Parkin Expression Research Articles

Upregulation of TIPE1 in tubular epithelial cell aggravates diabetic nephropathy by disrupting PHB2 mediated mitophagy

Renal tubular epithelial cells (RTECs) are one of the most mitochondria-rich cell types, and are thus vulnerable to mitochondrial dysregulation, which is defined as a pivotal event in tubular damage in diabetic nephropathy (DN). However, the underlying mechanisms remain largely unknown. Here, we investigated the role and mechanisms of tumor necrosis factor alpha-induced protein 8-like 1 (TNFAIP8L1/TIPE1) in high glucose (HG)-induced mitochondrial dysfunction in RTECs and DN progression. TIPE1 expression was predominantly upregulated in RTECs in patients with DN and mice with streptozotocin (STZ)-induced DN. Conditional knockout of Tipe1 in RTECs significantly decreased the urine protein creatinine ratio, renal tubular damage, epithelial-mesenchymal transition, and interstitial fibrosis in STZ-induced mice. RNA sequencing revealed that citrate cycle-related genes were positively enriched in the renal tissues of RTEC-specific Tipe1 knockout mice. Tipe1 deficiency upregulated ATP levels, mitochondrial membrane potential, and respiration rate, but downregulated mitochondrial ROS levels in RTECs. Furthermore, Tipe1 ablation led to enhanced mitophagy in RTECs, indicative of increased LC3II, PINK1, and Parkin expression, but decreased p62 expression in mitochondria. Mechanistically, mass spectrometry screening and co-immunoprecipitation assays revealed the interaction of TIPE1 with prohibitin 2 (PHB2), a crucial mitophagy receptor. Intriguingly, TIPE1 promoted the ubiquitination and proteasomal degradation of PHB2. Subsequently, PHB2 knockdown almost abrogated the improvement of Tipe1 loss on HG-induced tubular cell mitophagy and damage. Thus, TIPE1 disrupts mitochondrial homeostasis in RTECs and promotes tubular damage by destabilizing PHB2 under HG conditions. Hence, TIPE1 may act as a potential therapeutic target to prevent DN progression.

Impact of Organelle Transport Deficits on Mitophagy and Autophagy in Niemann-Pick Disease Type C.

Defective mitochondria are pathophysiological features of a number of neurodegenerative diseases. Here, we investigated mitochondrial dysfunction in the context of the rare lysosomal storage diseases Niemann–Pick disease type C1 and type C2 (NP-C1 and NP-C2). Mutations in either the NPC1 or NPC2 gene lead to cholesterol accumulation in late endosomes and lysosomes, resulting in impaired cholesterol homeostasis. The extent to which this may lead to mitochondrial dysfunction has been poorly studied so far. Therefore, we investigated the morphology, function, and transport of mitochondria, as well as their degradation via mitophagy, in a disease-associated human neural cell model of NP-C. By performing live cell imaging, we observed markedly reduced mitochondrial transport, although morphology and function were not appreciably altered. However, we observed a defective mitophagy induction shown by a reduced capability to elevate parkin expression and engulf mitochondria in autophagosomes after treatment with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). This was accompanied by defects in autophagy induction, exhibited by a hampered p62 expression and progression, shown by increased LC3BII levels and a defective fusion of autophagosomes and lysosomes. The latter might have been additionally influenced by the observed reduced lysosomal transport. Hence, we hypothesized that a reduced recycling of mitochondria contributes to the pathophysiology of NP-C.

Salvianolic acid B alleviates diabetic endothelial and mitochondrial dysfunction by down-regulating apoptosis and mitophagy of endothelial cells

