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Neurodegenerative disorders are among the most devastating human illnesses. They present a significant source of morbidity and mortality, and given an aging population, an impending public health crisis. Disease-modifying treatments remain sparse, with most current therapies focused on reducing symptom burden. The cellular stress response is intimately linked to energy management and has frequently been posited as playing a central role in neurodegeneration. Using two distinct neurodegenerative diseases as ‘case studies’, aberrant cellular stress and energy management are demonstrated as potential pathways contributing to neurodegeneration. First, the Huntington’s disease protein, huntingtin, is observed to rapidly localize to early endosomes, where it is associated with arrest in early-to-late and early-to-recycling endocytic trafficking. Given the energy-dependent nature of vesicular trafficking, this arrest is postulated to free substantial energy within the cell, which may subsequently be diverted to pathways that are critical for the initiation of longer-duration stress responses, such as the unfolded protein response. In the context of Huntington’s disease, impaired recovery from this stress response is observed, suggesting deficits in intracellular vesicular trafficking and energy regulation exist in disease states. In the second ‘case study’, a novel spinocerebellar ataxia variant is characterized, occurring as a result of point mutations within two genes: ATXN7 and TOP1MT, which encode ataxin-7 and the type I mitochondrial topoisomerase (top1mt), respectively. Ataxin-7 has previously been implicated in spinocerebellar ataxia type 7, which occurs as a result of a polyglutamine expansion in the first exon of the protein. Patient cells are noted to have substantially lower mitochondrial respiratory function in comparison to healthy controls and decreased levels of mitochondrial DNA, and ataxin-7 subcellular localization is observed to be abnormal. This suggests that there is important interplay between the mitochondria and proteins implicated in neurodegeneration and provides further support for aberrant cellular bioenergetics as a unifying pathway to neurodegeneration. In the concluding chapters, the nuclear localization signal of ataxin-7 is characterized, and there is analysis comparing conical ‘atraumatic’ lumbar puncture needles with bevel-tipped ‘conventional’ needles. Atraumatic needles are noted to be associated with significantly less patient complications and require fewer return visits to hospital. Moreover, atraumatic needles are demonstrated to have similar rates of success and failure when controlling for important variables like clinician specialty, dispelling common misconceptions surrounding their ease-of-use. As lumbar puncture is ubiquitous within the clinical neurosciences and is important for diagnosis, monitoring, and treatment of disease, as well as clinical trials, this work has far-reaching implications for patient care and future research. / Thesis / Doctor of Philosophy (PhD)

Parkinson's disease (PD) is the second most prevalent age-related neurodegenerative disease and currently affects over 8 million people worldwide. The primary features of PD include cognitive, behavioral, and motor function deficits induced primarily by the progressive loss of dopaminergic neurons within the substantia nigra of the basal ganglia (BG). Motor coordination becomes severely affected over the course of the disease, causing patients to experience tremors at rest, bradykinesia, and body rigidity. The availability of treatment options has increased the quality of life for patients experiencing the early stages of PD; however, there exists no cure and treatment options are limited for those experiencing severe, advanced disease symptoms. Genetic studies in PD patients have led to the identification of causative genes, but revealed that less than 20% of cases can be attributed to monogenic variations. Evidence strongly indicates that the majority of PD cases are idiopathic and likely driven due to gene by environmental interactions. Reflective of this idea, recent research efforts have turned to genome-wide association studies (GWAS) to provide indications of gene variations, that while not causative of PD, incur increased risk within patient populations. GWAS findings play a particularly crucial role in neurodegenerative interventions, as early identification of patient risk may allow for preventative therapeutics to delay disease onset or reduce symptom severity. Amongst the many gene variants identified as incurring increased PD risk, single-nucleotide polymorphisms (SNPs) in the loci for CD38 that cause reduced gene expression are consistently identified as increasing risk. The cluster of differentiation 38 (CD38) protein serves two major roles: one as a receptor for immunological response and a second as an ectoenzyme that modulates bioenergetic