In vitro models are often used both to characterize and test therapeutics for neurodevelopmental disorders (‘NDDs’). While in vitro models have extraordinary potential to develop therapies for patients, they have historically been confounded by absence of robust phenotypes and/or in vitro phenotypes that fail to translate from laboratory bench to bedside. Within this thesis work, we attempt to address three areas in which in vitro models may be improved – gene selection, model validation and identification of disease-relevant functional assays suited for therapeutic testing. Publicly available databases aggregating identified and annotated disease-causing variants for Mendelian diseases have rapidly expanded over the past two decades. Elucidating mechanisms of disease and developing therapies using in vivo model systems often is both time and cost intensive. Thus, determining which subsets of genes are more likely to generate addressable signals in a dish may lead to more effective drug development. In chapter 1, we identify a set of genes ideally suited for therapeutic inhibition. Specifically, we leverage the aforementioned large genetic databases to identify a set of genes likely to act through a gain-of-function mechanism that are both tolerant to loss-of-function mutations and in the druggable genome. In chapter 2, we aim to characterize the degree of conservation of transcriptomic dysregulation between a human in vitro cortical organoid (‘hCOs’) model, and two mouse models of a severe neurodevelopmental disorder resulting from HNRNPU deficiency. Human model systems may improve upon animal models when human pathogenesis and patient phenotypes are divergent from animal models due to species-specific etiology. However, human model systems often lack the heterogeneity and cell-type specificity and maturity seen in primary fetal samples. Importantly, some mouse models of HNRNPU deficiency have muted phenotypes compared with human patients. We hypothesized that while there are distinctions between humans and mice with HNRNPU deficiency, there will be overlap in transcriptomic dysregulation between human and mouse models. In fact, we find 45-day-old HNRNPU+/- hCOs have consistent transcriptomic dysregulation to embryonic mouse models, but not to perinatal mice. Our findings suggest hCOs are a viable model for characterizing HNRNPU deficiency; however, such models may only be appropriate for elucidating a transcriptomic disease state at a specific developmental time period. Functional assays for neurodevelopmental disorders can aid in understanding whether transcriptomic dysregulation is relevant to patient symptoms, as genomic findings may not always correlate to disease-relevant phenotypes. Further, relevant functional phenotypes can then be utilized for testing potential therapeutics. Importantly, seizures are commonly present in a significant subset of neurodevelopmental disorders and seizure phenotypes have been described as driven by aberrant synchrony in neuronal networks. Using a multielectrode array platform, investigators can use a variety of computational methods to quantify aspects of synchrony in vitro. In chapter 3a, we introduce topological approaches capable of identifying novel synchrony phenotypes in primary neuronal networks from mouse models of neurodevelopmental disorders. Certain mouse models will be confounded by species-specific pathogenesis and/or vastly different developmental timelines and fail to generalize to human patients, motivating the need for functionally active and physiologically relevant human in vitro models. In chapter 3b, we attempt to generate human networks with balanced levels of excitation and inhibition and find confounding lack of functional maturation of inhibitory neuronal subtypes in 90-day-old stem cell-derived neuronal networks. Future work generating in vitro human neuronal networks with functionally mature inhibitory neurons would complement the findings in chapters 1 and 2 and allow for more efficient therapeutic development strategies that may lead to improved patient outcomes.

Indiana University-Purdue University Indianapolis (IUPUI) / The main, underlying cause of neurodegenerative disease is the progressive loss of neuronal structure or function, whereby central and/or peripheral nervous system circuitry is severely and irreversibly damaged, resulting in the manifestation of clinical symptoms and signs. Neurodegenerative research has revealed many similarities among these diseases: although their clinical presentation and outcomes may differ, many parallels in their pathological mechanisms can be found. Unraveling these relationships and similarities could provide the potential for the discovery of therapeutic advances such that a treatment for one neurologic disease may also be effective for several other neurodegenerative disorders. There is growing awareness that due to the complexity of pathophysiological processes in human disease, specifically targeting or inactivating a single degenerative process or a discrete cellular molecular pathway may be ineffective in the treatment of these multifaceted disorders. Rather, potential therapeutics with a multi-target approach may be required to successfully and effectively control disease progression. Recent advances in neurodegenerative research involve the creation of animal disease models that closely mimic their human counterparts. The use of both toxin- exposure and genetic animal models in combination may give insight into the underlying pathologic mechanisms of neurodegenerative disorders (target identification) leading to the development and screening of prospective treatments and determination of their neuroprotective mechanism (target validation). Taken together, ideal candidates for the treatment of neurodegenerative disease would need to exert their neuroprotective effect on multiple pathological pathways. Previous studies from this laboratory and collaborators have shown that the naturally-occurring compound, caffeic acid phenethyl ester (CAPE), is efficacious for the treatment against neurodegeneration. Because of its versatile abilities, CAPE was chosen for this study as this compound may be able to target the pathogenic pathways shared by two different animal models of neurodegeneration and may exhibit neuroprotection. In addition, adipose-derived stem cell conditioned media (ASC-CM), a biologically-derived reagent containing a multitude of neuroprotective and neurotrophic factors, was selected as ASC-CM has been previously shown to be neuroprotective by using both animal and cell culture models of neurodegeneration.

