autophagy and protects cardiac cells from ischemia/reperfusion injury. This study aimed to test if zinc can induce mitophagy leading to attenuation of mitochondrial superoxide generation in the setting of hypoxia/reoxygenation (H/R) in cardiac cells. H9c2 cells were subjected to 4?h hypoxia followed by 2?h reoxygenation. Under normoxic conditions, treatments of cells with ZnCl₂ increased both the LC3-II/LC3-I ratio and GFP-LC3 puncta, implying that zinc induces autophagy. Further experiments showed that endogenous zinc is required for the autophagy induced by starvation and rapamycin. Zinc down-regulated TOM20, TIM23, and COX4 both in normoxic cells and the cells subjected to H/R, indicating that zinc can trigger mitophagy. Zinc increased ERK activity and Beclin1 expression, and zinc-induced mitophagy was inhibited by PD98059 and Beclin1 siRNA during reoxygenation. Zinc-induced Beclin1 expression was reversed by PD98059, implying that zinc promotes Beclin1 expression via ERK. In addition, zinc failed to induce mitophagy in cells transfected with PINK1 siRNA and stabilized PINK1 in mitochondria. Moreover, zinc-induced PINK1 stabilization was inhibited by PD98059. Finally, zinc prevented mitochondrial superoxide generation and dissipation of mitochondrial membrane potential (ΔΨ_m) at reoxygenation, which was blocked by both the Beclin1 and PINK1 siRNAs, suggesting that zinc prevents mitochondrial oxidative stress through mitophagy. In summary, zinc induces mitophagy through PINK1 and Beclin1 via ERK leading to the prevention of mitochondrial superoxide generation in the setting of H/R. Clearance of damaged mitochondria may account for the cardioprotective effect of zinc on H/R injury.     

How could Parkin-mediated ubiquitination of mitofusin promote mitophagy?

Much evidence links mitochondrial dysfunction to the death of neurons in Parkinson disease (PD), and is particularly emphasized by our growing understanding of the function of genes linked to recessively inherited PD such as PINK1, parkin and DJ-1. Recent work has revealed an exciting link between the PINK1-Parkin pathway and the autophagic turnover of dysfunctional mitochondrial (mitophagy). We have recently shown that mitofusin is ubiquitinated by Parkin when it is recruited to dysfunctional mitochondria. Recent work also shows that regulated fission and fusion events help segregate dysfunctional mitochondria prior to mitophagy. Here we hypothesize how Parkin-mediated ubiquitination of Mfn may play a role in this mechanism.     

Defending the mitochondria: The pathways of mitophagy and mitochondrial-derived vesicles

Mitochondria are the powerhouses for the cell, consuming oxygen to generate sufficient energy for the maintenance of normal cellular processes. However, a deleterious consequence of this process are reactive oxygen species generated as side-products of these reactions. As a means to protect mitochondria from damage, cells and mitochondria have developed a wide-range of mitochondrial quality control mechanisms that remove damaged mitochondrial cargo, enabling the mitochondria to repair the damage and ultimately restore their normal function. If the damage is extensive and mitochondria can no longer be repaired, a process termed mitophagy is initiated in which the mitochondria are directed for autophagic clearance. Canonical mitophagy is regulated by two proteins, PINK1 and Parkin, which are mutated in familial forms of Parkinson’s disease. In this review, we discuss recent work elucidating the mechanism of PINK1/Parkin-mediated mitophagy, along with recently uncovered PINK1/Parkin-independent mitophagy pathways. Moreover, we describe a novel mitochondrial quality control pathway, involving mitochondrial-derived vesicles that direct distinct and damaged mitochondrial cargo for degradation in the lysosome. Finally, we discuss the association between mitochondrial quality control, cardiac, hepatic and neurodegenerative disease and discuss the possibility of targeting these pathways for therapeutic purposes.     

Mitochondrial Ca2+ oscillation induces mitophagy initiation through the PINK1-Parkin pathway

Dysregulation of the PINK1/Parkin-mediated mitophagy is essential to Parkinson’s disease. Although important progress has been made in previous researches, the biochemical reagents that induce global and significant mitochondrial damage may still hinder deeper insights into the mechanisms of mitophagy. The origin of PINK1/Parkin pathway activation in mitophagy remains elusive. In this study, we develop an optical method, ultra-precise laser stimulation (UPLaS) that delivers a precise and noninvasive stimulation onto a submicron region in a single mitochondrial tubular structure. UPLaS excites localized mitochondrial Ca²⁺ (mitoCa²⁺) oscillations with tiny perturbation to mitochondrial membrane potential (MMP) or mitochondrial reactive oxygen species. The UPLaS-induced mitoCa²⁺ oscillations can directly induce PINK1 accumulation and Parkin recruitment on mitochondria. The Parkin recruitment by UPLaS requires PINK1. Our results provide a precise and noninvasive technology for research on mitophagy, which stimulates target mitochondria with little damage, and reveal mitoCa²⁺ oscillation directly initiates the PINK1-Parkin pathway for mitophagy without MMP depolarization.Subject terms: Mitophagy, Calcium signalling     

