Stroke results from disturbance in blood flow to an area of the brain, leading to neuronal dysfunction and loss. Mitochondrial dysfunction and oxidative stress are critical factors in neuropathology of stroke. They have also been implicated in Parkinson's disease (PD). Select cases of PD are caused by homozygous mutations in the PINK1 gene. Critically, this gene works with another PD gene, Parkin, to regulate mitochondrial quality control (MQC) mechanisms. Additionally, initial studies of the PINK1 protein have suggested that it plays a critical role in cellular pro-survival responses to oxidative stress though the mechanism by which it does so is unclear. In this dissertation, I explored the potential mechanisms through which PINK1 confers neuroprotection, particularly in the case of ischemic insult. I found that PINK1 deficiency sensitizes neurons to glutamate-induced excitotoxicity. I also found that the PINK1 kinase domain, but not the mitochondrial targeting motif, is essential for its protective effect. Additionally, PINK1 or Parkin deficiency significantly increases the infarct volume after middle cerebral artery occlusion, in vivo. Importantly, expression of Parkin reduces the sensitivity of neurons to cytotoxicity induced by PINK1 deficiency indicating that Parkin functionally interacts with PINK1 either through the same or on parallel survival pathways. Moreover, I investigated if PINK1 and Parkin confer neuroprotection against ischemia through PINK1/Parkin MQC pathways. However, I did not find any evidence indicating Parkin mitochondrial translocation following stroke insult suggesting that PINK1/Parkin MQC pathways are not involved in the protective functions of PINK1/Parkin. Interestingly, I found that PINK1 or Parkin deficiency decreases the level of phosphorylation of pro-survival protein AKT (pAKT) whereas expression of these genes enhances pAKT following glutamate treatment. My data also indicate that the mTORC2/AKT pathway partially mediates the neuroprotective effect of PINK1. Taken together, my data indicate that both PINK1 and Parkin play a critical neuroprotective role against ischemia and Ca2+ dysregulation in a fashion independent of mitochondrial control but dependent on AKT function.

Indiana University-Purdue University Indianapolis (IUPUI) / Mutations in PINK1 (PTEN-induced Kinase 1) are the second most common cause of early-onset Parkinson’s Disease (PD). PINK1 is believed to maintain mitochondrial integrity by orchestrating mitophagy of dysfunctional mitochondria through phosphorylation of its substrate, Parkin. However, the effects of PD-associated mutations remain unclear. To investigate this, a PINK1 orthologue, Tribolium castaneum PINK1 (TcPINK1), was genetically engineered and purified for biochemical studies. Then, TcPINK1 was reacted against the Ubiquitin-like domain (UBL1-76) of Parkin and other proteins with a similar beta-grasp fold including Ubiquitin, ATG8, NEDD8, and SUMO using an in vitro radioisotopic filter-based kinase assay. The data revealed that TcPINK1’s preferred substrate with the highest amount of activity was UBL followed by Ubiquitin, NEDD8, and SUMO, with no activity against ATG8, which lacks a Serine residue equivalent to the phosphorylated residue in UBL. NEDD8 and SUMO were phosphorylated even though they are not substrates which suggests that PINK1 is capable of nonspecific phosphorylation of proteins with a similar fold to UBL. In addition, it is possible that the phosphorylation of Ubiquitin as reported in the literature may be nonspecific as well. TcPINK1 point mutations equivalent to the PD-associated human PINK1 mutations were genetically engineered, purified, and reacted against UBL. The P374L mutant showed a similar activity to wild type, and the A194D, G285D, and S289M mutants showed a significant decrease in activity. Since P374 resides in the C-lobe of the kinase away from the active site, the data suggest that this residue may not be involved with catalysis or with UBL binding. As A194, G285, and S289 all reside in the N-lobe near the active site, the data suggest that these point mutations may be involved with catalysis. In conclusion, the data suggest that PINK1 specificity for Parkin may involve binding outside of the UBL domain. / 2024-05-26

