PTEN-induced Putative Kinase 1 Protein Research Articles

Effects of electroacupuncture on mitophagy mediated by SIRT3/PINK1/Parkin pathway in Parkinson's disease mice.

To observe the effects of electroacupuncture (EA) at "Fengfu"(GV16), "Taichong"(LR3), and "Zusanli"(ST36) on mitophagy mediated by silencing regulatory protein 3 (SIRT3)/ PTEN induced putative kinase 1 (PINK1)/PARK2 gene coding protein (Parkin) in the midbrain substantia nigra of Parkinson's disease (PD) mice, and to explore the potential mechanisms of EA in treating PD. C57BL/6 mice were randomly divided into the control, model, EA, and sham EA groups, with 12 mice in each group. The PD mouse model was established by intraperitoneal injection of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). The EA group received EA stimulation at GV16, LR3 and ST36, while the sham EA group received shallow needling 1 mm away from the above acupoints without electrical stimulation. The motor ability of mice in each group was evaluated using an open field experiment. Immunohistochemistry was used to detect the expression of tyrosine hydroxylase (TH) and α-synuclein (α-syn) in the substantia nigra of mice. The ultrastructure of neurons in substantia nigra was observed by transmission electron microscope (TEM). Immunofluorescence was used to detect the expression of the autophagy marker autophagy-associated protein light chain 3 (LC3). The expression levels of TH, α-syn, SIRT3, PINK1, Parkin, P62, Beclin-1, LC3Ⅱ mRNA and protein were detected by PCR and Western blot. Compared with the control group, mice in the model group showed a decrease in the total exercise distance, time, movement speed and times of crossing central region (P<0.01)；the positive expressions of TH and LC3 were decreased (P<0.01), while the positive expression of α-syn increased (P<0.01), accompanied by mitochondrial swelling, mitochondrial cristae fragmentation and decrease, and decreased lysosome count；the expression levels of TH, SIRT3, PINK1, Parkin, Beclin-1, and LC3Ⅱ mRNA and protein in the midbrain substantia nigra were decreased (P<0.01), while the expression levels of α-syn and P62 mRNA and protein were increased (P<0.01, P<0.05). Compared with the model group, the mice in EA group showed a significant increase in the total exercise distance, time, movement speed and times of crossing central region (P<0.01, P<0.05)；the positive expressions of TH and LC3 were increased (P<0.01, P<0.05), while the positive expression of α-syn was decreased (P<0.01), accompanied by an increase in mitochondrial count, appearance of autophagic va-cuoles, and a decrease in swelling, the expression levels of TH, SIRT3, PINK1, Parkin, Beclin-1 and LC3Ⅱ mRNA and protein in the midbrain substantia nigra were increased (P<0.01, P<0.05), while the mRNA and protein expression levels of α-syn and P62 were decreased (P<0.01)；the sham EA group showed an increase in the total exercise distance and time(P<0.05), with an increase in the positive expression of TH (P<0.05) and a decrease in the positive expression of α-syn (P<0.05)；some mitochondria exhibited swelling, and no autophagic vacuoles were observed；the protein expression levels of TH, SIRT3, Parkin and LC3Ⅱ were increased (P<0.01, P<0.05), and the expression levels of P62 mRNA, α-syn mRNA and protein were decreased (P<0.01, P<0.05), and LC3Ⅱ mRNA expression was increased (P<0.05). In comparison to the sham EA group, the EA group showed an extension in the total exercise time (P<0.01), the positive expression and mRNA expression levels of α-syn were decreased (P<0.01, P<0.05), while the expression levels of TH, SIRT3, PINK1, Parkin mRNA and SIRT3 protein were increased (P<0.05). EA at GV16, LR3, and ST36 can exert neuroprotective function and improve the motor ability of PD mice by activating the SIRT3/PINK1/Parkin pathway to enhance the expression of TH and reduce α-syn aggregation in the substantia nigra of PD mice.

Glycosides of Buyang Huanwu decoction inhibits inflammation associated with cerebral ischemia-reperfusion via the PINK1/Parkin mitophagy pathway

