Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). PD affects ~1% of the population over 65, as demonstrated by characteristic symptoms such as tremor, rigidity, and bradykinesia. While the majority of PD cases are idiopathic, some cases are familial, including those caused by homozygous loss-of-function mutations in DJ-1 (PARK7), which lead to early onset PD. Although the physiological role of DJ-1 is not fully understood, DJ-1’s neuroprotective role against oxidative stress is well documented. DJ-1 is required for AKT-mediated neuroprotective effects, however the mechanism by which DJ-1 affects membrane localization/activation of AKT is unknown and is likely a critical aspect of DJ-1 function. In this thesis we explore the mechanism through which DJ-1 confers neuroprotection through AKT membrane recruitment, particularly in the case of oxidative stress insult. We demonstrate here that DJ-1 interacts with PHLDA3, a negative regulator of AKT, and loss of DJ-1 leads to hypersensitivity of neurons to PHLDA3-mediated death. Additionally, we demonstrate that in the absence of DJ-1, PHLDA3 localization at the membrane is increased, and overexpression of PHLDA3 causes reduced AKT phosphorylation in DJ-1 KO MEFs in response to oxidative stress. Taken together, these studies provide a potential novel mechanism by which DJ-1 regulates the activity of AKT, a critical neuronal survival pathway. Elucidation of these mechanisms may provide insight into the design of neuroprotective therapies for PD.

Parkinson’s disease (PD) is the second most common neurodegenerative disease and is characterized by motor symptoms such as tremor, rigidity, akinesia and postural instability. Approximately 90% of the cases are due to unknown causes however a familial inheritance has been shown for about 10% of cases. Loss-of-function mutations in DJ-1 cause early-onset PD. Originally identified as an oncogene, DJ-1 has since had many functions attributed to it but its major role in the cell seems to be oxidative stress handling. We have previously demonstrated that DJ-1 deficiency results in hypersensitivity of cells to oxidative stress. Additionally, mitochondria from DJ-1 null mice are fragmented and produce more ROS. To better understand the relationship between DJ-1, cell survival and mitochondria, we investigated the possible interaction between DJ-1 and the mitochondrial protein voltage dependent anion channel 1 (VDAC1). Here we show mitochondrial translocation of DJ-1 following oxidative stress in murine embryonic fibroblasts (MEFs) and primary cortical neurons, a process dependent on Pink1 and Parkin. Additionally, we confirm that DJ-1 and VDAC1 interact and that stress-induced mitochondrial translocation of DJ-1 depends on VDAC1. Deficiency of VDAC1 in primary cortical neurons results in decreased survival, increased ROS production following extended stress, fragmented mitochondria and decreased mitochondrial ATP production. We also demonstrate that there is substantially less matrix-localized DJ-1 in VDAC1 deficient cells. Finally, we demonstrate that decreased mitochondria ATP production can signal for DJ-1 translocation to mitochondria. Taken together, we suggest that mitochondrial translocation of DJ-1 is a two-step process. First, a signal, perhaps decreased mitochondrial ATP production, induces DJ-1 translocation to mitochondria. Second, DJ-1 localizes to the matrix in a VDAC1-dependent manner. Our work suggests that stress-induced mitochondrial localization of DJ-1, specifically to the matrix, is regulated by VDAC1 to promote survival possibly by promoting ATP production.

