PTEN-induced Kinase 1 Knockout Research Articles

A190 HELICOBACTER PYLORI INDUCES AN AUTO-REACTIVE PHENOTYPE IN PINK1 DEFICIENT MICE THAT CORRELATES WITH MOTOR DYSFUNCTION

Abstract Background Parkinson’s disease (PD) is a chronic neurodegenerative disorder. Several genetic predispositions, including the PTEN-induced kinase 1 (PINK1) mutation, have been implicated in early onset family cases. Besides the loss of dopaminergic neurons in the brain, PD patients have a unique immune phenotype that includes increased inflammation, blood serum, brain levels of proinflammatory cytokines, brain infiltration with cytotoxic CD8 T cells, and loss of regulatory T cells (Treg) and their anti-inflammatory phenotype. The role of the gut microbiota and gastrointestinal infections are increasingly recognized as a cofactor in PD. One of pathogens associated with the risk of PD is Helicobacter pylori. The prevalence of this Gram-negative bacteria in PD patients is higher than in the general population and its eradication in PD patients improves motor function. Loss of the PD-associated PINK1 alters induced Treg function in vitro. We previously have shown that in PINK1 knock-out (KO) mice, gut infection with Gram-negative bacteria, Citrobacter rodentium, induces mitochondrial antigen presentation (MitAP) to the CD8 T cells that later infiltrate the brain. In this model, PINK1 KO mice develop a Parkinson-like L-DOPA-responsive motor phenotype after four C. rodentium infections. Aims Here we aimed to scrutinize potential role of the H. pylori infection in the induction of motor dysfunction and disbalanced immune tolerance in PINK1 KO mice. Methods In addition to standard behavioural testing, at 2- and 6-months post-infection we performed a multiplex cytokine analysis of gastric homogenates and spectral flow cytometry of gastric lamina propria, mesenteric lymph nodes, blood, spleen, and brain infiltrating immunocytes. As well as in vitro immunocytes response and function assays to H.pylori exposure in PINK1 KO and wild-type (WT) littermate controls. Results We show that infection with H. pylori causes a long-lasting inflammation in the stomach; and local and systemic immune phenotype that remains 6 months post-infection. This phenotype, though present in some WT infected mice, is higher in PINK1 KOs and similarly to PD patients includes a decrease in Treg proportion, FoxP3 downregulation in regulatory T cells, Gata3+ CD4 T cell loss, as well as increase of circulating mitochondrial antigen-specific CD8 T cells. Moreover, the immune phenotype in the H. pylori infected PINK1 KO mice correlates with motor dysfunction and CD8 T cell brain infiltration with no such association seen in the WT mice. Conclusions These results provide insight to the gut-immunity-brain axis in the pathogenesis of Parkinson’s disease, and further investigate the role of Gram-negative bacteria in the establishment of immune tolerance-autoreactivity balance. Funding Agencies ASAP