ABSTRACT Endothelial dysfunction is a critical mediator in the pathogenesis of vascular complications of diabetes. Herein, this study was conducted to investigate the therapeutic effects of Salvianolic acid B (Sal B) on diabetes-induced endothelial dysfunction and the underlying mechanisms. Diabetic models were established both in db/db mice and high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs). Moreover, HUVECs were exposed to carbonyl cyanide m-chlorophenyl hydrazone (CCCP) to induce endothelial cell damage. Following Sal B treatment, pathological changes of thoracic aorta were investigated by hematoxylin and eosin, alcian blue (AB), elastic fiber, Masson, and reticular fiber staining. BCL2-associated X (BAX), B-cell lymphoma-2 (Bcl-2), Beclin1, Parkin and PTEN Induced Kinase 1 (Pink1) expression was detected by Western blot, immunohistochemistry, and immunofluorescence in thoracic aorta, HG- and CCCP-induced HUVECs. Cell scratch test, MitoTracker Red CMXRos staining and Flou-4 AM staining were separately presented to detect migration, mitochondrial activity and intracellular Ca2+ in HUVECs. Our results showed that Sal B significantly ameliorated hyperlipidemia, hyperglycemia, hyperinsulinemia, and insulin resistance in db/db mice. Furthermore, it significantly alleviated diabetes-induced vascular endothelial dysfunction according to histopathology analysis. In diabetic thoracic aorta, HG- and CCCP-induced HUVECs, Sal B distinctly increased Bcl-2 expression and reduced BAX, Beclin1, Parkin and Pink1 expression, thereby protecting endothelial cells from apoptosis and mitophagy. Moreover, Sal B markedly enhanced migration, mitochondrial activity and intracellular Ca2+ levels both in HG- and CCCP-induced HUVECs. Collectively, Sal B exhibited a potential to improve diabetes-induced endothelial and mitochondrial dysfunction through down-regulating apoptosis and mitophagy of endothelial cells. Abbreviations: DM: diabetes mellitus; T2DM: type 2 diabetes mellitus; Sal B: Salvianolic acid B; HG: high glucose; FBG: fasting blood glucose; TC: total cholesterol; TG: triglycerides; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; FINS: fasting insulin; HOMA-IR: homeostasis model assessment insulin resistance; QUICKI: quantitative insulin-sensitivity check index; H&E: hematoxylin and eosin; HUVECs: human umbilical vein endothelial cells; IHC: immunohistochemistry; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; FCM: flow cytometry; CCK-8: cell counting kit-8

A Novel Method for Identifying Parkin Binding Agents in Complex Preparations of Herbal Medicines.

Parkin is a crucial E3 ubiquitin ligase for initiating mitophagy through the PINK1/Parkin pathway. Regulating the expression and activity of parkin can remedy mitophagy and human disease. We developed an efficient method to isolate natural parkin ligands from herbal medicines by combining centrifugal ultrafiltration and liquid chromatography/mass spectrometry. The heterologous expression technology identified functionally active and pure parkin proteins. After evaluating the reliability of the method using DL-selenomethionine and DL-dithiothreitol as positive controls, this method was successfully applied to capture parkin ligands from Polygoni Cuspidati Rhizoma et Radix and Sophorae Flavescentis Radix. LC/MS identified seven novel parkin-targeting compounds, namely, 7,4′-dihydroxy-5-methoxy-8-(γ, γ-dimethylallyl)-flavanone, kushenol I, kurarinone, sophoraflavanone G, torachrysone-8-O-glucoside, apigenin, and emodin, supported by the molecular docking analysis. Five of the seven novel compounds (kushenol I, kurarinone, sophoraflavanone G, apigenin, and emodin) can activate parkin in in vitro autoubiquitination assays. Meanwhile, kushenol I and kurarinone had antisteatosis activity in fat emulsion-damaged human hepatocytes. These results confirmed the effectiveness of the method for identifying parkin ligands from complex preparations, useful to advance drug discovery from medicinal herbs.

Ginsenoside Rg_1 protects PC12 cells against Aβ-induced injury through promotion of mitophagy by PINK1/parkin activation

Amyloid β-protein(Aβ) deposition in the brain is directly responsible for neuronal mitochondrial damage of Alzheimer's disease(AD) patients. Mitophagy, which removes damaged mitochondria, is a vital mode of neuron protection. Ginsenoside Rg_1(Rg_1), with neuroprotective effect, has displayed promising potential for AD treatment. However, the mechanism underlying the neuroprotective effect of Rg_1 has not been fully elucidated. The present study investigated the effects of ginsenoside Rg_(1 )on the autophagy of PC12 cells injured by Aβ_(25-35) to gain insight into the neuroprotective mechanism of Rg_1. The autophagy inducer rapamycin and the autophagy inhi-bitor chloroquine were used to verify the correlation between the neuroprotective effect of Rg_1 and autophagy. The results showed that Rg_1 enhanced the viability and increased the mitochondrial membrane potential of Aβ-injured PC12 cells, while these changes were blocked by chloroquine. Furthermore, Rg_(1 )treatment increased the LC3Ⅱ/Ⅰ protein ratio, promoted the depletion of p62 protein, up-regulated the protein levels of PINK1 and parkin, and reduced the amount of autophagy adaptor OPTN, which indicated the enhancement of autophagy. After the silencing of PINK1, a key regulatory site of mitophagy, Rg_1 could not increase the expression of PINK1 and parkin or the amount of NDP52, whereas it can still increase the LC3Ⅱ/Ⅰ protein ratio and promote the depletion of OPTN protein which indicated the enhancement of autophagy. Collectively, the results of this study imply that Rg_1 can promote autophagy of PC12 cells injured by Aβ, and may reduce Aβ-induced mitochondrial damage by promoting PINK1-dependent mitophagy, which may be one of the key mechanisms of its neuroprotective effect.

