Role Of PINK1 Research Articles

Formoterol attenuated mitochondrial dysfunction in rotenone-induced Parkinson's disease in a rat model: Role of PINK-1/PARKIN and PI3K/Akt/CREB/BDNF/TrKB axis

β2-adrenoreceptors (β2AR have been identified recently as regulators of the α-synuclein gene (SNCA), one of the key milieus endorsed in injury of dopamine neurons in Parkinson's disease (PD). Accumulation of α-synuclein leads to mitochondrial dysfunction via downregulation of mitophagy proteins (PINK-1 and PARKIN) and inhibition of mitochondria biogenesis (PGC-1α) along with an increase in the master inflammatory regulator NF-κB p65 production that provokes neurodegeneration and diminishes neuroprotective signaling pathway (PI3k/Akt/CREB/BDNF). Recently, formoterol exhibited a promising neuroprotective effect against neurodegenerative conditions associated with brain inflammation. Therefore, the present investigation aims to unveil the possible neuroprotective activity of formoterol, β2AR agonist, against rotenone-induced PD in rats. Rats received rotenone (1.5 mg/kg; s.c.) every other day for 3 weeks and cured with formoterol (25 μg/kg/day; i.p.) 1 hr. after rotenone administration, starting from day 11. Formoterol treatment succeeded in upregulating β2-adrenoreceptor expression in PD rats and preserving the function and integrity of dopaminergic neurons as witnessed by enhancement of muscular performance in tests, open field, grip strength-meter, and Rotarod, besides the increment in substantia nigra and striatal tyrosine hydroxylase immunoexpression. In parallel, formoterol boosted mitophagy by activation of PINK1 and PARKIN and preserved mitochondrial membrane potential. Additionally, formoterol stimulated the neuro-survival signaling axis via stimulation of PI3k/pS473-Akt/pS133-CREB/BDNF cascade to attenuate neuronal loss. Noteworthy formoterol reduces neuro-inflammatory status by decreasing NFκBp65 immunoexpression and TNF-α content. Finally, formoterol's potential as a stimulant therapy of mitophagy via the PINK1/PARKIN axis and regulation of mitochondrial biogenesis by increasing PGC-1α to maintain mitochondrial homeostasis along with stimulation of PI3k/Akt/CREB/BDNF axis.

PINK1 deficiency impairs osteoblast differentiation through aberrant mitochondrial homeostasis

BackgroundPTEN-induced kinase 1 (PINK1) is a serine/threonine-protein kinase in mitochondria that is critical for mitochondrial quality control. PINK1 triggers mitophagy, a selective autophagy of mitochondria, and is involved in mitochondrial regeneration. Although increments of mitochondrial biogenesis and activity are known to be crucial during differentiation, data regarding the specific role of PINK1 in osteogenic maturation and bone remodeling are limited.MethodsWe adopted an ovariectomy model in female wildtype and Pink1−/− mice. Ovariectomized mice were analyzed using micro-CT, H&E staining, Masson’s trichrome staining. RT-PCR, western blot, immunofluorescence, alkaline phosphatase, and alizarin red staining were performed to assess the expression of PINK1 and osteogenic markers in silencing of PINK1 MC3T3-E1 cells. Clinical relevance of PINK1 expression levels was determined via qRT-PCR analysis in normal and osteoporosis patients.ResultsA significant decrease in bone mass and collagen deposition was observed in the femurs of Pink1−/− mice after ovariectomy. Ex vivo, differentiation of osteoblasts was inhibited upon Pink1 downregulation, accompanied by impaired mitochondrial homeostasis, increased mitochondrial reactive oxygen species production, and defects in mitochondrial calcium handling. Furthermore, PINK1 expression was reduced in bones from patients with osteoporosis, which supports the practical role of PINK1 in human bone disease.ConclusionsIn this study, we demonstrated that activation of PINK1 is a requisite in osteoblasts during differentiation, which is related to mitochondrial quality control and low reactive oxygen species production. Enhancing PINK1 activity might be a possible treatment target in bone diseases as it can promote a healthy pool of functional mitochondria in osteoblasts.

Vps13D functions in a Pink1-dependent and Parkin-independent mitophagy pathway.