Ethnopharmacological relevanceA classic stroke formula is Buyang Huanwu Decoction (BYHWD), Glycosides are the pharmacological components found in BYHWD, which are utilized for the prevention and management of cerebral ischemia-reperfusion (CIR), as demonstrated in a previous study. Its neuroprotective properties are closely related to its ability to modulate inflammation, but its mechanism is as yet unclear. Aim of the studyA research was undertaken to investigate the impact of glycosides on the inflammation of CIR through the PTEN-induced putative kinase-1 (PINK1)/Parkin mitophagy pathway. Materials and methodsAnalyzing glycosides containing serum components was performed with ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS). Glycosides were applied to rat of Middle cerebral artery occlusion/reperfusion (MCAO/R) model and primary neural cell of Oxygen glucose deprivation/reperfusion (OGD/R) model. The neuroprotective effect and the regulation of mitophagy of glycosides were evaluated through neural damage and PINK1/Parkin mitophagy activation. Moreover, the assessment of the relationship between glycosides regulation of mitophagy and its anti-inflammatory effects subsequent to mitophagy blockade was conducted by examining neural damage, PINK1/Parkin mitophagy activation, and levels of pyroptosis. Results(1) It was observed that the administration of glycosides resulted in a decrease in neurological function scores, a reduction in cerebral infarction volume, an increase in mitochondrial autophagosome, and the maintenance of a high expression status of light chain 3 (LC3) II/LC3Ⅰ protein. Additionally, there was a significant inhibition of p62 protein expression and an enhancement of PINK1 and Parkin protein expression. Furthermore, it was found that the effect of glycosides at a dosage of 0.128 g · kg-1 was significantly superior to that of glycosides at a dosage of 0.064 g · kg-1. Notably, the neuroprotective effect and inhibition of pyroptosis protein of glycosides at a dosage of 0.128 g · kg-1 were attenuated when mitochondrial autophagy was blocked. (2)Glycosides repaired cellular morphological damage, enhanced cell survival, and reduced Lactate dehydrogenase (LDH) leakage, with glycosides (2.36 μg·mL-1 and 4.72 μg·mL−1) neuronal protection being the strongest. Glycosides (4.72 μg·mL−1) maintained LC3II/LC3Ⅰ protein high expression state, inhibited p62 protein expression, and promoted PINK1 and Parkin protein expression, which was stronger than glycosides (2.36 μg·mL-1). The blockade of mitophagy resulted in a reduction of neuroprotection and inhibition of pyroptosis protein exerted by glycosides. ConclusionGlycosides demonstrate the ability to hinder inflammation through the activation of the PINK1/Parkin mitophagy pathway, thereby leading to subsequent neuroprotective effects on CIR.

Phosphate and tension homology-induced kinase 1/Parkin signaling mediates cognitive dysfunction in sepsis-associated encephalopathy through activation of hippocampal mitochondrial autophagy

To investigate the effects of gene of phosphate and tension homology (PTEN)-induced putative kinase 1 (PINK1)/Parkin pathway on hippocampal mitophagy and cognitive function in mice with sepsis-associated encephalopathy (SAE) and its possible mechanism. A total of 80 male C57BL/6J mice were randomly divided into Sham group, cecal ligation puncture (CLP) group, PINK1 plasmid transfection pretreatment groups (p-PINK1+Sham group, p-PINK1+CLP group), empty vector plasmid transfection control group (p-vector+CLP group), with 16 mice in each group. The mice in CLP groups were treated with CLP to reproduce SAE models. The mice in the Sham groups were performed laparotomy only. Animals in the p-PINK1+Sham and p-PINK1+CLP groups were transfected with PINK1 plasmid through the lateral ventricle at 24 hours before surgery, while mice in the p-vector+CLP group were transfected with the empty plasmid. Morris water maze experiment was performed 7 days after CLP. The hippocampal tissues were collected, the pathological changes were observed under a light microscope after hematoxylin-eosin (HE) staining, and the mitochondrial autophagy was observed under a transmission electron microscopy after uranyl acetate and lead citrate staining. The expressions of PINK1, Parkin, Beclin1, interleukins (IL-6, IL-1β) and microtubule-associated protein 1 light chain 3 (LC3) were detected by Western blotting. Compared with the Sham group, CLP group mice in Morris water maze experiment had longer escape latency, shorter target quadrant residence time, and fewer times of crossing the platform at 1-4 days. Under the light microscope, the hippocampal structure of the mouse was injured, the neuronal cells were arranged in disorder, and the nuclei were pyknotic. Under the electron microscope, the mitochondria appeared swollen, round, and wrapped by bilayer or multilayer membrane structures. Compared with the Sham group, CLP group had higher expressions of PINK1, Parkin, Beclin1, LC3II/LC3I ratio, IL-6 and IL-1β in hippocampus, indicating that sepsis induced by CLP could activated inflammatory response and caused PINK1/Parkin-mediated mitophagy. Compared with the CLP group, p-PINK1+CLP group had shorter escape latencies, spent more time in the target quadrant and had more number of crossings in the target quadrant at 1-4 days. Under the light microscope, the hippocampal structures of mice was destroyed, the neurons were arranged disorderly, and the nuclei were pyknotic. Under transmission electron microscope, swollen and rounded mitochondria and mitochondrial structure wrapped by double membrane or multilayer membrane structure were observed. Compared with the CLP group, the levels of PINK1, Parkin, Beclin1 and LC3II/LC3 ratio in the p-PINK1+CLP group were significantly increased [PINK1 protein (PINK1/β-actin): 1.95±0.17 vs. 1.74±0.15, Parkin protein (Parkin/β-actin): 2.06±0.11 vs. 1.78±0.12, Beclin1 protein (Beclin1/β-actin): 2.11±0.12 vs. 1.67±0.10, LC3II/LC3I ratio: 3.63±0.12 vs. 2.27±0.10, all P < 0.05], while the levels of IL-6 and IL-1β were significantly decreased [IL-6 protein (IL-6/β-actin): 1.69±0.09 vs. 2.00±0.11, IL-1β protein (IL-1β/β-actin): 1.11±0.12 vs. 1.65±0.12, both P < 0.05], suggesting that overexpression of PINK1 protein could further activate mitophagy and reduce the inflammatory response caused by sepsis. There was no statistically significant difference in the above pathological changes and related indicators between Sham group and p-PINK1+Sham group, CLP group and p-vector+CLP group. PINK1 overexpression can further activate CLP-induced mitophagy by upregulating Parkin, thereby inhibiting inflammation response and alleviate cognitive function impairment in SAE mice.