High glucose induces renal mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which is inhibited by resveratrol

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. Hyperglycemia-induced oxidative stress is implicated in the etiology of diabetic nephropathy. Resveratrol has potent antioxidative and protective effects on diabetic nephropathy. We aimed to examine whether high glucose (HG)-induced NADPH oxidase activation and reactive oxygen species (ROS) production contribute to glomerular mesangial cell proliferation and fibronectin expression and the effect of resveratrol on HG action in mesangial cells. By using rat mesangial cell line and primary mesangial cells, we found that NADPH oxidase inhibitor (apocynin) and ROS inhibitor (N-acetyl cysteine) both inhibited HG-induced mesangial cell proliferation and fibronectin expression. HG-induced elevation of NADPH oxidase activity and production of ROS in mesangial cells was inhibited by apocynin. These results suggest that HG induces mesangial cell proliferation and fibronectin expression through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunits p22(phox) and p47(phox) expression through JNK/NF-κB pathway, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced mesangial cell proliferation and fibronectin expression through inhibiting HG-induced JNK and NF-κB activation, NADPH oxidase activity elevation and ROS production. These results demonstrate that HG enhances mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide novel therapeutic targets for diabetic nephropathy.     

Matrine promotes liver cancer cell apoptosis by inhibiting mitophagy and PINK1/Parkin pathways

Matrine is a natural alkaloid isolated from the root and stem of the legume plant Sophora. Its anti-proliferative and pro-apoptotic effects on several types of cancer have been well-documented. However, the role of matrine in regulating mitochondrial homeostasis, particularly mitophagy in liver cancer apoptosis, remains uncertain. The aim of our study was to explore whether matrine promotes liver cancer cell apoptosis by modifying mitophagy. HepG2 cells were used in the study and treated with different doses of matrine. Cell viability and apoptosis were determined by MTT assay, TUNEL staining, western blotting, and LDH release assay. Mitophagy was monitored by immunofluorescence assay and western blotting. Mitochondrial function was assessed by immunofluorescence assay, ELISA, and western blotting. The results of our study indicated that matrine treatment dose-dependently reduced cell viability and increased the apoptotic rate of HepG2 cells. Functional studies demonstrated that matrine treatment induced mitochondrial dysfunction and activated mitochondrial apoptosis by inhibiting protective mitophagy. Re-activation of mitophagy abolished the pro-apoptotic effects of matrine on HepG2 cells. Molecular investigations further confirmed that matrine regulated mitophagy via the PINK1/Parkin pathways. Matrine blocked the PINK1/Parkin pathways and repressed mitophagy, whereas activation of the PINK1/Parkin pathways increased mitophagy activity and promoted HepG2 cell survival in the presence of matrine. Together, our data indicated that matrine promoted HepG2 cell apoptosis through a novel mechanism that acted via inhibiting mitophagy and the PINK1/Parkin pathways. This finding provides new insight into the molecular mechanism of matrine for treating liver cancer and offers a potential target to repress liver cancer progression by modulating mitophagy and the PINK1/Parkin pathways.     

PARK2-mediated mitophagy is involved in regulation of HBEC senescence in COPD pathogenesis

Cigarette smoke (CS)-induced mitochondrial damage with increased reactive oxygen species (ROS) production has been implicated in COPD pathogenesis by accelerating senescence. Mitophagy may play a pivotal role for removal of CS-induced damaged mitochondria, and the PINK1 (PTEN-induced putative kinase 1)-PARK2 pathway has been proposed as a crucial mechanism for mitophagic degradation. Therefore, we sought to investigate to determine if PINK1-PARK2-mediated mitophagy is involved in the regulation of CS extract (CSE)-induced cell senescence and in COPD pathogenesis. Mitochondrial damage, ROS production, and cell senescence were evaluated in primary human bronchial epithelial cells (HBEC). Mitophagy was assessed in BEAS-2B cells stably expressing EGFP-LC3B, using confocal microscopy to measure colocalization between TOMM20-stained mitochondria and EGFP-LC3B dots as a representation of autophagosome formation. To elucidate the involvement of PINK1 and PARK2 in mitophagy, knockdown and overexpression experiments were performed. PINK1 and PARK2 protein levels in lungs from patients were evaluated by means of lung homogenate and immunohistochemistry. We demonstrated that CSE-induced mitochondrial damage was accompanied by increased ROS production and HBEC senescence. CSE-induced mitophagy was inhibited by PINK1 and PARK2 knockdown, resulting in enhanced mitochondrial ROS production and cellular senescence in HBEC. Evaluation of protein levels demonstrated decreased PARK2 in COPD lungs compared with non-COPD lungs. These results suggest that PINK1-PARK2 pathway-mediated mitophagy plays a key regulatory role in CSE-induced mitochondrial ROS production and cellular senescence in HBEC. Reduced PARK2 expression levels in COPD lung suggest that insufficient mitophagy is a part of the pathogenic sequence of COPD.     

