PINK1 Knockout Research Articles

PTEN-Induced Putative Kinase 1 Dysfunction Accelerates Synucleinopathy.

Background:Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive Parkinson’s disease (PD) and contribute to the risk of sporadic PD. However, the relationship between PD-related PINK1 mutations and alpha-synuclein (α-syn) aggregation—a main pathological component of PD—remains unexplored.Objective:To investigate whether α-syn pathology is exacerbated in the absence of PINK1 after α-syn preformed fibril (PFF) injection in a PD mouse model and its effects on neurodegeneration.Methods:In this study, 10-week-old Pink1 knockout (KO) and wildtype (WT) mice received stereotaxic unilateral striatal injection of recombinant mouse α-syn PFF. Then, α-syn pathology progression, inflammatory responses, and neurodegeneration were analyzed via immunohistochemistry, western blot analysis, and behavioral testing.Results:After PFF injection, the total α-syn levels significantly increased, and pathological α-syn was markedly aggregated in Pink1 KO mice compared with Pink1 WT mice. Then, earlier and more severe neuronal loss and motor deficits occurred. Moreover, compared with WT mice, Pink1 KO mice had evident microglial/astrocytic immunoreactivity and prolonged astrocytic activation, and a higher rate of protein phosphatase 2A phosphorylation, which might explain the greater α-syn aggravation and neuronal death.Conclusion:The loss of Pink1 function accelerated α-syn aggregation, accumulation and glial activation, thereby leading to early and significant neurodegeneration and behavioral impairment in the PD mouse model. Therefore, our findings support the notion that PINK1 dysfunction increases the risk of synucleinopathy.

PINK1-mediated Drp1S616 phosphorylation modulates synaptic development and plasticity via promoting mitochondrial fission

Dynamic change of mitochondrial morphology and distribution along neuronal branches are essential for neural circuitry formation and synaptic efficacy. However, the underlying mechanism remains elusive. We show here that Pink1 knockout (KO) mice display defective dendritic spine maturation, reduced axonal synaptic vesicles, abnormal synaptic connection, and attenuated long-term synaptic potentiation (LTP). Drp1 activation via S616 phosphorylation rescues deficits of spine maturation in Pink1 KO neurons. Notably, mice harboring a knockin (KI) phosphor-null Drp1S616A recapitulate spine immaturity and synaptic abnormality identified in Pink1 KO mice. Chemical LTP (cLTP) induces Drp1S616 phosphorylation in a PINK1-dependent manner. Moreover, phosphor-mimetic Drp1S616D restores reduced dendritic spine localization of mitochondria in Pink1 KO neurons. Together, this study provides the first in vivo evidence of functional regulation of Drp1 by phosphorylation and suggests that PINK1-Drp1S616 phosphorylation coupling is essential for convergence between mitochondrial dynamics and neural circuitry formation and refinement.

Pink1 promotes cell proliferation and affects glycolysis in breast cancer.

Phosphatase and tensin homolog (PTEN)-induced kinase 1 (Pink1) is regarded as a tumor suppressor and plays an important role in cancer cell biology, while relatively few studies have examined Pink1 in breast cancer, especially in vivo. The aims of this study were to investigate Pink1 expression in different subtypes of breast cancer tissues and cell lines and explore the effect of Pink1 protein on breast cancer. In these experiments, Pink1 expression was investigated using the tissue microarray immunohistochemistry (TMA-IHC) method in 150 samples of breast cancer tissues with different subtypes, and strong staining of Pink1 was significantly correlated with the histological grade of breast cancer (p = 0.015). In addition, Pink1 messenger RNA (mRNA) displayed much higher expression levels in breast cancer cell lines than in MCF-10A breast epithelial cells. Moreover, proteomic data obtained by isobaric tags for relative and absolute quantification (iTRAQ) showed that Pink1 deletion induced a distinct proteomic profile in MDA-MB-231 cells, and enrichment analysis showed that the differential proteins were concentrated mainly in energy metabolism-related pathways. Moreover, Seahorse XF analysis showed that Pink1 knockout reduced the glycolytic ability of MDA-MB-231 cells. Our findings indicated that Pink1 may be an indicator of malignancy in breast cancer and that it presents oncogenic properties in breast cancer, which raises another perspective for understanding the regulatory role of Pink1 in breast cancer.