Laminar Shear Stress Enhances Cytosolic PINK1 Expression: Increased Mitophagic Sensitivity Toward Mitochondrial Dysfunction

PURPOSEPhosphatase and tensin homolog (PTEN)‐induced putative kinase 1 (PINK1) is an essential molecule in mitophagy process and known to act as a cytoprotective protein in mammalian cells. Mutation or deficiency of PINK1 has been closely related to several disease conditions. The purpose of this study was to determine PINK1 expression levels and subcellular localization under exercise‐mimic laminar shear stress (LSS) condition in human aortic endothelial cells (HAECs) or in exercising mice, and its implication on endothelial homeostasis and cardiovascular disease (CVD) prevention.METHODS &amp; RESULTSFirst, we measured full‐length PINK1 (FL‐PINK1) mRNA and protein expression levels in HAECs under unidirectional laminar shear stress (LSS) at 15 dyne/cm2 for 48h. LSS significantly elevated both FL‐PINK1 mRNA and protein expressions in HAECs compared to static control. Mitochondrial fractionation assays showed a decrease in FL‐PINK1 accumulation in the mitochondria with a compensatory increase in the cytosolic FL‐PINK1 level under LSS. Confocal microscopic analysis confirmed these subcellular localization patterns suggesting downregulation of mitophagy induction. Also, mitophagy flux was decreased under LSS, determined by a mtKeima probe. Mitochondrial morphometric analysis and mitochondrial membrane potential (JC‐1) showed mitochondrial elongation and increased mitochondrial membrane potential, respectively, suggesting that an elevation of cytosolic PINK1 is not related to an immediate induction of mitophagy. Based on this observation, we further hypothesized that the elevation of the cytosolic pool of FL‐PINK1 would increase the sensitivity to mitophagic signals in pathological conditions. Preconditioned HAECs with LSS showed lower mtDNA lesions under angiotensin II stimulation (100 nM, 6h). Moreover, LSS‐preconditioned ECs showed rapid Parkin recruitment and mitophagy induction in response to mitochondrial toxin (i.e. CCCP) treatment compared to the control. We measured PINK1 expression at ECs of the thoracic aorta in exercised mice, a physiological LSS‐enhanced model, which was significantly elevated compared to sedentary animals. In addition, exercise‐preconditioned mice were more protective to angiotensin II‐induced mtDNA lesion formation in the mouse abdominal aorta than sedentary mice, suggesting a potential protective mechanism of exercise in a PINK1‐dependent manner.CONCLUSIONLSS increases a cytosolic pool of FL‐PINK1, which may elevate the mitophagic sensitivity toward dysfunctional mitochondria or activate other cytoprotective mechanisms in endothelial cells. Our data suggest that exercise may support mitochondrial homeostasis in vascular endothelial cells by enhancing PINK1‐dependent cell protection mechanisms.

PINK1-Dependent Mitophagy Regulates the Migration and Homing of Multiple Myeloma Cells via the MOB1B-Mediated Hippo-YAP/TAZ Pathway.

The roles of mitochondrial dysfunction in carcinogenesis remain largely unknown. The effects of PTEN‐induced putative kinase 1 (PINK1)‐dependent mitophagy on the pathogenesis of multiple myeloma (MM) are determined. The levels of the PINK1‐dependent mitophagy markers PINK1 and parkin RBR E3 ubiquitin protein ligase (PARK2) in CD138+ plasma cells are reduced in patients with MM and correlate with clinical outcomes in myeloma patients. Moreover, the induction of PINK1‐dependent mitophagy with carbonylcyanide‐m‐chlorophenylhydrazone (CCCP) or salinomycin, or overexpression of PINK1 leads to inhibition of transwell migration, suppression of myeloma cell homing to calvarium, and decreased osteolytic bone lesions. Furthermore, genetic deletion of pink1 accelerates myeloma development in a spontaneous X‐box binding protein‐1 spliced isoform (XBP‐1s) transgenic myeloma mouse model and in VK*MYC transplantable myeloma recipient mice. Additionally, treatment with salinomycin shows significant antimyeloma activities in vivo in murine myeloma xenograft models. Finally, the effects of PINK1‐dependent mitophagy on myeloma pathogenesis are driven by the activation of the Mps one binder kinase activator (MOB1B)‐mediated Hippo pathway and the subsequent downregulation of Yes‐associated protein (YAP)/transcriptional co‐activator with PDZ‐binding motif (TAZ) expression. These data provide direct evidence that PINK1‐dependent mitophagy plays a critical role in the pathogenesis of MM and is a potential therapeutic target.

