Master of Science in Biomedical Engineering (MSBME), Wright State University, 2024, Biomedical Engineering

This research explores how motor neurons (MNs) and Kv2.1 clustering change with age, emphasizing sex differences and MN subtypes. We found that MN density decreases with age in both sexes, while soma size increases in male mice. FF MNs were the most affected, and old weak mice had smaller MNs than their stronger counterparts, underscoring FF MN vulnerability. Baseline studies revealed that FF and FI MNs have larger Kv2.1 clusters compared to FR and S MNs. Female mice had smaller, denser Kv2.1 clusters than male mice, suggesting less clustering in females. With age, Kv2.1 clusters grew larger but became less dense and intense, indicating increased clustering. Old weak mice showed even more pronounced clustering than strong ones, linking Kv2.1 changes to age-related weakness. These findings highlight the susceptibility of FF MNs to aging and position Kv2.1 clustering as a key factor in motor neuron function and age-related decline.

Committee: Sherif Elbasiouny Ph.D. (Advisor); Jaime Ramirez-Vick Ph.D. (Committee Member); Keiichiro Susuki M.D., Ph.D. (Committee Member) Subjects: Aging; Biomedical Engineering; Gerontology; Neurobiology; Neurosciences

Master of Science in Biomedical Engineering (MSBME), Wright State University, 2024, Biomedical Engineering

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron (MN) death resulting in paralysis and eventually death. ALS has greater prevalence in older populations sharing characteristics with aging like muscle weakness and MN type specific degeneration. MNs innervate skeletal muscles and control muscle contraction through their excitability which is altered in both conditions. C-Boutons are a cholinergic, excitatory synaptic input to MNs and have been studied in ALS and aging but have produced inconsistent findings and undesired gaps. We used immunohistochemistry to label mouse lumbar spinal cord and separate MN types. 60x imaging and automated analysis was performed providing robust 3D measurements. Our results presented similar findings between two ALS mutations with differing changes in a third mutation. We also show C-Bouton input with age undergoes sex and MN type specific reductions aligning with age-related weakness. Finally, we identify C-Bouton similarities and differences between ALS and aging.

Committee: Sherif M. Elbasiouny Ph.D. (Advisor); David R. Ladle Ph.D. (Committee Member); Tarun Goswami D.Sc. (Committee Member) Subjects: Aging; Biomedical Engineering; Neurosciences

Doctor of Philosophy, Case Western Reserve University, 2024, Pharmacology

Globally, an estimated 420 million people today suffer from debilitating vision loss caused by age-related macular degeneration (AMD), diabetic retinopathy (DR), retinitis pigmentosa (RP), or glaucoma; a large majority of these cases (up to 90%) have only minimally effective or no treatment options available. These chronic, progressive retinal diseases arise from a complex interplay of genetic and environmental factors that disrupt, and eventually compromise, cellular and tissue stability. Such disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, effective treatments to preserve eyesight have remained elusive for the millions of patients suffering from these debilitating disorders, especially in the vast majority of cases that are in early stages of disease progression, wherein lies the greatest opportunity to slow or halt vision loss. In the coming decades, population aging will exacerbate the increase in global prevalence of vision impairment and blindness, thus underscoring a critical, unmet need for innovative, new ophthalmic medications. In pre-clinical studies, we demonstrated the efficacy of prototypical ‘stress resilience-enhancing drugs' (SREDs) that preserved both retinal morphology and function across a variety of genetic and environmental animal models of AMD, DR, RP, and glaucoma. These small-molecule therapies can be subdivided according to primary mechanism of action, resulting in two distinct subclasses of SREDs: 1) epigenetic modulators that include inhibitors of select histone deacetylases (HDACi) or methyltransferases (SUVi); and 2) selective inhibitors of cyclic nucleotide phosphodiesterases (PDEi). With pharmacological inhibition of histone deacetylase 11 (HDAC11) or suppressor of variegation 3-9 homolog 2 (SUV39H2), key histone-modifying enzymes involved in promoting reduced chromatin accessibility, stress-induced retinal (open full item for complete abstract)

Committee: Krzysztof Palczewski (Advisor); Philip Kiser (Advisor); Walter Boron (Committee Member); Johannes von Lintig (Committee Member); George Dubyak (Committee Member); John Mieyal (Committee Chair) Subjects: Medicine; Ophthalmology; Pharmaceuticals; Pharmacology