Autophagy and protein aggregation as a mechanism of dopaminergic degeneration in a primary human dopaminergic neuronal model

The pathophysiology underlying the loss of dopaminergic neurons in Parkinson’s disease (PD) is unclear. A gap of knowledge in the molecular and cellular events leading to degeneration of the nigrostriatal DA system is a major barrier to the development of effective therapies for PD. 1-methyl-4-phenylpyridinium (MPP+) is used as a reliable in vitro model of PD in dopaminergic neurons; however, the molecular mechanisms that lead to cell death with this model are not fully understood. Additionally, there is a lack of translational in vitro models to fully understand progressive dopaminergic neurotoxicity. Here, we propose cultures of primary human dopaminergic neuronal precursor cells (HDNPCs) as a model to study progressive dopaminergic toxicity and neuronal damage in PD. We evaluated the concentration-response of MPP+ (0–10 mM) at 24 h, using cell viability and mitochondrial activity assays (LDH, XTT, Live/Dead staining, and MitoTracker). Based on concentration-response data, we chose two concentrations (1.0 and 2.5 mM) of MPP+ to evaluate markers of autophagy and dopaminergic status [tyrosine hydroxylase (TH)] after a 24-h exposure. Exposure to MPP+ induced cytotoxicity, reduced cell viability, and decreased mitochondrial activity. MPP+ at 1.0 and 2.5 mM also induced expression of lysosome-associated membrane protein 1 (LAMP-1) and increased the ratio of light chain 3 (LC3), LC3BII/LC3BI. The expression of TH also decreased. Furthermore, α-synuclein (α-SYN) and parkin were evaluated by immunofluorescence (IF) at 1.0 and 2.5 mM MPP+ after 24 h. A qualitative analysis revealed decreased parkin expression while α-SYN aggregation was observed in the cytoplasm and the nucleus. These data suggest that in HDNPCs MPP+ can cause cytotoxicity and neuronal damage. This damage may be mediated by autophagy, dopamine synthesis, and protein aggregation. The combination of HDNPCs and MPP+ may serve as valuable in vitro model of progressive dopaminergic neurotoxicity for research into potential treatments for PD.

Melatonin and verteporfin synergistically suppress the growth and stemness of head and neck squamous cell carcinoma through the regulation of mitochondrial dynamics.

The prevalence of head and neck squamous cell carcinoma (HNSCC) has continued to rise for decades. However, drug resistance to chemotherapeutics and relapse, mediated by cancer stem cells (CSCs), remains a significant impediment in clinical oncology to achieve successful treatment. Therefore, we focused on analyzing CSCs in HNSCC and demonstrated the effect of melatonin (Mel) and verteporfin (VP) on SCC-25 cells. HNSCC CSCs were enriched in the reactive oxygen species-low state and in sphere-forming cultures. Combination treatment with Mel and VP decreased HNSCC viability and increased apoptosis without causing significant damage to normal cells. Sphere-forming ability and stem cell population were reduced by co-treatment with Mel and VP, while mitochondrial ROS level was increased by the treatment. Furthermore, the expression of mitophagy markers, parkin and PINK1, was significantly decreased in the co-treated cells. Mel and VP induced mitochondrial depolarization and inhibited mitochondrial function. Parkin/TOM20 was localized near the nucleus and formed clusters of mitochondria in the cells after treatment. Moreover, Mel and VP downregulated the expression of markers involved in epithelial-mesenchymal transition and metastasis. The migration capacity of cells was significantly decreased by co-treatment with Mel and VP, accompanied by the down-regulation of MMP-2 and MMP-9 expression. Taken together, these results indicate that co-treatment with Mel and VP induces mitochondrial dysfunction, resulting in the apoptosis of CSCs. Mel and VP could thus be further investigated as potential therapies for HNSCC through their action on CSCs.