Deletion of PINK1 Accelerates and Exacerbates High‐fat‐diet Induced Cardiomyopathy

RationaleMitochondrial dysfunction is a central event in the development of diabetic cardiomyopathy. Damaged mitochondria are a major source of reactive oxygen species (ROS) and pro‐apoptotic factors. Therefore, selective removal of dysfunctional mitochondria via autophagy (i.e., mitophagy) is an important mechanism to maintain mitochondrial homeostasis and preserve cell viability. Although mitochondrial dysfunction is implicated in diabetic cardiomyopathy, the molecular mechanisms by which diabetes impairs mitochondrial function remain elusive. PTEN‐induced putative kinase 1 (PINK1), a mitochondrial serine/threonine‐protein kinase encoded by the Pink1 gene, protects cells from stress‐induced mitochondrial dysfunction by activating mitophagy pathway.ObjectiveThis study was designed to evaluate the role of PINK1 in the development of cardiomyopathy in mice fed with high fat diet (HFD).ResultsWe observed that PINK1 protein levels were markedly reduced in HFD‐treated mouse heart. HFD‐treated mouse developed obvious cardiomyopathy, demonstrated by impaired diastolic function, which was evidenced by a decreased E/A‐wave ratio (E‐wave and A‐wave represent the left ventricle early filling and atrial contraction filling, respectively). Moreover, ejection fraction and fractional shortening were all decreased, indicating an impaired systolic function. The cardiac dysfunction was concurrent with increased cardiac fibrosis and apoptotic cells. Pink1−/− mouse also exhibited abnormal cardiac structure and function. Importantly, PINK1 ablation accelerated the development of diastolic cardiac dysfunction, as evidenced by a decreased E/A ratio, and exacerbated cardiomyopathy, as revealed by decreased ejection fraction, fractional shortening, E/A ratio, cardiomyocyte apoptosis and cardiac fibrosis. Autophagy‐dependent clearance of mitochondria was drastically reduced with a much more pronounced effect in Pink1−/− mouse, resulting in the accumulation of damaged mitochondria in the hearts. Thus, HFD feeding caused more severe mitochondrial injury, ROS generation, and apoptotic cell death in Pink1−/− mouse.ConclusionTaken together, our results suggested that PINK1‐mediated mitophagy plays an important role in the development of HFD‐induced cardiomyopathy.Support or Funding InformationThis work is supported by NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigation of key autophagy-and mitophagy-related proteins and gene expression in BALF cells from patients with IPF and RA-ILD.

Idiopathic pulmonary fibrosis (IPF) is a chronic and irreversible interstitial lung disease with a poor prognosis and limited therapeutic options. Over the past decade, research efforts have focused on the pathogenetic mechanisms involved in this enigmatic lung disease. Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease often complicated by the development of interstitial lung disease (ILD), leading to high mortality and morbidity. Autophagy is a process regulating the turnover of subcellular components and organelles, and represents a major cellular homeostatic mechanism. Recent evidence suggests a role of autophagy and mitochondrial dysfunction in the development of IPF, focusing on lung fibroblasts and epithelial cells. The aim of this study was to examine the mRNA levels of molecules involved inthe autophagy pathway in bronchoalveolar lavage fluid (BALF)-derived cellsfrom patients with IPF in comparison topatients with RA demonstrating lung involvement (ILD) by RT-qPCR. The significant upregulation of BECLIN1 was observed in patients with RA-ILD compared with those with IPF. Other genes involved in the autophagy pathway were also examined, such as Unc-51 like autophagy activating kinase 1 (ULK1), BCL2 interacting protein 3 (BNIP3) and p62. No differences in the mRNA expression levels of these genes were observed. As regards the selective degradation of mitochondria and mitophagy, similar PTEN-induced putative kinase 1 (PINK1) and PARKIN; E3 ubiquitin ligase (PRKN) expression, as well as PINK1 protein levels, were observed. On the whole, the findings of this study demonstrate an increased expression of BECLIN1 in BALF cells from patients with RA-ILD compared with those from patients with IPF, while similar levels in other key molecules implicating in the autophagy pathway were observed in patients with IPF and RA-ILD.