Pitavastatin activates mitophagy to protect EPC proliferation through a calcium-dependent CAMK1-PINK1 pathway in atherosclerotic mice

Statins play a major role in reducing circulating cholesterol levels and are widely used to prevent coronary artery disease. Although they are recently confirmed to up-regulate mitophagy, little is known about the molecular mechanisms and its effect on endothelial progenitor cell (EPC). Here, we explore the role and mechanism underlying statin (pitavastatin, PTV)-activated mitophagy in EPC proliferation. ApoE−/− mice are fed a high-fat diet for 8 weeks to induce atherosclerosis. In these mice, EPC proliferation decreases and is accompanied by mitochondrial dysfunction and mitophagy impairment via the PINK1-PARK2 pathway. PTV reverses mitophagy and reduction in proliferation. Pink1 knockout or silencing Atg7 blocks PTV-induced proliferation improvement, suggesting that mitophagy contributes to the EPC proliferation increase. PTV elicits mitochondrial calcium release into the cytoplasm and further phosphorylates CAMK1. Phosphorylated CAMK1 contributes to PINK1 phosphorylation as well as mitophagy and mitochondrial function recover in EPCs. Together, our findings describe a molecular mechanism of mitophagy activation, where mitochondrial calcium release promotes CAMK1 phosphorylation of threonine177 before phosphorylation of PINK1 at serine228, which recruits PARK2 and phosphorylates its serine65 to activate mitophagy. Our results further account for the pleiotropic effects of statins on the cardiovascular system and provide a promising and potential therapeutic target for atherosclerosis.

Inducing Energetic Switching Using Klotho Improves Vascular Smooth Muscle Cell Phenotype.

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox’d Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival. Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells. Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis. Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.

An Inducible and Vascular Smooth Muscle Cell-Specific Pink1 Knockout Induces Mitochondrial Energetic Dysfunction during Atherogenesis.

DNA damage and mitochondrial dysfunction are defining characteristics of aged vascular smooth muscle cells (VSMCs) found in atherosclerosis. Pink1 kinase regulates mitochondrial homeostasis and recycles dysfunctional organelles critical for maintaining energetic homeostasis. Here, we generated a new vascular-specific Pink1 knockout and assessed its effect on VSMC-dependent atherogenesis in vivo and VSMC energetic metabolism in vitro. A smooth muscle cell-specific and MHC-Cre-inducible flox’d Pink1f/f kinase knockout was made on a ROSA26+/0 and ApoE−/− C57Blk6/J background. Mice were high fat fed for 10 weeks and vasculature assessed for physiological and pathogical changes. Mitochondrial respiratory activity was then assessed in wild-type and knockout animals vessels and isolated cells for their reliance on oxidative and glycolytic metabolism. During atherogenesis, we find that Pink1 knockout affects development of plaque quality rather than plaque quantity by decreasing VSMC and extracellular matrix components, collagen and elastin. Pink1 protein is important in the wild-type VSMC response to metabolic stress and induced a compensatory increase in hexokinase II, which catalyses the first irreversible step in glycolysis. Pink1 appears to play an important role in VSMC energetics during atherogenesis but may also provide insight into the understanding of mitochondrial energetics in other diseases where the regulation of energetic switching between oxidative and glycolytic metabolism is found to be important.