Abstract 14210: Impaired Mitophagy and Reduced Parkin Expression in Human Pulmonary Arterial Smooth Muscle Cells (PASMCs) in Pulmonary Arterial Hypertension (PAH)

Introduction: Mitophagy is the key mitochondrial quality control process of selective removal of damaged mitochondria which is necessary for maintaining cellular homeostasis. While studies have demonstrated that abnormalities in mitochondrial metabolism drive a hyperproliferative phenotype of pulmonary vasculature in PAH, there is limited understanding of mitophagy dysregulation in PAH. Recent studies have shown that hypoxia induced Parkin/Pink1-mediated mitophagy promotes vascular remodeling, however another report suggests that loss of Pink1 and reduced mitophagy in hypoxia promotes PASMC proliferation. The role and mechanisms of altered mitochondrial dynamics/mitophagy dysregulation in driving pulmonary vascular remodeling in PAH remain incompletely understood. Hypothesis: We hypothesized that impaired mitophagy and reduced Parkin expression may drive the apoptosis-resistant, hyperproliferative phenotype in PAH PASMCs. Methods: Mitophagy was assessed in human PASMCs from control donors and PAH patients stably expressing mtKeima protein and in lungs from hypoxia-exposed mtKeima transgenic mice. Parkin expression was assessed by Western blotting and immunocytochemistry. Immunofluorescence staining for Parkin was performed in lungs from mice exposed to chronic hypoxia. Human PASMCs from control donors were transfected with siControl and siParkin and assessed for proliferation and apoptosis by Ki67 and Annexin V/PI flow cytometry and PCNA expression. Results: PASMCs from PAH patients showed reduced KeimaRed vs. KeimaGreen ratio compared to control donors indicating reduced mitophagy i.e. further confirmed by decreased Parkin in PAH PASMCs. Parkin deficient PASMCs demonstrated more Ki67 and less Annexin V/PI positive cells and increased PCNA. Interestingly, lungs from hypoxia-exposed mtKeima transgenic mice showed increased KeimaRed vs. KeimaGreen ratio and increased Parkin in lungs of hypoxia-exposed mice. Conclusions: PAH PASMCs demonstrate impaired mitophagy and reduced Parkin expression; Parkin deficiency promotes proliferation and inhibits apoptosis in PASMCs. Our findings suggest that Parkin may regulate remodeling phenotypes in PASMCs and thus may represent a potential therapeutic target in PAH.

Globular adiponectin protects hepatocytes against intermittent hypoxia-induced injury via Pink1/Parkin-mediated mitophagy induction.

This study sought to determine the effect of Pink1/Parkin-mediated mitophagy on liver cells exposed to intermittent hypoxia (IH) and the roles of globular adiponectin (gAPN). The hepatocyte model of IH was established. Cell apoptosis was assessed using flow cytometry. Mitochondrial membrane potential (MMP) level was determined using JC-1, and mitophagy was assessed using a confocal laser. Mitochondrial injury associated protein levels of bax and bcl-2, and protein levels of Pink1 and Parkin were evaluated via western blotting. We downregulated Parkin expression by transfecting the cells with Parkin siRNA. Pink1 and Parkin protein levels, mitophagy, and cell apoptosis rate were high, while the MMP level and protein level ratio of bcl-2/bax were low in IH-treated hepatocyte. gAPN upregulated Pink1 and Parkin protein levels, MMP level, protein level ratio of bcl-2/bax, and mitophagy while it reduced the rate of cell apoptosis in IH-treated hepatocytes. Inhibiting Parkin expression significantly reduced mitophagy and increased mitochondrial injury andthe rate of hepatocyte apoptosis under IH or IH with gAPN. gAPN alleviated IH-induced mitochondrial injury and hepatocyte apoptosis by upregulating Pink1/Parkin-mediated mitophagy.

