Doctor of Philosophy, Miami University, 2023, Chemistry and Biochemistry

The local environment of a protein determines whether it can perform its functions properly. Studying proteins in their native environment provides the most accurate information on their structure, function, dynamics, and interactions with other proteins. Membrane proteins are especially sensitive to their environment. In vitro studies require solubilization in a compatible membrane mimetic to allow membrane proteins to adopt native conformations and interact with binding partners. In this work, protein-protein interactions, aggregation and novel membrane mimetics are studied with EPR spectroscopy complemented by biochemical techniques. KCNQ1 is a six pass transmembrane protein that forms a potassium channel responsible for the regulation of many physiological processes including heartbeat by interacting with an accessory protein, KCNE1. In this dissertation the interactions between KCNQ1 monomers are further clarified by demonstrating the role of the transmembrane region in tetramer formation, and the interactions of KCNQ1 and KCNE1 are observed from the perspective of KCNE1 side chain dynamics with CW EPR spectroscopy. The novel membrane mimetic SMALPs were used to purify KCNE1 directly from inclusion bodies, expanding the potential applications for SMALPs to be used with proteins that denature upon removal from the native membrane. EPR spectroscopy can also be used to track protein-protein interactions that are detrimental to an organism, such as tau aggregation. Tau is an intrinsically disordered protein that is prone to degradation and has proven to be difficult to study in its full-length form. In this work, a method for spin labeling full-length Tau was developed as a screening tool to quickly and cost effectively estimate the location of aggregate cores. Lastly, the spectroscopic methods used in this work were used to spin label and observe the dynamics and interhelical distances of the human TRPV1 channel for the first time. This method can be used to obser (open full item for complete abstract)

Committee: Gary Lorigan (Advisor); Carole Dabney-Smith (Committee Chair); Tracy Haynes (Committee Member); Kevin Yehl (Committee Member); Andrea Kravats (Committee Member) Subjects: Biochemistry; Biophysics; Chemistry

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2021, Photochemical Sciences

The anisotropic nature of the force fluctuation inside cells makes it very biologically relevant to study the dynamics of the structure-function relationship of protein molecules under force. The ability to manipulate individual molecules under near-physiological conditions makes the Atomic force microscope a very widely used technique. Force applied by AFM can be either compressive force or pulling force. Here in the thesis, we have explored the compressive force response of the protein molecules and the impact of the compressive force using AFM. The abrupt ruptures of protein native structures under compressive force were demonstrated by single-molecule AFM-FRET spectroscopic nanoscopy. The simultaneous measurement of the force curve and the FRET efficiency showed a temporal correlation between the compressive force drop and the FRET efficiency drop which indicated a spontaneous and abrupt rupture of the protein native tertiary structure. Furthermore, a similar compressive force experiment was done on targeted calmodulin molecules to characterize two different forms of CaM, the Ca2+-ligated activated form, and the Ca2+ free non-activated form (Apo-Calmodulin). A sudden and spontaneous rupture of Apo-CaM molecules was observed under the compressive force applied by an AFM tip, though no such events were recorded in the case of the Ca2+-ligated form. To further prove that this kind of compressive force rupture of Apo-CaM can trigger new chemistry inside the cell, a similar experiment was carried out where we observed compressive force rupture of apo-CaM molecules and successive binding of C28W peptide to the ruptured protein, a typical protein signaling activity that only a Ca2+-activated CaM has. This observation demonstrates that both chemical activation and force activation can play a vital role in biology, such as cell-signaling protein dynamics and function. Lastly, we further explored the entangled protein state formed following the events of the multiple (open full item for complete abstract)

Committee: H Peter Lu Ph.D. (Advisor); Virginia L. Dubasik Ph.D. (Other); Liangfeng Sun Ph.D. (Committee Member); Mikhail A. Zamkov Ph.D. (Committee Member) Subjects: Biochemistry; Biophysics; Chemistry; Physical Chemistry

