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

Doctor of Philosophy (PhD), University of Toledo, 2023, Experimental Therapeutics

Triple-negative breast cancer (TNBC), the most lethal and aggressive subtype of breast cancer, lacks estrogen receptors, progesterone receptors, and human epidermal receptors, rendering it unsuitable with targeted-based treatment. TNBC has higher relapse rate, worst prognosis and higher metastasis rate compared to non-TNBC because of their tendency to resist to apoptosis, a form of programmed cell death, induced by chemotherapy. Hence, non-apoptotic cell death inducers could be a potential alternative to circumvent the apoptotic drug resistance. In this study, we discovered two novel compounds, TPH104c and TPH104m, which induce non-apoptotic cell death in TNBC cells. These lead compounds were 15 to 30-fold more selective in TNBC cell lines and significantly decreased the proliferation of TNBC cells compared to normal mammary epithelial cell lines. TPH104c and TPH104m induced a unique type of non-apoptotic cell death characterized by no cellular shrinkage, absence of nuclear fragmentation and f apoptotic blebs. Although TPH104c and TPH104m produced the loss of the mitochondrial membrane potential, TPH104c- and TPH104m-induced cell death did not increase total cytochrome c and intracellular ROS, lacked caspases activation, and was not rescued by pan-caspase inhibitor, zVAD-FMK. Moreover, TPH104c and TPH104m significantly downregulated mitochondrial fission protein, Drp1 and its levels determined their cytotoxic efficacy. Studies have shown that protein, Bcl-2 interacting protein 3 (BNIP3), mediates a non-apoptotic, necrosis-like cell death similar to that produced by TPH104c and TPH104m that lacked activation of caspases and reduced mitochondrial transmembrane potential. Therefore, we determined the effect of TPH104c and TPH104m on various mitochondrial functions, in triple negative breast cancer (TNBC) cells, BT-20 and MDA-MB-231. TPH104c and TPH104m (2 and 5 μM), compared to vehicle, significantly increased the levels of reactive oxygen species (ROS) BNIP3 and c-Jun (open full item for complete abstract)

Committee: Amit K. Tiwari (Committee Chair); Aniruddha Ray (Committee Member); Ana Maria Oyarce (Committee Member); Frederick E. Williams (Committee Member) Subjects: 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, 2022, Biomedical Engineering

Endothelial cells (ECs) line all blood vessels and vasculature in the human body and are exposed to mechanical shear stress from blood flow. EC dysfunction has been shown to be one of the root causes of cardiovascular atherosclerotic disease. Atherosclerosis is a site-specific phenomenon and occurs in vessels at areas of disturbed blood flow (atheroprone regions). It is known that hemodynamic forces and mechanochemical environments alter the intracellular signal responses in ECs and these alterations are known to contribute to EC dysfunction. Calcium (Ca2+) is a key intracellular signalling ion and is a ubiquitous secondary messenger that is responsible for many intracellular signalling mechanisms. Intracellular calcium concentration ([Ca2+]i) oscillations are an important signaling response to hemodynamic forces and fluid shear stress. Oscillatory changes in [Ca2+]i occur due to inositol 1,4,5 trisphosphate (IP3)-regulated Ca2+ release from the endoplasmic reticulum (ER), and require Ca2+ transport between the ER and mitochondria. Ca2+ uptake by mitochondria is a major determinant of bioenergetics, adenosine triphosphate (ATP) production and EC fate. Mitochondrial Ca2+ uptake occurs via the mitochondrial Ca2+ uniporter (MCU) complex, an inner mitochondrial membrane (IMM) protein assembly consisting of a core MCU Ca2+ channel and MCU complex regulatory/auxiliary subunit proteins. The MCU is a Ca2+-selective channel that is responsible for the influx of cytosolic Ca2+ into the mitochondria. In vivo and in vitro evidence showed upregulated MCU expression in vascular ECs under conditions associated with cardiovascular disease (CVD). However, the critical role of MCU in regulating the mitochondrial Ca2+ concentration ([Ca2+]m) in ECs exposed to fluid shear stress and the regulation of the MCU complex expression by hemodynamic forces are currently unknown. This work examined the role of MCU in cultured human ECs exposed to hemodynamic shear stresses. This included mon (open full item for complete abstract)

Committee: Jonathan Song (Advisor); Yi Zhao (Committee Member); Shaurya Prakash (Committee Member) Subjects: Biomedical Engineering; Cellular Biology

Master of Science in Biomedical Sciences (MSBS), University of Toledo, 2022, Biomedical Sciences (Bioinformatics and Proteomics/Genomics)