Pterostilbene alleviates liver ischemia/reperfusion injury via PINK1-mediated mitophagy

Hepatic ischemia/reperfusion (I/R) injury contributes to morbidity and mortality during liver resection or transplantation, with limited effective treatments available. Here, we investigated the potential benefits and underlying mechanisms of pterostilbene (Pt), a natural component of blueberries and grapes, in preventing hepatic I/R injury. Male C57BL/6 mice subjected to partial warm hepatic I/R and human hepatocyte cell line L02 cells exposed to anoxia/reoxygenation (A/R) were used as in vivo and in vitro models, respectively. Our findings showed that pretreatment with Pt ameliorated hepatic I/R injury by improving liver histology, decreasing hepatocyte apoptosis, and reducing plasma ALT and AST levels. Likewise, cell apoptosis, mitochondrial membrane dysfunction, and mitochondrial ROS overproduction in L02 cells triggered by the A/R challenge in vitro were reduced due to Pt administration. Mechanistically, Pt treatment efficiently enhanced mitophagy and upregulated PINK1, Parkin, and LC3B expression. Notably, the protective effect of Pt was largely abrogated after cells were transfected with PINK1 siRNA. Moreover, Pt pretreatment promoted hepatocyte proliferation and liver regeneration in the late phase of hepatic I/R. In conclusion, our findings provide evidence that Pt exerts hepatoprotective effects in hepatic I/R injury by upregulating PINK1-mediated mitophagy.

Abstract MP06: Insights Into The Role Of Anp In Hfpef During Ss Hypertension

Background: Atrial natriuretic peptide ( ANP) is a hormone produced primarily in the heart, which induces vasodilation and natriuresis. We showed previously that a knockout of ANP in Dahl SS rat results in hypertension and HFpEF phenotype. There is a gap in knowledge regarding the role of inflammation and mitochondrial function in HFpEF that accompanies SSH. We hypothesize that the ANP deficiency in SSH leads to pressure overload, which affects cardiac inflammation and mitochondrial function. Methods: At 8 weeks of age, male wild type (SS WT ) and Nppa knockout (ANP knockout, SS Nppa-/- ) Dahl SS rats were placed on normal salt ( NS , 0.4% NaCl) or a high salt ( HS , 4% NaCl) for 21 days. Tissues were harvested for histological and Western analysis. TEM was used to obtain electron micrographs. EPR spectroscopy was performed in snap-frozen tissues post injection of POBN. 2-way ANOVA was employed for statistical comparison. Results: Heart-to-body-weight ratio was higher in SS Nppa-/- rats (p<0.001), on NS and HS diet ( p <0.001). SS Nppa-/- rats displayed increases in cardiac fibrosis following HS diet ( p <0.01). EPR demonstrated that SS Nppa-/- hearts exhibit a trend for increased lipid peroxidation compared to SS WT . Cardiac cytokine profile demonstrated an increase in IL-1α, IL-1β, L-6, IL-17, TNF-α, sICAM-1, MIP-1α, MIP-3α, in the SS WT rats fed a HS diet vs NS diet, and lower levels of these in the HS-diet fed SS Nppa-/- rats vs SS WT . HS diet was sufficient to induce increases in mitochondrial area ( p <0.001), and decreases in density ( p 0.001), and overall health score ( p <0.001) in both SS WT and SS Nppa-/- rats, but no genotype-specific differences were reported. We observed changes in fission/fusion proteins: Opa1 expression was elevated in SS Nppa-/- vs SS WT ( p <0.01), and Parkin expression was lower in all rats on a HS diet ( p <0.05). Conclusions: Although SS Nppa-/- rats develop a HFpEF phenotype, exacerbated cardiac fibrosis and higher lipid peroxidation vs SS WT rats, inflammation and overall mitochondrial damage are diminished in the hearts of these animals at day 21 of the HS diet. Further studies are needed to elucidate the mechanisms by which inflammation may affect the development of HFpEF during SS hypertension in a cell-specific and time-dependent manner.

Propofol protects hippocampal neurons in sleep-deprived rats by inhibiting mitophagy and autophagy.

BackgroundSleep deprivation (SD) causes a disturbance in the cognitive function of rats. While propofol has a powerful sedative and hypnotic effect and is an antioxidant, its effect on the cognitive function of rats following SD remains unknown. The purpose of this study was to explore the protective effects of propofol on excessive autophagy and mitophagy in the hippocampus of rats after SD.MethodsAdult male rats were intraperitoneally injected with 30 mg/kg of propofol after 96 hours of SD. Then we evaluated the effect of propofol on the cognitive function of sleep deprived rats by the Morris water maze. Transmission electron microscopy, Western blotting, PCR, immunohistochemistry, autophagy enhancer and autophagy inhibitor were used to study the effect of propofol on hippocampal neurons of rat with excessive autophagy and mitophagy.ResultsThe behavioral experimental results of the Morris water maze showed that propofol improved the learning and memory ability of sleep-deprived rats. The expression of Beclin1, PINK1, parkin, p62, and LC3 protein increased significantly after sleep deprivation. While the intervention of propofol could significantly reduce the expression of these proteins, rapamycin treatment eliminated this effect.ConclusionsOur findings showed that propofol could reduce the impairment of learning and memory in sleep-deprived rats by inhibiting excessive autophagy and mitophagy in hippocampal neurons. This strategy may provide an application basis for the clinical use of propofol in patients with chronic insomnia.