Les mitochondries sont la principale source d’énergie dans les neurones. Les défauts mitochondriaux participent à l’apparition de maladies neurodégénératives, cependant ils peuvent être contrés par un système de contrôle qualité. Le but de ma thèse a été de déterminer si ce système est dérégulé dans la maladie de Huntington (MH) et la sclérose latérale amyotrophique (SLA) et si sa restauration est neuroprotectrice, en utilisant principalement des modèles drosophile. La MH, caractérisée par une atteinte des neurones du striatum, est due à la protéine Huntingtin mutée (mHtt). Nous avons montré que la mHtt induit une accumulation des mitochondries dans la rétine. Ceci pourrait être dû à un défaut de la mitophagie, un mécanisme qui permet l’élimination des mitochondries défectueuses et qui est orchestré par la protéine PINK1. De manière intéressante, la surexpression de PINK1 corrige le phénotype pathologique des drosophiles exprimant la mHtt. Je me suis aussi intéressé à la SLA, chez laquelle les motoneurones dégénèrent, plus exactement au gène TDP-43 qui est un contributeur majeur à la maladie. Nous avons montré que la surexpression de TDP-43 dans les neurones de drosophiles entraîne une fragmentation des mitochondries liée à une sous-expression du gène mitofusin. Ce dernier contrôle le processus de fusion entre les mitochondries saines et endommagées et donc l’intégrité de cet organite. La surexpression de Mitofusin améliore les défauts locomoteurs et l’activité neuronale altérée chez les drosophiles exprimant TDP-43. Nos résultats montrent l’importance du contrôle qualité mitochondrial dans la pathogenèse de ces maladies, et que de le renforcer pourrait être bénéfique. / Mitochondria are the main energy source in neurons. Mitochondrial defects contribute to the development of neurodegenerative diseases, however they can be countered by a quality control system. The purpose of my thesis has been to determine if this system is dysregulated in Huntington’s disease (HD) and in amyotrophic lateral sclerosis (ALS) and if restoring it can be neuroprotective, by mainly using Drosophila models. HD, which is characterized by loss of striatal neurons, is caused by the mutant Huntingtin protein (mHtt). We showed that mHtt induces the accumulation of mitochondria in the retina. This could be due to a defect in mitophagy, a mechanism which allows the elimination of defective mitochondria and which is orchestrated by the protein PINK1. Interestingly, PINK1 overexpression ameliorates the abnormal phenotype of flies expressing mHtt. I also got interested in ALS, in which motor neurons degenerate, and mainly in the TDP-43 gene which is a major contributor to the disease. We showed that TDP-43 overexpression in Drosophila neurons leads to fragmentation of mitochondria due to decreased expression levels of the mitofusin gene. The latter controls the fusion process between healthy and damaged mitochondria and therefore the organelle integrity. We show that Mitofusin overexpression ameliorates locomotor defects and abnormal neuronal activity in flies expressing TDP-43. Our results show the importance of mitochondrial quality control in the pathogenesis of these diseases, and that reinforcing it can be beneficial.

Un tiers de la population européenne est touché par au moins un trouble du cerveau. En effet, la maladie d’Alzheimer, et les gliomes, représentent respectivement le syndrome de démence et les tumeurs cérébrales les plus fréquentes chez l’homme. Plusieurs études épidémiologiques ont montré l’existence d’une corrélation inverse entre le risque de développer une maladie neurodégénérative et un cancer cérébral. Ceci suggère la présence de dénominateurs moléculaires communs entre ces pathologies. Dans les deux cas, un dysfonctionnement mitochondrial est rapporté, représentant une caractéristique partagée par ces deux troubles neurologiques. La protéine kinase mitochondriale PINK1 responsable, lorsqu’elle est mutée, d’une forme précoce et familiale de Parkinson, est particulièrement impliquée dans les processus de maintien de l’homéostasie mitochondriale. Par conséquent, les mécanismes moléculaires régulant PINK1 ainsi que leurs impacts au cours des désordres mitochondriaux répertoriés dans la maladie d’Alzheimer et les tumeurs cérébrales, ont suscité un intérêt central, lors de ma thèse. Au cours de ce travail, nous avons examiné certaines des fonctions mitochondriales de PinK1, associées au maintien de l’homéostasie mitochondriale dans un contexte « Alzheimerisé ». Nous mettons en évidence le rôle de la γ-secretase dans la physiologie mitochondriale en contrôlant la régulation transcriptionnelle de PINK1 par l’AICD, le fragment généré conjointement avec le peptide amyloïde toxique Aβ, à partir du précurseur βAPP. Nous montrons de surcroît, l’initiation de cette régulation par la parkine. / One third of the European populations is affected by a brain disorder. Thus, Alzheimer’s disease and gliomas represent the most frequent human brain dementia syndrome and tumor type, respectively. Several epidemiological studies have shown an inverse relationship between the risk of developing a neurodegenerative disease and a brain tumor, suggesting the existence of common molecular denominators between these pathologies. Interestingly, both pathologies are characterized by a mitochondrial dysfunction. The mitochondrial kinase associated to autosomal recessive Parkinson’s disease, PINK1, is particularly implicated in the control of mitochondrial homeostasis. The main objective of my thesis was to study the molecular mechanisms underlying PINK1 gene regulation and their link with the mitochondrial dysfunction observed in either Alzheimer’s disease or gliomas. Thus, during my thesis we have examined the ability of PINK1 to control mitochondria homeostasis in an Alzheimer’s pathological context. We demonstrate that AICD, a cleavage product of the trans-membrane protein βAPP by γ-secretase, impacts mitochondrial physiology via its ability of positively controlling PINK1 transcription. In addition, we show that the signaling cascade linking γ-secretase and PINK1 is initiated by parkin transcriptional regulation of presenilins, the main component of γ-secretase catalytic complex. Finally, we also establish that the tumor suppressor p53 can negatively regulate PINK1 transcription in vitro and in vivo suggesting that the misregulated autophagic response associated to brain tumors development may be caused by defective p53-PINK1 interplay.