Doctor of Philosophy, Case Western Reserve University, 2015, Molecular Medicine

Alzheimer's disease (AD) is a devastating neurodegenerative disease that represents an immense challenge to public health and wellness. AD is defined by extracellular accumulations of amyloid beta (Aß), a protein heavily associated with AD pathogenesis, into senile plaques, and intracellular accumulations of tau into neurofibrillary tangles. The primary focus in the field has been on how Aß accumulation exhibits toxicity and contributes to AD. However, increasing evidence suggests Aß is not the only effector mediating AD pathogenesis. Aß is released from proteolytic processing of a larger protein known as Amyloid Precursor Protein (APP), which is heavily implicated in AD. The proteolytic process that releases Aß from APP also releases increased functional levels of the APP intracellular domain (AICD). AICD is cytotoxic in vitro, and in vivo can mediate many AD-like pathologies in transgenic mice overexpressing AICD (AICD-Tg mice). The cell signaling mechanisms mediating this pathogenesis are unknown. AICD binds many other cell signaling proteins. One of these proteins is c-Jun N-terminal Kinase (JNK)-Interacting Protein-1 (JIP1). JIP1 mediates stress-specific JNK activation. This work demonstrates increased presence of AICD can contribute to AD pathogenesis in vivo through JIP1. Increased JNK activation can also result in increased inflammatory activation. Inflammation is also heavily implicated in AD pathogenesis. Therefore, AICD's effects on JIP1-mediated JNK activation may contribute to AD pathogenesis by serving to activate increasing inflammation. Anti-inflammatory treatments administered peripherally to AICD-Tg mice alleviate pathogenesis. Intravenous immunoglobulin (IVIg) is a robustly anti-inflammatory reagent being investigated as a clinical treatment for AD. To confirm if neuroinflammation can mediate pathogenesis in AICD-Tg mice IVIg was administered both prophylactically (before pathology development) and therapeutically (after pathology had already deve (open full item for complete abstract)

Committee: Sanjay Pimplikar PhD (Advisor); Brian McDermott PhD (Committee Chair); Michael Parsons PhD (Committee Member); Bruce Lamb PhD (Committee Member); Hoonkyo Suh PhD (Committee Member) Subjects: Biology; Cellular Biology; Health Sciences; Medicine; Neurobiology; Neurosciences

Doctor of Philosophy, The Ohio State University, 2004, Biophysics

In vitro tau polymerization in the presence of inducers is a good model for the fibrillization of the protein in Alzheimer's disease, a process believed to lead to neurodegeneration. Here we characterize the mechanism of tau polymerization induction by free fatty acids and newly identified inducers (anionic surfactants and microspheres and dyes). We show that detergent micelles and phospholipid vesicles nucleate filament formation and this parallels observations from biopsy samples of Alzheimer's disease patients. We parallel these results with another natively unstructured amyloid forming protein, α-synuclein, and with a homologous microtubule associated protein, MAP2c. Finally, we show that tau polymerization proceeds via a partially folded assembly intermediate and that dye molecules can modulate tau polymerization, by affecting the propensity or the nature of this intermediate. The latter observation has relevance for the therapeutics of Alzheimer's disease. Drugs that affect tau polymerization in certain ways can be tested for their efficiency and the pathogenicity of the intermediate versus the filament can be assessed.

Committee: Jeffrey Kuret (Advisor) Subjects:

Doctor of Philosophy, Case Western Reserve University, 2023, Physiology and Biophysics

Tau is a microtubule associated protein that is involved in neurodegenerative disorders collectively classified as tauopathies, including Alzheimer's disease and frontotemporal dementia. In tauopathies, tau protein aggregates into insoluble filaments that form cytoplasmic inclusions that have properties of amyloid fibrils. Recently, it was reported that, akin to many other natively unstructured proteins, tau undergoes liquid-liquid phase separation (LLPS) in vitro as well as in cells. This finding could have potentially important implications for understanding the pathogenic process in tauopathies, since emerging evidence supports the hypothesis that aberrant phase separations may play an important role in neurodegenerative diseases. In studies described in my thesis, we determined that tau LLPS is driven by attractive electrostatic intermolecular interactions between the negatively charged N-terminal and positively charged middle/C-terminal regions of the protein. Consistent with the electrostatic model of tau LLPS, we found that phosphorylation patterns that increase the polarization of charge distribution promote tau LLPS, whereas those that decrease charge distribution polarization have the opposite effect. Furthermore, we determined that disease-related point mutations in tau do not have any effect on the ability of tau to undergo LLPS. Pathogenic mutations, however, greatly accelerate the liquid-to-solid phase transition within the droplets, leading to rapid formation of fibrillar aggregates. Finally, we uncovered a novel and unique mechanism by which LLPS can regulate the fibrilization rate of mixtures containing tau isoforms with different aggregation propensities. This mechanism is operational only under the condition of LLPS, where total concentration of all tau variants in the condensed phase is constant. Therefore, increasing the proportions of a slowly aggregating tau isoform gradually lowers the concentration of the isoform with high aggregation propen (open full item for complete abstract)