Doctor of Philosophy, Case Western Reserve University, 2024, Pathology

Neurodegenerative diseases influence millions of people worldwide, characterized by progressive loss of neurons. These diseases include age-related as well as childhood-onset neurodegeneration such as amyotrophic lateral sclerosis (ALS) and pontocerebellar hypoplasia (PCH). Despite the diverse etiologies, shared cellular and molecular features in neurodegeneration are observed, including mitochondrial abnormalities and aberrant RNA processing. This dissertation explored these features within the context of the conditions mentioned, elucidating their roles in disease pathogenesis. Fine-tuned control of mRNA isoform expression plays a crucial role in isoform diversity essential to neuronal function. Alternative polyadenylation results in diversity at the 3' end of the transcriptome, which demonstrates strong tissue-specific manner. Here, we demonstrated that the RNA kinase CLP1 regulates mRNA isoform expression by suppressing proximal cleavage and polyadenylation (CPA) events. Stem-cell-derived motor neurons carrying CLP1 variants exhibit imbalanced mRNA isoform expression, resulting from altered expression of components of the CPA complex and their interaction with the RNA polymerase II (RNAPII) co-transcriptional machinery. Interestingly, similar alterations in alternative polyadenylation (APA) and reduced 3' mRNA isoform diversity are also observed in other neurodegenerative disorders, suggesting a common underlying mechanism. Despite the established regulatory patterns of APA in various cell types, the mechanism remains incompletely understood. We propose a potential model termed the “CPA code,” where not only the expression levels of CPA factors but also their interaction with the RNAPII contribute to distinct APA patterns in different cell types, including stem cells and motor neurons. Additionally, we introduce a novel sequencing method that enables the detection of APA in nascent mRNA. Furthermore, we have developed a system for targeted CLP1 degradation to (open full item for complete abstract)

Committee: Ashleigh Schaffer (Advisor); Xiongwei Zhu (Committee Chair); Andrew Pieper (Committee Member); Polyxeni Philippidou (Committee Member); Clive Hamlin (Committee Member) Subjects: Cellular Biology; Molecular Biology; Neurosciences; Pathology

PHD, Kent State University, 2024, College of Arts and Sciences / Department of Chemistry and Biochemistry

Mitochondria are the major sites of cellular energy production. Electrons from reduced metabolites flow through the ETC consisting of complexes I through IV that creates the mitochondrial membrane potential (MMP) which is harnessed by complex V, culminating in ATP synthesis. Any disruption in the activity of the ETC complexes hampers formation of MMP and consequently ATP synthesis. ETC in the inner mitochondrial membrane during oxidative phosphorylation also serves as the primary source of reactive oxygen species (ROS), which if not fully neutralized causes oxidation of major biomolecules. While the consequences of DNA, protein and lipid oxidation have been explored, the effect of RNA oxidation on cellular processes in general is rather poorly understood. It's all the more important because the mechanisms for repair of oxidized RNA are still under debate. Various diseases including major neurological ailments and certain cancers are associated with RNA oxidation. Also, mitochondrial dysfunction is a hallmark of major neurodegenerative diseases including, Parkinson's disease (PD), Alzheimer's disease (AD), and Multiple Sclerosis (MS). However, the connection between the mitochondrial dysfunction and RNA oxidation is yet to be investigated. We hypothesize that “mitochondrial dysfunction can be a result of oxidation of the mRNAs encoding the subunits of the ETC complexes, which would impede subunit production leading to compromised ETC and decreased ATP production.” To systematically and comprehensively address the hypotheses, we focused on addressing three specific aims in this study: i) identify the oxidized mitochondrial mRNAs in neuronal cells, and determine if they lead to lowering of the cognate protein subunit levels and measure function of the ETC complexes and ATP level, ii) investigate if nuclear mRNAs encoding ETC complex subunits are oxidized and consequence of that on function of ETC complexes and ATP synthesis, and iii) detect oxidized mRNAs in EAE mice, (open full item for complete abstract)

Committee: Soumitra Basu (Committee Chair); Sanjaya Abeysisrigunawardena (Committee Member); Yaorong Zheng (Committee Member); John Johnson (Committee Member); Jennifer McDonough (Committee Member) Subjects: Biochemistry; Molecular Biology

Master of Science (MS), Ohio University, 2023, Food and Nutrition Sciences (Health Sciences and Professions)