Defects in autophagy cause problems in metabolism, development, and disease. The autophagic clearance of mitochondria, mitophagy, is impaired by the loss of Vps13D. Here, we discover that Vps13D regulates mitophagy in a pathway that depends on the core autophagy machinery by regulating Atg8a and ubiquitin localization. This process is Pink1 dependent, with loss of pink1 having similar autophagy and mitochondrial defects as loss of vps13d. The role of Pink1 has largely been studied in tandem with Park/Parkin, an E3 ubiquitin ligase that is widely considered to be crucial in Pink1-dependent mitophagy. Surprisingly, we find that loss of park does not exhibit the same autophagy and mitochondrial deficiencies as vps13d and pink1 mutant cells and contributes to mitochondrial clearance through a pathway that is parallel to vps13d. These findings provide a Park-independent pathway for Pink1-regulated mitophagy and help to explain how Vps13D regulates autophagy and mitochondrial morphology and contributes to neurodegenerative diseases.

Abstract 2400: PINK1 is a novel regulator of mitochondria and iron metabolism in colon cancer

Abstract Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover and cellular health. In particular, PINK and Parkin mediate one of the canonical mitophagy pathways. The mechanisms behind PINK1-Parkin mediated mitophagy is well established, yet its role in cancer remains equivocal. Based on differential gene expression analyses on colon cancer patients, higher PINK1 expressions are correlated with worsened survival. To confirm increased mitophagy in colon cancer cells, we assessed lysosomal proteome under nutrient stress and observed an enrichment of mitochondrial peptides. Then, we generated inducible shRNA knockdown (KD) models for PINK1 in a panel of colon cancer cell lines. Preliminary data suggest loss of PINK1 hinders colon cancer cell growth and induces cell death. We showed that PINK1 KD hinders mitophagy assessed by decreased phospho-ubiquitin, a marker of PINK1 activity. We observed high levels of basal mitophagy in colon cancer cells, and PINK1 KD can efficiently inhibit mitophagy using a fluorescent mitophagy reporter. Interestingly, we demonstrated PINK KD disrupts mitochondria dynamics by decreasing MFN2 protein level, suggesting mitochondrial fusion could be inhibited. In summary, we demonstrated colon cancer cells have high levels of mitophagy and PINK1 KD decreases mitophagy and disrupts mitochondria network. Mitochondria are important organelles that integrates cellular metabolic pathways. With PINK1's role as an overall mitochondrial health sensor, we hypothesized disrupting PINK1 could affect mitochondria metabolism in colon cancer cells. We demonstrated alterations in mitochondrial metabolites as assessed by metabolomics, NAD/NADH imbalance, membrane hyperpolarization, and decreased oxygen consumption rate. It is known that TCA cycle and electron transport chain require iron sulfur clusters (ISC) to maintain proper function. Although the exact quantification of mitochondrial iron fraction is elusive, some studies suggested that mitochondria could harbor up to 20% of cellular iron. These evidences suggest mitochondria play pivotal roles in cellular iron homeostasis. Upon PINK1 KD, we saw an accumulation of cytosolic and mitochondrial iron storage protein, ferritin. Interestingly, we also observed a decrease in ISC-containing ETC proteins upon PINK1 KD. We believe PINK1 could have a non-canonical function in regulating mitochondria iron and play a profound role in regulating cellular iron balance. Overall, these results indicate that PINK1 is essential for maintaining mitochondria metabolism and survival in colon cancers. Here, we showcased that PINK has a unique role in regulating mitochondrial metabolism. In conclusion, the growth and mitochondrial defects we observed from PINK1 KD cells suggest a potential oncogenic role of PINK1 in colorectal cancer that can be targeted for future therapeutic purposes. Citation Format: Brandon Chen, Samantha Devenport, Costas Lyssiotis, Yatrik Shah. PINK1 is a novel regulator of mitochondria and iron metabolism in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2400.

PINK1 alleviates thermal hypersensitivity in a paclitaxel-induced Drosophila model of peripheral neuropathy.