Committee: Witold K. Surewicz (Advisor); Sudha Chakrapani (Committee Chair); Allison Kraus (Committee Member); Xin Qi (Committee Member); Matthias Buck (Committee Member) Subjects: Biochemistry; Biology; Biophysics

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

Doctor of Philosophy (PhD), Ohio University, 2019, Chemistry and Biochemistry (Arts and Sciences)

Tau protein plays a crucial role in stabilizing microtubules inside neuronal axons and maintaining the structural integrity of neurons. Binding of tau to microtubules at tau repeat domains (R) is regulated by phosphorylation. This phosphorylation is regulated by a family of enzymes called kinases. Under pathological conditions, tau is hyperphosphorylated by elevated activity of kinases such as the microtubule affinity-regulating kinase (MARK) proteins, leading to complete detachment of tau, microtubule collapse and ultimately, neuronal cell death. The free, hyper-phosphorylated tau proteins aggregate into insoluble prion-like oligomers which have been implicated in neurodegenerative diseases, including Alzheimer's disease (AD) and frontotemporal dementia. There is currently no treatment to prevent the progression of AD; all medications available today only reduce the symptoms of the disease. Moreover, using small molecule kinase inhibitors as treatment can cause serious negative side effects because of their lack of specificity. The research outlined in this work aims to develop a metabolically stable, selective peptide-based MARK kinase inhibitor that targets MARK proteins. This peptide-based inhibitor, designated tR1, was designed as a direct sequence memetic of the microtubule-binding R1 repeat domain of tau. Here, we show that tR1 peptides can inhibit MARK2 activity and reduce the level of tau phosphorylation in vitro and in cultured rat primary cortical neurons. In the second segment of this project, we attempted to inhibit tau aggregation in vitro using peptide-based aggregation inhibitors. Here, we synthesized peptides designated (an-R3, PHF6, and PHF6*) which mimic nucleating sites in the microtubule binding repeat domain of full-length tau. We hypothesized that these peptides would associate with tau protein and block further tau aggregation. We assessed the ability of these three peptides to inhibit tau aggregation using in vitro heparin-induced tau (open full item for complete abstract)

Committee: Justin Holub M. (Advisor); Marcia Kieliszewski (Committee Member); Robert Colvin (Committee Member); Jana Houser (Committee Member); Jixin Chen (Committee Member) Subjects: Biochemistry

Master of Science in Mechanical Engineering, Cleveland State University, 2018, Washkewicz College of Engineering

Traumatic injuries and neurodengerative disease are two primary and debilitating causes of cell damage and death in the nervous system. Understanding the mechanobiological behavior of the neuron, and in particular the influence of the mechanical environment on microtubules, is integral to the prevention and treatment of many of these conditions. Within the axon, microtubules are the primary filament that provide structural support and cargo transport. The central nervous system has been a primary focus, where axonal microtubules have been studied for both traumatic brain injury [1] and many neurodegenerative diseases. Beyond the many invaluable experimental tools, modeling is a complementary tool to study the mechanics of a “bundle” of microtubules. Previous work adopted a discrete element formulation [1], though it is unclear how these assumed dynamic simulations affect modeling capabilities. A finite element (FE) based approach offers the possibility to model potentially important bending behavior (i.e. beam elements), may result in more efficient simulations, and when implemented with a co-rotational formulation [2] can account for large deformations. Hence, the goal of this work was to 1. verify implementation of a co-rotational formulation for traditional beam and truss finite elements and 2. apply the framework to predict the mechanical response of a typical microtubule bundle.