Suicide is defined as the act of self-injurious behavior that results in an outcome of death. Nearly 800,000 people are estimated to die by suicide each year. Approximately 90% of those that die by suicide are suspected to have a comorbid neuropsychiatric condition, such as major depressive disorder (MDD), schizophrenia (SCZ), or bipolar disorder (BP). Given this clear correlation, studies of suicide have been largely conducted in the context of gene transcription associated with neuropsychiatric disorders. Such approaches, like RNAseq and microarray technologies, have been invaluable in assessing gene expression changes in the brain in subjects diagnosed with a neuropsychiatric disorder, offering significant insight into the biological processes that are perturbed in these disorders and therefore associated with an outcome of suicide. In this analysis, we utilized public postmortem brain tissue datasets from frontal cortical regions (i.e. DLPFC, frontal cortex, prefrontal cortex) to identify transcriptional gene signature specific to suicide across multiple neuropsychiatric disorders (MDD, SCZ, BP). Three RNAseq (150 total subjects) and four microarray (322 total subjects) datasets were collected from 4 NCBI GEO to construct a meta-analysis of various neuropsychiatric disorders. We assessed differentially expressed genes (DEGs) between suicide and natural death groups. We further explored DEG results using full transcriptome (GSEA) and targeted transcriptome (EnrichR) pathway analysis. As chronic psychological stress is associated with an increase in suicidal risk, we identified DEGs from mouse models of chronic stress. We explored these DEGs using similar pathway analyses as human data for comparison. Results in human data show dysfunction in mitochondrial pathways, purine metabolism and purinergic signaling pathways, as well as neuroinflammation associated pathways which have prior implications in suicide risk and underlying pathology of MDD, SCZ, and BP. We ad (open full item for complete abstract)

Committee: Robert Smith (Committee Co-Chair); Cheryl McCullumsmith (Committee Member); Rammohan Shukla (Committee Member); Sinead O'Donovan (Committee Co-Chair) Subjects: Bioinformatics; Biology; Biomedical Research; Neurosciences

Master of Sciences, Case Western Reserve University, 2021, Physiology and Biophysics

The abnormal hyperphosphorylation and aggregation of tau proteins is a hallmark of tauopathies. These neurodegenerative diseases impact millions of people worldwide and yet there are no adequate therapies currently available. Mitochondrial dysfunction and mitophagy impairment have more recently been identified as contributors to the progression of age-related diseases, but their exact roles in the pathogenesis of tauopathy-type neurodegenerative diseases remains unknown. Preliminary investigations in the field yielded conflicting results that supported two different hypotheses: One identifying pathological tau as the driving force, and the other identifying mitochondrial dysfunction and mitophagy impairment as the driving force of disease progression. While the two different theories have been consolidated into the “vicious cycle” model, the field is left with many questions related to what causes the onset of tauopathies. This review summarizes the connections between mitochondrial dysfunction and the pathogenesis of tauopathy-type neurodegenerative diseases. Additionally, this review covers how the roles of mitochondrial dysfunction and mitophagy impairment are currently being applied to the development of therapeutics.

Committee: Xin Qi Ph.D. (Advisor); George Dubyak Ph.D. (Committee Chair); William Schilling Ph.D. (Committee Member); Corey Smith Ph.D. (Committee Member) Subjects: Biophysics; Philosophy

Doctor of Philosophy (PhD), Wright State University, 2019, Biomedical Sciences PhD

Parkinson's disease is the second most common neurodegenerative disease. This disease is caused by the degeneration of dopaminergic neurons, leading to debilitating motor symptoms and early mortality. The protein α-synuclein (α-syn), encoded by SNCA, misfolds and forms inclusions in Parkinson's disease brains. When α-syn is overexpressed in yeast, it causes cellular toxicity and an increased number of aggregates, recapitulating the toxic phenotypes observed in humans and animal models. Yeast models are a powerful tool to perform high-throughput overexpression screening to identify modifiers of α-syn toxicity. α-syn causes mitochondrial dysfunction by inhibiting complex I and inducing mitochondrial fragmentation. Prior screening of α-syn were limited to only the galactose condition, where mitochondrial function is dispensable. Previous screening was performed exclusively with the GAL1 promoter, restricting the genes to only those induced by galactose. We have validated an overexpression system using GAL3 alleles that can induce genes under mitochondrial-dependent glycerol-ethanol condition and other non-galactose conditions (calorie restriction, nitrogen starvation and raffinose). α-syn showed discrepancy in the correlation of toxicity and aggregation in non-galactose conditions. Compared to galactose, under glycerol-ethanol condition, α-syn exhibited higher toxicity, formed more aggregates, and decreased viability and respiratory competency despite having similar expression under the two conditions. We screened 14,827 human gene clones and identified 87 that can suppress α-syn toxicity in glycerol-ethanol. Genes involved in RNA polymerase II function, anterior-posterior axis and nucleoplasm were overrepresented. Among the suppressor hits, we identified four 14-3-3 protein isotypes (β, γ, θ, and ζ). None of the four suppressors suppressed the toxicity under galactose. However, the 14-3-3 suppressors did not reduce aggregates under glycerol-ethanol. No increase in res (open full item for complete abstract)