Paclitaxel is a representative anticancer drug that induces chemotherapy-induced peripheral neuropathy (CIPN), a common side effect that limits many anticancer chemotherapies. Although PINK1, a key mediator of mitochondrial quality control, has been shown to protect neuronal cells from various toxic treatments, the role of PINK1 in CIPN has not been investigated. Here, we examined the effect of PINK1 expression on CIPN using a recently established paclitaxel-induced peripheral neuropathy model in Drosophila larvae. We found that the class IV dendritic arborization (C4da) sensory neuron-specific expression of PINK1 significantly ameliorated the paclitaxel-induced thermal hyperalgesia phenotype. In contrast, knockdown of PINK1 resulted in an increase in thermal hypersensitivity, suggesting a critical role for PINK1 in sensory neuron-mediated thermal nociceptive sensitivity. Interestingly, analysis of the C4da neuron morphology suggests that PINK1 expression alleviates paclitaxel-induced thermal hypersensitivity by means other than preventing alterations in sensory dendrites in C4da neurons. We found that paclitaxel induces mitochondrial dysfunction in C4da neurons and that PINK1 expression suppressed the paclitaxel-induced increase in mitophagy in C4da neurons. These results suggest that PINK1 mitigates paclitaxel-induced sensory dendrite alterations and restores mitochondrial homeostasis in C4da neurons and that improvement in mitochondrial quality control could be a promising strategy for the treatment of CIPN.

The Parkinson's disease gene PINK1 activates Akt via PINK1 kinase-dependent regulation of the phospholipid PI(3,4,5)P3.

Akt signalling is central to cell survival, metabolism, protein and lipid homeostasis, and is impaired in Parkinson's disease (PD). Akt activation is reduced in the brain in PD, and by many PD-causing genes, including PINK1 This study investigated the mechanisms by which PINK1 regulates Akt signalling. Our results reveal for the first time that PINK1 constitutively activates Akt in a PINK1-kinase dependent manner in the absence of growth factors, and enhances Akt activation in normal growth medium. In PINK1-modified MEFs, agonist-induced Akt signalling failed in the absence of PINK1, due to PINK1 kinase-dependent increases in PI(3,4,5)P3 at both plasma membrane and Golgi being significantly impaired. In the absence of PINK1, PI(3,4,5)P3 levels did not increase in the Golgi, and there was significant Golgi fragmentation, a recognised characteristic of PD neuropathology. PINK1 kinase activity protected the Golgi from fragmentation in an Akt-dependent fashion. This study demonstrates a new role for PINK1 as a primary upstream activator of Akt via PINK1 kinase-dependent regulation of its primary activator PI(3,4,5)P3, providing novel mechanistic information on how loss of PINK1 impairs Akt signalling in PD.This article has an associated First Person interview with the first author of the paper.

PINK1 and BECN1 relocalize at mitochondria-associated membranes during mitophagy and promote ER-mitochondria tethering and autophagosome formation

ABSTRACTMitophagy is a highly specialized process to remove dysfunctional or superfluous mitochondria through the macroautophagy/autophagy pathway, aimed at protecting cells from the damage of disordered mitochondrial metabolism and apoptosis induction. PINK1, a neuroprotective protein mutated in autosomal recessive Parkinson disease, has been implicated in the activation of mitophagy by selectively accumulating on depolarized mitochondria, and promoting PARK2/Parkin translocation to them. While these steps have been characterized in depth, less is known about the process and site of autophagosome formation upon mitophagic stimuli. A previous study reported that, in starvation-induced autophagy, the proautophagic protein BECN1/Beclin1 (which we previously showed to interact with PINK1) relocalizes at specific regions of contact between the endoplasmic reticulum (ER) and mitochondria called mitochondria-associated membranes (MAM), from which the autophagosome originates. Here we show that, following mitophagic stimuli, autophagosomes also form at MAM; moreover, endogenous PINK1 and BECN1 were both found to relocalize at MAM, where they promoted the enhancement of ER-mitochondria contact sites and the formation of omegasomes, that represent autophagosome precursors. PARK2 was also enhanced at MAM following mitophagy induction. However, PINK1 silencing impaired BECN1 enrichment at MAM independently of PARK2, suggesting a novel role for PINK1 in regulating mitophagy. MAM have been recently implicated in many key cellular events. In this light, the observed prevalent localization of PINK1 at MAM may well explain other neuroprotective activities of this protein, such as modulation of mitochondrial calcium levels, mitochondrial dynamics, and apoptosis.