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.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder. Its cause(s) are predominantly unknown; however, a subset of cases has a genetic origin. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of PD. Cases are clinically indistinguishable from idiopathic PD and display incomplete penetrance. Thereby, it is predicted that genetic vulnerability combined with environmental factors cause pathogenesis. However, the identity of these factors is unknown. Unfortunately, LRRK2`s native and pathogenic biological function(s) remain to be defined; owing to obstacles including a complex protein structure and the lack of pathological phenotypes in LRRK2 research models. To address the knowledge gap in LRRK2 biology, we set out to investigate the role of LRRK2 in the central nervous system (CNS). We generated and characterized a disease-mimicking D. melanogaster model of LRRK2-linked PD. This system was utilized to perform an in vivo, unbiased, high-throughput genetic screen to identify candidate interactors of LRRK2. Successful identification of a discrete number of genetic interactors was accomplished and, coupled with published evidence, highlighted the pursuit of subsequent mitochondrial-related investigations of LRRK2. These studies were performed using the M. musculus model system. Since LRRK2 murine models lack disease-relevant phenotypes, and LRRK2’s incomplete penetrance is predicted to be the result of gene-environment interaction, we employed the mitochondrial-targeting exogenous neurotoxin – MPTP/MPP+, to investigate neuronal mitochondrial phenotypes and subsequent survival in the context of LRRK2. Using the pathogenic R1441 GTPase-linked mutation, we did not observe altered neuronal mitochondrial length phenotypes or enhanced CNS sensitization to MPTP/MPP+-induced death; highlighting that MPTP-mediated, mitochondrial-centered mechanisms of action should be approached cautiously in the context of R1441-LRRK2. Collectively, the work presented herein has unveiled novel targets for the exploration of LRRK2 biological function and encourages the investigation of alternative pathogenic trigger mechanisms in the context of LRRK2-linked PD.

Parkinson’s disease is a highly prevalent multifactorial neurodegenerative disorder caused by a complex cascade of interactions between various genetic and environmental factors. Due to this, the majority of cases are termed idiopathic. However, about 10% of PD cases are due to defined genetic factors. Interestingly, both idiopathic and familial cases of PD share mitochondrial dysfunction as a central component in the pathology of the disease. The mitochondrial protease, presenilin-associated rhomboid-like (PARL), is one such Parkinson's disease-linked gene, and is associated with diverse processes including mitochondrial dynamics, active inhibition of unnecessary apoptosis and mitophagy in Drosophila and yeast. Here, I investigated the role of the two zebrafish parl paralogs, parla and parlb, through stable CRISPR-Cas9 mediated mutagenesis. I injected wild type embryos with sgRNAs targeting parla and parlb loci, successfully producing indel mutations in parlb and multi-exon deletions in parla at mutation efficiencies of 74% and 40%, respectively. Through whole mount in situ hybridization experiments against th1, I saw no change in dopaminergic (DA) neuron development displayed by parlb mutants compared to wild types. Injection of parla splice blocking morpholinos into parlb mutants indicates that proper DA neuron development may depend principally on Parla function and loss of both Parla and Parlb function increases larval mortality. These results suggest a negative epistatic relationship between the parl paralogs as seen by the more severe phenotype observed in the loss of both Parla and Parlb function compared to the individual effects.

Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. The mechanism by which these DA neurons die is still unclear and under investigation. Although mostly idiopathic, about 10% of PD cases have shown familial inheritance. Mutations in leucine-rich repeat kinase 2 (LRRK2), a large multi-domain protein with unknown physiological and pathological roles, have been linked to PD cases of autosomal dominant inheritance. A PD Drosophilamelanogaster model over expressing the human LRRK2(I2020T) kinase mutant using the GAL4/UAS system has shown a loss of DA neurons and locomotor deficiency. Additionally, ectopic overexpression of human LRRK2 in the eye caused a damaged eye phenotype characterized by roughness of the surface, loss of pigmentation and presence of black lesions (Venderova Ket. al., 2009). The presence of this identifiable eye phenotype has allowed us to perform a suppressor/enhancer screen to identify possible genetic interactors of LRRK2. The LRRK2(I2020T) transgenic flies were crossed with genomic deficiency lines and the eye phenotype screened for either suppression or enhancement. Twenty-two genes, which are implicated in a variety of biological processes, have been identified thus far. Fourteen of these 22 interacting genes were assessed in the DA neurons of the D.melanogaster model. This functional screen is a rapid method to provide us with potential genetic interactions between LRRK2 and other genes, which will in turn, aid in elucidating the functional role of LRRK2 in PD pathology.