Neurodegenerative diseases (NDs) are a broad range of disorders characterized by the chronic and progressive degeneration of neurons and synaptic connections of the nervous system. After Alzheimer's disease (AD), Parkinson's disease (PD) is the most common ND. PD affects ~0.3% of the general population and accounts for an estimated $15.5 billion in medical and non-medical costs each year. PD is a movement disorder characterized by the gradual loss of gross and fine motor control, impaired speech, dyskinesias, postural instability, and a resulting decrease in quality of life. As the disease progresses, interrupted sleep, depression, and cognitive impairment may also arise. Clinically, these symptoms have been associated with the loss of dopamine (DA) production due to the progressive degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNc) region of the midbrain. Another hallmark of PD is the presence of insoluble inclusions called Lewy bodies in neurons due to the pathological aggregation of a protein called α-synuclein. The molecular mechanisms governing the aggregation of α-synuclein and formation of Lewy bodies are not well understood. Emerging research suggests that excitotoxicity, oxidative stress, and neuroinflammation may be important factors in the α-synuclein aggregation-mediated formation of Lewy bodies and associated loss of dopaminergic neurons, and therefore may serve as promising therapeutic targets for PD. The current pharmaceutical therapies available for PD are only effective in providing short-term management of symptoms and are unable to slow the progression of the disease. Furthermore, these treatments have been associated with many adverse side effects, limiting patient compliance and long-term effectiveness. Natural products such as plants, fungi, and microbes have played a pivotal role in pharmaceutical drug discovery for a variety of pathologies. More recently, molecules derived from natural products have been exten (open full item for complete abstract)

Committee: Dhiraj Vattem PhD (Advisor); Vatsala Maitin PhD (Committee Member); Robert Brannan PhD (Committee Member) Subjects: Aging; Animal Diseases; Food Science; Health; Health Sciences; Medicine; Neurology; Neurosciences; Nutrition; Pharmacology

Master of Science (MS), Wright State University, 2023, Physiology and Neuroscience

Environmental exposure to organophosphate (OP) pesticides, such as malathion, is a risk factor for neuropathy and neurodegeneration. Toxic levels of OPs irreversibly inhibit acetylcholinesterase (AchE) activity, leading to acute paralysis and even death as the neurotransmitter acetylcholine accumulates at cholinergic synapses. In addition, there is compelling evidence that repeated low-level “occupational-like” exposure to OPs is associated with somatosensory defects but the cellular mechanisms for this effect are unclear. We show sensory neuron cell size in the dorsal root ganglia is significantly reduced in rats exposed to occupational level malathion. However, co-administration of a reversible AchE inhibitor, galantamine, prevented this effect.

Committee: David R. Ladle Ph.D. (Advisor); Kathrin Engisch Ph.D. (Committee Member); Mark M. Rich M.D., Ph.D. (Committee Member) Subjects: Neurosciences

Master of Science (M.S.), University of Dayton, 2023, Interdisciplinary Studies

The SARS-CoV2 virus was responsible for the COVID-19 Pandemic, one of the most fatal international public health emergencies experienced in the past century. SARS-CoV2 induces symptoms like increased inflammatory response, severe acute respiratory syndrome (SARS), cognitive dysfunction like brain fog, and cardiovascular defects. Prolonged or long-term infection led to the emergence of Post-COVID-19 Syndrome, or PCS. PCS is characterized by chronic cardiovascular, autoimmune, and neurological manifestations and remains understudied. Individuals with pre-existing neurological insult like those with neuroinflammatory or neurodegenerative diseases are likely more vulnerable to such PCS effects. Furthermore, individuals with pre-existing neurological conditions often have comorbidities like obesity, hypertension, hyperlipidemia, and low activity levels. However, little is understood about the molecular effects of SARS-CoV2 on neuron in both healthy and neuro-compromised individuals. Currently, many individuals experiencing PCS-related neurological symptoms require management of their symptoms even though our knowledge in this area is still limited. Therefore, this study utilized an interdisciplinary approach to better understand how SARS-CoV2 impacts both neurons at a cellular level and clinically in neurologically compromised populations such as Multiple Sclerosis (MS). This interdisciplinary approach sheds light on how translational work is being done where basic science efforts complement efforts made clinically to make connections and identify relationships between observed effects and known science. To do so, SARS-CoV2 proteins were misexpressed in the Drosophila eye and through a forward genetic screen evaluated for changes to cellular structure or function. To corroborate these findings, SARS-CoV2 proteins were also transfected into Neuro-2a cells to assess how these proteins affected cellular functioning. Furthermore, SARS-CoV2 protein structure-function analys (open full item for complete abstract)

Committee: Kurt Jackson (Advisor); Mrigendra Rajput (Advisor) Subjects: Biomedical Research; Neurology; Neurosciences; Physical Therapy; Virology

Doctor of Philosophy, The Ohio State University, 2023, Neuroscience Graduate Studies Program