#5403 PTEN-INDUCED KINASE 1 HAS ASSOCIATION WITH RENAL AGING PROCESS THROUGH THE CGAS -STING PATHWAY

Abstract Background and Aims The global increase of an aging population has encouraged the research on renal aging, as the kidney is one of the organs that shows the greatest change as aging progresses. Although the mechanism of renal aging has not been clearly identified, dysfunctional mitochondria has been suggested as one of the factors that induce inflammation of the kidney, which is the major mediator of the pro-aging process of chronic kidney disease. PTEN-induced kinase 1(PINK1) is a protein involved in the quality control of mitochondria and regulates mitochondrial dysfunction. Although it is known that the mitochondrial DNA release promoted by PINK1 deficiency stimulates cyclic GMP–AMP synthase (cGAS) - stimulator of interferon genes (STING) pathway eventually resulting in the inflammatory response, the role of PINK1 and cGAS-STING pathway in renal aging has not yet been clarified. This study aimed to investigate the relationship between PINK1 and renal aging, especially through the cGAS-STING pathway. Method To determine the role of PINK1 on the renal aging process, renal fibrosis, and tubular injury were compared in 4- and 24-month-old wild-type (Pink1+/+) and PINK1 knockout (Pink1-/-) mice. To establish in vitro senescence model, hydrogen peroxide (H2O2) treatment on human renal proximal cells (HKC-8) was used. The changes in gene expression levels related to PINK1 were analyzed by RNA sequencing, applying transcriptomic and metabolomic analyses. To validate the results of RNA sequencing, we measured mitochondrial oxygen consumption rate (OCR) by Seahorse Mito Stress Test. To investigate the relationship between PINK1 and renal aging through the cGAS-STING pathway, we explored the change of cGAS-STING expression on senescence-induced HKC-8 cells and additionally used H-151 treatment, a specific STING inhibitor. Results The renal fibrosis and tubular injury were significantly aggravated in 24-month-old Pink1-/- mice compared to 24-month-old Pink1+/+ mice. Western blot and RT-qPCR confirmed remarkably increased senescence markers and senescence-associated secretory phenotype (SASPs) in 24-month Pink1-/- mice and senescence-induced HKC-8 cells. The RNA sequencing of mice kidneys showed that inflammation-related pathways significantly increased in 24 months Pink1-/-mice, and transcriptomic and metabolomic analyses showed that PINK1 has an association with mitochondrial metabolism dysregulation. On OCR measurement, the basal respiration, maximal respiration, ATP production, and respiratory capacity significantly declined in H2O2-treated siPINK1 cells, suggesting that PINK1 deficiency might have effects on mitochondrial dysfunction. Finally, the STING pathway was significantly activated in 24-month Pink1-/- mice and senescence-induced HKC-8 cells, which was inhibited by a specific inhibitor of STING, H-151. Conclusion In conclusion, PINK1 is associated with renal aging, and the dysregulation of mitochondria caused by PINK1 deficiency might lead to aging-related inflammatory responses through the cGAS-STING pathway.

PTEN-induced kinase 1 is associated with renal aging, via the cGAS-STING pathway.

Mitochondrial dysfunction is considered to be an important mediator of the pro-aging process in chronic kidney disease, which is continuously increasing worldwide. Although PTEN-induced kinase 1 (PINK1) regulates mitochondrial function, its role in renal aging remains unclear. We investigated the association between PINK1 and renal aging, especially through the cGAS-STING pathway, which is known to result in an inflammatory phenotype. Pink1 knockout (Pink1-/- ) C57BL/6 mice and senescence-induced renal tubular epithelial cells (HKC-8) treated with H2 O2 were used as the renal aging models. Extensive analyses at transcriptomic-metabolic levels have explored changes in mitochondrial function in PINK1 deficiency. To investigate whether PINK1 deficiency affects renal aging through the cGAS-STING pathway, we explored their expression levels in PINK1 knockout mice and senescence-induced HKC-8 cells. PINK1 deficiency enhances kidney fibrosis and tubular injury, and increases senescence and the senescence-associated secretory phenotype (SASP). These phenomena were most apparent in the 24-month-old Pink1-/- mice and HKC-8 cells treated with PINK1 siRNA and H2 O2 . Gene expression analysis using RNA sequencing showed that PINK1 deficiency is associated with increased inflammatory responses, and transcriptomic and metabolomic analyses suggested that PINK1 deficiency is related to mitochondrial metabolic dysregulation. Activation of cGAS-STING was prominent in the 24-month-old Pink1-/- mice. The expression of SASPs was most noticeable in senescence-induced HKC-8 cells and was attenuated by the STING inhibitor, H151. PINK1 is associated with renal aging, and mitochondrial dysregulation by PINK1 deficiency might stimulate the cGAS-STING pathway, eventually leading to senescence-related inflammatory responses.

Intraperitoneal Administration of Forskolin Reverses Motor Symptoms and Loss of Midbrain Dopamine Neurons in PINK1 Knockout Rats.

