The aberrant accumulation of aggregated proteins is a pathologic hallmark across adult-onset neurodegenerative diseases, the majority of which have no effective treatment. Although the relative importance of these structures to pathogenesis has been proposed in several diseases, there is little understanding of how we might accelerate the turnover of aggregated proteins, and in turn, a lack of consensus about whether targeting them would provide any therapeutic benefit. The overarching goal of my dissertation is to address both of these questions by focusing on how the pathway macroautophagy might handle protein aggregates in the adult brain. Aggregation-prone proteins are preferentially degraded through the lysosome-mediated degradation pathway macroautophagy (referred to hereafter as autophagy) (Ravikumar 2002; Iwata 2005; Yamamoto 2006). Although studies suggest that aggregates are degraded in bulk by autophagy (Ravikumar 2002; Iwata 2005), studies show that they are more likely degraded in an adaptor-protein dependent manner (Lemasters, 2005; Kraft, 2008; Hanna, 2012; Isakon, 2012; Filimonenko, 2010). In the Yamamoto lab, we have found that the adaptor, the Autophagy-linked FYVE protein (Alfy/WDFY3), is required for the degradation of detergent-insoluble aggregated proteins through selective autophagy in cell-based systems and the adult brain (Simonsen, 2004; Eenjes, 2016; Filimonenko, 2010; Fox, 2020). Through immunohistochemical and loss-of-function studies, Alfy has been implicated in the turnover of disease-relevant protein aggregates including mHtt, α-synuclein, SOD1, and TDP-43, as well as protein complexes such as the midbody ring (Filimonenko, 2010; Clausen, 2010; Han, 2014; Hocking, 2010; Isakson, 2013; Kadir, 2016). Here, I present a potential strategy to suppress disease progression across neurodegenerative disorders by increasing the levels, and thereby the function, of Alfy. I hypothesized that genetically augmenting Alfy levels in the brain will be sufficient to alleviate aggregate burden and delay the onset of proteotoxic stress in different mouse models of neurodegeneration. Using biochemical and genetic approaches, I conducted an extensive in vivo study, demonstrating that augmenting Alfy expression levels in mice can be neuroprotective, and that Alfy may be an influential genetic modifier of neurodegenerative disease. Using two independent genetic approaches that upregulate Alfy expression, I found that they both dramatically delay the onset of disease phenotypes in mouse models of Huntington’s disease, synucleinopathy and TDP-43 proteinopathy. First, I found that ectopic overexpression of Alfy has a pronounced, neuroprotective effect on reducing aggregation, improving motor function, and extending survival in disease models. In parallel, I used mouse genetics to verify the potency of a rare Alfy variant identified in a large Venezuelan cohort of Huntington’s disease that correlated with delayed onset in Huntington’s disease by 10-20 years. Excitingly, in support of our hypothesis, I found that the presence of this single nucleic acid polymorphism led to an increase in steady state levels of Alfy in both humans and in mice, and it was sufficient to recapitulate the benefits of ectopic Alfy overexpression. Taken together, these studies demonstrate that increasing Alfy levels in the brain are sufficient to augment the turnover of aggregated proteins, and may be an effective therapeutic strategy that can be beneficial across neurodegenerative diseases.

<p>Huntington's Disease brains display a striking accumulation of huntingtin in the nucleus of striatal and cortical neurons, suggesting that nuclear functions may be key to the onset of cellular pathogenesis. Entry to the nucleus is tightly regulated by a family of import receptors called karyopherins that limit their binding to proteins bearing specific nuclear localization signals (NLS). Although huntingtin is primarily cytoplasmic in healthy neurons, it is also found in the nucleus at low levels and contains a nuclear export signal (NES), suggesting that shuttling to and from the nucleus is part of the protein's normal function. Indeed, recent publications from our lab (Atwal <em>et al.</em> 2007 and 2011, and Munsie <em>et al</em>. 2011) describe huntingtin's ability to enter the nucleus in response to cell stress, and localize to cofilin-actin rods. We have identified an active NLS near the amino terminus of huntingtin between amino acids 174-207, which is recognized by both karyopherin β2 and β1. While functional in ST<em>Hdh </em>and NIH 3T3 cells, the huntingtin NLS lacks activity in HEK 293 and MCF-7 cell lines. This surprising observation suggests that additional levels of regulation exist amongst cell types. We have isolated a shorter sequence within the NLS that localizes to membrane structures throughout the cell (such as the plasma membrane and vesicles). This thesis explores several putative secondary regulatory mechanisms, such as, nuclear export activity, transcriptional dependence, palmitoylation and acetylation. However, the most promising mechanism thus far is masking of the NLS by HAP1 and HMGB1. The functions of these proteins, in vesicular trafficking and autophagy/apoptosis, respectively, may offer further insight into huntingtin’s nuclear function. By understanding the underlying regulatory mechanisms of huntingtin nuclear import, we hope to gain further insight into why huntingtin accumulates in the nucleus of specific neurons in HD, and whether or not this mislocalization directly contributes to disease.</p> / Doctor of Philosophy (PhD)

