Double-stranded RNA (dsRNA) is a key molecule that initiates the immune response to viral infection, but increasingly endogenous (self) dsRNA has been found to be central to the pathology of diverse non-infectious diseases, from neurodegenerative disease to autoimmunity to cancers. Therefore, it is critical to understand the mechanisms that regulate endogenous dsRNA and the pattern recognition receptors that sense dsRNA. In this dissertation, I address three main questions pertaining to this. First, why is the brain so prone to dsRNA-mediated non-infectious disease, especially considering that dsRNA sensors are expressed in almost all tissues. Using stem cell differentiation, gene expression manipulation, and microscopy, I determined that neurons are a special cell type that express high levels of endogenous dsRNA. This high dsRNA burden in neurons is driven by global lengthening of 3`untranslated regions (3`UTRs) and induces tonic inflammation. Second, I examined the mechanism through which the dsRNA regulator ADAR1 controls endogenous dsRNA levels. I employed heavy use of tissue culture and visualization of dsRNA by confocal microscopy to determine that both the dsRNA-binding and dsRNA-editing activities of ADAR1 are required to suppress global endogenous dsRNA levels. Third, after identifying the existence of transcript isoforms of the key dsRNA sensor PKR, I explored their regulatory potential on the PKR protein itself. By genetically altering human cells, I identify that 3`UTR isoforms of PKR regulate transcript localization, translation efficiency, and PKR protein activatability. Overall, the studies described herein demonstrate novel regulatory roles of endogenous dsRNA and underscore the importance of dsRNA in neurological disease.

Progressive disorders of airway protection, namely cough (dystussia) and swallowing (dysphagia) dysfunction, are highly prevalent in Parkinson’s disease (PD), impacting quality of life and contributing to the development of aspiration pneumonia – a leading cause of death in this population. To date, dysphagia rehabilitation has remained the primary (and often only) treatment target of choice by clinicians managing dysphagia in patients with PD and other neurodegenerative disease. This is a major concern since the progressive nature of PD makes it somewhat unreasonable to expect that treatments can fully rehabilitate swallowing dysfunction and eliminate chronic aspiration. Instead, rehabilitating cough dysfunction can serve as an adjunctive approach to promote pulmonary health. Considering that impairments in motor control and organization are primary features of PD, skill training may have a necessary role in cough rehabilitation. Despite a growing body of research supporting the feasibility and effectiveness of cough skill training, a significant gap remains in our understanding of optimal skill training parameters that maximize treatment outcomes through motor learning. This document addresses this gap in the literature in a series of three research studies. Chapter 1 will begin by reviewing the current body of literature related to normal and disordered mechanisms of airway protection dysfunction in PD, skill training as an efficacious approach to rehabilitate cough dysfunction, and motor learning considerations to maximize treatment outcomes. Chapter 2 will characterize motor performance and learning during a voluntary cough skill training paradigm, and evaluate the contributions of physiologic (i.e., lung volume) and treatment-specific (i.e., biofeedback) factors to treatment response in PD. Chapter 3 will then characterize trajectories of motor performance during multiple sessions of sensorimotor cough skill training and explore the role of task-specific predictors (i.e., variability, motor learning) on motor performance. Chapter 4 will examine the effects of cough skill training with variable practice on motor performance and motor learning and characterize contributions of laryngeal and respiratory subsystems to cough strength. This document will then conclude (Chapter 5) by synthesizing results from these studies and discussing clinical implications, limitations, and potential directions for future research.