Background:Parkinson’s disease (PD) is a relentless, chronic neurodegenerative disease characterized by the progressive loss of substantia nigra (SN) neurons that leads to the onset of motor and non-motor symptoms. Standard of care for PD consists of replenishing the loss of dopamine through oral administration of Levodopa; however, this treatment is not disease-modifying and often induces intolerable side effects. While the etiology that contributes to PD is largely unknown, emerging evidence in animal models suggests that a significant reduction in neuroprotective Protein Kinase A (PKA) signaling in the SN contributes to PD pathogenesis, suggesting that restoring PKA signaling in the midbrain may be a new anti-PD therapeutic alternative.Objective:We surmised that pharmacological activation of PKA via intraperitoneal administration of Forskolin exerts anti-PD effects in symptomatic PTEN-induced kinase 1 knockout (PINK1-KO), a bona fide in vivo model of PD.Methods:By using a beam balance and a grip strength analyzer, we show that Forskolin reverses motor symptoms and loss of hindlimb strength with long-lasting therapeutic effects (> 5 weeks) following the last dose.Results:In comparison, intraperitoneal treatment with Levodopa temporarily (24 h) reduces motor symptoms but unable to restore hindlimb strength in PINK1-KO rats. By using immunohistochemistry and an XF24e BioAnalyzer, Forskolin treatment reverses SN neurons loss, elevates brain energy production and restores PKA activity in SN in symptomatic PINK1-KO rats.Conclusion:Overall, our collective in vivo data suggest that Forskolin is a promising disease-modifying therapeutic alternative for PD and is superior to Levodopa because it confers long-lasting therapeutic effects.

Knockout of PINK1 altered the neural connectivity of Drosophila dopamine PPM3 neurons at input and output sites.

Impairment of the dopamine system is the main cause of Parkinson disease (PD). PTEN-induced kinase 1 (PINK1) is possibly involved in pathogenesis of PD. However, its role in dopaminergic neurons has not been fully established yet. In the present investigation, we have used the PINK1 knockout Drosophila model to explore the role of PINK1 in dopaminergic neurons. Electrophysiological and behavioral tests indicated that PINK1 elimination enhances the neural transmission from the presynaptic part of dopaminergic neurons in the protocerebral posterior medial region 3 (PPM3) to PPM3 neurons (which are homologous to those in the substantia nigra in humans). Firing properties of the action potential in PPM3 neurons were also altered in the PINK1 knockout genotypes. Abnormal motor ability was also observed in these PINK1 knockout animals. Our results indicate that knockout of PINK1 could alter both the input and output properties of PPM3 neurons.

Enhanced Susceptibility of PINK1 Knockout Rats to α-Synuclein Fibrils

The main neuropathological hallmarks of Parkinson’s disease (PD) are loss of dopaminergic neurons in the substantia nigra and intraneuronal protein aggregates immunoreactive for α-synuclein phosphorylated at serine 129 (pS129). Most cases of PD are idiopathic; however, genetic mutations have been identified in several genes linked to familial PD. Mutations in the gene encoding α-synuclein are causally linked to dominantly inherited forms of PD and mutations in the PTEN-induced kinase-1 (PINK1) gene are linked to recessively inherited forms of PD. Because abnormal α-synuclein protein aggregates appear spontaneously in PINK1 knockout (KO) rats, we hypothesize that PINK1-deficiency causes endogenous α-synuclein to be more prone to aggregation. α-Synuclein aggregation does not normally occur in mice or rats, however, it can be induced by intracranial injection of α-synuclein pre-formed fibrils (PFFs), which also induces loss of dopaminergic nigral neurons 3–6 months post-injection. Because PINK1-deficiency is linked to early-onset PD, we further hypothesize that PINK1 KO rats will show earlier PFF-induced neurodegeneration compared to wild-type (WT) rats. Herein, we report that intracranial injection of α-synuclein PFFs into the dorsal striatum induced more abundant pS129 α-synuclein in PINK1 KO rat brains compared to WT littermate controls. Moreover, the synuclein extracted from the brains of PFF-injected PINK1 KO rats was more insoluble compared to PFF-injected WT littermates, suggesting greater progression of α-synuclein pathology in PINK1 KO rats. Four weeks post-injection, PFFs caused significant loss of dopaminergic neurons in the substantia nigra of PINK1 KO rats, but not WT controls. Together, our results indicate that PINK1 deficiency increases vulnerability to α-synuclein aggregation and dopaminergic neurodegeneration in vivo.