Single nucleotide variants (SNVs) in the gene encoding Kinesin Family Member 5A (KIF5A), a neuronal motor protein subunit involved in transport along microtubules, have been associated with amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive and fatal neurodegenerative disease that primarily affects motor neurons. Numerous ALS-associated KIF5A SNVs are clustered near splice site junctions of the penultimate exon 27 and are predicted to alter the carboxy-terminal (C-term) cargo-binding domain of KIF5A. Mis-splicing of exon 27, resulting in exon exclusion, is proposed to be the mechanism by which these SNVs cause ALS. Whether all KIF5A SNVs proximal to exon 27 result in exon exclusion is unclear. To address this question, we designed an in vitro minigene splicing assay in HEK293 cells which revealed heterogeneous site-specific effects on splicing: only 5´ splice site (5´ss) SNVs resulted in exon skipping. We also quantified splicing in select CRISPR-edited human stem cells differentiated to motor neurons and in neuronal tissues from a 5´ss SNV knock-in mouse (Mouse: c.3005+1G>A; Human homolog: c.3020+1G>A), which showed the same result. Moreover, survival of representative 3´ splice site (3´ss), 5´ss, and truncated C-term (ΔC) variant KIF5A (v-KIF5A) motor neurons was significantly reduced compared to wildtype (WT) motor neurons, and overt morphological changes were apparent. While total KIF5A mRNA levels were comparable across cell lines, total KIF5A protein levels were decreased for v-KIF5A lines, suggesting an impairment of protein synthesis or stability. Thus, despite the heterogeneous effect on RNA splicing, KIF5A SNVs near exon 27 similarly reduce the availability of the KIF5A protein, leading to axonal transport defects and motor neuron pathology.

Committee: Stephen Kolb MD, PhD (Advisor); Michael Kearse PhD (Committee Member); Karin Musier-Forsyth PhD (Committee Member); Arthur Burghes PhD (Committee Member) Subjects: Biochemistry; Chemistry; Experiments; Genetics; Molecular Biology; Molecular Chemistry; Neurobiology; Neurology; Neurosciences; Pharmaceuticals; Pharmacology

Doctor of Philosophy, Case Western Reserve University, 2023, Neurosciences

Traumatic brain injury (TBI) is highly prevalent and frequently transitions into a chronic neurodegenerative disease sharing numerous pathologic features with Alzheimer's disease (AD). Patients often present with progressive neurocognitive impairment, psychiatric symptoms, and metabolic shift. One driving feature of AD is excessive mitochondrial fission due to increased expression and activity of dynamin-related protein 1 (Drp1), which binds fission 1 protein (Fis1). Here, we show that aberrant mitochondrial fission and fragmentation also occur in both acute and chronic TBI. However, with TBI this is associated with increased expression of Fis1 rather than Drp1. We also report elevated Fis1 in human brain from subjects with both TBI and AD, but not with AD alone. We show that elevated mitochondrial fission after TBI is associated with acutely and chronically impaired mitochondria, oxidative damage, blood-brain barrier (BBB) deterioration, neurodegeneration, and cognitive impairment. Remarkably, two weeks of daily treatment after brain injury with a selective pharmacologic agent that prevents Drp1-Fis1 interaction completely normalized mitochondrial fission and bioenergetics, and protected mice from oxidative damage, BBB deterioration, neurodegeneration, and cognitive impairment. Neuroprotection occurred acutely and lasted chronically 9 and 17 months after TBI in mice, which is the equivalent of many decades in people. However, there was no protective effect when the same treatment was delayed until 8 months after TBI. Additionally, we have demonstrated that a metabolic shift in favor of fatty acid oxidation occurs in our model of TBI, and acute P110 treatment is able to revert it. Our results demonstrate that early transient inhibition of excessive mitochondrial fission after some forms of brain injury may prevent development of chronic neurodegenerative disease in some patients.

Committee: Andrew Pieper (Advisor); Xin Qi (Committee Member); Heather Broihier (Committee Member); Wen-Cheng Xiong (Committee Chair) Subjects: Neurosciences