Human antigen R regulates hypoxia-induced mitophagy in renal tubular cells through PARKIN/BNIP3L expressions

Mitochondrial dysfunction contributes to the pathophysiology of acute kidney injury (AKI). Mitophagy selectively degrades damaged mitochondria and thereby regulates cellular homeostasis. RNA-binding proteins (RBPs) regulate RNA processing at multiple levels and thereby control cellular function. In this study, we aimed to understand the role of human antigen R (HuR) in hypoxia-induced mitophagy process in the renal tubular cells. Mitophagy marker expressions (PARKIN, p-PARKIN, PINK1, BNIP3L, BNIP3, LC3) were determined by western blot analysis. Immunofluorescence studies were performed to analyze mitophagosome, mitolysosome, co-localization of p-PARKIN/TOMM20 and BNIP3L/TOMM20. HuR-mediated regulation of PARKIN/BNIP3L expressions was determined by RNA-immunoprecipitation analysis and RNA stability experiments. Hypoxia induced mitochondrial dysfunction by increased ROS, decline in membrane potential and activated mitophagy through up-regulated PARKIN, PINK1, BNIP3 and BNIP3L expressions. HuR knockdown studies revealed that HuR regulates hypoxia-induced mitophagosome and mitolysosome formation. HuR was significantly bound to PARKIN and BNIP3L mRNA under hypoxia and thereby up-regulated their expressions through mRNA stability. Altogether, our data highlight the importance of HuR in mitophagy regulation through up-regulating PARKIN/BNIP3L expressions in renal tubular cells.     

High glucose induces mitochondrial dysfunction and apoptosis in human retinal pigment epithelium cells via promoting SOCS1 and Fas/FasL signaling

Diabetic retinopathy (DR) is one of the most serious complications of diabetes mellitus (DM), however, the contribution of high glucose (HG) or hyperglycemia to DR is far from fully understanding. In the present study, we examined the expression of Fas/FasL signaling and suppressors of cytokine signaling (SOCS)1 and 3 in HG-induced human retinal pigment epithelium cells (ARPE-19 cells). And then we investigated the regulatory role of both Fas and SOCS1 in HG-induced mitochondrial dysfunction and apoptosis. Results demonstrated that HG with more than 40 mM induced mitochondrial dysfunction via reducing mitochondrial membrane potential (MMP) and via inhibiting the Bcl-2 level, which is the upstream signaling of mitochondria in ARPE-19 cells. HG also upreuglated the Fas signaling and SOCS levels probably via promoting JAK/STAT signaling in ARPE-19 cells. Moreover, the exogenous Fas or entogenous overexpressed SOCS1 accentuated the HG-induced mitochondrial dysfunction and apoptosis, whereas the knockdown of either Fas or SOCS1 reduced the HG-induced mitochondria dysfunction and apoptosis. Thus, the present study confirmed that both Fas/FasL signaling and SOCS1 promoted the HG-induced mitochondrial dysfunction and apoptosis. These results implies the key regulatory role of Fas signaling and SOCS in DR.     

PINK1/Parkin-mediated mitophagy promotes apelin-13-induced vascular smooth muscle cell proliferation by AMPKα and exacerbates atherosclerotic lesions

Aberrant proliferation of vascular smooth muscle cells (VSMC) is a critical contributor to the pathogenesis of atherosclerosis (AS). Our previous studies have demonstrated that apelin-13/APJ confers a proliferative response in VSMC, however, its underlying mechanism remains elusive. In this study, we aimed to investigate the role of mitophagy in apelin-13-induced VSMC proliferation and atherosclerotic lesions in apolipoprotein E knockout (ApoE-/-) mice. Apelin-13 enhances human aortic VSMC proliferation and proliferative regulator proliferating cell nuclear antigen expression in dose and time-dependent manner, while is abolished by APJ antagonist F13A. We observe the engulfment of damage mitochondria by autophagosomes (mitophagy) of human aortic VSMC in apelin-13 stimulation. Mechanistically, apelin-13 increases p-AMPKα and promotes mitophagic activity such as the LC3I to LC3II ratio, the increase of Beclin-1 level and the decrease of p62 level. Importantly, the expressions of PINK1, Parkin, VDAC1, and Tom20 are induced by apelin-13. Conversely, blockade of APJ by F13A abolishes these stimulatory effects. Human aortic VSMC transfected with AMPKα, PINK1, or Parkin and subjected to apelin-13 impairs mitophagy and prevents proliferation. Additional, apelin-13 not only increases the expression of Drp1 but also reduces the expressions of Mfn1, Mfn2, and OPA1. Remarkably, the mitochondrial division inhibitor-1(Mdivi-1), the pharmacological inhibition of Drp1, attenuates human aortic VSMC proliferation. Treatment of ApoE-/- mice with apelin-13 accelerates atherosclerotic lesions, increases p-AMPKα and mitophagy in aortic wall in vivo. Finally, PINK1-/- mutant mice with apelin-13 attenuates atherosclerotic lesions along with defective in mitophagy. PINK1/Parkin-mediated mitophagy promotes apelin-13-evoked human aortic VSMC proliferation by activating p-AMPKα and exacerbates the progression of atherosclerotic lesions.     