Loss of Non-Apoptotic Role of Caspase-3 in the PINK1 Mouse Model of Parkinson's Disease.

Caspases are a family of conserved cysteine proteases that play key roles in multiple cellular processes, including programmed cell death and inflammation. Recent evidence shows that caspases are also involved in crucial non-apoptotic functions, such as dendrite development, axon pruning, and synaptic plasticity mechanisms underlying learning and memory processes. The activated form of caspase-3, which is known to trigger widespread damage and degeneration, can also modulate synaptic function in the adult brain. Thus, in the present study, we tested the hypothesis that caspase-3 modulates synaptic plasticity at corticostriatal synapses in the phosphatase and tensin homolog (PTEN) induced kinase 1 (PINK1) mouse model of Parkinson’s disease (PD). Loss of PINK1 has been previously associated with an impairment of corticostriatal long-term depression (LTD), rescued by amphetamine-induced dopamine release. Here, we show that caspase-3 activity, measured after LTD induction, is significantly decreased in the PINK1 knockout model compared with wild-type mice. Accordingly, pretreatment of striatal slices with the caspase-3 activator α-(Trichloromethyl)-4-pyridineethanol (PETCM) rescues a physiological LTD in PINK1 knockout mice. Furthermore, the inhibition of caspase-3 prevents the amphetamine-induced rescue of LTD in the same model. Our data support a hormesis-based double role of caspase-3; when massively activated, it induces apoptosis, while at lower level of activation, it modulates physiological phenomena, like the expression of corticostriatal LTD. Exploring the non-apoptotic activation of caspase-3 may contribute to clarify the mechanisms involved in synaptic failure in PD, as well as in view of new potential pharmacological targets.

Analysis of α-Synuclein Pathology in PINK1 Knockout Rat Brains.

Mutations in PTEN induced kinase 1 (PINK1) cause autosomal recessive Parkinson’s disease (PD). The main pathological hallmarks of PD are loss of dopaminergic neurons in the substantia nigra pars compacta and the formation of protein aggregates containing α-synuclein. Previous studies of PINK1 knockout (PINK1-/-) rats have reported mitochondrial dysfunction, locomotor behavioral deficits, loss of neurons in the substantia nigra and α-synuclein aggregates in various brain regions. We sought to characterize PINK1-/- rats in more detail specifically with respect to α-synuclein pathology because abnormal α-synuclein has been implicated genetically, biophysically and neuropathologically as a mechanism of PD pathogenesis. Moreover, the spontaneous formation of α-synuclein aggregates without α-synuclein overexpression, injection or toxin administration is a rare and important characteristic for an animal model of PD or other synucleinopathies, such as dementia with Lewy bodies and multiple system atrophy. We observed α-synuclein-immunoreactive aggregates in various brain regions of PINK1-/- rats including cortex, thalamus, striatum and ventral midbrain, but nowhere in wild-type (WT) rats. Co-immunofluorescence showed that the α-synuclein-immunoreactive aggregates are both thioflavin S and ubiquitin positive. Many cells in the brains of PINK1-/- rats but not WT rats contained protease-resistant α-synuclein. Total synuclein protein levels were unchanged; however, biochemical fractionation showed a significant shift of α-synuclein from the cytosolic fraction to the synaptic vesicle-enriched fraction of PINK1-/- brain homogenates compared to WT. This data indicates that PINK1 deficiency results in abnormal α-synuclein localization, protease resistance and aggregation in vivo. The PINK1-/- rat could be a useful animal model to study the role of abnormal α-synuclein in PD-related neurodegeneration.

Loss of PINK1 causes age-dependent decrease of dopamine release and mitochondrial dysfunction.