Dopaminergic but not cholinergic neurodegeneration is correlated with gait disturbances in PINK1 knockout rats

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by gait dysfunction in later stages of the disease. PD hallmarks include a decrease in stride length, run speed, and swing time; an increase in stride time, stance time, and base of support; dopaminergic degeneration in the basal ganglia; and cholinergic degeneration in the pedunculopontine nucleus (PPN). A progressive animal model of PD is needed to identify treatments for gait dysfunction. The goal of this study was to quantify progressive gait degeneration in PTEN-induced putative kinase 1 knockout (P1KO) rats and investigate neurodegeneration as potential underlying mechanisms. Gait analysis was performed in male P1KO and wild-type rats at 5 and 8 months of age and immunohistochemical analysis at 8 months. Multiple parameters of volitional gait were measured using a runway system. P1KO rats exhibited significant gait deficits at 5 months, but not 8 months. Gait abnormalities improved over time suggesting compensation during behavioral testing. At 8 months a 15% loss of tyrosine hydroxylase (TH) in the striatum, a 27% loss of TH-positive cells in the substantia nigra pars compacta, and no significant loss of choline acetyltransferase-positive cells in the PPN was found. Dopaminergic cell loss may contribute to gait deficits in the P1KO model, but not cholinergic cell loss. The P1KO rat with the greatest dopamine loss exhibited the most pronounced PD-like gait deficits, highlighting variability within the model. Further analysis is required to determine the suitability of the P1KO rat as a progressive model of gait abnormalities in PD.

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Cytosolic PINK1 orchestrates protein translation during proteasomal stress by phosphorylating the translation elongation factor eEF1A1.

Mutations in PINK1 (PTEN-induced putative kinase 1) are associated with autosomal recessive early-onset Parkinson's disease. Full-length PINK1 (PINK1-l) has been extensively studied in mitophagy; however, the functions of the short form of PINK1 (PINK1-s) remain poorly understood. Here, we report that PINK1-s is recruited to ribosome fractions after short-term inhibition of proteasomes. The expression of PINK1-s greatly inhibits protein synthesis even without proteasomal stress. Mechanistically, PINK1-s phosphorylates the translation elongation factor eEF1A1 during proteasome inhibition. The expression of the phosphorylation mimic mutation eEF1A1S396E rescues protein synthesis defects and cell viability caused by PINK1 knockout. These findings implicate an important role for PINK1-s in protecting cells against proteasome stress through inhibiting protein synthesis.

Increased glutamate transmission onto dorsal striatum spiny projection neurons in Pink1 knockout rats

Loss-of-function PTEN Induced Kinase 1 (PINK1) mutations cause early-onset familial Parkinson's disease (PD) with similar clinical and neuropathological characteristics as idiopathic PD. While Pink1 knockout (KO) rats have mitochondrial dysfunction, locomotor deficits, and α-synuclein aggregates in several brain regions such as cerebral cortex, dorsal striatum, and substantia nigra, the functional ramifications on synaptic circuits are unknown. Using whole cell patch clamp recordings, we found a significant increase in the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) onto striatal spiny projection neurons (SPNs) in Pink1 KO rats at ages 4 and 6 months compared to wild-type (WT) littermates, suggesting increased excitability of presynaptic neurons. While sEPSC amplitudes were also increased at 2 and 4 months, no changes were observed in AMPAR/NMDAR ratio or receptor expression. Further analysis revealed increased glutamate release probability and decreased recovery of the synaptic vesicle pool following a train of stimulation in Pink1 KO rats. Ultrastructural analysis revealed increased excitatory and inhibitory synapse number and increased levels of presynaptic α-synuclein, while the number and structure of striatal mitochondria appeared normal. Lastly, we found that Pink1 KO rats have altered striatal dopamine tone, which together with the abnormal α- synuclein distribution and dysfunctional mitochondria, could contribute to the increase in excitatory transmission. Together, these studies show that PINK1 is necessary for normal glutamatergic transmission onto striatal SPNs and reveal possible mechanisms underlying striatal circuit dysfunction in PD.