PINK1 Is Dispensable for Mitochondrial Recruitment of Parkin and Activation of Mitophagy in Cardiac Myocytes

Myocyte function and survival relies on the maintenance of a healthy population of mitochondria. The PINK1/Parkin pathway plays an important role in clearing defective mitochondria via autophagy in cells. However, how the PINK1/Parkin pathway regulates mitochondrial quality control and whether it coordinates with other mitophagy pathways are still unclear. Therefore, the objective of this study was to investigate the effect of PINK1-deficiency on mitochondrial quality control in myocytes. Using PINK1-deficient (PINK1-/-) mice, we found that Parkin is recruited to damaged cardiac mitochondria in hearts after treatment with the mitochondrial uncoupler FCCP or after a myocardial infarction even in the absence of PINK1. Parkin recruitment to depolarized mitochondria correlates with increased ubiquitination of mitochondrial proteins and activation of mitophagy in PINK1-/- myocytes. In addition, induction of mitophagy by the atypical BH3-only protein BNIP3 is unaffected by lack of PINK1. Overall, these data suggest that Parkin recruitment to depolarized cardiac mitochondria and subsequent activation of mitophagy is independent of PINK1. Moreover, alternative mechanisms of Parkin activation and pathways of mitophagy remain functional in PINK1-/- myocytes and could compensate for the PINK1 deficiency.     

Dual role of USP30 in controlling basal pexophagy and mitophagy

USP30 is an integral protein of the outer mitochondrial membrane that counteracts PINK1 and Parkin‐dependent mitophagy following acute mitochondrial depolarisation. Here, we use two distinct mitophagy reporter systems to reveal tonic suppression by USP30, of a PINK1‐dependent component of basal mitophagy in cells lacking detectable Parkin. We propose that USP30 acts upstream of PINK1 through modulation of PINK1‐substrate availability and thereby determines the potential for mitophagy initiation. We further show that a fraction of endogenous USP30 is independently targeted to peroxisomes where it regulates basal pexophagy in a PINK1‐ and Parkin‐independent manner. Thus, we reveal a critical role of USP30 in the clearance of the two major sources of ROS in mammalian cells and in the regulation of both a PINK1‐dependent and a PINK1‐independent selective autophagy pathway.     

Genome-wide RNAi screen identifies ATPase inhibitory factor 1 (ATPIF1) as essential for PARK2 recruitment and mitophagy

Mitochondrial dysfunction is a hallmark of aging and numerous human diseases, including Parkinson disease (PD). Multiple homeostatic mechanisms exist to ensure mitochondrial integrity, including the selective autophagic program mitophagy, that is activated during starvation or in response to mitochondrial dysfunction. Following prolonged loss of potential across the inner mitochondrial membrane (ΔΨ), PTEN-induced putative kinase 1 (PINK1) and the E3-ubiquitin ligase PARK2 work in the same pathway to trigger mitophagy of dysfunctional mitochondria. Mutations in PINK1 and PARK2, as well as PARK7/DJ-1, underlie autosomal recessive Parkinsonism and impair mitochondrial function and morphology. In a genome-wide RNAi screen searching for genes that are required for PARK2 translocation to the mitochondria, we identified ATPase inhibitory factor 1 (ATPIF1/IF1) as essential for PARK2 recruitment and mitophagy in cultured cells. During uncoupling, ATPIF1 promotes collapse of ΔΨ and activation of the PINK-PARK2 mitophagy pathway by blocking the ATPase activity of the F₁-F_o ATP synthase. Restoration of ATPIF1 in Rho0 cells, which lack mtDNA and a functional electron transport chain, lowers ΔΨ and triggers PARK2 recruitment. Our findings identified ATPIF1 and the ATP synthase as novel components of the PINK1-PARK2 mitophagy pathway and provide genetic evidence that loss of ΔΨ is an essential trigger for mitophagy.