PTEN induced putative kinase 1 (PINK1) alleviates angiotensin II-induced cardiac injury by ameliorating mitochondrial dysfunction

BackgroundMitochondrial quality control is crucial to the development of angiotensin II (AngII)-induced cardiac hypertrophy. PTEN induced putative kinase 1 (PINK1) is rapidly degraded in normal mitochondria but accumulates in damaged mitochondria, triggering autophagy to protect cells. PINK1 mediates mitophagy in general, but whether PINK1 mediates AngII-induced mitophagy and the effects of PINK1 on AngII-induced injury are unknown. This study was designed to investigate the function of PINK1 in an AngII stimulation model and its regulation of AngII-induced mitophagy. MethodsWe studied the function of PINK1 in mitochondrial homeostasis in AngII-stimulated cardiomyocytes via RNA interference-mediated knockdown and adenovirus-mediated overexpression of the PINK1 protein. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, adenosine triphosphate (ATP) content, cell apoptosis rates and cardiomyocyte hypertrophy were measured. The expression of LC3B, Beclin1 and p62 was measured. Mitochondrial morphology was examined via electron microscopy. Mitophagy was detected by confocal microscopy based on the co-localization of lysosomes and mitochondria. Additionally, endogenous PINK1, phosphorylated PINK1, mito-PINK1, total Parkin, cyto-Parkin, mito-Parkin and phosphorylated Parkin protein levels were measured. ResultsCardiomyocytes untreated by AngII had very low levels of total and phosphorylated PINK1. However, in the AngII stimulation model, the MMP was decreased, and the levels of total and phosphorylated PINK1 were increased. After PINK1 was knocked down, Parkin translocation to the mitochondria was inhibited. Moreover, levels of phosphorylated Parkin were reduced, and autophagy markers were downregulated. MMP and ATP contents were further reduced, ROS production and the apoptotic rate were further increased, and myocardial hypertrophy was further aggravated compared with those in the AngII group. However, PINK1 overexpression promoted Parkin translocation and phosphorylation, autophagy markers were upregulated, and myocardial injury was reduced. In addition, the effects of PINK1 overexpression were reversed by autophagy inhibitors. ConclusionDecreased MMP induced by AngII maintains the stability of PINK1, causing PINK1 autophosphorylation. PINK1 activation promotes Parkin translocation and phosphorylation and increases autophagy to clear damaged mitochondria. Thus, PINK1/Parkin-mediated mitophagy has a compensatory, protective role in AngII-induced cytotoxicity.

RhoA Regulates PINK1 Accumulation and Mitophagy through PKD Activation

RhoA is a small GTPase that regulates myriad cellular events, including adhesion, migration, invasion, cell cycle progression, gene expression, and cellular protection. Our previous research demonstrated that expression of RhoA in the heart protects cardiomyoctes from ischemia/reperfusion injury. RhoA is activated by various GPCRs, including those for sphingosine‐1‐phosphate (S1P); activation of RhoA contributes to S1P‐mediated cardioprotection. We recently determined that RhoA activation in the heart increases mitochondrial autophagy (mitophagy), a process that eliminates compromised mitochondria and thereby diminishes induction of mitochondrial death pathways. The involvement of RhoA induced mitophagy in the cardioprotective response to RhoA has not been examined. The accumulation of PTEN‐induced putative kinase 1 (PINK1) at damaged mitochondria plays an important role in induction of mitophagy. Increasing RhoA expression through adenovirus in neonatal rat ventricular myocytes (NRVMs), or in the mouse heart by AAV9 RhoA injection, leads to increased PINK1 expression at mitochondria. Importantly, this also increases Parkin recruitment to mitochondria, ubiquitination of mitochondrial proteins, and induction of autophagosomes containing mitochondria. Interestingly, there is no concomitant mitochondrial membrane depolarization associated with these responses. qRT‐PCR analysis also showed no difference in PINK1 mRNA levels in control and RhoA‐expressing cells. Degradation of exogenously expressed PINK1 protein in the presence of cycloheximide was, however, greatly inhibited by RhoA expression. ROCK is one of the most established RhoA downstream effectors, but inhibition of ROCK did not affect PINK1 accumulation induced by RhoA. We previously showed that RhoA activates PKD to confer cardioprotection. Studies using siRNA‐mediated knockdown of PKD or pharmacological inhibition showed that this largely attenuated RhoA mediated PINK1 stabilization. These results suggest that RhoA increases PINK1 levels at mitochondria by preventing its degradation through PKD and in a mitochondrial membrane depolarization‐independent fashion. We plan to further investigate the functional significance of RhoA‐dependent PINK1 induction and mitophagy in cardioprotection against sustained ischemia in vivo (e.g. myocardial infarction).Support or Funding InformationThis work was supported by the ASPET SURF Program and NIH HL028143.

Heterozygous PINK1 p.G411S increases risk of Parkinson's disease via a dominant-negative mechanism.