Parkinson's disease (PD) is a progressive neurological disorder resulting from loss of dopaminergic neurons of the substantia nigra, in part due to mitochondrial dysfunction. The E3 ubiquitin ligase, PARKIN, and mitochondrial kinase, PINK1, found mutated in familial early onset recessive forms of PD play central roles in mitochondrial homeostasis, thus maintaining control of a diversity of cellular processes, including energy metabolism, calcium buffering, and cell death. Together, PARKIN and PINK1 control mitochondrial homeostasis via a signaling cascade in which depolarization-induced PINK1 stabilization and activation on the mitochondrial outer membrane (MOM) promotes recruitment of PARKIN. Consequently, the outer mitochondrial membrane is extensively decorated with ubiquitin, ultimately resulting in removal of the damaged organelles via mitophagy, the selective autophagic removal of mitochondria. While PARKIN has been demonstrated to ubiquitylate Porin, Mitofusin, and Miro proteins on the MOM, the full repertoire of PARKIN substrates - the PARKIN-dependent ubiquitylome - remains poorly defined. Here, large-scale quantitative diGlycine (diGly) capture proteomics was used to identify PARKIN-dependent ubiquitylation on lysine residues in proteins modified upon mitochondrial depolarization. Hundreds of ubiquitylation sites in dozens of proteins were identified, with strong enrichment for MOM proteins, indicating that PARKIN activity has the capacity to dramatically alter the ubiquitylation status of the mitochondrial proteome. Complementary interaction proteomics identified physical association of PARKIN with a cohort of MOM ubiquitylation targets, autophagy receptors, and the proteasome, interactions which were completely dependent upon mitochondrial damage and drastically reduced upon mutation of the active site residue, C431, found mutated in PD patients. Furthermore, structural and evolutionary analysis of PARKIN-dependent ubiquitylation events revealed extensive conservation of target sites on cytoplasmic domains in vertebrate and D. melanogaster MOM proteins. Parallel PINK1 interaction proteomics identified numerous subunits of the translocase of the outer mitochondrial membrane (TOMM) and a novel interactor, CLU1, shown to regulate mitochondrial morphology in lower eukaryotes. Positive genetic interaction between CLU1, PINK1, and PARKIN suggests the potential of a newly identified node of regulation for the PINK1/PARKIN pathway. These studies define how PARKIN and PINK1 re-sculpt the proteome to support mitochondrial homeostasis, ultimately contributing toward an improved understanding of their role in the progression of disease.

