Parkin gene is associated with the development of autosomal recessive juvenile parkinsonism (Kitada et al., 1998) which is a common form of familial Parkinson’s disease (Klein and Schlossmacher, 2006). Since Parkin has multiple cell protective effects, increasing the expression level of Parkin in the brain might be able to rescue cells in danger, which in turn might prevent or slow down the development of Parkinson’s disease (Ulusoy and Kirik, 2008). In order to increase Parkin expression, it is important to understand the transcriptional mechanisms regulating Parkin expression (Maston et al., 2006). Since human Parkin is very big (~1.4 Mb) (Asakawa et al., 2001), in this study we use the smaller Fugu parkin gene, which is an ortholog of human Parkin (Yu et al., 2005), to search for the transcriptional factors and signaling pathways regulating Parkin expression. We have cloned vertebrate constructs that allow for the monitoring of an entire genomic Fugu parkin gene tagged with a reporter (eGFP or luciferase) in mammalian cells; and have established cellular model for studying the expression. According to the “TRANSFAC” transcription factor database, as well as “TFBIND” and “TFSEARCH” softwares (Wingender et al., 1996; Heinemeyer et al., 1998; Heinemeyer et al., 1999; Tsunoda and Takagi, 1999; Akiyama 1995), potential Nrf2 binding sites are conserved in the promoters of mammalian parkin (including human Parkin and mouse parkin) and in Fugu parkin. In this study, we could not find a link between the presence of the potential Nrf2 binding site(s) in the parkin promoter and the up-regulation of parkin; and we could not find an association between the Nrf2 pathway activation and the induction of parkin under the specific experimental conditions.

A leading cause of male infertility is genetic variation in genes required for sperm formation and/or function. There is evidence to suggest PACRG is involved in mammalian spermatogenesis. Specifically, the loss of Pacrg function causes a spermatogenic defect and male infertility in mice. To investigate if PACRG plays a similar role in human spermatogenesis, the localisation of PACRG was determined in human testis. Using an immunohistochemical approach, this study demonstrated that PACRG is localised to the human sperm flagella. To investigate a potential role for PACRG in human male infertility, sequence analysis and an association study were performed. Sequence analysis did not identify any pathological alterations. However, 1 of 3 variants identified (rs9347683) was shown to be significantly associated with male infertility by association analysis (p=0.009, Odds Ratio=1.6, n=766). / A high degree of structural and functional conservation exists between different types of motile cilia/flagella. Evidence from studies in C.reinhardtii and T.brucei indicate Pacrg is necessary for axoneme formation and microtubule stability. To test the role of the mammalian homologue, this study characterised the Pacrg knockout mouse, quakingviable (qkv) and generated Pacrg transgenic qkv mice (qkv-Tg). Using immunohistochemistry and immunoelectron microscopy this study demonstrated that Pacrg was localised to the axonemal microtubule doublets of sperm and ependymal cilia. The absence of Pacrg was associated with compromised sperm flagella formation and male infertility. In addition, histological and MRI analysis of qkv mutant mice revealed hydrocephalus. Specifically, qkv mutant mice showed a ~2.5 fold expansion of the lateral ventricle area compared to wildtype mice. The hydrocephalus phenotype was associated with a reduction in ependymal cilial beat frequency (CBF). Transgenic expression of Pacrg was sufficient to rescue the hydrocephalus and infertility phenotypes. In conclusion, this study has demonstrated that Pacrg is a novel axonemal protein in a subset of motile cilia and loss of Pacrg function results in spermiogenic defects and hydrocephalus in mice. Further, this study has shown that variations in the human PACRG promoter are a risk factor in human male infertility. Collectively these data provide evidence for a conserved role of PACRG in the cilial axoneme. This suggests the protein may be a candidate for a variety of human diseases characterised by cilial dysfunction.