Focused ultrasound (FUS) can modulate activity in the central nervous system of animals, however the mechanism of action is not yet fully understood. FUS is a promising technique for clinical use in treating both physiological and psychological pathology of the nervous system. FUS can noninvasively penetrate the skull deep into the brain and modulate brain targets with millimeter-scale resolution. FUS is less invasive than deep brain stimulation (DBS) and can target deeper structures with greater resolution than transcranial magnetic stimulation (TMS). Functional ultrasound imaging (fUSI) is an emerging modality for monitoring stimulus-evoked brain activity. However, the thick skull of large animals poses a significant obstacle for the noninvasive translation of the technique to nonhuman primates and humans. In this dissertation, FUS is performed in mice and nonhuman primates and an fUSI technique is developed for transcranially imaging FUS-evoked responses in both species. The first aim of this dissertation established a procedure for performing high-resolution FUS in mice in vivo. FUS-evoked motor responses were evaluated using four-limb electromyography (EMG). A detailed quantitative analysis of several EMG characteristics demonstrated that observed motor responses exhibited brain target-specific differences. FUS in the brain was also shown to modulate cardiorespiratory activity. However, simulations conceded that intracranial reverberations may activate brain structures outside acoustic foci, suggesting that direct detection of brain activity is preferable to responses like EMG and cardiorespiratory activity. The second aim of this dissertation developed an fUSI system for monitoring FUS-evoked responses in mice in vivo. fUSI was validated using electrical peripheral nerve stimulation to elicit somatosensory-evoked responses, a well-characterized approach in established techniques like functional magnetic resonance imaging (fMRI). fUSI was later integrated into an ultrasoundbased optogenetic stimulation procedure. Lastly, a dual FUS-fUSI transducer system for performing neuromodulation and functional activity monitoring was developed and successfully demonstrated in mice in vivo. The final aim of this dissertation was to adapt the FUS-fUSI procedure developed in mice for use in nonhuman primates. Two approaches were developed and tested in vivo. The first approach employed a low-frequency ultrasound array for both neuromodulation and activity monitoring. The second approach implemented a dual FUS-fUSI transducer system similar to that used in mice. Preliminary evidence indicated that the adapted dual transducer system can successfully perform fully noninvasive neuromodulation and functional activity monitoring transcranially in nonhuman primates in vivo. The findings presented in this dissertation provide a framework for performing fully noninvasive ultrasound-mediated neuromodulation and functional activity monitoring in non human primates and describes a road map for further translating the technique for clinical use in human subjects. A fully noninvasive FUS-fUSI technique can provide an invaluable tool for clinicians to treat diseases of the nervous system not indicated for invasive procedures, opening the door to a wide range of therapeutic applications.

Utilization of airway protective rehabilitation among individuals with neurodegenerative disease is astoundingly low. Yet, due to progressive decline in airway protective function and resulting health consequences such as aspiration pneumonia, the need for rehabilitation is clear. Moreover, a growing literature supports the benefit of airway protective rehabilitation in neurodegenerative populations. Therefore, it is a healthcare priority to increase treatment utilization in order to improve health and quality of life for individuals with neurodegenerative disease. Improving treatment accessibility and relevance are two approaches that have the potential to improve utilization. Despite the need to increase treatment accessibility and the growing evidence base to support the use of telehealth to increase access, a significant gap remains in our understanding of the feasibility and acceptability of telehealth to manage dysphagia in neurodegenerative disease. Moreover, little is known about patient perspectives which are critical to refine person-centered models of care that are relevant to patient’s needs. To address this important clinical research gap, this dissertation includes a series of three research studies aimed at improving accessible and relevant rehabilitation for airway protective dysfunction in neurodegenerative disease. Chapter 1 will provide an overview of the current literature as it relates to airway protective dysfunction in neurodegenerative disease, existing rehabilitation approaches, telehealth to manage dysphagia, and the need for personalized care. Chapter 2 will examine the feasibility of rehabilitating airway protection via telehealth in individuals with neurodegenerative movement disorders. Chapter 3 will then explore speech language pathologists’ (SLPs) perspectives and experiences using telehealth to manage dysphagia. Chapter 4 will characterize patient perspectives on airway protective dysfunction and treatment experience following cough skill training (CST). I will conclude (Chapter 5) by synthesizing the findings from chapters 2-4 and suggesting directions for future research.

Parkinson’s disease (PD) is characterized by the degeneration of midbrain dopaminergic neurons. Genetic studies have revealed causative and risk loci associated with a proportion of PD cases, such as PRKN/PARK2, encoding parkin and when mutated causes a rare familial form of autosomal recessive PD. Cell-based studies have linked parkin to mitochondrial turnover by autophagy, but to date, manipulating this gene in rodents has not robustly recapitulated core features of PD. Reconciling these results is essential to determine parkin’s role in mitochondrial biology, brain physiology, and PD pathogenesis. Here, we find that global, inducible deletion of Prkn/Park2 (parkin iKO) in the adult mouse leads to age-dependent motor impairments that are responsive to levodopa treatment. We report that these behavioral defects are associated with progressive pathology in dopaminergic neurons, regional gliosis and lipid oxidation changes, culminating in the selective degeneration of nigrostriatal dopaminergic neurons. We also present a new, in vivo mitophagy reporter system to investigate the relationship of parkin’s described roles in mitochondrial homeostasis to the observed phenotypes. These results give critical insight into parkin’s contribution to dopaminergic neuron stability in the mammalian brain, and provide two distinct and novel organismal tools to investigate mitochondrial homeostasis and PD pathogenesis.