SEE GANDHI AND PLUN-FAVREAU DOI101093/AWW320 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: It has been postulated that heterozygous mutations in recessive Parkinson's genes may increase the risk of developing the disease. In particular, the PTEN-induced putative kinase 1 (PINK1) p.G411S (c.1231G>A, rs45478900) mutation has been reported in families with dominant inheritance patterns of Parkinson's disease, suggesting that it might confer a sizeable disease risk when present on only one allele. We examined families with PINK1 p.G411S and conducted a genetic association study with 2560 patients with Parkinson's disease and 2145 control subjects. Heterozygous PINK1 p.G411S mutations markedly increased Parkinson's disease risk (odds ratio = 2.92, P = 0.032); significance remained when supplementing with results from previous studies on 4437 additional subjects (odds ratio = 2.89, P = 0.027). We analysed primary human skin fibroblasts and induced neurons from heterozygous PINK1 p.G411S carriers compared to PINK1 p.Q456X heterozygotes and PINK1 wild-type controls under endogenous conditions. While cells from PINK1 p.Q456X heterozygotes showed reduced levels of PINK1 protein and decreased initial kinase activity upon mitochondrial damage, stress-response was largely unaffected over time, as expected for a recessive loss-of-function mutation. By contrast, PINK1 p.G411S heterozygotes showed no decrease of PINK1 protein levels but a sustained, significant reduction in kinase activity. Molecular modelling and dynamics simulations as well as multiple functional assays revealed that the p.G411S mutation interferes with ubiquitin phosphorylation by wild-type PINK1 in a heterodimeric complex. This impairs the protective functions of the PINK1/parkin-mediated mitochondrial quality control. Based on genetic and clinical evaluation as well as functional and structural characterization, we established p.G411S as a rare genetic risk factor with a relatively large effect size conferred by a partial dominant-negative function phenotype.

Expression and purification of the kinase domain of PINK1 in Pichia pastoris

PTEN-induced putative kinase 1 (PINK1) is a Ser/Thr kinase that specifically localizes on the mitochondrial membrane. It cooperates with Parkin to regulate mitochondrial quality control. Mutations in PINK1 protein which account for 8–15% of Parkinson's disease (PD), are the second most common cause of early-onset Autosomal Recessive Parkinson's disease (AR-PD). The lack of methods for PINK1 heterologous expression and purification has slowed progress in the AR-PD research field. To pave the way for direct structural study of this important protein, in this study, we developed an efficient expression system of recombinant PINK1 kinase domain (rPINK1) using Pichia pastoris (P. pastoris). Our results showed that rPINK1 is best expressed in P. pastoris at 25 °C induction. Additionally, we determined that the optimal induction time was 72 h and the optimal induction methanol concentration was 1% for the expression of rPINK1 in P. pastoris. Subsequent purification by Ni affinity chromatography (Ni-NTA) and cation-exchange chromatography (Mono S) produced the protein with purity higher than 95%. The pure rPINK1 was active to phosphorylate ubiquitin in a substrate phosphorylation assay. Overall, these studies provide the first effective method for heterologous expression and purification of the rPINK1 with a high purity. These findings can help contribute to further researches on the interactions study and biochemical characterization of PINK1.

Cytoprotection against Hypoxic and/or MPP⁺ Injury: Effect of δ-Opioid Receptor Activation on Caspase 3.

The pathological changes of Parkinson’s disease (PD) are, at least partially, associated with the dysregulation of PTEN-induced putative kinase 1 (PINK1) and caspase 3. Since hypoxic and neurotoxic insults are underlying causes of PD, and since δ-opioid receptor (DOR) is neuroprotective against hypoxic/ischemic insults, we sought to determine whether DOR activation could protect the cells from damage induced by hypoxia and/or MPP+ by regulating PINK1 and caspase 3 expressions. We exposed PC12 cells to either severe hypoxia (0.5%–1% O2) for 24–48 h or to MPP+ at different concentrations (0.5, 1, 2 mM) and then detected the levels of PINK1 and cleaved caspase 3. Both hypoxia and MPP+ reduced cell viability, progressively suppressed the expression of PINK1 and increased the cleaved caspase 3. DOR activation using UFP-512, effectively protected the cells from hypoxia and/or MPP+ induced injury, reversed the reduction in PINK1 protein and significantly attenuated the increase in the cleaved caspase 3. On the other hand, the application of DOR antagonist, naltrindole, greatly decreased cell viability and increased cleaved caspase 3. These findings suggest that DOR is cytoprotective against both hypoxia and MPP+ through the regulation of PINK1 and caspase 3 pathways.