Doctor of Philosophy, The Ohio State University, 2023, Biomedical Sciences

Alzheimer's Disease (AD) is a debilitating neurodegenerative disorder characterized by the progressive and selective accumulation of neurofibrillary tangles in specific areas of the brain over the course of disease. Composed of aggregated tau protein, these tangles appear to spread from the earliest affected regions to networked brain regions across the synapse, templating additional pathology in a prion-like manner. However, the cerebellum appears to resist this prion-like insult, despite connectivity to early and profoundly affected regions. The selective vulnerability and resistance of specific brain regions and cell types to prion-like tau pathology offers a window into disease etiology and endogenous mechanisms of neuroprotection. The objective of this work was to untangle the adaptive changes to disease that respond in parallel and in contrast between differentially vulnerable tissues to provide new insight into disease etiology and new targets for biological validation in disease models. First, we define a unique gene expression approach termed Ratio of Ratios that tests differential gene expression across AD and control in the vulnerable prefrontal cortex and the resistant cerebellum. We apply this along with Desirability Function Analysis to a publicly available microarray data set to sort genes into priority groups demonstrating contrasting differential expression between regions that associates with selective vulnerability, and parallel differential expression between regions that is nonspecific to vulnerability. Among contrasting genes, we find a neuronal and endothelial proteostasis signature where chaperones are selectively upregulated in the cerebellum. Among parallel genes, we find a microglial, astrocytic, and endothelial signature of immune and stress activation. Using transcription factor interaction network analysis, we report potential key regulators of these contrasting and parallel responses. We also show that the identified chaperone p (open full item for complete abstract)

Committee: Jeffrey Kuret (Advisor); Karl Obrietan (Committee Member); Andrea Tedeschi (Committee Member); Hongjun Fu (Committee Member) Subjects: Bioinformatics; Biology; Biomedical Research; Neurosciences

Master of Sciences, Case Western Reserve University, 2023, Physiology and Biophysics

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with no cure. ALS causes degeneration of both upper and lower motor neurons leading to progressive paralysis. Life expectancy after diagnosis averages 3-5 years and the current disease modifying therapies can only extend life by about 6 months (1). The exact mechanism of the pathology of ALS is unknown (2). However, the majority of cases have aggregates of TAR DNA-binding protein 43 (TDP-43), which is an RNA-binding protein (RBP), in the brain and spinal cord (3). RBPs play a special role during cellular stress by forming stress granules (SGs) through liquid-liquid phase separation. SGs are a transient, membraneless organelle that regulate RNA metabolism by separating stalled translation machinery, RNA, and RBPs from the general cytosol (4). This is a protective reaction, but in conditions of chronic stress, can become pathogenic. The TDP-43 inclusions found in patient samples co-localized with known markers of SGs, leading to the hypothesis that SGs are seeding mechanisms for protein aggregation (4). In this study, I performed qPCR analysis to determine the changes of SG-related genes in ALS patient postmortem spinal cords. I found a significant increase in DEAD-Box Helicase 3 X-Linked (DDX3X) and significant decrease in Tudor domain containing protein 3 (TDRD3) mRNA levels. Moreover, immunofluorescence staining showed that DDX3X and TDRD3 are expressed in motor neurons using Choline acetyltransferase (ChAT) as a marker for motor neurons and western blot analysis suggested that protein changes followed the mRNA changes. These findings suggest DDX3X and TDRD3 may have a role in the pathology of ALS. Thus, the overarching goal of this study is to determine the role of DDX3X and TDRD3 in ALS models, their involvement in SG mechanics, and the therapeutic viability of their manipulation. My hypothesis is that DDX3X and TDRD3 contribute to ALS pathology through their participation in patholo (open full item for complete abstract)

Committee: XIn Qi (Advisor); George Dubyak (Committee Chair); Rajesh Ramachandran (Committee Member); Tingwei Mu (Committee Member); Andrew Pieper (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, Case Western Reserve University, 2023, Molecular Medicine

Multiple sclerosis (MS) is a demyelinating and neurodegenerative disease affecting 2.5 million people globally. Immunomodulatory therapies reduce central nervous system (CNS) inflammatory events, but do not directly offer neuroprotection. This can lead to progressive decline, warranting a better understanding of how neuronal loss occurs in MS and improved therapeutic options. The visual pathway offers a distinct system to study neurodegeneration in MS and preclinical models. Due to the organization of retinal ganglion cells (RGCs) in the retina and their anatomically separate myelinated axons that form the optic nerve, we can investigate how axonal demyelination affects their respective neuronal cell bodies. We hypothesize that visual system pathology reflects CNS demyelination and neurodegeneration in MS, and can be used to monitor neurodegeneration and evaluate neuroprotective strategies. Here, we describe the spatio-temporal timeline of neurodegeneration relative to axonal loss in the experimental autoimmune encephalomyelitis (EAE) mouse model of demyelinating disease. We show that T cell infiltration, demyelination and axonal loss, and neurodegeneration occur similarly in the optic nerve and spinal cord white matter. Therefore, the anterior visual pathway serves as a pathological substrate to more clearly track neurodegeneration. The retina is also easily accessible to monitor neurodegeneration longitudinally in rodent models and in human disease. We show that retinal imaging significantly correlated with thalamic volume in MS, a strong predictor of disease progression. We also established that myelinated axon loss was concurrent with synaptic loss in the thalamus, which receives presynaptic input from the optic nerve and spinal cord. This preceded retrograde loss of neurons in the retina and spinal cord gray matter. After establishing this model, the visual system was used to investigate neuroprotective strategies. We previously showed that reducing glut (open full item for complete abstract)