Mutations and deletions in PTEN-induced kinase 1 (PINK1) cause autosomal recessive Parkinson's disease (PD), the second most common neurodegenerative disorder. PINK1 is a nuclear-genome encoded Ser/Thr kinase in mitochondria. PINK1 deletion was reported to affect dopamine (DA) levels in the striatum and mitochondrial functions but with conflicting results. The role of PINK1 in mitochondrial function and in PD pathogenesis remains to be elucidated thoroughly. In this study, we measured DA release using fast-scan cyclic voltammetry in acute striatal slices from both PINK1 knockout (KO) and wild-type (WT) mice at different ages. We found that single pulse-evoked DA release in the dorsal striatum of PINK1 KO mice was decreased in an age-dependent manner. Furthermore, the decrease was because of less DA release instead of an alteration of DA transporter function or DA terminal degeneration. We also found that PINK1 KO striatal slices had significantly lower basal mitochondria respiration compared with that of WT controls, and this impairment was also age-dependent. These results suggest that the impaired DA release is most likely because of mitochondrial dysfunction and lower ATP production.

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.

Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser(65)) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser(111)) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser(111) of each of the Rabs, we demonstrate that Rab Ser(111) phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser(111) phosphorylation is not directly regulated by PINK1 invitro and demonstrate in cells the time course of Ser(111) phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser(65). We further show mechanistically that phosphorylation at Ser(111) significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser(111) may represent novel biomarkers of PINK1 activity invivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.

Quantitative expression proteomics and phosphoproteomics profile of brain from PINK1 knockout mice: insights into mechanisms of familial Parkinson's disease.

Parkinson's disease (PD) is an age-related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α-synuclein-containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN-induced kinase 1 (PINK1) are associated with an autosomal recessive familial form of early-onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy-lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2-D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6-month-old PINK1-deficient mice and wild-type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD. Mutations in PINK1 are associated with an early-onset form of Parkinson's disease (PD). This study examines changes in the proteome and phosphoproteome of the PINK1 knockout mouse brain. Alterations were noted in several key proteins associated with: increased oxidative stress, aberrant cellular signaling, altered neuronal structure, decreased synaptic plasticity, reduced neurotransmission, diminished proteostasis networks, and altered metabolism. 14-3-3ε, 14-3-3 protein epsilon; 3-PGDH, phosphoglycerate dehydrogenase; ALDOA, aldolase A; APT1, acyl-protein thioesterase 1; CaM, calmodulin; CBR3, carbonyl reductase [NADPH] 3; ENO2, gamma-enolase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HSP70, heat-shock-related 70kDa protein 2; IDHc, cytoplasmic isocitrate dehydrogenase [NADP+]; MAPK1, mitogen-activated protein kinase 1; MEK1, MAP kinase kinase 1; MDHc, cytoplasmic malate dehydrogenase; NFM, neurofilament medium polypeptide; NSF, N-ethylmaleimide-sensitive fusion protein; PHB, prohibitin; PINK1, PTEN-induced putative kinase 1; PPIaseA, peptidyl-prolyl cis-trans isomerase A; PSA2, proteasome subunit alpha type-2; TK, transketolase; VDAC-2, voltage-dependent anion-selective channel protein 2.

Early Expression of Parkinson's Disease-Related Mitochondrial Abnormalities in PINK1 Knockout Rats.

PTEN-induced kinase 1 (PINK1) mutations are responsible for an autosomal recessive, familial form of Parkinson's disease. PINK1 protein is a Ser/Thr kinase localized to the mitochondrial membrane and is involved in many processes including mitochondrial trafficking, mitophagy, and proteasomal function. Using a new PINK1 knockout (PINK1 KO) rat model, we found altered brain metabolomic markers using magnetic resonance spectroscopy, identified changes in mitochondrial pathways with quantitative proteomics using sequential window acquisition of all theoretical spectra (SWATH) mass spectrometry, and demonstrated mitochondrial functional alterations through measurement of oxygen consumption and acidification rates. The observed alterations included reduced creatine, decreased levels of complex I of the electron transport chain, and increased proton leak in the electron transport chain in PINK1 KO rat brains. In conjunction, these results demonstrate metabolomic and mitochondrial alterations occur during the asymptomatic phase of Parkinson's disease in this model. These results indicate both potential early diagnostic markers and therapeutic pathways that can be used in PD.