The PINK1/PARK2/optineurin pathway of mitophagy is activated for protection in septic acute kidney injury

Sepsis is the major cause of acute kidney injury (AKI) associated with high mortality rates. Mitochondrial dysfunction contributes to the pathophysiology of septic AKI. Mitophagy is an important mitochondrial quality control mechanism that selectively eliminates damaged mitochondria, but its role and regulation in septic AKI remain largely unknown. Here, we demonstrate the induction of mitophagy in mouse models of septic AKI induced by lipopolysaccharide (LPS) treatment or by cecal ligation and puncture. Mitophagy was also induced in cultured proximal tubular epithelial cells exposed to LPS. Induction of mitophagy under these experimental setting was suppressed by pink1 or park2 knockout, indicating the role of the PINK1/PARK2 pathway of mitophagy in septic AKI. In addition, sepsis induced more severe kidney injury and cell apoptosis in pink1 or park2 knockout mice than in wild-type mice, suggesting a beneficial role of mitophagy in septic AKI. Furthermore, in cultured renal tubular cells treated with LPS, knockdown of pink1 or park2 inhibited mitochondrial accumulation of the autophagy adaptor optineurin (OPTN) and silencing Optn inhibited LPS-induced mitophagy. Taken together, these findings suggest that the PINK1/PARK2 pathway of mitophagy plays an important role in mitochondrial quality control, tubular cell survival, and renal function in septic AKI.

HO-1/PINK1 Regulated Mitochondrial Fusion/Fission to Inhibit Pyroptosis and Attenuate Septic Acute Kidney Injury.

Background Endotoxin-associated acute kidney injury (AKI), a disease characterized by marked oxidative stress and inflammation disease, is a major cause of mortality in critically ill patients. Mitochondrial fission and pyroptosis often occur in AKI. However, the underlying biological pathways involved in endotoxin AKI remain poorly understood, especially those related to mitochondrial dynamics equilibrium disregulation and pyroptosis. Previous studies suggest that heme oxygenase- (HO-) 1 confers cytoprotection against AKI during endotoxic shock, and PTEN-induced putative kinase 1 (PINK1) takes part in mitochondrial dysfunction. Thus, in this study, we examine the roles of HO-1/PINK1 in maintaining the dynamic process of mitochondrial fusion/fission to inhibit pyroptosis and mitigate acute kidney injury in rats exposed to endotoxin. Methods An endotoxin-associated AKI model induced by lipopolysaccharide (LPS) was used in our study. Wild-type (WT) rats and PINK1 knockout (PINK1KO) rats, respectively, were divided into four groups: the control, LPS, Znpp+LPS, and Hemin+LPS groups. Rats were sacrificed 6 h after intraperitoneal injecting LPS to assess renal function, oxidative stress, and inflammation by plasma. Mitochondrial dynamics, morphology, and pyroptosis were evaluated by histological examinations. Results In the rats with LPS-induced endotoxemia, the expression of HO-1 and PINK1 were upregulated at both mRNA and protein levels. These rats also exhibited inflammatory response, oxidative stress, mitochondrial fission, pyroptosis, and decreased renal function. After upregulating HO-1 in normal rats, pyroptosis was inhibited; mitochondrial fission and inflammatory response to oxidative stress were decreased; and the renal function was improved. The effects were reversed by adding Znpp (a type of HO-1 inhibitor). Finally, after PINK1 knockout, there is no statistical difference in the LPS-treated group and Hemin or Znpp pretreated group. Conclusions HO-1 inhibits inflammation response and oxidative stress and regulates mitochondria fusion/fission to inhibit pyroptosis, which can alleviate endotoxin-induced AKI by PINK1.