Fused in sarcoma (FUS) is an RNA binding protein involved in DNA repair and RNA metabolism, including mRNA transcription, splicing, transport and translation. FUS is genetically and pathologically associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To explore the mechanisms by which pathogenic mutations in FUS cause neurodegeneration in ALS-FUS, we generated a series of FUS knock-in mouse lines that express the equivalent of the ALS-associated mutant proteins FUSP525L and FUSΔEX14 at physiological levels from the FUS locus. We demonstrate that heterozygous mutant FUS mice show progressive, age-dependent loss of vulnerable subpopulations of spinal motor neurons. While ALS-associated mutations in FUS lead to partial loss of function, we provide genetic evidence that the motor neuron phenotype observed is a consequence of a dose-dependent gain of function, associated with the insolubility of FUS and related RNA binding proteins (RBPs). Furthermore, we show that motor neuron degeneration is driven by cell autonomous mechanisms, associated with mutant FUS-independent inflammatory changes. In this faithful mouse model of ALS-FUS, we demonstrate that an antisense oligonucleotide (ASO) targeting the FUS transcript (ION363) results in the efficient silencing of both wild type and mutant FUS alleles, and that postnatal reduction of FUS protein levels in the brain and spinal cord delays disease onset in this mouse model of ALS-FUS. In a first-in-human trial of ION363, we demonstrate that repeated, intrathecal injections of this candidate therapeutic in an ALS patient with a FUSP525L mutation leads to the efficient silencing of both wild type and mutant FUS in the central nervous system, and a reduction in the burden of FUS aggregates that are a pathological hallmark of ALS-FUS. In mouse genetic and human clinical studies, we provide evidence in support of a therapeutic strategy by which silencing of the FUS gene may be used to prevent or delay disease onset in pre-symptomatic carriers of pathogenic FUS mutations, or to slow disease progression in symptomatic ALS- and FTD-FUS patients. In addition, we use this newly generated model to investigate the role of potential modifiers of FUS toxicity, including hnRNP U and UPF1, and study the role of chronic neuroinflammation in the disease progression that could lead to the development of novel therapeutics to provide immediate clinical benefit to patients with ALS-FUS.

No description available.

Alzheimer’s disease (AD) is a neurodegenerative disease and the most common form of dementia associated with amyloid-beta peptide deposition and loss of mitochondrial function and regulation. Currently, there is no cure for AD, thus, there is a need to continuously develop therapeutic strategies that could address the complex multifactorial causes of AD development. Due to this necessity, this study has investigated the role of vitamin B2 as a disease modifying drug for AD by employingamyloid-beta and mitochondrial based AD therapeutic strategies. Using a transgenic C. elegans AD worm model expressing amyloid-beta (Aβ1-42) in muscle cells at temperature upshift to 25°C, we screened for protective effect of dose-dependent concentrations of active forms of vitamin B2, FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), against amyloid-beta mediated paralysis. Protective concentrations were then assayed for improvement of mitochondrial metabolic functions by performing ATP, oxygen consumption and reactive oxygen species (ROS) production assays. Consequently, we investigated for drug protective mechanisms of FMN and FAD using RNAi genetic screening technique. FMN and FAD significantly delayed amyloid-beta mediated paralysis and improved mitochondrial metabolic functions at final concentrations of 0.74mM and 0.74µM respectively. More so, both compounds induced activation of stress response FOXO transcription factor, daf-16. Specifically, FMN treatment induced mitochondrial unfolded protein response (UPRmt) pathway through ubiquitin-like protein (ubl-5) activation as well as other stress response pathway signature such as Activating Transcription Factor Associated with Stress (atfs-1). This study will be useful in understanding the importance of micronutrients such as vitamin B2 in normal cellular function as related to neurodegenerativediseases and aging. Therefore, vitamin B2 supplementation could be an important source of Alzheimer’s disease therapeutic strategy.

Alzheimer’s disease (AD) is associated with amyloid-beta peptide deposition and loss of mitochondrial function. Using a transgenic C. elegans AD worm model expressing amyloid-beta in body wall muscle, we determined that supplementation with either of the forms of vitamin B2, flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) protected against amyloid-beta mediated paralysis. FMN and FAD were then assayed to determine effects on ATP, oxygen consumption, and reactive oxygen species (ROS) with these compounds not significantly improving any of these mitochondrial bioenergetic functions. Knockdown of the daf-16/FOXO transcriptional regulator or the FAD synthase enzyme completely abrogated the protective effects of FMN and FAD, while knockdown of the mitochondrial unfolded protein response factors ubl-5 or atfs-1 also blocked the protective effects. Therefore, vitamin B2 supplementation could lead to the activation of conserved signaling pathways in humans to delay the onset and progression of neurodegenerative diseases such as AD.