Mitochondrial and cytosolic roles of PINK1 shape induced regulatory T‐cell development and function

Mutations in PTEN-induced kinase 1 (PINK1), a serine/threonine kinase linked to familial early-onset Parkinsonism, compromise mitochondrial integrity and metabolism and impair AKT signaling. As the activation of a naïve T cell requires an AKT-dependent reorganization of a cell's metabolic machinery, we sought to determine if PINK1-deficient T cells lack the ability to undergo activation and differentiation. We show that CD4(+) T cells from PINK1 knockout mice fail to properly phosphorylate AKT upon activation, resulting in reduced expression of the IL-2 receptor subunit CD25. Following, deficient IL-2 signaling mutes the activation-induced increase in respiratory capacity and mitochondrial membrane potential. Under polarization conditions favoring the development of induced regulatory T cells, PINK1(-/-) T cells exhibit a reduced ability to suppress bystander T-cell proliferation despite normal FoxP3 expression kinetics. Our results describe a critical role for PINK1 in integrating extracellular signals with metabolic state during T-cell fate determination, and may have implications for the understanding of altered T-cell populations and immunity during the progression of active Parkinson's disease or other immunopathologies.

Mitochondrially localized PKA reverses mitochondrial pathology and dysfunction in a cellular model of Parkinson's disease

Mutations in PTEN-induced kinase 1 (PINK1) are associated with a familial syndrome related to Parkinson's disease (PD). We previously reported that stable neuroblastoma SH-SY5Y cell lines with reduced expression of endogenous PINK1 exhibit mitochondrial fragmentation, increased mitochondria-derived superoxide, induction of compensatory macroautophagy/mitophagy and a low level of ongoing cell death. In this study, we investigated the ability of protein kinase A (PKA) to confer protection in this model, focusing on its subcellular targeting. Either: (1) treatment with pharmacological PKA activators; (2) transient expression of a constitutively active form of mitochondria-targeted PKA; or (3) transient expression of wild-type A kinase anchoring protein 1 (AKAP1), a scaffold that targets endogenous PKA to mitochondria, reversed each of the phenotypes attributed to loss of PINK1 in SH-SY5Y cells, and rescued parameters of mitochondrial respiratory dysfunction. Mitochondrial and lysosomal changes in primary cortical neurons derived from PINK1 knockout mice or subjected to PINK1 RNAi were also reversed by the activation of PKA. PKA phosphorylates the rat dynamin-related protein 1 isoform 1 (Drp1) at serine 656 (homologous to human serine 637), inhibiting its pro-fission function. Mimicking phosphorylation of Drp1 recapitulated many of the protective effects of AKAP1/PKA. These data indicate that redirecting endogenous PKA to mitochondria can compensate for deficiencies in PINK1 function, highlighting the importance of compartmentalized signaling networks in mitochondrial quality control.

PARK6 PINK1 mutants are defective in maintaining mitochondrial membrane potential and inhibiting ROS formation of substantia nigra dopaminergic neurons

Mutations in PTEN-induced kinase 1 (PINK1) gene cause recessive familial type 6 of Parkinson's disease (PARK6). PINK1 is believed to exert neuroprotective effect on SN dopaminergic cells by acting as a mitochondrial Ser/Thr protein kinase. Autosomal recessive inheritance indicates the involvement of loss of PINK1 function in PARK6 pathogenesis. In the present study, confocal imaging of cultured SN dopaminergic neurons prepared from PINK1 knockout mice was performed to investigate physiological importance of PINK1 in maintaining mitochondrial membrane potential (Δ Ψ m) and mitochondrial morphology and test the hypothesis that PARK6 mutations cause the loss of PINK1 function. PINK1-deficient SN dopaminergic neurons exhibited a depolarized Δ Ψ m. In contrast to long thread-like mitochondria of wild-type neurons, fragmented mitochondria were observed from PINK1-null SN dopaminergic cells. Basal level of mitochondrial superoxide and oxidative stressor H 2O 2-induced ROS generation were significantly increased in PINK1-deficient dopaminergic neurons. Overexpression of wild-type PINK1 restored hyperpolarized Δ Ψ m and thread-like mitochondrial morphology and inhibited ROS formation in PINK1-null dopaminergic cells. PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 failed to rescue mitochondrial dysfunction and inhibit oxidative stress in PINK1-deficient dopaminergic neurons. Mitochondrial toxin rotenone-induced cell death of dopaminergic neurons was augmented in PINK1-null SN neuronal culture. These results indicate that PINK1 is required for maintaining normal Δ Ψ m and mitochondrial morphology of cultured SN dopaminergic neurons and exerts its neuroprotective effect by inhibiting ROS formation. Our study also provides the evidence that PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 is defective in regulating mitochondrial functions and attenuating ROS production of SN dopaminergic cells.

