Metabotropic glutamate receptor 5 (mGluR5) is a key regulator of numerous brain functions including memory, cognition, and motor behavior. Dysregulation of mGluR5 signaling is evident in Huntington's disease (HD) neuropathology, an inherited, neurodegenerative disease characterized with progressive deterioration in motor, cognitive, and psychiatric functions. In this context, two cellular proteins draw particular interest for this thesis: vesicular glutamate transporter 3 (VGLUT3) and optineurin (OPTN). VGLUT3 modulates glutamate release from selected neurons that are affected by HD, while OPTN is a mGluR5-interacting protein and contributes to neuronal vulnerability in HD. However, current evidence on their involvement in mGluR5 signaling and HD pathogenesis is still lacking. Using VGLUT3 knockout (VGLUT3⁻ᐟ⁻) mice, we showed that this transporter dynamically regulated glutamate receptor densities in different brain regions. Of note, VGLUT3 deletion upregulated mGluR5 in the cerebral cortex and the striatum, unlike the hippocampus which exhibited reduced mGluR5 cell surface densities. We then crossed VGLUT3⁻ᐟ⁻ mice with the zQ175 knock-in mouse model of HD (zQ175:VGLUT3⁻ᐟ⁻) to assess the impact of VGLUT3 transmission loss on HD progression. The longitudinal behavioral assessment revealed that VGLUT3 ablation rescued the deficits in motor coordination and short-term memory in both male and female zQ175 mice throughout 15 months of age. Furthermore, VGLUT3 deletion rescued striatal cell loss likely via activation of Akt and ERK1/2 cellular pathways, with no impact on total mutant huntingtin aggregation or the associated microgliosis. To delineate the role of OPTN in mGluR5 signaling, we employed a CRISPR/Cas9 OPTN-deficient cell line and global OPTN knockout mice. We demonstrated that OPTN was essential for mGluR5-mediated canonical signaling and ERK1/2 activation in both the striatal cell line, STHdh^Q7/Q7, and acute hippocampal slices. We then showed that OPTN deletion impaired autophagic machinery via GSK3β/ZBTB16 and mTOR/ULK1 signaling pathways downstream of mGluR5. This work offers novel insights into the molecular roles of VGLUT3 transmission and OPTN in mGluR5 signaling and provides a rationale for their targeting to therapeutically mitigate pathological mGluR5 signaling in HD.

Caspases are cysteine proteases best known for their controlling roles in apoptosis and inflammation. Caspase-6 has recently been shown to play a key role in the cleavage of neurodegenerative substrates that causes Huntington and Alzheimer's Disease, heightening interest in caspase-6 and making it a drug target. All thirteen human caspases have related specificities for binding and cleaving substrate, so achieving caspase-specific regulation at the active site has been extremely challenging if not impossible. We have determined the structures of four unliganded forms of caspase-6, which attain a novel helical structure not observed in any other caspases. In this conformation, rotation of the 90's helix results in formation of a cavity that can function as an allosteric site, locking caspase-6 into an inactive conformation. We are using this cavity to look for chemical ligands that target this cavity and maintain caspase-6 in the inactive, helical conformation. We found that known allosteric inhibitors of caspase-3 and -7 also inhibit caspase-6 through a cavity at the dimer interface. We have determined new structures of a phosphomimetic state and a zinc-bound conformation of caspase-6, which show the molecular details of two additional allosteric sites. The phosphomimetic form of caspase-6 inactivates caspase-6 by disrupting formation of the substrate binding-groove by steric clash of the phosphorylated residue with P201 in the L2' loop. Another allosteric site was found on the "back" of caspase-6 that coordinates a zinc molecule that leads to inactivation. In total we have uncovered four independent allosteric sites in caspase-6, structurally characterized inhibition from these sites and demonstrated that each of these sites might be targeted for caspase-6 specific inhibition by synthetic or natural-product ligands.

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.

Adult neurogenesis is affected in neurodegenerative diseases and also represents an important therapeutic target. The goal of this dissertation research was to test the hypothesis that regeneration of neurons and glia in the adult brain can be manipulated by neurotrophic drugs in the context of two mouse models of neurodegenerative disease : Parkinson's disease and Huntington's disease.... These findings have implications for the pathophysiology of Huntington's disease and neurodegeneration in general. Specific alterations to the SVZ neurogenic niche parallel some of the pre-motor symptoms of Parkinson's disease and Huntington's disease. This dissertation research contributes to the growing body of literature concerning the pharmacological modulation of SVZ-derived neurogenesis designed to attenuate the progressive loss of neurons in neurodegenerative diseases and perhaps delay the onset of symptoms. / by Mark Harvey McCollum. / Vita. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

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.