Geranylgeraniol Increases Autophagy And Mitophagy Gene Expression In Soleus Of Rats With Diabetes

The autophagy and mitophagy (selective autophagy for mitochondria) processes are important in maintaining muscle homeostasis, e.g., removing damaged mitochondria. With diabetes, skeletal muscle autophagy decreases. Geranylgeraniol (GG) has been shown to reduce mitochondrial damage; however, the effect of GG on basal autophagy and mitophagy in diabetic rats is not known. PURPOSE: To determine the effect of GG on selective autophagy and mitophagy genes in rats with diabetes induced by a high-fat diet (HFD) with streptozotocin (STZ). METHODS: Sprague-Dawley rats were divided into three groups: normal diet (CON; n = 11), HFD with 35 mg/kg body weight of STZ (HFD; n = 9), and HFD/STZ with 800 mg/kg body weight of GG (GG; n = 9). On the 7th week, soleus muscles were collected and analyzed for gene expression of LC3A, LC3B, P62, PINK1, PARKIN, DRP, and MFN. Gene data were normalized to CON. RESULTS: A significant (p < 0.05) condition effect was found for autophagy (LC3A, LC3B, and P62) and mitophagy (PINK, DRP, and MFN) gene expression. For autophagy, HFD (0.14 ± 0.03-fold) had significantly lower LC3A than CON (1.00 ± 0.22-fold), lower LC3B (0.67 ± 0.26-fold vs. 2.37 ± 0.72-fold) and P62 (0.44 ± 0.13-fold vs. 1.70 ± 0.35-fold) than GG. HFD trended to have lower LC3A than GG (0.14 ± 0.03-fold vs. 0.86 ± 0.26-fold; p = 0.066) while CON trended to have lower LC3B than GG (1.00 ± 0.07-fold vs. 2.37 ± 0.72-fold; p = 0.078). For mitophagy, HFD (0.32 ± 0.07-fold) and GG (0.51 ± 0.15-fold) had significantly lower PINK1 than CON (1.00 ± 0.13-fold). Further, HFD had lower MFN than GG (0.31 ± 0.08-fold vs. 1.46 ± 0.25-fold) and lower DRP than CON (0.48 ± 0.11-fold vs. 1.00 ± 0.15-fold). HFD trended to have lower PARKIN than GG (0.46 ± 0.08-fold vs. 1.38 ± 0.38-fold; p = 0.053) with no difference between GG and CON. CONCLUSION: In comparison to HFD, GG consumption improved the basal transcript abundance of the selective skeletal muscle autophagic and mitophagic genes, which could indicate an increased capacity to remove damaged mitochondria in diabetic rats.

Histone deacetylase inhibitors inhibit cervical cancer growth through Parkin acetylation-mediated mitophagy

Parkin, an E3 ubiquitin ligase, plays a role in maintaining mitochondrial homeostasis through targeting damaged mitochondria for mitophagy. Accumulating evidence suggests that the acetylation modification of the key mitophagy machinery influences mitophagy level, but the underlying mechanism is poorly understood. Here, our study demonstrated that inhibition of histone deacetylase (HDAC) by treatment of HDACis activates mitophagy through mediating Parkin acetylation, leading to inhibition of cervical cancer cell proliferation. Bioinformatics analysis shows that Parkin expression is inversely correlated with HDAC2 expression in human cervical cancer, indicating the low acetylation level of Parkin. Using mass spectrometry, Parkin is identified to interact with two upstream molecules, acetylase acetyl-CoA acetyltransferase 1 (ACAT1) and deacetylase HDAC2. Under treatment of suberoylanilide hydroxamic acid (SAHA), Parkin is acetylated at lysine residues 129, 220 and 349, located in different domains of Parkin protein. In in vitro experiments, combined mutation of Parkin largely attenuate the interaction of Parkin with PTEN induced putative kinase 1 (PINK1) and the function of Parkin in mitophagy induction and tumor suppression. In tumor xenografts, the expression of mutant Parkin impairs the tumor suppressive effect of Parkin and decreases the anticancer activity of SAHA. Our results reveal an acetylation-dependent regulatory mechanism governing Parkin in mitophagy and cervical carcinogenesis, which offers a new mitophagy modulation strategy for cancer therapy.