Neurons are metabolically active cells that depend on mitochondria for ATP production and calcium homeostasis. Within a single neuron, the demand for mitochondrial function is highly variable both spatially and temporally. This need-based distribution is reflected in high local density of mitochondria at presynaptic endings, post-synaptic densities, nodes of Ranvier, and in growth cones, where mitochondrial function is required to sustain neuronal activity. To meet local demand, mitochondria are mobile organelles that move along microtubule cytoskeleton in axons and dendrites. Due to their role in oxidative phosphorylation, mitochondria are prone to oxidative damage that can in turn jeopardize the cell. To minimize cellular damage, an autophagic process, known as mitophagy, has evolved to clear dysfunctional mitochondria. Defects in mitochondrial clearance are implicated in neurodegenerative diseases such as Parkinson's disease (PD). In neurons, it was thought that mitochondria with reduced membrane potential are retrogradely transported to the soma where they are degraded. In this dissertation, I present a new paradigm where damaged mitochondria are arrested and undergo mitophagy locally in axons. In chapter 2 we report that mitochondrial damage causes arrest of mitochondrial motility in neuronal axons through the action of Parkin, an E3 ubiquitin ligase implicated in PD. Parkin accumulates on the surface of depolarized mitochondria and triggers proteosomal degradation of the mitochondrial motor adaptor protein, Miro, thereby detaching mitochondria from the kinesin and dynein motor complex. This arrest of mitochondria would serve to quarantine them in preparation for their subsequent degradation. In chapter 3, I demonstrate that damage to a small population of axonal mitochondria triggers a pathway of mitophagy that occurs locally in distal axons. Two PD-associated proteins, PINK1, a mitochondrial kinase mutated, and Parkin are both required for axonal mitophagy. In chapter 4, I present preliminary studies examining the turnover rate of neuronal PINK1 in order to characterize its mechanism of activation in distal axons. In conclusion, I have characterized a pathway for quality control of mitochondria in neuronal axons showing that clearance of defective mitochondria oocurs locally in distal axons without a need for their retrograde transport to the soma.

Mitochondrial dysfunction has been implicated as one of the primary causes of Parkinson's disease (PD). The proteins PINK1, a serine-threonine kinase, and Parkin, an E3 ubiquitin ligase, are mutated in many genetic cases of PD. In healthy individuals, Parkin is recruited to damaged mitochondria and leads to autophagic degradation of mitochondria in a process termed mitophagy. Following depolarization of the mitochondrial membrane, PINK1 is stabilized on the outer mitochondrial membrane, and triggers Parkin translocation from the cytosol to mitochondria. Precisely how this phenomenon is regulated is still unclear. We employed RNA interference (RNAi) technology in a 384-well format to identify novel genes that are required for Parkin recruitment to mitochondria. We identified ATPase inhibitory factor 1 (IF1) as the strongest hit required for Parkin recruitment following treatment with the protonophore CCCP. We show that IF1 is upstream of PINK1 and Parkin, and required to sense mitochondrial damage by allowing the loss of membrane potential. In cells treated with CCCP, the absence of IF1 permits the ATP synthase to run freely in reverse, consuming ATP to maintain potential across the inner mitochondrial membrane, thus blocking PINK1 and Parkin activation. Interestingly, Rho0 cells, that lack mitochondrial DNA, have downregulated endogenous expression of IF1 in order to maintain mitochondrial function. Overexpression of IF1 in Rho0 cells results in the depletion of mitochondrial membrane potential and the initiation of mitophagy. These data demonstrate a unique role for IF1 in the regulation of mitochondrial quality control that has not been explored in the etiology of PD.

Ubiquitin phosphorylation by PINK1 (PTEN-induced Putative Kinase 1) is crucial for mitochondrial quality control and loss or mutation of PINK1 can lead to autosomal recessive juvenile parkinsonism (AR-JP). PINK1 is an unusual kinase, as it is characterised by three unique insertions in its kinase N lobe and a C-terminal region after the kinase domain. Despite great effort, a structure of PINK1 could not be determined and the molecular mechanism of ubiquitin phosphorylation and the effect of the PINK1 AR-JP patient mutations remained elusive. The versatile modifier ubiquitin (Ub) is also an unusual kinase substrate, as its phosphorylation site (Ser65) is not exposed, but protected by the Ub fold. Hence, it was not clear how a kinase would be able to target Ser65 of Ub. This work shows that Ub needs to adopt a previously described conformation in order to be efficiently phosphorylated by PINK1. NMR experiments revealed that in a small population of Ub the last β-strand is retracted, resulting in a more accessible Ser65 loop. It could be shown that PINK1 binds the Ser65 loop in this C-terminally retracted conformation (Ub-CR), but not in the ‘common’ conformation. In addition, it could be shown that Ub trapped in the Ub-CR conformation by point mutations (Ub TVLN) is phosphorylated significantly faster than Ub wt, which only adopts the Ub-CR conformation at very low frequency. To further elucidate how PINK1 binds and phosphorylates Ub, the kinase domain of Pediculus humanus corporis (Ph)PINK1 was crystallised in complex with Ub TVLN stabilised by a nanobody. The structure revealed many peculiarities of PINK1, such as the architecture of the unique insertions and the C-terminal region. Together with NMR and mass spectrometry studies, the structure explains how PINK1 interacts with ubiquitin via insertion-3 and its activation segment, and how PINK1 utilises the Ub- CR conformation for efficient Ser65 phosphorylation. In addition, the structure shows that two autophosphorylation sites in the N lobe regulate PINK1, by stabilising the functionally important insertions. The structure helped our understanding of the molecular basis of over 40 AR-JP patient mutations and may guide the design of ARJP therapeutics in the future.