Myeloid cells of the innate immune system have been strongly implicated in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Similarly, several lines of evidence call on the adaptive immune system as a critical driver of disease, particularly in PD. The immune dynamics in both of these diseases are complex, and span across not only the innate and adaptive immune systems, but also across the periphery local action in the central nervous system (CNS). This thesis aims to address critical gaps in our knowledge regarding molecular and functional alterations of immune cells in AD and PD. We apply tools including single nucleus RNA – and ATAC – sequencing as well as protein – level and functional studies to advance our understanding of molecular pathways involved in the innate and adaptive immune dysfunction in these diseases, including both immune cells in the CNS as well as in the periphery. Chapter 1 provides an overview of the evidence implicating myeloid cell dysfunction in AD and PD, including microglia as well as peripheral myeloid cells such as monocytes. It also describes the features of immune dysregulation in both diseases, and evidence implicating the adaptive immune system in PD. Chapter 2 aims to address our currently limited understanding of microglial molecular phenotypes and diversity in PD, by characterizing microglial transcriptomic and chromatin signatures in disease. We demonstrate microglial subpopulation-specific effects, including the focal depletion of a microglial population in the substantia nigra in PD, which open novel avenues for targeted neuroimmune interventions in PD. Chapter 3 aims to identify interactions regulating the infiltration and retention of peripheral immune cells into the CNS in PD; a process which is implicated in the progression of this disease, but the mechanisms of which are not fully understood. We characterized transcriptomic signatures of infiltrating lymphocytes and blood brain barrier cells, and found increased T cell infiltration in PD as well as fibroblast and endothelial populations associated with disease. We further identified transcriptional shifts suggestive of a proinflammatory and profibrotic milieu in disease, in which chemokines and extracellular matrix elements produced by fibroblasts may influence T cell trafficking and retention in the substantia nigra in PD. Chapter 4 aims to address the gap in our knowledge of how myeloid dysfunction in the periphery contributes to AD. While genetics implicate all myeloid cells in AD and PD, contributions of peripheral myeloid cells, such as monocytes, have been largely overlooked in place of microglia, which are resident in the CNS. We evaluate the convergence of the AD genetic risk loci on functional outcomes in monocytes, in the context of Aβ as an immune stimulus. We identified functional convergence of the CD33 and SPI1 AD risk variants in the context of Aβ stress, including reduced phagocytosis and loss of surface TREM2 expression, demonstrating an interaction between genetics and environment to reduce myeloid cell fitness. Finally, Chapter 5 concludes with a summary of key findings from this work, and discusses future directions for modulating innate and adaptive immune populations, both in the CNS and in the periphery, as therapeutic approaches for these neurodegenerative diseases.

Human microglia play a pivotal role in neurological diseases, but few targeted therapies that directly modulate microglial state or function exist due to an incomplete understanding of microglial heterogeneity. This thesis aims to advance our understanding of microglial heterogeneity by using single-cell RNA sequencing to profile live human microglia from autopsies or surgical resections across diverse neurological diseases and using computational tools to infer chemical and genetic regulators of specific microglial substates. Chapter 1 provides an overview of microglial ontogeny, function, and known heterogeneity, especially in disease contexts. It also describes the steadily increasing disease burden seen in neurological disease as well as the lack of efficacious treatments and future directions for microglia-targeted therapies. Chapter 2 focuses on microglial heterogeneity in an understudied disease, ALS, describing population structure shifts seen in ALS across cortex and spinal cord. Chapter 3 instead focuses on exploring underlying cross-disease microglial population structure, identifying subsets with metabolic and functional properties, as well as subsets enriched in susceptibility genes for neurodegenerative disease. We then demonstrate applications of this type of data by using our resource to annotate other datasets. Chapter 4 leverages this data in another way, by identifying and validating candidates for chemically and genetically inducing subtype-specific states in vitro. Notably, we show that Camptothecin downregulates the transcriptional signature of disease-enriched subsets and upregulates a signature previously shown to be depleted in Alzheimer’s. Finally, I review our findings and discuss future directions for the field.