Gene expression within the periaqueductal gray is linked to vocal behavior and early-onset parkinsonism in Pink1 knockout rats

BackgroundParkinson’s disease (PD) is a degenerative disease with early-stage pathology hypothesized to manifest in brainstem regions. Vocal deficits, including soft, monotone speech, result in significant clinical and quality of life issues and are present in 90% of PD patients; yet the underlying pathology mediating these significant voice deficits is unknown. The Pink1−/− rat is a valid model of early-onset PD that presents with analogous vocal communication deficits. Previous work shows abnormal α-synuclein protein aggregation in the periaqueductal gray (PAG), a brain region critical and necessary to the modulation of mammalian vocal behavior. In this study, we used high-throughput RNA sequencing to examine gene expression within the PAG of both male and female Pink1−/− rats as compared to age-matched wildtype controls. We used a bioinformatic approach to (1) test the hypothesis that loss of Pink1 in the PAG will influence the differential expression of genes that interact with Pink1, (2) highlight other key genes that relate to this type of Mendelian PD, and (3) catalog molecular targets that may be important for the production of rat vocalizations.ResultsKnockout of the Pink1 gene resulted in differentially expressed genes for both male and female rats that also mapped to human PD datasets. Pathway analysis highlighted several significant metabolic pathways. Weighted gene co-expression network analysis (WGCNA) was used to identify gene nodes and their interactions in (A) males, (B) females, and (C) combined-sexes datasets. For each analysis, within the module containing the Pink1 gene, Pink1 itself was the central node with the highest number of interactions with other genes including solute carriers, glutamate metabotropic receptors, and genes associated with protein localization. Strong connections between Pink1 and Krt2 and Hfe were found in both males and female datasets. In females a number of modules were significantly correlated with vocalization traits.ConclusionsOverall, this work supports the premise that gene expression changes in the PAG may contribute to the vocal deficits observed in this PD rat model. Additionally, this dataset identifies genes that represent new therapeutic targets for PD voice disorders.

Dual effects of thyroid hormone on neurons and neurogenesis in traumatic brain injury

Thyroid hormone (TH) plays a crucial role in neurodevelopment, but its function and specific mechanisms remain unclear after traumatic brain injury (TBI). Here we found that treatment with triiodothyronine (T3) ameliorated the progression of neurological deficits in mice subjected to TBI. The data showed that T3 reduced neural death and promoted the elimination of damaged mitochondria via mitophagy. However, T3 did not prevent TBI-induced cell death in phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (Pink1) knockout mice suggesting the involvement of mitophagy. Moreover, we also found that T3 promoted neurogenesis via crosstalk between mature neurons and neural stem cells (NSCs) after TBI. In neuron cultures undergoing oxygen and glucose deprivation (OGD), conditioned neuron culture medium collected after T3 treatment enhanced the in vitro differentiation of NSCs into mature neurons, a process in which mitophagy was required. Taken together, these data suggested that T3 treatment could provide a therapeutic approach for TBI by preventing neuronal death via mitophagy and promoting neurogenesis via neuron–NSC crosstalk.

Mitophagy deficiency increases NLRP3 to induce brown fat dysfunction in mice

ABSTRACT Although macroautophagy/autophagy deficiency causes degenerative diseases, the deletion of essential autophagy genes in adipocytes paradoxically reduces body weight. Brown adipose tissue (BAT) plays an important role in body weight regulation and metabolic control. However, the key cellular mechanisms that maintain BAT function remain poorly understood. in this study, we showed that global or brown adipocyte-specific deletion of pink1, a Parkinson disease-related gene involved in selective mitochondrial autophagy (mitophagy), induced BAT dysfunction, and obesity-prone type in mice. Defective mitochondrial function is among the upstream signals that activate the NLRP3 inflammasome. NLRP3 was induced in brown adipocyte precursors (BAPs) from pink1 knockout (KO) mice. Unexpectedly, NLRP3 induction did not induce canonical inflammasome activity. Instead, NLRP3 induction led to the differentiation of pink1 KO BAPs into white-like adipocytes by increasing the expression of white adipocyte-specific genes and repressing the expression of brown adipocyte-specific genes. nlrp3 deletion in pink1 knockout mice reversed BAT dysfunction. Conversely, adipose tissue-specific atg7 KO mice showed significantly lower expression of Nlrp3 in their BAT. Overall, our data suggest that the role of mitophagy is different from general autophagy in regulating adipose tissue and whole-body energy metabolism. Our results uncovered a new mitochondria-NLRP3 pathway that induces BAT dysfunction. The ability of the nlrp3 knockouts to rescue BAT dysfunction suggests the transcriptional function of NLRP3 as an unexpected, but a quite specific therapeutic target for obesity-related metabolic diseases. Abbreviations: ACTB: actin, beta; BAPs: brown adipocyte precursors; BAT: brown adipose tissue; BMDMs: bone marrow-derived macrophages; CASP1: caspase 1; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; ChIP: chromatin immunoprecipitation; EE: energy expenditure; HFD: high-fat diet; IL1B: interleukin 1 beta; ITT: insulin tolerance test; KO: knockout; LPS: lipopolysaccharide; NLRP3: NLR family, pyrin domain containing 3; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RD: regular diet; ROS: reactive oxygen species; RT: room temperature; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); WT: wild-type.