LRPPRC regulates metastasis and glycolysis by modulating autophagy and the ROS/HIF1-α pathway in retinoblastoma

Retinoblastoma (RB) is the most common intraocular tumor among children. Leucine-rich pentatricopeptide repeat (PPR)-motif-containing protein (LRPPRC), a suppressor gene of autophagy, has been proven to play a regulatory role in tumor progression. However, little is known about functional roles and mechanisms of LRPPRC in RB progression. First, we performed a detailed analysis for RB and normal control. The expression of LRPPRC in the RB tissues was significantly higher than that in normal tissues. Moreover, LRPPRC suppression could repress tumor cell migration, invasion, glycolysis, and reactive oxygen species (ROS)/hypoxia-inducible factor-1α (HIF1-α) pathway activation by mediating autophagy. Furthermore, overexpression of HIF1-α partially reversed the above changes induced by LRPPRC knockdown. The regulation of LRPPRC on tumor metastasis and glycolysis was also validated by a xenograft tumor assay. In summary, LRPPRC could regulate metastasis and glycolysis of RB by mediating autophagy suppression and further activating the ROS/HIF1-α pathway, and LRPPRC could be a promising prognostic biomarker for RB.

Protective effects of melatonin on the white matter damage of neonatal rats by regulating NLRP3 inflammasome activity.

The aim of the study was to investigate the protective effects and relevant mechanisms of melatonin on the white matter damage (WMD) caused by endotoxin and ischemic hypoxia in neonatal rats. Seventy-two female neonatal rats (postnatal day 3) were randomly divided into the sham, melatonin-treated, and control groups (n = 24 for each group). The periventricular white matter was collected to evaluate the WMD and apoptosis. In addition, the reactive oxygen species (ROS) level was measured. The expression levels of nucleotide-binding domain-like receptor protein 3 (NLRP3), interleukin (IL)-1β, IL-18, pink1, parkin, Toll-like receptor (TLR)-4, and nuclear factor (NF)-κB were detected. Hematoxylin and eosin and terminal-deoxynucleoitidyl transferase mediated nick end labeling staining showed that the WMD, as well as cell degeneration, necrosis, and apoptosis in the control group, were more severe than those in the melatonin-treated group. Endotoxin and ischemic hypoxia upregulated the expression of NLRP3 and downstream inflammatory factors such as IL-1β and IL-18, which could be reversed by melatonin treatment. Melatonin increased mitochondrial autophagy marker (pink1 and parkin) expression in the white matter and reduced ROS production. Moreover, melatonin-reduced TLR4 and NF-κB expression. Melatonin can inhibit the hyperactivity of NLRP3 inflammasomes by enhancing mitochondrial autophagy and inhibiting TLR4/NF-κB pathway activity. Thus, melatonin may be a promising treatment for alleviating the WMD caused by endotoxin and ischemic hypoxia in neonatal rats.

Protective effect of mitophagy against aluminum-induced MC3T3-E1 cells dysfunction

Aluminum (Al) is a ubiquitous environmental metal toxicant that causes osteoblast (OB) damage which leads to Al-related bone diseases. Mitochondrial damage plays a key role in Al-related bone diseases, and while mitophagy can clear damaged mitochondria and improve OB function, the relationship between mitophagy and Al-induced OB dysfunction is unknown. To explore the role of mitophagy in Al-induced OB dysfunction in vitro, we used 2 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 0.4 μM Cyclosporin A (CsA) to activate and inhibit mitophagy, respectively. MC3T3-E1 cells were treated with 0 mM AlCl3 (control group); 2 mM AlCl3 (Al group); 2 μM CCCP (CCCP group); 2 μM CCCP and 2 mM AlCl3 (CCCP + Al group); 0.4 μM CsA (CsA group); 0.4 μM CsA and 2 mM AlCl3 (CsA + Al group). The results showed that Al induced ultrastructural and functional impairment of MC3T3-E1 cells. Compared to the Al group, mitophagy activation caused mitochondrial membrane potentials to collapse, up-regulated PINK1, Parkin, and LC3 expression, down-regulated p62 expression, and increased mitophagosome numbers. Mitophagy activation also reduced Al-induced oxidative stress and MC3T3-E1 cell functional damage, as seen in improvement in cell viability, cellular calcium and phosphorus contents, and collagen I, osteocalcin, and bone alkaline phosphatase gene expression. Mitophagy inhibition had the opposite effects on activation. Overall, these results show that mitophagy can protect against Al-induced OB dysfunction.