Committee: Jason Halloran (Advisor); Richter Hanz (Committee Member); Kothapalli Chandra (Committee Member) Subjects: Biomechanics; Biomedical Engineering; Biomedical Research; Mechanical Engineering

Doctor of Philosophy, Case Western Reserve University, 2015, Molecular Medicine

Tauopathies are a class of neurodegenerative diseases, including Alzheimer's disease, characterized by progressive cognitive decline and widespread brain atrophy following hyperphosphorylation and aggregation of microtubule-associated protein tau (MAPT). Presently, there is no treatment, and the underlying source of pathogenesis has not been identified. One of the symptoms commonly found in tauopathies is a reduction in neurogenesis, the birth of new neurons. However, the relation between neurogenesis and MAPT has never been examined. In these studies, we used a mouse model of human tauopathy known as the hTau mouse, which expresses human MAPT and develops an age-related tauopathy, to examine adult neurogenesis. Our characterization of adult neurogenesis in the hTau mouse found that hTau mice exhibit reduced neurogenesis as early as 2 months of age, a substantially earlier marker than previously described pathology in the model. This alteration was found to be due to reduced proliferation and correlated with increased MAPT phosphorylation in the neurogenic precursors. Neurospheres grown in vitro further revealed an inherent proliferative defect. These findings suggest that altered adult neurogenesis is a strong early marker of alterations in MAPT in the hTau mouse model of tauopathy, and that further research should be conducted in examining adult neurogenesis as a marker and therapeutic target for human tauopathies.

Committee: Bruce Lamb (Advisor); Gary Landreth (Committee Chair); Sanjay Pimplikar (Committee Member); Alexander Rae-Grant (Committee Member); Thomas Egelhoff (Committee Member) Subjects: Biology; Biomedical Research

Doctor of Philosophy, The Ohio State University, 2012, Biophysics

Alzheimer's disease is a debilitating, progressive neurodegenerative disorder that affects a large percentage of the elderly population. Currently, there is no definitive premortem diagnosis and no cure. These protein aggregates accumulate for many years before the onset of clinical symptoms; therefore, their in situ detection would be an invaluable tool for early premortem diagnosis. Because radiolabeled small molecules used for whole brain imaging, such as those used for PET imaging, have the advantage of tracking the spatiotemporal pattern of molecular targets, this approach has tremendous utility for neurodegenerative disorders. More specifically, the detection of tau-bearing neurofibrillary tangles is especially promising as the amount and spatiotemporal pattern of tau-aggregate deposition is the gold standard of postmortem disease assessment, as it correlates with loss of neurons. The main challenge in the development and optimization of a tau-selective imaging agent is the structure of these aggregates, which adopts a cross-beta-sheet of interdigitating monomers. Although multiple scaffold classes have been reported to bind cross-ß-sheet structure, their mechanism of binding and their ability to selectively bind different aggregates of varying protein composition are not well understood. There are no crystal or NMR structures that would reveal the atomic-level binding modes of this interaction. Most small molecule development studies focus on iterative structure-activity relationship modifications and testing of known scaffolds, such as the benzothiazole dye and commonly used tissue staining agent Thioflavin T. Even though quantitative structure activity relationship studies have been employed to investigate amyloid-binding compounds, these retrospective studies have not elucidated any novel compound molecular properties that could explain the mechanism of interaction. Moreover, these studies have not rationalized the binding activity or selectivity of cross (open full item for complete abstract)

Committee: Jeffrey Kuret PhD (Advisor); Christopher Hadad PhD (Committee Member); Pui-Kai Li PhD (Committee Member); Michael Tweedle PhD (Committee Member) Subjects: Biochemistry; Biology; Biophysics; Medical Imaging; Molecular Biology; Molecular Chemistry; Molecules; Neurobiology; Neurosciences; Physical Chemistry

Doctor of Philosophy, The Ohio State University, 2007, Neuroscience

Alzheimer's disease and related tauopathies are characterized by the loss of neurons occurring in parallel with the formation of filamentous lesions composed of the tau protein. Though macroscopic analysis has provided insights into the development of tau lesions, the mechanism by which filaments assemble has remained unknown. The first stage in clarifying the reaction pathway was to characterize the partially folded intermediate species and determine conditions which promote intermediate stabilization. Data indicate that intermediate species contain increased secondary and tertiary structure. Several planar aromatic dyes including Congo red, thiazin red, thioflavin S known to bind β-structure, were capable of triggering filament formation. They were also capable of dramatically reducing critical concentration and nucleation rate relative to other inducers. In the presence of β sheet binding dyes assembly time courses were sigmoidal and reached plateau within 8 hours. Reaction kietics were utilized to estimate nucleus cluster size as well as several of the rate contstants governing the reaction. These estimates along with data from polymerization time courses were used to create a mathematical model of the tau polymerization reaction. These methods were further utilized to examine the effects of alternative splicing on tau polymerization. The effects of individual exons on both nucleation and elongation rates were determined through analysis of reaction kinetics. Finally, the effects of a previously described inhibitor of tau polymerization, N744 were examined over a wide concentration range. Results revealed a biphasic effect with inhibitory activity at low dye concentrations followed first by relief of inhibition, and then enhancement. Greater than 50% inhibition was seen over only a narrow concentration range. Changes in activity parallel changes in dye aggregation state, with dimers predominant under inhibitory concentrations. In the presence of tau protein the (open full item for complete abstract)