Parkinson's disease is a progressively debilitating disease that affects about 1% of the world's population, and does not differentiate between genders or races. The disease is caused by the death of the dopaminergic neurons in the basal ganglia nuclei, especially those in the substantia nigra pars compacta. Subsequent loss of dopamine production engenders the cardinal symptoms of bradykinesia, rigidity, akinesia, and postural instability found in all patients with Parkinson's disease. While there are several types of Parkinson's disease, the majority of the cases are made up of the idiopathic and Levodopa responsive type. The current consensus on treatment is to use medications until the patient becomes refractory to all medicines. It is only at this point will the surgical option deep brain stimulation be considered. while this procedure comes with a higher risk of post surgery complications, the benefits it offers patients with advanced Parkinson's disease are far superior to those offered patients by medications. It reasons then that patients would benefit more if they received this treatment earlier in the course of the disease. The mechanisms, side effects, costs, cost-effectiveness, and long term effects on quality of life of deep brain stimulation will be compared with those of medications to assess whether it is worthwhile to use this treatment for patients with mild Parkinson's disease.

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

Novel advances in engineering and data analytics are revolutionizing both our ability to monitor Parkinson’s disease (PD) patient symptoms and our understanding of neuropathology. Despite promise, key challenges exist before patient monitoring technologies become standard in clinical settings, including 1) industry standardization of sensor-based analytical approaches; 2) validation of endpoint sensitivity to degree of impairment and medication state; and 3) consensus regarding appropriate devices, algorithms, data requirements, and statistical analysis requirements for symptom measurement outside of the clinic. In addition to the need for better patient monitoring, no disease-modifying therapeutics currently exist and thorough understanding of the neuropathology of PD remains elusive. To this end, large network brain simulations that leverage efficient computational frameworks are beginning to provide insight into mechanisms that facilitate pathological oscillations and may serve to identify new therapeutic targets. To address current limitations in patient monitoring and our understanding of neuropathology, in this dissertation I 1) develop and evaluate validity and reliability of an open-source, wearable sensor-based algorithm for measuring gait in PD patients, 2) evaluate and compare sensitivity of at-home measurements relative to in-clinic measurements, 3) evaluate sensitivity of wearable-derived features for measuring degree of gait impairment and treatment response in PD patients, and 4) investigate the effect of synaptic parameters on beta synchrony and entrainment in a large-scale spiking model of the subthalamic nucleus-globus pallidus externa (STN-GPe) network of the basal ganglia. Importantly, I find that sensor-derived features derived from the at-home environment differ from and are more sensitive to small changes compared to in-clinic, traditional assessments. Furthermore, I demonstrate the capacity for a single, lower back sensor-based algorithm to estimate gait features with sufficient sensitivity to detect degree of gait impairment and treatment effect in a mild-to-moderate PD population. Lastly, I demonstrate that weak synaptic connections between STN and GPe allows the STN-GPe network to entrain to a wide range of frequencies outside of the beta range, thus elucidating constraints on conditions required for beta production. Together, my work provides new insights into the feasibility and benefits of sensor-based symptom monitoring and PD-related neuropathology.

Indiana University-Purdue University Indianapolis (IUPUI) / Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive loss of motor function resulting from dopaminergic neuronal death in the substantia nigra pars compacta leading to subsequent decreased striatal dopamine levels. The majority of PD cases are diagnosed as sporadic in nature, however 10% - 15% of patients show a positive family history of the disease. While many genes have been found to be implicated in the familial form of PD, early-onset autosomal recessive PD has been associated with mutations in PARK7, a gene which codes for the protein DJ-1. While there are many proposed roles of DJ-1 across numerous systems, the function of DJ-1 in relation to the development and progression of PD remains largely unclear. A first step towards determining this function is the creation of biologically relevant cell models of PD. The goal of this work was to design a representative cell model of DJ-1-associated PD in order to further study DJ-1 with the intention of elucidating its relevant function in relation of PD pathogenesis.