Committee: Tara DeSilva (Advisor); Robert Fairchild (Committee Chair); Jeffrey Cohen (Committee Member); Bela Anand-Apte (Committee Member) Subjects: Molecular Biology; Neurology; Neurosciences; Ophthalmology

Doctor of Philosophy, Case Western Reserve University, 0, Physiology and Biophysics

Neurodegenerative diseases are nervous system disorders characterized by selective types of neuronal cell death. The causal factors for the different diseases vary, and clear molecular and cellular pathways underlying the pathogeneses remain elusive. However, accumulating evidence has suggested that mitochondrial dysfunction plays a critical role in disease-associated cell death due to the enormous energy demands of neurons. To improve the efficiency of oxidative respiration and generate more ATP, proteins on mitochondrial inner membrane form a complex adjacent to cristae junction. This complex, called the mitochondrial contact site and cristae organizing system (MICOS), maintains proper cristae morphology and stabilizes the intracristal environment for important mitochondrial functions, such as oxidative respiration. Previous studies have revealed that disruption of the MICOS leads to similar mitochondrial defects as in neurodegenerative diseases. This suggests MICOS loss as an upstream event of mitochondrial dysfunction and neuronal cell death in these disorders. In the present study, MICOS disruption was found in both Alzheimer's disease (AD) and Huntington's disease (HD) with different target components and molecular mechanisms. CHCHD6 was specifically connected to AD through a feedback loop that lowered CHCHD6 levels and increased APP processing. In cellular and animal AD models and human AD brains, the APP intracellular domain fragment inhibited CHCHD6 transcription by binding its promoter. CHCHD6 and APP bound to and stabilized each other. Reduced CHCHD6 enhanced APP accumulation on mitochondria-associated ER membranes and accelerated APP processing and induced mitochondrial dysfunction and neuronal cholesterol accumulation, promoting the amyloid pathology. Compensation for CHCHD6 loss in an AD mouse model ameliorated the AD-associated neuropathology and cognitive impairment. In contrast to AD, the specific MICOS component contributing to MICOS loss in HD wa (open full item for complete abstract)

Committee: George Dubyak (Committee Chair); Corey Smith (Committee Member); Xin Qi (Advisor); Wencheng Xiong (Committee Member); Sudha Chakrapani (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Molecular Biology; Neurosciences; Pathology; Physiology

Doctor of Philosophy, The Ohio State University, 2022, Biomedical Sciences

Batten Disease is a rare, neurodegenerative disorder that causes vision loss, cognitive and behavioral abnormalities, motor impairment, and early death. Mutations in one of 13 ceroid lipofuscin neuronal (CLN) genes can cause varying subtypes, of Batten Disease. Vision loss is a common first presenting symptom in several Batten Disease forms which quickly progresses to complete blindness. We previously established gene replacement strategies for CLN6 and CLN3 Batten Disease using adeno-associated viral vector serotype 9 (AAV9) delivered into the cerebrospinal fluid (CSF). In accordance with previous studies, I confirmed that AAV9 retinal transduction is achieved after CSF administration in mice and non-human primates, which might allow partial rescue of the vision phenotype. However, it is unclear if all necessary cell-types required for the prevention of vision loss are targeted via this delivery route. Hence, we propose that the best strategy to prevent vision loss would be to design an optimized intraocular gene replacement approach that can be combined with the current in-clinic AAV9 CSF-based gene therapies. In my thesis work, I characterized, for the first time, the expression profile of CLN3, CLN6, and CLN8 in the murine, primate, and porcine retina via single-cell RNA sequencing. We found that CLN transcripts are broadly expressed and therefore the success of an intraocular therapy would be maximized by targeting several of these cell types for gene replacement. In the first part of my thesis work, I compared the transduction profiles of AAV9 and Anc80L65, a newly engineered retina-tropic vector, following subretinal and intravitreal administration in wildtype mice. Through the development of a novel AI retinal quantification method, we found overall similar transduction profiles between the two capsids. In addition, preliminary results measuring both AAV9 and Anc80 capsid cross-reactivity with AAV2 in human serum indicates Anc80 may not be as immunologically (open full item for complete abstract)