<p>Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin protein. The huntingtin protein has many roles in vesicular and endocytic trafficking, which can be modified in HD cells. When mutant huntingtin is expressed in HD, protein levels of the huntingtin interacting protein, Huntingtin-associated protein of 40kDa (HAP40) are increased. This increase in HAP40 protein levels causes the formation of a complex between carboxyl terminal huntingtin, HAP40 and active Rab5 on the early endosome. This complex induces a switch from early endosomal movements on microtubules to movements on actin filaments, greatly reducing both the speed and distance of movement. The main objective of this research is to determine where the interaction occurs between huntingtin and HAP40 on the huntingtin protein. Here we show that HAP40 interacts with the amino terminus of huntingtin, specifically the first 17 amino acids (N17). In co-localization studies, we found that HAP40 co-localizes with the carboxyl terminal fragment of huntingtin corresponding to amino acids 2570-2634; however, GFP trap immunoprecipitation analysis revealed no interaction between the carboxyl terminal fragments of huntingtin and HAP40. An interaction was discovered between HAP40 and N17, which was then confirmed by far western blot. These results demonstrate that HAP40 interacts with N17. By identifying the interaction site between HAP40 and huntingtin, modifications can be explored to prevent the interaction of HAP40 with huntingtin. This would restore motility on microtubules reinstating fast, long-range movements of early endosomes.</p> / Master of Science (MSc)

Huntington’s disease is an autosomal dominantly inherited neurodegenerative disorder characterized by degeneration of striatal and cortical neurons. The neurons in these regions are particularly energy-demanding and need to maintain high levels of oxidative phosphorylation to support cellular activities. Reactive oxygen species are generated as a byproduct of oxidative phosphorylation and can damage DNA and other biomolecules if not properly metabolized. In HD, there is elevated oxidative DNA damage and impaired DNA damage repair, likely due to impaired function of the mutant huntingtin protein in base excision repair (BER). Previous studies have shown that mutant huntingtin is hypo-phosphorylated at serines 13 and 16 in the N17 domain, and that restoring phosphorylation can reestablish normal protein function and is protective in HD. In this thesis, we show that a metabolite of the DNA damage product N6- furfuryladenine (N6FFA), kinetin triphoshate (KTP) increases N17 phosphorylation through casein kinase 2 (CK2) by acting as an ATP analog, with protective effects in cell and animal models of disease. We additionally show N6FFA increases the activity of CK2 on other substrates, specifically p53. We hypothesize that in times of ATP stress CK2 can utilize KTP as an alternate energy source, promoting DNA repair and cell viability. In HD, inefficient BER inhibits generation of KTP and promotes hypo- phosphorylation of CK2 substrates, which can be overcome by exogenous addition of N6FFA. Additionally, we show that another DNA-responsive kinase, PKCζ, can also phosphorylated N17, potentially priming this domain for CK2 phosphorylation. Finally, we propose that the protective effects of N6FFA may be via a two-pronged pathway, involving both CK2 and the mitochondrial quality control kinase, PINK1. Thus, this thesis presents a novel mechanism where a product of DNA damage acts as a phosphate source for critical kinases in DNA repair and mitochondrial maintenance in conditions where ATP levels are low. / Thesis / Doctor of Philosophy (PhD)

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)

Yes / Background: Past research has found cancellation tasks to be reliable markers of cognitive decline in Huntington’s disease (HD). Objective: The aim of this study was to extend previous findings by adopting the use of a dual task paradigm that paired cancellation and auditory tasks. Methods: We compared performance in 14 early stage HD participants and 14 healthy controls. HD participants were further divided into groups with and without cognitive impairment. Results: Results suggested that HD participants were not slower or less accurate compared with controls; however, HD participants showed greater dual task interference in terms of speed. In addition, HD participants with cognitive impairment were slower and less accurate than HD participants with no cognitive impairment, and showed greater dual task interference in terms of speed and accuracy. Conclusions: Our findings suggest that dual task measures may be a better measure of cognitive processing in HD compared with more traditional measures. / Supported by the School of Psychological Sciences, Monash University.

Intrastriatal injections of L-Pyroglutamate (L-PGA) in mice produced behavioral and neuropathological effects that resemble in part the kainate-injected rat striatal model of Huntington's Disease (HD). The behavioral responses induced after unilateral injections of L-PGA included circling, postural asymmetry of head and trunk and possible dyskinesias. The neuropil in the injected striatum contained dilated profiles, degenerating neurons and oligodendroglia, and numerous phagocytic microglial-like cells. A dose response relation existed. The size of the lesion (expressed as a percent volume of the striatum destroyed) ranged from 1±0.18% at 0.02 μmoles to 20.2±3.97% at 200 μmoles L-PGA (pH=7.3). L-PGA is a weak neurotoxin when compared to kainic acid. Several factors raise interest in the possible role of L-PGA in HD, including the recently reported elevated plasma levels of L-PGA in some HD patients [51,52], and these are considered in the discussion.

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.

Huntington's disease (HD) is an autosomal dominant hereditary neurodegenerative disease characterized by motor, cognitive and behavioral disorders. HD is caused by expansion of CAG triplet (cytosine-adenosine-guanine) located in a gene on the short arm of the fourth chromosome. This expansion encodes an aberrant polyglutamine chain in the protein huntingtin. Physiological and mutated huntingtin (in case of HD) are expressed in almost all tissues and influences many cellular functions. The prevalence of HD in population is about 1 per 10.000. The disease is currently incurable and its mechanisms are not sufficiently understood. Besides affecting the central nervous system HD also affects peripheral tissues, including skeletal muscles. HD disrupts mitochondrial function and damages oxidative phosphorylation system, which has the task of producing energy in the form of ATP in cells. Research of transgenic minipig model for HD could help elucidate the mechanisms of disease's pathogenesis and potential therapeutic strategy. In this diploma thesis, immunodetection with help of specific antibodies to detect changes in amount of 14 selected mitochondrial proteins in skeletal muscle tissue of three age groups of transgenic HD minipigs - 24, 36 and 48 months old was used. Gradual progression in reduced...

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.