Committee: Quan Zhong Ph.D. (Advisor); David R. Cool Ph.D. (Committee Member); Paula Ann Bubulya Ph.D. (Committee Member); David R. Ladle Ph.D. (Committee Member); Weiwen Long Ph.D. (Committee Member) Subjects: Biology; Cellular Biology; Molecular Biology

Master of Sciences, Case Western Reserve University, 2016, Physiology and Biophysics

Huntington's disease (HD) is an incurable neurodegenerative disease with autosomal dominant inheritance caused by expanded CAG repeats in the huntingtin gene that confers a toxic gain-of-function on mutant huntingtin (mtHtt) protein. Mitochondrial dysfunction is a major cytopathology in HD. However, the molecular mechanisms by which mtHtt affect mitochondrial function remains elusive. This project explores the role MAPK1 plays in the over-activation of Drp1, the mitochondrial fission protein, which leads to the mitochondrial dysfunction and neurodegeneration seen in HD. Hdh striatal cells, treated with U0126, a potent inhibitor of MEK1/2, showed a decrease in mitochondrial fragmentation, and restored mitochondrial function. We show MAPK1 binding to and phosphorylating Drp1 at Ser616 in HD, as well as U0126 treatment abolishing this phosphorylation. A phosphorylation-deficient mutant of Drp1, S616A, also corrected the mitochondrial fragmentation associated with HD. This study suggests that MAPK1 activation of the mitochondrial fission machinery leads to the aberrant mitochondrial dysfunction and neurodegeneration seen in HD; and that inhibition of Drp1-mediated excessive mitochondrial fission can be used as a treatment for HD.

Committee: Xin Qi (Advisor); William Schilling (Committee Chair); George Dubyak (Committee Member); Joseph LaManna (Committee Member); Charles Hoppel (Committee Member) Subjects: Neurology; Physiology

PhD, University of Cincinnati, 2015, Medicine: Toxicology (Environmental Health)

The focus of this dissertation is the characterization of the effects of developmental (in utero) aryl hydrocarbon receptor (AHR) disruption in the embryo and its consequences to the adult, primarily regarding the role of this receptor in cardiogenesis, cardiac homeostasis, and cardiac function. Perturbations in cardiogenesis may result in structural and functional defects broadly categorized as congenital heart disease (CHD). With about 2 cases per 1,000 live births, CHDs are the most frequent type of congenital disease in newborns, resulting in a major burden with direct social and economic impacts in the developed and developing world. Considering that the vast majority of CHD cases cannot be directly attributed to specific disease-causing mutations, gene-gene and gene-environment interactions have emerged as significant contributors in the etiology of most non-syndromal forms of CHD. Epidemiological studies have identified links between environmental exposures to AHR ligand pollutants and congenital or adult cardiovascular disease. In this context, as a mediator of environmental sensing, the AHR has a potential role in the pathogenesis of CHD. Ultimately, furthering our understanding of the roles of the AHR in heart development in health and disease, and as an etiological nexus for CHD, will contribute to the understanding, treatment and prevention of the majority of CHD cases. Chapter One summarizes the current body of knowledge regarding CHD, the AHR, heart development, and mitochondria as it relates to heart development and function. Chapter Two describes a mouse model of environmental in utero AHR disruption, and characterizes the effects to cardiogenesis at key developmental windows at the molecular, structural, ultrastructural, and functional levels. Chapter Three further investigates the phenotypes produced by in utero AHR disruption to the young and adult heart in the pre-established mouse model. Finally, Chapter Four discusses the findings of the studie (open full item for complete abstract)

Committee: Alvaro Puga Ph.D. (Committee Chair); Brad Newland Bolon Ph.D. (Committee Member); Jack Rubinstein M.D. (Committee Member); Mary Beth Genter Ph.D. (Committee Member); Ying Xia Ph.D. (Committee Member) Subjects: Toxicology