Committee: Kathrin Meyer (Advisor); Andrew Fischer (Committee Member); Arthur Burghes (Committee Member); Kevin Flanigan (Committee Member) Subjects: Biomedical Research; Genetics; Neurosciences; Ophthalmology

Doctor of Philosophy, Case Western Reserve University, 2022, Genetics

Balanced mRNA isoform diversity and abundance are spatially and temporally regulated throughout cellular differentiation. The proportion of expressed isoforms contribute to cell type specification and determine key properties of the differentiated cells. Neurons are unique cell types with intricate developmental programs, characteristic cellular morphologies, and electrophysiological potential. Neuron-specific gene expression programs establish these distinctive cellular characteristics and drive diversity among neuronal subtypes. Genes with neuron-specific alternative processing are enriched in key neuronal functions, including synaptic proteins, adhesion molecules, and scaffold proteins. Despite the similarity of neuronal gene expression programs, each neuronal subclass can be distinguished by unique alternative mRNA processing events. Alternative processing of developmentally-important transcripts alters coding and regulatory information, including interaction domains, transcript stability, subcellular localization, and targeting by RNA binding proteins. Here, we investigate the role of CLP1, a component of the 3' end processing complex, and THOC6, a component of the transcription/export complex, in mRNA transcription. We find CLP1 suppresses proximal polyadenylation and disease-associated variants exhibit toxic gain-of-function properties. We establish a regulatory pattern for CLP1 in motor neuron 3' end diversity and identify neuronal gene overexpression as a possible mechanism of disease. Furthermore, we find THOC6 is involved in regulation of splicing and is essential for neurodevelopment in the mammalian brain. Fine-tuning of mRNA processing is essential for neuronal activity and maintenance. Thus, the focus of neuronal RNA biology research is to dissect the transcriptomic mechanisms that underlie neuronal homeostasis, and consequently, pre-dispose neuronal subtypes to disease.

Committee: Ashleigh Schaffer (Advisor); Peter Scacheri (Committee Chair); Polyxeni Philippidou (Committee Member); Helen Miranda (Committee Member); Donny Licatalosi (Committee Member) Subjects: Biochemistry; Genetics; Neurosciences

Doctor of Philosophy, Case Western Reserve University, 2022, Neurosciences

Neurons develop remarkably long, complex processes that integrate into circuitry critical for neuronal communication. Once mature, postmitotic neurons must survive for decades of life and continuously function to ensure normal, lifelong cognition, perception, behavior and movement. Due to their long and complex fibers, neurons face a unique challenge to supply distal synapses and axons with materials and energy needed to compensate for turnover, conferring a vulnerability to aging and neurodegenerative diseases should the mechanisms controlling these processes break down. Remarkably little is known regarding the transcriptional regulation of postmitotic neuronal survival and maintenance of long range axons. Here we found an adult stage specific transcriptional program controlled by terminal selectors Lmx1b and Pet1 for the survival of serotonergic synapses and axons in vivo. Adult conditional targeting of Lmx1b/Pet1 in mouse serotonergic neurons resulted in dysregulation of hundreds of genes enriched for functions related to synapses, axonal transport and structure, and mitochondrial functions. Mutant neurons presented with fragmented mitochondria, and progressive degeneration of serotonergic synaptic baskets and fiber pathology characterized by dramatically thinned axons with swollen varicosities and spheroids containing unusual aggregates of amyloid precursor protein, alpha-synuclein, and phosphorylated neurofilament. Lmx1b/Pet1 targeting induced fiber degeneration without cell body loss, is independent of 5-HT levels, and is accompanied by neuroinflammation and altered physiological and behavioral responses. This work provides substantial evidence for transcriptional programs required in the adult mammalian brain for survival of long range axonal architectures.

