Ferroptosis is a non-apoptotic, regulated form of cell death that is characterized by the iron-dependent lethal accumulation of lipid peroxides and lipid peroxide byproducts. Huntington’s disease (HD) is an autosomal neurodegenerative disease with characteristic motor, psychiatric, and cognitive signs and symptoms caused by the expansion of CAG repeats in the Huntingtin gene, resulting in the production of pathogenic protein with an extended polyglutamine tract that is prone to aggregation. Accumulating evidence has identified links between ferroptosis and HD suggesting that ferroptosis inhibition may provide therapeutic benefit. However, the ability to evaluate this potential has been limited by the unavailability of potent, brain-penetrant specific ferroptosis inhibitors. This dissertation evaluates two different types of ferroptosis inhibitors and increases the available molecular tools to investigate the role of ferroptosis in the etiology of HD. In the second and third chapters, two new classes of ferrostatin analogs, termed fourth and fifth generation ferrostatins, are developed and their in vitro and in vivo properties characterized. These efforts identify three, fifth generation analogs that are potent, brain-penetrant, and stable and can be administered chronically to symptomatic HD mice. The fourth chapter provides molecular insights into the mechanism of action of the hypocholesterolemic drug probucol in inhibiting ferroptosis and identifies cellular cholesterol levels and cholesterol import as regulators of ferroptosis. In sum, this work provides new molecular tools and insights that can be utilized to elucidate the contribution of ferroptosis to HD and other disease states.

Dysfunction of the N-methyl-D-aspartate (NMDA) receptor is thought to contribute to the cognitive deficits of many neurodegenerative diseases and psychiatric disorders. Cognitive symptoms of Alzheimer's disease can be treated with NMDA receptor antagonists or drugs targeting the cholinergic system; however, there are no effective treatments for cognitive deficits of schizophrenia or Huntington's disease. With the discovery of a potent and selective allosteric modulator of the NMDA receptor, there is the possibility of new treatments based on NMDA receptor functional-enhancement through neuroactive steroids, closely related in structure to the endogenous neurosteroid, cerebrosterol. The aim of this thesis was to examine steroidal modulation of the NMDA receptor both in vitro and in vivo. In chapter 2, NMDA receptor enhancement of both the synthetic and endogenous neuroactive steroids was assessed in neurons maintained in cell culture using calcium imaging techniques. Sulphation of the steroids greatly increased the efficacy of NMDA receptor enhancement compared to the unsulphated steroids. Chapters 3 and 4 investigate the potential for neuroactive steroids to treat cognitive impairments of Huntington's disease. Using a mouse model, tests were selected that were analogous to those in which patients are impaired; however, no impairments were found in the mouse model. Chapter 5, therefore, used a different model of cognitive impairment – namely, rats with a set-shifting impairment, as is seen in many psychiatric and neurological disorders, including Huntington's disease – to assess the effect of the synthetic steroid administration. Unfortunately, the rats did not show the expected impairment. The lack of reliable animal models compromised testing the efficacy of these promising NMDA receptor positive allosteric modulators. Nevertheless, the promising in vitro results suggest that there could still be therapeutic potential. In addition, the compound is a useful research tool for exploring NMDA receptor function in health and disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Huntington’s Disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder. There is no cure for HD and the existing therapies only alleviate HD symptoms without eliminating the cause of this neuropathology. HD is linked to a mutation in the huntingtin gene, which results in an elongation of the poly-glutamine stretch in the huntingtin protein (Htt). A major hypothesis is that mutant Htt (mHtt) leads to aberrant Ca2+ homeostasis in affected neurons. This may be caused by increased Ca2+ influx into the cell via the N-methyl-Daspartate (NMDA)-subtype of glutamate receptors. The contribution of two major Ca2+ removal mechanisms, mitochondria and plasmalemmal Na+/Ca2+ exchangers (NCX), in neuronal injury in HD remains unclear. We investigated Ca2+ uptake capacity in isolated synaptic (neuronal) and nonsynaptic mitochondria from the YAC128 mouse model of HD. We found that both Htt and mHtt bind to brain mitochondria and the amount of mitochondriabound mHtt correlates with increased mitochondrial Ca2+ uptake capacity. Mitochondrial Ca2+ accumulation was not impaired in striatal neurons from YAC128 mice. We also found that expression of the NCX1 isoform is increased with age in striatum from YAC128 mice compared to striatum from wild-type mice. Interestingly, mHtt and Htt bind to the NCX3 isoform but not to NCX1. NCX3 expression remains unchanged. To further investigate Ca2+ homeostasis modulation, we examined the role of collapsin response mediator protein 2 (CRMP2) in wild-type neurons. CRMP2 is viewed as an axon guidance protein, but has been found to be involved in Ca2+ signaling. We found that CRMP2 interacts with NMDA receptors (NMDAR) and disrupting this interaction decreases NMDAR activity. CRMP2 also interacts with and regulates NCX3, resulting in NCX3 internalization and decreased activity. Augmented mitochondrial Ca2+ uptake capacity and an increased expression of NCX1 in the presence of mHtt suggest a compensatory reaction in response to increased Ca2+ influx into the cell. The role of NCX warrants further investigation in HD. The novel interactions of CRMP2 with NMDAR and NCX3 provide additional insight into the complexity of Ca2+ homeostasis regulation in neurons and may also be important in HD neuropathology.

The purpose of this thesis was to develop methodology by which treatments for the cognitive impairments in Huntington's disease (HD) could be tested. As such, the thesis focused mainly on evaluating rats with quinolinic acid (QA) lesions of the striatum, as this manipulation mimics some aspects of the neural damage in Huntington's disease, to try to identify cognitive deficits of HD resulting from cell loss in the striatum. In the first part (Chapters 3-5), the role of the striatum in implicit memory was investigated. Chapter 3 compared the performance of rats and humans on a reaction time task that evaluated implicit memory by presenting visual stimuli with differing probabilities which change over time. Although rats made higher percentage of incorrect responses and late errors, both groups showed a similar pattern of reaction times. Chapter 4 investigated whether implicit memory (the computation of probabilities to predict the location of a stimulus) was affected by selective blockade of dopaminergic transmission at the D1 or D2 receptors by SCH-23390 and raclopride, respectively. Reaction times were slower with SCH-23390 and raclopride, but only SCH-23390 reduced errors to the least probable target location. Chapter 5 used the same task to evaluate implicit memory in rats with QA lesions of the dorsomedial striatum (DMS). Implicit memory was not affected by lesions of the DMS, which suggested that once a task that requires implicit memory has been learned, the DMS was not involved in sustaining the performance of the task. The second part of this thesis (Chapter 6), explored the contribution of the DMS in habit formation. DMS lesioned rats did not show habitual responding, and were not impaired in learning a new goal-directed behaviour. The third part (Chapters 7 and 8), investigated the role of the dorsal striatum in reversal learning, attentional set-formation, and set-shifting. Dorsal striatum lesioned rats were not impaired in reversal learning, but had a diminished shift-cost, which suggested that dorsal striatum lesions disrupted the formation of attentional sets. These results showed that although QA lesions of the dorsal striatum mimic some aspects of the neural damage in HD, they did not result in the same cognitive deficits observed in patients with HD, at least using the tasks presented in this thesis. However, other animal models of HD could be evaluated using the different tasks presented in this thesis to continue the search of a reliable animal model of HD in which treatments for the disease could be evaluated.