PINK1/Parkin-mediated mitophagy inhibits warangalone-induced mitochondrial apoptosis in breast cancer cells.

Breast cancer is the most common malignancy in women all around the world, especially in many countries in Asia. However, antitumor drugs with unique curative effects and low toxic side-effects have not been found yet. Warangalone is an isoflavone extracted from the Cudrania tricuspidata fruit, and is reported to possess anti-inflammatory and anti-cancer activity. The purpose of this study was to determine the effects of warangalone on breast cancer cells. In this study, we found that warangalone decreased the viability of breast cancer cells by increasing the generation of reactive oxygen species (ROS) resulting in mitochondrial damage and decreased mitochondrial membrane potential (MMP). Warangalone induced mitochondrial apoptosis by increasing the BAX/BCL-2 ratio. Warangalone activated mitophagy via upregulation of PINK1 and Parkin expression and co-localization. The combination of warangalone and autophagy inhibitors or PINK1 siRNA increased the degree of cell apoptosis compared to treatment with warangalone alone. Warangalone damages mitochondria via ROS, thereby triggering PINK1/Parkin-mediated mitophagy and inducing mitochondrial apoptosis. However, autophagy/mitophagy protects against warangalone-induced mitochondrial apoptosis. A combination of warangalone and autophagy/mitophagy inhibitors may be a potential treatment for breast cancer.

DUSP1 overexpression attenuates renal tubular mitochondrial dysfunction by restoring Parkin-mediated mitophagy in diabetic nephropathy

Diabetic nephropathy (DN) is the primary cause of end-stage renal disease, and renal tubular cell dysfunction contributes to the pathogenesis of many kidney diseases. Our previous study demonstrated that dual-specificity protein phosphatase 1 (DUSP1) reduced hyperglycemia-mediated mitochondrial damage; however, its role in hyperglycemia-driven dysfunction of tubular cells is still not fully understood. In this study, we found that DUSP1 is reduced in human proximal tubular epithelial (HK-2) cells under high-glucose conditions. DUSP1 overexpression in HK-2 cells partially restored autophagic flux, improved mitochondrial function, and reduced reactive oxygen species generation and cell apoptosis under high-glucose conditions. Surprisingly, overexpressing DUSP1 abolished the decrease in mitochondrial parkin expression caused by high-glucose stimulation. In addition, knockdown of parkin in HK-2 cells reversed the effects of DUSP1 overexpression on mitophagy and apoptosis under high-glucose conditions. Overall, these data indicate that DUSP1 plays a defensive role in the pathogenesis of DN by restoring parkin-mediated mitophagy, suggesting that it may be considered a prospective therapeutic strategy for the amelioration of DN.

Parkin-independent mitophagy via Drp1-mediated outer membrane severing and inner membrane ubiquitination.

Here, we report that acute reduction in mitochondrial translation fidelity (MTF) causes ubiquitination of the inner mitochondrial membrane (IMM) proteins, including TRAP1 and CPOX, which occurs selectively in mitochondria with a severed outer mitochondrial membrane (OMM). Ubiquitinated IMM recruits the autophagy machinery. Inhibiting autophagy leads to increased accumulation of mitochondria with severed OMM and ubiquitinated IMM. This process occurs downstream of the accumulation of cytochrome c/CPOX in a subset of mitochondria heterogeneously distributed throughout the cell ("mosaic distribution"). Formation of mosaic mitochondria, OMM severing, and IMM ubiquitination require active mitochondrial translation and mitochondrial fission, but not the proapoptotic proteins Bax and Bak. In contrast, in Parkin-overexpressing cells, MTF reduction does not lead to the severing of the OMM or IMM ubiquitination, but it does induce Drp1-independent ubiquitination of the OMM. Furthermore, high-cytochrome c/CPOX mitochondria are preferentially targeted by Parkin, indicating that in the context of reduced MTF, they are mitophagy intermediates regardless of Parkin expression. In sum, Parkin-deficient cells adapt to mitochondrial proteotoxicity through a Drp1-mediated mechanism that involves the severing of the OMM and autophagy targeting ubiquitinated IMM proteins.