Recently, the zebrafish has been considered an important animal model that can be used to investigate human diseases and drug development. Parkinson¡¦s disease (PD), an important neurodegenerative disorder, is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra and movement defects, including bradykinesia, tremor, and postural imbalance. However, current treatments for PD are limited and mainly improve only the clinical symptoms of the disease. Thus, a neurodegenerative rat model has been widely used for a long while to search for a new treatment for PD. However, the use of rats as an animal model has certain limitations such as breeding, efficiency, and high dosage. Recently, researchers indicated that neurotoxins such as rotenone, 6-hydroxydopamine (6-OHDA), and paraquat can induce Parkinson¡¦s-like symptoms in zebrafish, and this may be a useful PD model because of the complete development of the zebrafish nervous system, low costs, and low dosage. In this study, we treated zebrafish with 6-OHDA and analyzed their locomotor activity to establish an in vivo animal model of PD. Then, we analyzed the mRNA expression of parkin and PINK1 by reverse transcription¡Vpolymerase chain reaction (RT-PCR).Moreover, we observed tyrosine hydroxylase (TH) expression by immunohistochemical (IHC) staining to confirm if this can be used as a PD model. Finally, we found that treatment with 6-OHDA significantly reduced TH expression. We observed a similar declining trend in the case of mammals. Likewise, parkin and PINK1 mRNA expressions were also decreased after treatment with 6-OHDA. In summary, our study provides a feasible in vivo Parkinson¡¦s model, and a small volume of drugs or compounds can be screened using this model.

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

Parkinson disease (PD) is an incurable disease, second only to Alzheimer’s disease as the most common neurodegenerative disease in adults. Unfortunately, the course of disease is significantly longer for individuals diagnosed at an early age (20-40 years of age). Although early-onset, recessively inherited cases represent a small subset of individuals with PD (~5- 10%), their clinical presentation is unique, with symptoms being almost exclusively motor-related. The expressivity of early-onset PD is partially explained by post-mortem neuropathological findings, which demonstrate a specific loss of dopamine synthesizing cells in brainstem nuclei that also produce neuromelanin (i.e. Substantia nigra and Locus coeruleus). With the majority of early-onset PD cases being caused by homozygous and biallelic heterozygous mutations in the PRKN gene, its gene product, parkin, has been extensively studied. It is generally accepted that loss of its E3 ligase function leads to neurodegeneration by either one of the following two mechanisms: i) toxic substrate accumulation from the loss of target protein ubiquitination (and related degradation), or ii) accumulation of dysfunctional mitochondria due to impaired mitophagy initiation. However, whether these mechanisms lead to selective neuronal loss within the human brain remains unknown. This thesis represents a body of work that supports a novel role for parkin as a thiol-based anti-oxidant and redox homeostasis regulator, which helps explain the cell-specificity observed in recessive, PRKN-linked PD. These findings include: i) evidence that human brain parkin uniquely and natively undergoes age-associated aggregation beginning at 40 years of age (Chapter 2); ii) identification of multiple, reversible and irreversible oxidative modifications of parkin cysteines, both in cells and tissues, including dopamine-adduct conjugation on primate sequence-specific cysteine 95 (Chapter 2); iii) the demonstration that irreversible oxidation of parkin cysteines causes aggregate formation ii in cells and mice exposed to exogenous and/or endogenous sources of oxidative and dopamine stress (Chapter 2 and 3); iv) evidence that parkin functions as a thiol-dependent anti-oxidant similar to glutathione (Chapter 2), which lowers oxidation state in cells and tissues under native and stress conditions (Chapter 2 and 3); v) the demonstration that parkin cysteines, notably C95, directly bind glutathione and regulate glutathione redox homeostasis in cells and tissues in a dynamic fashion (Chapter 3); and vi) the development of novel, human-specific, anti-parkin monoclonal antibodies that preferentially detect oxidized and aggregated forms of parkin found associated with neuromelanin and lysosomal storage vesicles within neurons of human Substantia nigra (Chapter 2 and 4). Future studies focusing on further validation of in situ oxidative modifications of parkin cysteines and their effect on protein structure, notably the poorly studied linker region that contains C95, will provide insight into how these oxidative modifications affect the function of parkin in vivo, including in adult human brain. Also, identifying the bona fide intracellular redox partners of parkin will be crucial to understanding how this protein regulates cellular redox state. Of clinical importance, the findings presented here indicate a potential, human-specific link between parkin and neuromelanin formation, which deserves to be further explored, such as with parkin mouse models engineered to produce neuromelanin. Finally, designing clinical trials using anti-oxidants specifically in individuals affected by PRKN-associated PD represents a logical, translational treatment approach to explore.