functions. The particular functions of CD38 are highly relevant to neurodegenerative contexts, as changes in central nervous system (CNS) inflammatory status and means of cellular energy production typically precede pathological indications. In the brain, CD38 expression is most enriched in astrocytes in BG regions, including substantia nigra, midbrain, and striatum. However, it is not known how CD38 deficiency may contribute to astrocytic dysfunction and neuropathological features of PD. This dissertation describes how CD38 influences astrocytic gene expression and cellular bioenergetics. Astrocyte RNA was sequenced from the BG of one-year old male Cd38+/+, Cd38+/-, and Cd38-/- mice by magnetic-activated cell sorting (MACS) to acquire astrocyte isolates. Numerous differentially expressed genes (DEGs) were identified in Cd38 Cd38+/- and Cd38-/- astrocytes that relate to regulation of cellular health, responses to stress, and bioenergetic functions. GO analysis further suggested mitochondrial dysfunction in both Cd38+/- and Cd38-/- astrocytes. In a subsequent set of experiments evaluating mitochondrial function by Seahorse XF96 platform, Cd38+/- and Cd38-/- astrocytes displayed altered bioenergetic function. The results herein demonstrate that astrocytic Cd38 expression regulates cellular function and implicates transcriptional changes associated with the hallmarks of neurodegeneration. These findings serve to provide future direction for studies evaluating the relationship between CD38 function and astrocytes as it relates to neurodegenerative PD risk. / Doctor of Philosophy / Parkinson's disease (PD) is the second most common age-related neurodegenerative disease and currently affects over 8 million people worldwide. The primary features of PD include cognitive, behavioral, and motor function deficits induced primarily by the progressive loss of specialized neurons within the substantia nigra of the basal ganglia (BG) brain region. Motor coordination becomes severely affected over the course of the disease, causing patients to experience body tremors, slowness, and rigidity. The availability of treatment options has increased the quality of life for patients experiencing the early stages of PD; however, there exists no cure and little treatment options for those experiencing severe, advanced disease symptoms. Genetic studies in PD patients have led to the identification of causative genes, but revealed that less than 20% of cases can be attributed to specific, individual variations. Evidence strongly indicates that the majority of PD cases are likely caused by small gene changes that interact with environmental factors. Recent research efforts have turned to genome-wide association studies (GWAS) to identify these small changes, that while not causative of PD, may increase risk within patient populations. GWAS findings play a particularly crucial role in treating neurodegenerative diseases, as early identification of patient risk may allow for preventative therapeutics to slow disease onset or reduce symptom severity. Amongst the many small gene changes identified as increasing PD risk, changes in the gene CD38 that cause reduced gene expression are consistently identified as increasing risk. The cluster of differentiation 38 (CD38) protein serves two major roles: one as a receptor for immune responses and a second as an enzyme that impacts how cells produce energy. The functions of CD38 are highly relevant to neurodegenerative contexts, as changes in central nervous system (CNS) inflammatory status and means of cellular energy production typically precede disease pathology. In the brain, CD38 expression is most enriched in astrocytes, specialized brain cells that supports neurons, in regions affected by PD. However, it is not known how CD38 deficiency may contribute to astrocytic dysfunction and neuropathological features of PD. This dissertation describes how CD38 influences astrocytic gene expression and cellular bioenergetics. Astrocyte RNA was sequenced from the BG of one-year old male Cd38+/+, Cd38+/- (50% CD38 loss), and Cd38-/- (100% CD38 loss) mice by magnetic-activated cell sorting (MACS) to acquire astrocytes. Numerous changes in gene expression were identified in Cd38 Cd38+/- and Cd38-/- astrocytes that relate to regulation of cellular health, responses to stress, and energy functions. Further analysis looking at functions, suggested mitochondrial abnormalities in both Cd38+/- and Cd38-/- astrocytes. In a subsequent set of experiments evaluating mitochondrial function by Seahorse XF96 platform, Cd38+/- and Cd38-/- astrocytes displayed altered energetic function. The results herein demonstrate that astrocytic Cd38 expression regulates cellular function and implicates transcriptional changes associated with the hallmarks of neurodegeneration. These findings serve to provide future direction for studies evaluating the relationship between CD38 function and astrocytes as it relates to neurodegenerative PD risk.