PINK1 alleviates palmitate induced insulin resistance in HepG2 cells by suppressing ROS mediated MAPK pathways

Oxidative stress is an important pathogenesis of insulin resistance (IR) and Type 2 diabetes mellitus (T2DM). Studies have shown that knockdown of PTEN-induced putative kinase 1 (PINK1) causes oxidative stress and mitophagy. In db/db mice, PINK1 protein level is down-regulated. However, little is known regarding the mechanism by which PINK1 modulates IR in response to reactive oxygen species (ROS) induced stress. In our study, PINK1 expression decreased during palmitate (PA) induced IR in HepG2 cells and the hepatic tissues of high fat diet (HFD) fed mice. Additionally, free fatty acids (FFAs) could increase ROS and suppress insulin signaling pathway, which was indicated by reduced phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3β (GSK-3β). In addition, insulin induced glucose uptake decreased and the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), two key gluconeogenic enzymes, was up-regulated after PA treatment. Intriguingly, PINK1 overexpression could lead to opposite results. Moreover, PA induced hepatic IR through C-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways, which were rescued by PINK1 overexpression. In summary, our results demonstrate that PINK1 promoted hepatic IR via JNK and ERK pathway in PA treated HepG2 cells, implying a novel molecular target for the therapy of diabetes.

Phosphatase and tensin homologue (PTEN)-induced putative kinase 1 reduces pancreatic β-cells apoptosis in glucotoxicity through activation of autophagy

Chronic elevated glucose is harmful to pancreatic β-cells, resulting in pancreatic β-cells dysfunction and apoptosis. Understanding the molecular mechanisms associated with β-cells survival is pivotal for the prevention of β-cells injury caused by glucotoxicity. The role of Phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1) in the fate of pancreatic β-cells constantly exposed to high glucose was studied. Sustained high glucose increased PINK1 protein expression both in rat pancreatic β-cells and INS-1 β-cells, and that this increase can be inhibited by PINK1 knockdown and further enhanced by PINK1 over-expression. PINK1 deficiency aggravated glucotoxicity-induced pancreatic β-cells apoptosis and inhibition of autophagy whereas PINK1 could reverse these adverse effects. This study provides fundamental data supporting the potential protective role of PINK1 as a new therapeutic target necessary to preserve β-cells survival under non-physiological hyperglycemia conditions.

PKA Regulates PINK1 Stability and Parkin Recruitment to Damaged Mitochondria through Phosphorylation of MIC60

A mitochondrial kinase, PTEN-induced putative kinase 1 (PINK1), selectively recruits the ubiquitin ligase Parkin to damaged mitochondria, which modifies mitochondria by polyubiquitination, leading to mitochondrial autophagy. Here, we report that treatment with an adenylate cyclase agonist or expression of protein kinase A (PKA) impairs Parkin recruitment to damaged mitochondria and decreases PINK1 protein levels. We identified a mitochondrial membrane protein, MIC60 (also known as mitofilin), as a PKA substrate. Mutational and mass spectrometric analyses revealed that the Ser528 residue of MIC60 undergoes PKA-dependent phosphorylation. MIC60 transiently interacts with PINK1, and MIC60 downregulation leads to a reduction in PINK1 and mislocalization of Parkin. Phosphorylation-mimic mutants of MIC60 fail to restore the defect in Parkin recruitment in MIC60-knocked down cells, whereas a phosphorylation-deficient MIC60 mutant facilitates the mitochondrial localization of Parkin. Our findingsindicate that PKA-mediated phosphorylation of MIC60 negatively regulates mitochondrial clearance that is initiated by PINK1 and Parkin.

NRF2 Regulates PINK1 Expression under Oxidative Stress Conditions.

Mutations of the PTEN-induced putative kinase 1 (PINK1) gene are a cause of autosomal recessive forms of Parkinson’s disease. Recent studies have revealed that PINK1 is an essential factor for controlling mitochondrial quality, and that it protects cells from oxidative stresses. Although there has been considerable progress in the elucidation of various aspects of PINK1 protein regulation such as activation, stability and degradation, the transcriptional regulation of PINK1 mRNA under stress conditions remains unclear. In this study, we found that nuclear factor (erythroid-derived 2)-like 2 (NRF2), an antioxidant transcription factor, regulates PINK1 expression under oxidative stress conditions. Damaged mitochondria arising from stress conditions induced NRF2-dependent transcription of the PINK1 gene through production of reactive oxygen species (ROS). Either an ROS scavenger or forced expression of KEAP1, a potent inhibitory partner to NRF2, restricted PINK1 expression induced by activated NRF2. Transcriptionally up-regulated PINK1 diminished oxidative stress-associated cell death. The results indicate that PINK1 expression is positively regulated by NRF2 and that the NRF2-PINK1 signaling axis is deeply involved in cell survival.