Paget's disease of the bone is characterized by a focal increase in the rate of bone turnover, which goes through phases of activity and quiescence. Most patients are asymptomatic. The two cardinal features are pain and deformities, and many complications may arise. Diphosphonates and calcitonin are the main therapeutic modalities.

One of the most foundational and personal philosophical questions one can ask is what makes you, you? In large part, you are made up of your relationships, experiences, and memories. The hippocampus, a brain region which is critical for the formation of memories, has been the focus of neuroscience research for decades due partially to this function, which is foundational to our individuality. In Alzheimer’s disease (AD), one of the most common and well-researched neurodegenerative diseases in the world, the hippocampus is one of the earliest targets. Despite extensive work on AD, we still lack a coherent understanding of what is causing the disease, the mechanisms by which it is causing neuronal dysfunction and death within the hippocampus and other brain regions, and how it ultimately causes deficits in cognition and behavior, leading to an erosion of our selves. In this thesis, I explore three independent but related questions: 1) what molecular mechanisms are causing early synaptic loss in AD, specifically within the hippocampus, 2) what molecular effectors are responsible for establishing and maintaining intracellular architecture in hippocampal neurons, which are exploited in early AD, and 3) how and when does the hippocampal circuit dysfunction in AD progression? Using a variety of experimental techniques, ranging from in utero and ex utero electroporation, primary murine and human neuronal cell culture, longitudinal confocal microscopy, immunohistochemistry, biochemistry, cell and molecular biology, in vivo two-photon calcium imaging, and behavioral assays, I have found that, within CA1 of the hippocampus, synapse loss requires degradation of the dendritic mitochondrial network, activity and input specificity are driving mitochondrial compartmentalization within CA1 neurons through the same pathway that is aberrantly overactivated in AD, and the hippocampal circuit is overly rigid in encoding the environment as the disease progresses.

<p>Multiple sclerosis (MS) is an immune-mediated neurodegenerative disease that results in a myriad of physical and mental symptoms. Current disease-modifying therapies do not prevent long-term disability accumulation and are particularly ineffective for patients with a progressive disease onset. Exercise may represent an alternative strategy for managing symptoms and disability accumulation, particularly in progressive MS.</p> <p>Whereas the benefits of exercise have been established primarily in ambulatory MS patients with a relapsing disease course, few studies have investigated the benefits of exercise for patients with progressive MS with greater impairment. Therefore, the purpose of this dissertation was to determine the short-term, long-term, and maintenance effects of adapted exercise interventions for patients with progressive MS of high disability which was addressed by conducting two adapted exercise interventions.</p> <p>The first intervention examined the effects of 24 weeks of body weight supported treadmill training (BWSTT) on outcomes of physical and mental functioning, fatigue, quality of life, and brain health. Outcomes were evaluated at baseline, 12, and 24 weeks following the intervention, and again 12 weeks post-intervention. The second intervention evaluated and compared the effects of 12 weeks of total-body recumbent stepper training (TBRST) to BWSTT on outcomes of safety, physical and mental functioning, fatigue, quality of life, and equipment preference.</p> <p>Safety of BWSTT and TBRST was established. Significant improvements in fatigue and QoL were observed with both training modalities; however, neither significantly improved physical function. There was some evidence to suggest long-term BWSTT may improve cognitive performance and brain health, and that TBRST was the preferred exercise modality. Furthermore, most beneficial effects of long-term BWSTT tended not to be maintained when exercise was discontinued.</p> <p>This dissertation established evidence for the potential benefits of BWSTT and TBRST in patients with progressive MS with high disability. BWSTT and TBRST may represent viable alternative strategies for disease management.<strong></strong></p> / Doctor of Philosophy (PhD)

<p>Huntington’s disease (HD) is a progressive, neurodegenerative disorder that leads to the selective loss of neurons in the striatum and the cerebral cortex. HD is caused by a CAG trinucleotide repeat expansion beyond the normal length in the <em>IT15 </em>(<em>Htt</em>) gene. The CAG stretch codes for an elongated polyglutamine tract within the amino‐terminus of the huntingtin protein. Polyglutamine tracts with lengths exceeding 37 repeats cause HD whereas repeat lengths below do not. This phenomenon has plagued the HD community since the discovery of the gene in 1993. In this thesis, we sought to elucidate the molecular mechanism by which huntingtin becomes toxic at polyglutamine lengths above 37. Using Förster resonance energy transfer (FRET) techniques, we describe an intramolecular proximity between the first 17 residues (N17) and the proline-rich regions, which flank the polyglutamine tract of huntingtin. We report that we can precisely measure differences between the conformations adopted by the huntingtin protein with polyglutamine tracts below and above the pathogenic repeat threshold of 37 repeats. Our data supports the hypothesis that polyglutamine tracts below the pathogenic threshold can act as a flexible hinge allowing the N17 domain to freely fold back upon huntingtin and come into close 3D proximity with the polyproline region. This flexibility is lost in polyglutamine tracts with >37 repeats resulting in a diminished spatial proximity between N17 and the polyproline domain.</p> / Doctor of Philosophy (PhD)