Psychological Stress Phenocopies Brain Mitochondrial Dysfunction and Motor Deficits as Observed in a Parkinsonian Rat Model.

Psychological distress is a public health issue as it contributes to the development of human diseases including neuropathologies. Parkinson's disease (PD), a chronic, progressive neurodegenerative disorder, is caused by multiple factors including aging, mitochondrial dysfunction, and/or stressors. In PD, a substantial loss of substantia nigra (SN) neurons leads to rigid tremors, bradykinesia, and chronic fatigue. Several studies have reported that the hypothalamic-pituitary-adrenal (HPA) axis is altered in PD patients, leading to an increase level of cortisol which contributes to neurodegeneration and oxidative stress. We hypothesized that chronic psychological distress induces PD-like symptoms and promotes neurodegeneration in wild-type (WT) rats and exacerbates PD pathology in PINK1 knockout (KO) rats, a well-validated animal model of PD. We measured the bioenergetics profile (oxidative phosphorylation and glycolysis) in the brain by employing an XF24e Seahorse Extracellular Flux Analyzer in young rats subjected to predator-induced psychological distress. In addition, we analyzed anxiety-like behavior, motor function, expression of antioxidant enzymes, mitochondrial content, and neurotrophic factors brain-derived neurotrophic factor (BDNF) in the brain. Overall, we observed that psychological distress diminished up to 50% of mitochondrial respiration and glycolysis in the prefrontal cortex (PFC) derived from both WT and PINK1-KO rats. Mechanistically, the level of antioxidant proteins, mitochondrial content, and BDNF was significantly altered. Finally, psychological distress robustly induced anxiety and Parkinsonian symptoms in WT rats and accelerated certain symptoms of PD in PINK1-KO rats. For the first time, our collective data suggest that psychological distress can phenocopy several aspects of PD neuropathology, disrupt brain energy production, as well as induce ataxia-like behavior.

PINK1 phosphorylates ubiquitin predominantly in astrocytes

Loss-of-function mutations in PINK1 are causally linked to recessively inherited Parkinson’s disease (PD), with marked loss of dopaminergic neurons in the substantia nigra that are required for normal movement. PINK1 is a nuclear-encoded mitochondrial-targeted kinase that phosphorylates a conserved serine at amino acid 65 (pS65) in ubiquitin as well as Parkin, another gene with loss-of-function mutations linked to recessive parkinsonism. The steady-state levels of PINK1 protein are very low, even in cells that express PINK1, because PINK1 is normally targeted for degradation after mitochondrial import by a process that is dependent upon mitochondrial membrane potential. Dissipation of the mitochondrial membrane potential with ionophores, such as CCCP and valinomycin, causes the accumulation of PINK1 on the outer mitochondrial membrane, a marked increase of pS65-ubiquitin and the recruitment of Parkin, which targets dysfunctional mitochondria for degradation by autophagy. While the high penetrance of PINK1 mutations establish its critical function for maintaining neurons, the activity of PINK1 in primary neurons has been difficult to detect. Mounting evidence implicates non-neuronal cells, including astrocytes and microglia, in the pathogenesis of both idiopathic and inherited PD. Herein we used both western analysis and immunofluorescence of pS65-ubiquitin to directly compare the activity of PINK1 in primary neurons, astrocytes, microglia, and oligodendrocyte progenitor cells cultured from the brains of wild-type (WT) and PINK1 knockout (KO) rat pups. Our findings that PINK1-dependent ubiquitin phosphorylation is predominantly in astrocytes supports increased priority for research on the function of PINK1 in astrocytes and the contribution of astrocyte dysfunction to PD pathogenesis.