Some organophosphorus (OP) esters induce a central-peripheral distal axonopathy called organophosphorus ester-induced delayed neurotoxicity (OPIDN). In the chicken model neurological deficits and microscopic lesions develop 7-21 days after exposure. Neurotoxic esterase (NTE) is thought to be the initial target in OPIDN. Evidence indicates that neuropathic OP esters have to bind NTE and chemically ?age? for OPIDN induction. It was hypothesized that phenyl saligenin phosphate (PSP), a neuropathic OP ester that essentially irreversibly inhibits NTE as it undergoes the chemical aging process, results in changes in spinal cord gene expression that do not occur with phenylmethylsulfonyl fluoride (PMSF), a non-neuropathic compound that inhibits NTE without aging. This hypothesis was tested in Gallus gallus in experiments designed to detect differences in spinal cord gene expression between PSP, PMSF and vehicle-treated birds 24 hours after exposure. Two approaches were used. Targeted display was developed and used to screen approximately 15000 gel bands. Three candidate genes were identified by targeted display. One, designated P1 has 100% homology with expressed sequence tag pgp1n.pk010.m23, another, P2, is homologous to human KIAA1307, and a third, P3, is unidentified. Northern blotting was used to measure spinal cord expression of a-tubulin and other genes previously reported to be differentially expressed following exposure to di-isopropryl phosphorofluoridate, another agent causing OPIDN. Only expression of a-tubulin was altered in PSP-treated hens. Time course experiments were undertaken to determine spinal cord expression changes of P1, P2, P3 and a-tubulin transcripts at 12, 24, 36 and 48 hours post-exposure. Findings indicated decreases and increases, respectively, of P1 (22%, p=0.0011) and P2 (26%, p=0.0055) transcripts at 12 hours in PSP treated hen spinal cord compared to DMSO controls. An ~2.5 kb a-tubulin transcript was decreased across most time points with maximum change at 48 hours (33%, p=0.0479); an ~4.5 kb a-tubulin transcript was upregulated at 12 hours (38%, p=0.0125) and down regulated at 48 hours (28%, p=0.0576). Responses to PMSF were different than responses to PSP. Spinal cord in-situ hybridization experiments revealed, 1.) mainly neuronal expression of P1, P2 and a-tubulin transcripts, and, 2.) decreased expression of neuronal P1 and a-tubulin transcripts at 12 and 48 hours, respectively. Results indicate that PSP can induce changes in gene expression distinct from those induced with the non-neuropathic NTE inhibitor, PMSF. However, expression changes were low in frequency and magnitude, and their mechanistic importance remains to be fully established. / Ph. D.

Alzheimer's disease is the leading cause of senile dementia in the elderly, and as life expectancy increases across the globe, incidence of the disease is continually increasing. Current estimates place the number of cases at 25-30 million worldwide, with more than 5.4 million of these occurring in the United States. While the exact cause of the disease remains a mystery, it has become clear that the amyloid β-peptide (Aβ) is central to disease pathogenesis. The aggregation and deposition of this peptide in the brain is known to give rise to the hallmark lesions associated with Alzheimer's disease, but its exact mechanism of toxicity remains largely uncharacterized. Molecular dynamics (MD) simulations have achieved great success in exploring molecular events with atomic resolution, predicting and explaining phenomena that are otherwise obscured from even the most sensitive experimental techniques. Due to the difficulty of obtaining high-quality structural data of Aβ and its toxic assemblies, MD simulations can be an especially useful tool in understanding the progression of Alzheimer's disease on a molecular level. The work contained herein describes the interactions of Aβ monomers and oligomers with lipid bilayers to understand the mechanism by which Aβ exerts its toxicity. Also explored is the mechanism by which flavonoid antioxidants may prevent Aβ self-association and destabilize toxic aggregates, providing insight into the chemical features that give rise to this therapeutic effect. / Ph. D.

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