Committee: Jeff Kuret (Advisor); Jiyan Ma (Other); Andrej Rotter (Other); Dale Vandre (Other) Subjects:

Doctor of Philosophy, Case Western Reserve University, 2006, Chemistry

Many neurological diseases are linked to the aberrant folding of proteins and the production of amyloid fibrils. The misfolding and aggregation of the protein alpha-synuclein into Lewy bodies is thought to play a key role in the pathogenesis of Parkinson's disease. Despite extensive studies on this protein, very little is known about the structures and molecular mechanisms associated with the aggregation events. This work unravels detailed structural data about the aggregation pathways of alpha-synuclein, using an array of spectroscopic techniques, including circular dichroism, Raman spectroscopy, atomic force microscopy, nuclear magnetic resonance and thioflavin-T fluorescence. Previous studies established that alpha-synuclein is predominantly unfolded in aqueous solution at neutral pH. My work revealed that under these conditions, alpha-synuclein adopts an ensemble of secondary structures (extended, alpha-helical and beta-sheet). This interpretation was based on the analysis of the Raman amide I band of the monomeric alpha-synuclein. The morphology and the secondary structure of the soluble protein aggregates (spheroidal and chainlike oligomers) observed as probable intermediates that form during alpha-synuclein fibrillization, were characterized by Raman and atomic force microscopies. During the formation of alpha-synuclein oligomers under physiological conditions or in methanol-water solutions, a significant change in the ensemble of structures present took place; beta-sheet structure was enhanced and the extended structure was diminished. Importantly, these aggregates retained a significant amount of alpha-helical secondary structure. Moderate (15 - 20%) concentrations of methanol encouraged alpha-synuclein aggregation into chainlike oligomers that were indistinguishable from those formed in the absence of methanol. In many sporadic neurodegenerative disorders, co-occurrence of alpha-synuclein and tau proteins as amyloid-like fibrils occurs, and it is thought t (open full item for complete abstract)

Committee: Michael Zagorski (Advisor) Subjects: Biophysics, Medical

Doctor of Philosophy, The Ohio State University, 2013, Biophysics

The aggregation of tau protein in the brain is a defining pathological characteristic of several diseases, known as tauopathies, the most common of which is Alzheimer's disease. Because the temporal and spatial progression of tau lesions correlates with neurodegeneration and cognitive decline, they are an attractive target for diagnostic and therapeutic strategies. Whole-brain imaging is a promising strategy for pre-mortem detection of tau lesions, but the approach is complicated by the high concentrations of potentially confounding binding sites presented by beta-amyloid plaques. Inhibition of tau aggregation using small molecules is a promising strategy for the treatment of tauopathies, but the field lacks an understanding of the inhibitory mechanism of these molecules. Towards the diagnosis of Alzheimer's disease, a nonlinear, four-tissue-compartmental pharmacokinetic model of diffusion-mediated radiotracer uptake and distribution was developed. This model was used to predict the contributions of relative binding affinity and binding site density to the imaging dynamics and selectivity of a hypothetical tau-directed radiotracer. Initial estimates of nonspecific binding and brain uptake parameters were made by fitting data from a previously published kinetic study of Pittsburgh Compound B, an established amyloid-directed radiotracer. The resulting estimates were then used to guide simulations of tau binding selectivity while assuming early-stage accumulation of disease pathology. The simulations suggest that for tau aggregates to represent at least 80% of specific binding signal, binding affinity or binding site density selectivities for tau over beta-amyloid should be at least 20- or 50- fold, respectively. The simulations also suggest, however, that overcoming nonspecific binding will be an additional challenge for tau-directed imaging agents owing to low concentrations of available binding sites. Overall, nonlinear modeling can provide insight into t (open full item for complete abstract)

Committee: Jeff Kuret Ph.D. (Advisor); Gunjan Agarwal Ph.D. (Committee Member); Richard Swenson Ph.D. (Committee Member); Michael Tweedle Ph.D. (Committee Member) Subjects: Biophysics