<i>Pink</i>1 Rescues <i>Gal</i>4-Induced Developmental Defects in the <i>Drosophila</i> Eye

Parkinson disease pathology often includes the presence of ubiquitin-positive, α-synuclein-enriched inclusions in the remaining neurons. Pink1 (also identified as PARK6) encodes a serinethreonine kinase involved in mitochondrial protection that works with parkin to ubiquitinate various proteins, promoting mitophagy. The parkin protein works to tag cystolic proteins for degradation, and previous work in our laboratory has shown the ability of parkin to rescue a Gal4-induced phenotype. To further investigate the role of Pink1 in protection against toxic proteins, we have performed expression studies to determine the effects of increases and decreases in Pink1 on the Gal4-induced phenotype consisting of developmental defects in the Drosophila eye. Our results show that Pink1 is able to rescue the Gal4-induced phenotype, highlighting a protective role for Pink1 against toxic proteins. When expressing low levels of Gal4, reductions in Pink1 or parkin are not able to induce a phenotype. This suggests that Pink1 or parkin may counter Gal4 effects despite reductions, or that the effects of low level Gal4 may be alleviated by an alternative mechanism. Moreover, the Pink1 mechanism of action during differing types of cell stress, including degradation of toxic proteins, warrants further investigation.

Abstract 207: Unc-51-like Kinase 1 (ULK1) Plays A Crucial Role In Mitophagy In Cardiomyocytes

The mitochondrion is an essential organelle that supplies ATP in cardiomyocytes (CMs). However, damaged mitochondria are harmful via the production of reactive oxygen species and induction of apoptosis in pathological conditions. Therefore, quality of mitochondria should be controlled tightly through various mitochondrial quality control mechanisms. Mitochondrial autophagy (mitophagy) is considered an integral part of this mechanism, and recent investigations uncovered the role of PINK1 and Parkin in mitophagy. However, these observations were made under artificial conditions, such as over-expression of Parkin or treatment with CCCP, and thus the precise mechanism has not been fully elucidated in more pathophysiologically relevant conditions. Recent evidence suggests that mitophagy can take place independently of ATG7, a molecule essential for the conventional form of autophagy, and that this form of autophagy is ULK1-dependent. We investigated the role of ULK1 and ATG7 in mediating mitophagy using mitochondria-targeted Keima (Mito-Keima) in cultured rat neonatal CMs. Keima has a bimodal excitation spectrum peaking at 440 and 560 nm, corresponding to the neutral and acidic pH, respectively. In CMs transfected with Mito-Keima, the fluorescent dots with a high 560nm/440nm ratio represent the mitochondria incorporated into autolysosomes which indicate mitophagy. Here we report that ULK1 plays a more predominant role in glucose deprivation (GD) -induced mitophagy than ATG7. Control CMs exhibited 8.7±1.0 % of the area of high-ratio dots per cells after GD. Knockdown of ULK1 significantly reduced the area to 2.3±0.9 % in CMs after GD (p<0.01, vs sh-Control). The reduction was significantly greater in CMs with knockdown of ULK1 than that of ATG7 (7.0±1.6 %, p<0.05, sh-ULK1 vs sh-ATG7). In addition, knockdown of Beclin1 and Drp1 also significantly decreased the area of high-ratio dots (about 1.0 % and 0.5 %, respectively). Overexpression of ULK1 was sufficient to induce mitophagy without starvation, whereas that of ATG7 was not. These results suggest that ULK1, Beclin1 and Drp1 play an essential role in mediating GD-induced mitophagy in CMs.