PHD, Kent State University, 2012, College of Arts and Sciences / School of Biomedical Sciences

PANDIT, ASHISH V., Ph.D., May 2012, BIOMEDICAL SCIENCES/CELL AND MOLECULAR BIOLOGY REGULATION OF MITOCHONDRIAL GENE EXPRESSION IN MULTIPLE SCLEROSIS CORTEX Director of Dissertation: Jennifer McDonough Multiple Sclerosis [MS] is a CNS disorder with unknown etiology. Deficient mitochondrial energy production is implicated in primary neurodegeneration seen in MS cortex. Previous studies have identified decreased expression of 26 electron transport chain [ETC] genes in MS motor cortex. In order to understand if this change was more global, the expression and transcriptional regulation of select ETC genes was studied here. Using RT-PCR, a significant decrease in the expression of transcripts for subunits of Complexes I, III, IV and V was observed in MS parietal and frontal cortices. Additionally, all the genes under study were transcriptionally regulated by NRF-2. Using EMSA studies, NRF-2 binding, but not its expression, was seen to be decreased in the presence of oxidative stress in MS. In order to better study the effects of oxidative stress on NRF-2 function, two possible models for MS, namely the human neuroblastoma SH-SY5Y cell line and the EAE mouse were studied, but both were found to be unsuitable. A comparative study of protein expression between MS and EAE showed that while it models inflammatory demyelinating events well, EAE does not model the respiratory deficits observed in MS and other factors such as oxidative stress and epigenetic influences may play a role in the evolution of MS. In line with this thought, DNA methylation was studied using ChIP and PCR for its effects on the regulation of electron transport chain gene expression in MS. A significant increase in the methylation of the promoter regions of the genes associated with respiration, namely GAD67 and ATP5I was seen. This indicates that it may play a role in the decreased expression of the ETC proteins and contribute to mitochondrial dysfunction seen in MS. Overall this study has found that ther (open full item for complete abstract)

Committee: Jennifer McDonough Dr (Advisor); Ernest Freeman Dr (Committee Member); Soumitra Basu Dr (Committee Member); James Blank Dr (Committee Member); Derek Damron Dr (Committee Member); Diane Stroup Dr (Committee Member) Subjects: Biology; Biomedical Research; Cellular Biology; Health Sciences; Molecular Biology; Neurobiology; Neurology; Neurosciences; Pathology

Doctor of Philosophy, Case Western Reserve University, 2009, Pathology

Alzheimer's disease (AD) is the leading cause of dementia in the elderly, characterized by neurofibrillary tangles, senile plaques and a progressive loss of neuronal cells in selective brain regions. Mitochondrial dysfunction is a prominent and early feature of the disease, although the reason for this is unclear. Nonetheless, emerging evidence suggest that mitochondrial function is dependent on the dynamic balance of fission and fusion events which are regulated by mitochondria fission and fusion proteins (i.e. DLP1/OPA1/Mfn1/Mfn2/Fis1). While an impaired balance of mitochondria fission and fusion is being increasingly implicated in neurodegenerative diseases, few studies have examined this aspect in AD. To address this issue, in this study, we investigated mitochondria morphology and distribution in primary fibroblasts and neurons from normal subjects and those from AD patients. We found disease-related changes in mitochondrial morphology and distribution as well as changes in expression levels and distribution of mitochondrial fission and fusion proteins. To understand the underlying mechanisms of these mitochondria alterations in AD, we overexpressed or knocked down functional mitochondrial fission and fusion proteins in primary human fibroblasts, M17 cells (derived from a human neuroblastoma) and rat E18 primary hippocampal neurons. Interestingly, functional protein changes in AD fibroblasts or neurons mimicking that found in AD, are correlated with similar changes in mitochondrial morphology and distribution to that observed in AD fibroblasts or neurons. We further demonstrated that ROS or amyloid-β are likely the potential pathogenic factor that causes an impaired balance of mitochondrial fission/fusion, mitochondria dysfunction and even synaptic abnormalities. Taken together, this is the first study to show that ROS or amyloid-β might induce mitochondria dynamic abnormalities in mitochondria, mitochondrial dysfunction and further neuronal dysfunction through (open full item for complete abstract)

Committee: Mark A. Smith PhD (Committee Chair); Xiongwei Zhu PhD (Advisor); Robert B. Petersen PhD (Committee Member); Shigemi Matsuyama PhD (Committee Member); Clive R. Hamlin PhD (Committee Member); George Perry PhD (Committee Member) Subjects: Pathology