Novel P-TEN-induced putative kinase 1 (PINK1) variant in Indian Parkinson’s disease patient

Loss-of-function mutation in PINK1 is known for causing autosomal recessive early onset Parkinsonism accounting approximately 6.5% of PD cases. Recently, PINK1 has also been shown to cause Parkinson’s disease (PD) in eastern India. Present study is aimed to see its contribution in north-Indian PD patients. A total of 106 PD patients and 60 ethnically matched healthy controls were included in the study. All the patients were screened for mutation in PINK1 by direct DNA sequence analysis of the PCR amplicons covering all exons and exon–intron boundaries. Identified novel variant was reconfirmed by DNA sequencing of 10 randomly selected TA clones containing the variant amplicon. In vitro functional assay of the mutant protein was performed by transfecting COS-7 cell line with wild type and mutant (created by site-directed-mutagenesis) cDNA construct of PINK1 fused to N′ terminal GFP followed by western blot analysis. Two potentially pathogenic, one being novel (p.Q267X) and 6 other apparently non-pathogenic variants were identified. Western blot analysis reveals production of truncated PINK1 fusion protein of ∼55kDa in p.Q267X mutant instead of 82/93kDa of wild type PINK1 fusion protein (molecular weight of GFP is ∼27kDa). Our study concludes that PINK1 variants are prevalent for causing Parkinson’s disease (PD) in India, as revealed by the occurrence of 1.8% (2/106) in PD patients from north Indian population. The novel homozygous variant of PINK1 (c.799C>T) reported here is the plausible cause for disease manifestation in this patient. Future study, however, would be helpful to understand the functional mechanism how this premature PINK1 protein (p.Q267X) responds to cellular stress leading to the PD pathophysiology.

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.

Early-onset Parkinson's disease due to PINK1 p.Q456X mutation--clinical and functional study.

Recessive mutations in the PTEN-induced putative kinase 1 (PINK1) gene cause early-onset Parkinson's disease (EOPD). The clinical phenotype of families that have this PINK1-associated disease may present with different symptoms, including typical PD. The loss of the PINK1 protein may lead to mitochondrial dysfunction, which causes dopaminergic neuron death. The clinical phenotypes of a large Polish family with EOPD and an identified PINK1 homozygous nonsense mutation were assessed. Ubiquitination and degradation of mitochondrial parkin substrates as well as mitochondrial bioenergetics were investigated as direct functional readouts for PINK1's kinase activity in biopsied dermal fibroblasts. A four-generation family was genealogically evaluated. Genetic screening identified two affected subjects who were both homozygous carriers of the pathogenic PINK1 p.Q456X substitution. Both patients presented with dystonia and gait disorders at symptom onset. Seven heterozygous mutation carriers remained unaffected. Functional studies revealed that the PINK1 p.Q456X protein is non-functional in activating the downstream ubiquitin ligase parkin and priming the ubiquitination of its substrates, and that the RNA levels of PINK1 were significantly reduced. The PINK1 p.Q456X mutation leads to a decrease in mRNA and a loss of protein function. The foot dystonia and gait disorders seen at disease onset in affected members of our family, which were accompanied by parkinsonism had a similar clinical presentation to what has been described in previous reports of PINK1 mutation carriers.

Abstract 19756: Association of Increased PTEN-Induced Putative Kinase 1 (PINK1) Expression and Oxidative Damage in Gastrocnemius Myofibers of Patients with Peripheral Arterial Disease

Introduction: Oxidative damage (OxD) determined as protein adducts of 4-hydroxy-2-nonenal (HNE) is increased in gastrocnemius myofibers of patients with peripheral arterial disease (PAD) and correlates with disease progression. Mitochondria in PAD gastrocnemius exhibit structural damage and respiratory dysfunction; however, the extent of mitochondrial damage and its relationship to OxD have not been determined. PINK1 is a glycoprotein normally expressed in low abundance on the mitochondrial external membrane. Mitochondrial damage signaled as loss of ion potential across the external membrane produces a marked increase in PINK1, which initiates mitophagy. In this study, we tested the hypothesis that mitochondrial damage is 1) prevalent among gastrocnemius myofibers of PAD patients and 2) linked to OxD. Methods: Gastrocnemius biopsies were collected from five PAD patients with claudication and five controls matched on the basis of age, gender, smoking history and type II diabetes. Biopsy specimens were fixed in methacarn, embedded in paraffin, sectioned and mounted to glass slides. Fluorescence labeled PINK1 protein and HNE adducts in individual myofibers, were quantified by quantitative fluorescence microscopy and expressed as mean pixel intensity in gray scale units (gsu) corrected for background. Results: PINK1 protein expression was increased (p=0.008) in PAD (mean ± S.E.M.; 494.78 ± 0.02 gsu) compared to control myofibers (102.45 ± 0.02 gsu) of the gastrocnemius. PINK1 was increased in 93% of the PAD myofibers. The association of increased (greater than two standard deviations above the control means) PINK1 expression and HNE adducts was determined for PAD myofibers. HNE adducts were increased in 67% of a random sample of PAD fibers expressing increased PINK1. Conclusions: Increased expression of PINK1 protein in the gastrocnemius myofibers of PAD patients with claudication, has established the presence of irreversible mitochondrial damage in a large proportion of myofibers. Elevation of OxD in 67% of PAD myofibers expressing increased PINK1 links OxD and mitochondrial damage. The data are consistent with a contribution of OxD to myofibers in PAD gastrocnemius, by damaged mitochondria.