Mitochondrial DNA heteroplasmy rises in substantial nigra of aged PINK1 KO mice

Mutations in PINK1 and Parkin result in early-onset autosomal recessive Parkinson’s disease (PD). PINK1/Parkin pathway maintain mitochondrial function by mediating the clearance of damaged mitochondria. However, the role of PINK1/Parkin in maintaining the balance of mtDNA heteroplasmy is still unknown. Here, we isolated mitochondrial DNA (mtDNA) from cortex, striatum and substantia nigra of wildtype (WT), PINK1 knockout (PINK1 KO) and Parkin knockout (Parkin KO) mice to analyze mtDNA heteroplasmy induced by PINK1/Parkin deficiency or aging. Our results showed that the Single Nucleotide Variants (SNVs) of late-onset somatic variants mainly increased with aging. Conversely, the early-onset somatic variants exhibited significant increase in the cortex and substantia nigra of PINK1 KO mice than WT mice of the same age. Increased average variant allele frequency was observed in aged PINK1 KO mice and in substantial nigra of aged Parkin KO mice than in WT mice. Cumulative variant allele frequency in the substantia nigra of PINK1 KO mice was significantly higher than that in WT mice, further supporting the pivotal role of PINK1 in mtDNA maintenance.This study presented a new evidence for PINK1 and Parkin in participating in mitochondrial quality control and provided clues for further revealing the role of PINK1 and Parkin in the pathogenesis of PD.

Loss of PTEN-induced kinase 1 (Pink1) reduces hippocampal tyrosine hydroxylase and impairs learning and memory

Phosphatase and tensin homolog (PTEN)-induced kinase 1 (Pink1) is involved in mitochondrial quality control, which is essential for maintaining energy production and minimizing oxidative damage from dysfunctional/depolarized mitochondria. Pink1 mutations are the second most common cause of autosomal recessive Parkinson's disease (PD). In addition to characteristic motor impairments, PD patients also commonly exhibit cognitive impairments. As the hippocampus plays a prominent role in cognition, we tested if loss of Pink1 in mice influences learning and memory. While wild-type mice were able to perform a contextual discrimination task, age-matched Pink1 knockout (Pink1−/−) mice showed an impaired ability to differentiate between two similar contexts. Similarly, Pink1−/− mice performed poorly in a delayed alternation task as compared to age-matched controls. Poor performance in these cognitive tasks was not the result of overt hippocampal pathology. However, a significant reduction in hippocampal tyrosine hydroxylase (TH) protein levels was detected in the Pink1−/− mice. This decrease in hippocampal TH levels was also associated with reduced DOPA decarboxylase and dopamine D2 receptor levels, but not post-synaptic dopamine D1 receptor levels. These presynaptic changes appeared to be selective for dopaminergic fibers as hippocampal dopamine beta hydroxylase, choline acetyltransferase, and tryptophan hydroxylase levels were unchanged in Pink1−/− mice. Administration of the dopamine D1 receptor agonist SKF38393 to Pink1−/− mice was found to improve performance in the context discrimination task. Taken together, our results show that Pink1 loss may alter dopamine signaling in the hippocampus, which could be a contributing mechanism for the observed learning and memory impairments.