Altered profile and D2-dopamine receptor modulation of high voltage-activated calcium current in striatal medium spiny neurons from animal models of Parkinson's disease

In the present work we analyzed the profile of high voltage-activated (HVA) calcium (Ca2+) currents in freshly isolated striatal medium spiny neurons (MSNs) from rodent models of both idiopathic and familial forms of Parkinson's disease (PD). MSNs were recorded from reserpine-treated and 6-hydroxydopamine (6-OHDA)-lesioned rats, and from DJ-1 and PINK1 (PTEN induced kinase 1) knockout (−/−) mice. Our analysis showed no significant changes in total HVA Ca2+ current. However, we recorded a net increase in the L-type fraction of HVA Ca2+ current in dopamine-depleted rats, and of both N- and P-type components in DJ-1−/− mice, whereas no significant change in Ca2+ current profile was observed in PINK1−/− mice. Dopamine modulates HVA Ca2+ channels in MSNs, thus we also analyzed the effect of D1 and D2 receptor activation. The effect of the D1 receptor agonist SKF 83822 on Ca2+ current was not significantly different among MSNs from control animals or PD models. However, in both dopamine-depleted rats and DJ-1−/− mice the D2 receptor agonist quinpirole inhibited a greater fraction of HVA Ca2+ current than in the respective controls. Conversely, in MSNs from PINK1−/− mice we did not observe alterations in the effect of D2 receptor activation. Additionally, in both reserpine-treated and 6-OHDA-lesioned rats, the effect of quinpirole was occluded by the selective L-type Ca2+ channel blocker nifedipine, while in DJ-1−/− mice it was mostly occluded by ω-conotoxin GVIA, blocker of N-type channels. These results demonstrate that both dopamine depletion and DJ-1 deletion induce a rearrangement in the HVA Ca2+ channel profile, specifically involving those channels that are selectively modulated by D2 receptors.

PINK1 cleavage at position A103 by the mitochondrial protease PARL

Mutations in PTEN-induced kinase 1 (PINK1) cause early onset autosomal recessive Parkinson's disease (PD). PINK1 is a 63 kDa protein kinase, which exerts a neuroprotective function and is known to localize to mitochondria. Upon entry into the organelle, PINK1 is cleaved to produce a ∼53 kDa protein (ΔN-PINK1). In this paper, we show that PINK1 is cleaved between amino acids Ala-103 and Phe-104 to generate ΔN-PINK1. We demonstrate that a reduced ability to cleave PINK1, and the consequent accumulation of full-length protein, results in mitochondrial abnormalities reminiscent of those observed in PINK1 knockout cells, including disruption of the mitochondrial network and a reduction in mitochondrial mass. Notably, we assessed three N-terminal PD-associated PINK1 mutations located close to the cleavage site and, while these do not prevent PINK1 cleavage, they alter the ratio of full-length to ΔN-PINK1 protein in cells, resulting in an altered mitochondrial phenotype. Finally, we show that PINK1 interacts with the mitochondrial protease presenilin-associated rhomboid-like protein (PARL) and that loss of PARL results in aberrant PINK1 cleavage in mammalian cells. These combined results suggest that PINK1 cleavage is important for basal mitochondrial health and that PARL cleaves PINK1 to produce the ΔN-PINK1 fragment.