Early-onset Parkinson disease, of which the best studied and most common cause are biallelic mutations in the PRKN gene, is characterized by an age of onset before 40 years. Parkin-deficient patients show slow progression, excellent responsiveness to L-dopa therapy, and are generally spared cognitive decline. At autopsy, PRKN-linked Parkinson disease is further distinguished by relative selectivity in cell loss, namely of dopamine producing neurons in the human brainstem, and the general absence of Lewy body inclusions. Since its discovery two decades ago, the field has focused on the function of parkin as an E3 ubiquitin ligase and its related role in mitophagy. However, its essential, neuroprotective function in ageing human midbrain and the mechanisms by which wildtype parkin preserves dopamine cell health in aged humans are yet to be elucidated. I hypothesized that parkin confers neuroprotection due to a redox function by its many cysteines (7.5%). We first focused on parkin’s biochemistry, reporting a shift from solubility to a nearly insoluble, aggregated state in adult control brain after age 40 years. We detected cysteine-based, post-translational modifications of parkin in response to oxidative stress, and characterized a novel, redox chemistry-based function: Through its own oxidation parkin reduced hydrogen peroxide in vitro. I validated this finding by showing its elevation in parkin-deficient, human brain. Wild-type parkin also participated in dopamine metabolism through the conjugation of reactive radicals at several of its cysteines, which augmented the generation of melanin. (Chapter 2). In addition, we demonstrated parkin’s heretofore unknown, antioxidant function in the cytosol using cellular paradigms and select genetic as well as toxin-based mouse models that featured elevated oxidative stress. Moreover, we uncovered parkin’s contribution to the wider thiol network, namely through an apparent feedback loop with glutathione metabolism (Chapter 3). Lastly, I developed and investigated a bi-genic (prkn-/-//Sod2+/-) model in an attempt to restage the pathogenesis of early-onset parkinsonism in mice; there, we detected a rise in systemic, oxidative stress and in total nitrotyrosination profiles of the brain, but did not observe any dopamine neuron loss in the midbrain. In accordance, the behavioural characterization of these animals did not reveal any motor abnormalities (Chapter 4). Based on this previously unknown function for parkin in redox biology, I envision three future research directions: a) additional studies to delineate the full range of oxidative modifications of parkin versus neutralization of radicals; this, to better define the distinct pathogenesis of parkin-linked Parkinson’s when compared to other forms of the disorder; b) structural studies of its oxidative modifications in vitro and renewed attempts of staging parkin-linked dopamine cell death in vivo; and c) and importantly, the exploration of cause-directed therapy based on parkin’s redox functions to preserve dopamine neurons of the human midbrain throughout adulthood.

<p>Advances in modern medicine have helped to prolong human life. These advancements coupled with an ever-increasing population means that diseases associated with aging will become more prevalent in the coming years. As such, it is critical to understand the pathogenesis of disease where aging is the main risk factor. While not widely known, age is in fact a large risk factor in development of obesity and metabolic syndrome. More widely known and discussed are the neurodegenerative diseases that occur late in life. While age as a risk factor is a common point between these types of pathology, there are other similarities, such as the interaction between lipid metabolism and mitochondrial health. </p><p>To study the overlap between obesity and neurodegeneration, we investigated two pathways that regulate both. First, we find that loss of cytoplasmic deacetylase HDAC6 leads to aberrant accumulation of lipid in vitro and in vivo. HDAC6 knock-out (KO) mice gain more weight than WT counterparts after a high-fat diet regimen. Additionally, the intermediary metabolism of cells lacking HDAC6 is disrupted as they increase glucose uptake while downregulating fatty acid oxidation. HDAC6 not only plays a role in lipid metabolism, but regulates mitochondrial dynamics. Upon glucose-withdrawal, HDAC6 KO cells fail to elongate their mitochondria and display increased levels of mitochondrial toxic by-products. Therefore, HDAC6 has critical roles in lipid homeostasis and mitochondrial health. </p><p>The other pathway we investigated is critical in neurodegenerative disease, Parkinson's disease. Parkin, an E3 ubiquitin ligase, flags damaged mitochondria for destruction so they do not poison the other functional organelles. We found that Parkin promotes lipid remodeling at the surface of the mitochondria. Phosphatidic acid (PA) accumulates shortly after mitochondrial damage while diacylglycerol (DAG) appears several hours later. This lipid accumulation is dependent upon Parkin's translocation and E3 ligase activity. Additionally, we found that lipin-1, a PA phosphatase, and endophilin B1 (EndoB1) are critical for DAG accumulation and effective mitochondrial clearance. </p><p>Through this work, we show that two proteins critical in quality control mechanisms also play significant roles in energy homeostasis. We aim to highlight this overlap and posit that common diseases of aging, though presenting differently, might have disruptions in the same basic process.</p> / Dissertation

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.