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.

Parkin is a cytosolic E3 ubiquitin ligase that is recruited to the mitochondria during cellular stress and has been suggested to be involved in a variety of biological processes such as mitophagy. The recruitment of Parkin (PARK2) to the mitochondria is dependent upon the kinase activity and the accumulation of PINK1 on damaged mitochondria. Mutations in either PINK1 or Parkin genes disrupt this protective pathway and lead to the accumulation of damaged mitochondria. From a clinical standpoint, mutations in the PARK2 gene have been associated with the progression and onset of autosomal recessive juvenile parkinsonism. Without the presence of a quality control system such as that of the PINK1/Parkin pathway, the accumulation of damaged mitochondria could lead to increased levels of oxidative stress, a decrease in ATP, and the progression towards cellular death. However, many of the details regarding the mechanism of Parkin-mediated ubiquitination and its involvement in mitophagy are not fully established. The intent of this thesis is to further explore the function of Parkin by utilizing the yeast-two hybrid system to identify novel Parkin interactors/substrates. A HeLa (cervical cell carcinoma) cDNA library was screened using Parkin124-465 as the "bait" protein. From this screening, six positive Parkin interactors were isolated and characterized. Using this approach it is possible to gain a better understanding of the function of Parkin in regulating cellular processes such as mitophagy.