Neuromyelitis Optica (NMO) is a rare, autoimmune, neurodegenerative disease selectively affecting the optic nerves and spinal cord. Relapsing NMO is nine times more prevalent in women than in men and approximately one-quarter of NMO patients have symptoms of another autoimmune disorder (National Institute of Health, 2019). NMO has not been linked to any genetic mutations and the cause of the disorder is unknown beyond the general understanding that the body produces anti-aquaporin-4 antibodies (AQP4) which mistakenly attack cells in the nervous system. NMO affects roughly one percent of that of Multiple Sclerosis (4000-8000 patients total) in the United States, but prevalence rates are abnormally high in a handful of regions around the world, particularly among Latin America, where rates can reach up to 5/100,000 individuals. The results of this study predict that there is a connection between African genetics and NMO, but further studies will need to be conducted in more Latin America nations and other regions to determine prevalence rates as well as genetic analysis of affected individuals.

Schwannomatosis (SWN) is a genetic disorder that predisposes affected individuals to develop multiple Schwannomas anywhere in the peripheral nervous system. This can be due to a mutation in the LZTR1 or SMARCB1 genes on chromosome 22. SWN has the defining clinical symptom of chronic pain and a lack of vestibular schwannomas, which sets it apart from other, related disorders such as Neurofibromatosis Type II (NF2). Currently, it is unknown what causes the chronic pain of SWN patients but it is hypothesized that cytokines may have promote the neuropathic pain experienced by patients. This study investigates the presence of the chemokine CCL2 and the cytokine IL6 in human SWN schwannomas and non-SWN schwannomas to determine if there is a difference in the presence of these cytokines between the two tumor types. It was demonstrated that all of the SWN schwannomas expressed both CCL2 and IL6 whereas the non-SWN schwannomas expressed only one or the other protein if either. These results indicate that the presence of these cytokines within the SWN schwannomas is different from non-SWN schwannomas and could be a potential contributing factor in the occurrence of neuropathic pain experienced by SWN which is part of the differential diagnosis for NF2 and SWN.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an X-linked late-onset neurodegenerative disorder caused by a noncoding trinucleotide repeat expansion in the FMR1 gene. This gene produces fragile x mental retardation protein (FMRP), an RNA binding protein whose targets are involved in brain development and synaptic plasticity. One of the proposed mechanisms of FXTAS pathogenesis is an RNA gain-of-function in which the repeat expansion causes toxic mRNA that sequesters important proteins in the cell, interfering with their functions. Another suggested method of pathogenesis is through a mutant protein called FMRpolyG. This protein results from repeat-associated non-AUG (RAN) translation, in which the expanded repeats are translated where they otherwise would not be. This protein co-localizes with intranuclear inclusions and nuclear membrane proteins, causing disorganization of the nuclear lamina in FXTAS patient brain samples and neurons differentiated from FXTAS patient-derived induced pluripotent stem cells (iPSCs). iPSC technology involves reprogramming an adult somatic cell back to an embryonic-like state, allowing it to be differentiated into all cell types. A limit with iPSCs, though, is modeling late-onset disorders because the cells lose all age-related features during reprogramming. Progerin, a truncated form of the lamin A protein, has been used to age neurons differentiated from Parkinson Disease (PD) patient-derived iPSCs. Progerin-mediated aging was found to unmask PD-like phenotypes in those neurons, making it a promising technology for modeling late-onset disorders such as FXTAS. In this study, we investigated the link between the aging process and FXTAS pathogenesis in neurons differentiated from FXTAS patient-derived iPSCs with the use of progerin. Progerin transduction was successful in aging the FXTAS neurons. The presence of FMRpolyG was confirmed and an interaction with Lap2b was observed. In some neurons, there was also an observed interaction between FMRpolyG and progerin. Overall, this data suggests that there is an interaction between the mutant FMRpolyG protein and the nuclear membrane during aging, which may contribute to the cell death that causes neurodegeneration in FXTAS patients.