Abstract 1117: Investigating the role of PTEN-induced kinase 1 in cancer development and pathogenesis

Abstract PTEN-induced kinase 1 (PINK1) was first identified in cancer cells as a gene up-regulated by overexpression of the central tumour suppressor, PTEN. Loss-of-function mutations in PINK1 were subsequently discovered to cause autosomal recessive Parkinson's disease (ARPD). While much research has focused on the proposed mechanism(s) through which loss of PINKI function causes neurodegeneration, some studies indicate a potential role for this serine/threonine kinase in cancer cell biology. PINK1 is known to be a pro-survival kinase, protecting cells from several stressors, and is a key controller of mitochondrial integrity, morphology and function. This study aimed to understand the function of PINK1 in the development of cancer. Using SV40 immortalised PINK1 -/- mouse embryonic fibroblasts (MEFs), we show that loss of PINK1 results in reduced cell proliferation and foci formation, as well as decreased invasiveness. Overexpression of human PINK1 in these cells rescues these phenotypes and increases migratory capacity, indicating a tumour promoting role for PINK1. Furthermore, PINK1 loss increases autophagy and levels of reactive oxygen species, as well as enhancing mitochondrial fission. Notably, we identify PINK1 as a novel controller of cell cycle progression events which are critical to tumourigenesis. These results indicate an important function for PINK1 in cancer cell development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1117. doi:1538-7445.AM2012-1117

Role of PINK1 in mitochondrial function and mitophagy in dopaminergic neuroblastoma cells (SH-SY5Y)

Role of PINK1 in mitochondrial function and mitophagy in dopaminergic neuroblastoma cells (SH-SY5Y)

Pink1 forms a multiprotein complex with Miro and Milton, linking Pink1 function to mitochondrial trafficking.

Recessive mutations in Pink1 lead to a selective degeneration of dopaminergic neurons in the substantia nigra that is characteristic of Parkinson disease. Pink1 is a kinase that is targeted in part to mitochondria, and loss of Pink1 function can alter mitochondrial morphology and dynamics, thus supporting a link between mitochondrial dysfunction and Parkinson disease etiology. Here, we report the unbiased identification and confirmation of a mitochondrial multiprotein complex that contains Pink1, the atypical GTPase Miro, and the adaptor protein Milton. Our screen also identified an interaction between Pink1 and Mitofilin. Based on previously established functions for Miro and Milton in the trafficking of mitochondria along microtubules, we postulate here a role for Pink1 in mitochondrial trafficking. Using subcellular fractionation, we show that the overexpression of Miro and Milton, both of which are known to reside at the outer mitochondrial membrane, increases the mitochondrial Pink1 pool, suggesting a function of Pink1 at the outer membrane. Further, we document that Pink1 expressed without a mitochondrial targeting sequence can still be targeted to a mitochondria-enriched subcellular fraction via Miro and Milton. The latter finding is important for the interpretation of a previously reported protective effect of Pink1 expressed without a mitochondrial targeting sequence. Finally, we find that Miro and Milton expression suppresses altered mitochondrial morphology induced by loss of Pink1 function in cell culture. Our findings suggest that Pink1 functions in the trafficking of mitochondria in cells.

PINK1 in mitochondrial function

Rare, inherited mutations causing familial forms of Parkinson's disease (PD) have provided much insight into some of the molecular mechanisms that underlie both the genetic and sporadic forms of the disease. The role of mitochondria in sporadic PD has been debated for a little over 20 years, since the identification of complex I deficiency in the substantia nigra pars compacta (SNpc) (1). However, it was the identification of loss-of-function recessive mutations in the PINK1 gene, encoding a mitochondrial, putative protein kinase, that reignited interest in the pathophysiology of mitochondria and their potential role in parkinsonism (PARK6) (2). In Drosophila models of PINK1, it has been shown that PINK1 and parkin (the PARK2 locus) (3) act, at least in part, in a common pathway. These studies strongly suggested a role for PINK1 in normal mitochondrial function and implied that parkin was downstream of PINK1. Nevertheless, the role of PINK1 in mammalian mitochondrial function remained unclear. The role of DJ-1 a third recessive parkinsonism locus (PARK7) (4) is also likely to relate to mitochondrial function (5), although whether it too maps to the same pathway as PINK and parkin is not yet clear. In any event, these genetic data clearly show an important role for the mitochondrion in the recessive parkinsonisms, although the extent to which these findings will be relevant to sporadic PD remains unclear (1, 6).