Committee: Evan Deneris (Advisor) Subjects: Neurobiology; Neurosciences

Doctor of Philosophy, The Ohio State University, 2022, Biomedical Sciences

The projects described within address a knowledge gap regarding specific epigenetic regulatory mechanisms underlying the dysfunction of microglia in Alzheimer's disease. AD is the 6th leading cause of death in the US. Of the top 10 causes of death, AD is the only one which currently cannot be prevented or cured. AD brain pathology is characterized by build-up of amyloid-β (Aβ) plaques and neurofibrillary tangles of tau protein. Previous research has established that neuroinflammation also contributes to the synaptic loss, neuronal death, and symptomatic decline of AD patients. Accumulating evidence suggests a critical role for microglia, innate immune phagocytes of the central nervous system, in AD pathogenesis. For instance, microglia are unable to effectively phagocytose and degrade Aβ aggregates, and they instead respond by releasing pro-inflammatory cytokines which are implicated in AD neurodegeneration. How microglia initiate these inflammatory responses without simultaneous clearance of Aβ is unknown. Autophagy is a normal, degradative process that discards non-functional cellular organelles, internalized microbes, protein aggregates, and regulates release of inflammatory proteins. Extensive research in AD established that diseased neurons exhibit dysfunctional autophagy, which contributes to neuronal death. However, autophagy function in other brain cells in AD, such as microglia, remains overlooked. This work uncovered the first-described mechanism underlying dysfunctional autophagy in AD microglia. The MicroRNA (miR) Mirc1/Mir17-92 cluster, known to diminish autophagy effectors, is elevated in AD microglia from human patient samples. Microglia from a mouse model of AD fail to clear Aβ due to the elevation of the Mirc1/Mir17-92 cluster. However, autophagy function and clearance of Aβ can be recovered by inhibiting miR-17 in diseased mouse microglia. Moreover, this work established a nanoparticle-based therapeutic (open full item for complete abstract)

Committee: Amal Amer (Advisor); Amy Lovett-Racke (Committee Chair); Douglas Scharre (Committee Member); Stephanie Seveau (Committee Member); Kathryn Lenz (Committee Member); Ruth Barrientos (Committee Member) Subjects: Immunology; Neurosciences

Doctor of Philosophy, Case Western Reserve University, 2022, Clinical Translational Science

The translation of medical therapies from basic and preclinical research to efficacious human interventions is challenging. The majority of candidate therapies fail in early-stage human trials, after showing promise in preclinical work. The primary aim of the research presented herein is to explore the potential role that poor statistical practice in preclinical animal trials might play in contributing to translational failure. First, a comprehensive appraisal of current statistical practice in one area of preclinical neuroscience research was carried out. A close review of the current related literature is presented, and the appraisal includes a tutorial to explain how certain statistical mistakes might result in overly optimistic results, as well as practical recommendations for improvement. A majority of articles included in this appraisal failed to account for sources of non-independence in the data (74-93%) and/or did not analytically account for mid-treatment animal attrition (78%). Ordinal variables were often treated as continuous (37%), outliers were predominantly not mentioned (83%), and plots often concealed the distribution of the data (51%). Next, a sample including both successful and failed human trials for neurologic targets was identified, and rates of statistical mistakes in the associated preceding rodent trials were compared. Failed human trials were found to have higher rates of select sources of potential statistical bias in preceding rodent trials, compared to successful trials. This research provides evidence that a contributing factor to translational failure is statistical misapplication in preclinical animal research in the neurosciences. It provides the groundwork for future research that will provide practical solutions to translational researchers and funders, facilitating preclinical experimental validity to increase the translational success rate.

Committee: Mary Dolansky PhD RN FAAN (Committee Chair); Kenneth Baker PhD (Committee Member); Nancy Obuchowski PhD (Committee Member); Jill Barnholtz-Sloan PhD (Advisor) Subjects: Animal Sciences; Biostatistics; Neurosciences; Statistics

Master of Science (MS), Ohio University, 2021, Biological Sciences (Arts and Sciences)

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by the loss of dopaminergic (DA) neurons in substantia nigra pars compacta and the formation of Lewy Bodies (LBs), cytoplasmic protein deposits of α-Synuclein (αSyn). In recent years, an intriguing concept of prion-like spreading of pathogenic proteins such as αSyn has emerged. Released αSyn spreads between neurons causing neurodegeneration, but the actual propagation mechanism is still under investigation. In order to test cell-to-cell propagation of αSyn, I investigate αSyn release. In my project, I develop a larval neuromuscular junction (NMJ) model in order to study αSyn release mechanisms. I hypothesize that neuronal activity regulates pathological αSyn release. Thus, using optogenetics to stimulate neurons that co-express αSyn and Channel Rhodopsin (ChR2) in Drosophila melanogaster larvae, I examine αSyn release induced by neuronal depolarization. I use ELISA technique to detect and compare released αSyn levels in the hemolymph of different fly lines. Results show activity-dependent αSyn release. This activity-dependent αSyn release is also influenced by synaptic transmission, mutations, and phosphorylation of αSyn. Hence, αSyn release might be induced in some regions of PD brain in response to excitability, and this αSyn release might underlie the disease progression. Therefore, targeting αSyn release could be further studied in hope of establishing new therapeutic interventions to stop or slow PD pathology.

Committee: Daewoo Lee (Advisor); Corinne Nielsen (Committee Member); Robert Colvin (Committee Member) Subjects: Biology; Neurobiology; Neurosciences