Mitochondrial dysfunction has been implicated as one of the primary causes of Parkinson's disease (PD). The proteins PINK1, a serine-threonine kinase, and Parkin, an E3 ubiquitin ligase, are mutated in many genetic cases of PD. In healthy individuals, Parkin is recruited to damaged mitochondria and leads to autophagic degradation of mitochondria in a process termed mitophagy. Following depolarization of the mitochondrial membrane, PINK1 is stabilized on the outer mitochondrial membrane, and triggers Parkin translocation from the cytosol to mitochondria. Precisely how this phenomenon is regulated is still unclear. We employed RNA interference (RNAi) technology in a 384-well format to identify novel genes that are required for Parkin recruitment to mitochondria. We identified ATPase inhibitory factor 1 (IF1) as the strongest hit required for Parkin recruitment following treatment with the protonophore CCCP. We show that IF1 is upstream of PINK1 and Parkin, and required to sense mitochondrial damage by allowing the loss of membrane potential. In cells treated with CCCP, the absence of IF1 permits the ATP synthase to run freely in reverse, consuming ATP to maintain potential across the inner mitochondrial membrane, thus blocking PINK1 and Parkin activation. Interestingly, Rho0 cells, that lack mitochondrial DNA, have downregulated endogenous expression of IF1 in order to maintain mitochondrial function. Overexpression of IF1 in Rho0 cells results in the depletion of mitochondrial membrane potential and the initiation of mitophagy. These data demonstrate a unique role for IF1 in the regulation of mitochondrial quality control that has not been explored in the etiology of PD.

Skeletal muscles have a remarkable capacity to repair and regenerate in response to injury by virtue of their unique population of resident muscle stem cells (MuSCs). Recently, several studies have reported that mitochondria are important regulators of fate and function in various types of stem cells including MuSCs. Furthermore, emerging evidence has shown that accumulation of dysfunctional mitochondria leads to stem cell aging, premature commitment and impaired self-renewal. Preliminary evidence from publicly available transcriptomics datasets processed by our lab showed that Phosphatase and tensin homolog (PTEN)-induced putative kinase 1(PINK1) and Parkin/PARK2 genes, two key regulators of mitophagy are expressed in quiescent MuSCs and are transiently down-regulated as MuSCs activate. This led us to hypothesize that maintenance of an optimally functioning population of mitochondria through mitophagy would be important for self-renewal and muscle repair. In vitro single myofiber cultures isolated from mitophagy reporter mice (mito-QC mice), show that mitophagy is active in quiescent MuSCs and is transiently decreased upon MuSCs activation. We also show that mitophagy is re-activated in differentiating and self-renewing MuSCs. To further study muscle regeneration, we used a cardiotoxin (CTX) injury model of the Tibialis anterior (TA) muscle in mouse models harboring a knockout (KO) of PINK1 and Parkin. We show that loss of PINK1 in vivo promotes commitment of MuSCs in response to acute injury and ultimately leads to depletion of the MuSC pool and impaired muscle regeneration compared to wild type (WT) mice following repetitive injuries. Similarly, loss of Parkin in MuSCs in vivo impaired their self-renewal capacity. Consistent with these results, in vitro single myofiber cultures isolated from PINK1-deficient mice showed increased MuSCs commitment and impaired self-renewal. In vitro preliminary results from MuSCs-specific KO of Parkin revealed altered lineage progression, differentiation and self-renewal of MuSCs. Together, these findings suggest that PINK1/Parkin-dependent mitophagy acts as an important mitochondrial quality control mechanism which could be required for regulating MuSCs fate and function during muscle regeneration.