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

Dynamin superfamily proteins (DSPs) are present in all organisms, mediating critical membrane remodeling events throughout the cell. Despite the decades of structural and functional studies across the superfamily, no structure has been determined of a DSP demonstrating the conformational changes required to transition from a cytosolic, solution state to a helical assembly. This gap in knowledge limits the field's understanding of the mechanisms required for regulation and functional assembly into membrane remodeling complexes. Dynamin-related protein 1 (Drp1) is the master regulator of outer membrane fission for mitochondria. Proper mitochondrial dynamics is essential for cellular health, and an imbalance in this cycle has been implicated in many diseases, from heart failure to prion-related neurodegeneration. Drp1 has been identified as a potential therapeutic target; however, the underlying mechanisms governing its regulation are largely unclear. Drp1 exists predominantly as a mixture of dimers and tetramers in solution, but the specific interactions that stabilize these solution forms and prevent the assembly of larger complexes are not known. Using cryo-EM, we have observed significant conformational rearrangements in native solution structures for both dimer and tetramer states when compared to existing DSP crystal structures. Additionally, we have identified a helical lattice which demonstrates unique GTPase domain assembly between adjacent filaments, providing insight into the mechanisms of constriction. Finally, we studied the impact of terminal tags on the structure and function of Drp1 and identified a newly appreciated intrinsically disordered region of regulation. Together, these observations provide insight into regulatory interactions that stabilize oligomer states and mediate the activation of assembly into a functional fission machinery.

Committee: Jason Mears (Advisor); Edward Yu (Committee Chair); Marcin Golczak (Committee Member); Rajesh Ramachandran (Committee Member); Beata Jastrzebska (Committee Member) Subjects: Biology; Biophysics; Molecular Biology; Pharmacology

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

Mitochondria form dynamic networks and need to maintain a delicate balance between fission and fusion to satisfy the cell's energetic and metabolic requirements. Fission is necessary to ensure mitochondria are properly distributed throughout the cell and to remove damaged mitochondrial components. The dysregulation of mitochondrial dynamics resulting in either an abnormally fragmented or interconnected mitochondrial network is associated with a variety of pathologies. Mutations in dynamin-related protein 1 (Drp1), the master regulator of mitochondrial fission, have been identified in patients presenting with severe neurological defects. Patient-derived fibroblasts exhibit hyperfused mitochondria, indicating mitochondrial dysregulation. Thus, these clinically relevant mutations impair Drp1 function, but the mechanism by which these mutations disrupt mitochondrial fission was undetermined. To address this lack of knowledge, the overarching objective of this research was to elucidate the specific Drp1 functional defects that are caused by these disease-associated mutations to better understand the relationship between impaired Drp1 function and disease. Drp1 self-assembles around the outer mitochondrial membrane (OMM) and, subsequently, hydrolyzes GTP which provides the mechanical force required to cleave apart the mitochondrion. The recruitment of Drp1 to the OMM is mediated in part by lipid interactions. A mitochondria-specific lipid, cardiolipin, promotes Drp1 self-assembly, enhances its GTPase activity, and is believed to facilitate membrane constriction. Disease-associated mutations in Drp1 are predominantly located within the GTPase and middle domains, which mediate its capabilities for hydrolysis and self-assembly, respectively. We have employed an ensemble of biochemical and EM-based techniques to investigate the impact of these mutations on the self-assembly, lipid recognition, and enzymatic capabilities of Drp1. Ultimately, we have shown that even mutatio (open full item for complete abstract)

Committee: Jason Mears (Advisor); Marvin Nieman (Committee Chair); Phoebe Stewart (Committee Member); Danny Manor (Committee Member); Edward Yu (Committee Member) Subjects: Biochemistry; Biomedical Research; Biophysics; Molecular Biology

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, Case Western Reserve University, 2022, Pathology

Mitochondrial dynamics, the fission and fusion of mitochondria, are vital to cellular health and homeostasis. Altered dynamics are associated with a number of diseases, one of which is the most aggressive and prevalent brain cancer, glioblastoma (GBM). Once diagnosed, patients live a median of less than 15 months with nearly all experiencing tumor recurrence, highlighting the need for therapeutic advancement. Also, increased mitochondrial fragmentation is often observed within the GBM stem cells (GSCs), known to be resistant to conventional therapies and believed to contribute to tumor recurrence. The mitochondrial fission machinery, dynamin-related protein-1 (Drp1) and mitochondrial fission factor (Mff), have both been implicated in contributing to the GBM disease state. To understand the altered regulation of these proteins in GBM, we investigate the post-translation modifications, O-GlcNAcylation and phosphorylation on them, respectively. We found that Drp1 O-GlcNAcylation and Mff phosphorylation were both increased in patient GSCs. Specifically, elevated O-GlcNAcylation of Drp1 correlated with increased mitochondrial fragmentation and mass, as well as altered mitochondrial ETC complex function. We also found that adenosine monophosphate (AMP)-activated protein kinase (AMPK) was the primary kinase phosphorylating Mff in one, but not all, of the GSC patient samples examined, demonstrating the importance of individual patient tumor profiling. Altogether, these studies demonstrate the importance of proper PTM regulation of Drp1and Mff in GBM, which may serve as future therapeutic points of intervention.

Committee: Jason Mears (Advisor); Brian Cobb (Committee Chair); Clive Hamlin (Committee Member); Justin Lathia (Committee Member); Ruth Keri (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Cellular Biology; Molecular Biology; Morphology; Science Education

Doctor of Philosophy, Case Western Reserve University, 2018, Pharmacology

The complex processes of mitochondrial fission and fusion are opposing functions whose proper balance ensures optimal mitochondrial function in eukaryotic cells. Aberrant mitochondrial morphology where one of these mitochondria-shaping processes dominates the other are commonly found in diverse pathologies, highlighting the importance of maintaining appropriate rates of both fission and fusion. Both of these competing processes are mediated by members of the dynamin superfamily of membrane-remodeling GTPases. Scission of the mitochondrial membranes is carried out by an ancient member of this protein superfamily called dynamin-related protein 1 (Drp1). While its function is required for fission, Drp1 alone is unable to mediate this complex process, and requires interaction with one or more partner proteins of the mitochondrial outer membrane to ensure fission. Chordates express several such proteins whose genetic interaction with Drp1 has been proven to be crucial for maintenance of 2 appropriate mitochondrial morphology. These include mitochondrial fission protein 1 (Fis1), mitochondrial fission factor (Mff), and mitochondrial dynamics proteins of 49 and 51 kilodaltons (MiD 49/51). Of these proteins, the first that was proposed to contribute significantly to the maintenance of mitochondrial morphology in man was Mff. Due to its relatively recent discovery, its specific role(s) in this function remain unclear. To address this lack of knowledge, the primary objective of these studies was to better understand the various factors that control the association of Drp1 and Mff, and to shed light on the regulatory mechanisms that underlie this interaction. We have shown that the interaction between Drp1 and Mff is mediated by the stalk of Drp1, and not the variable domain (VD) as was previously thought. We also demonstrated the utility of mitochondrial outer membrane-like scaffolding liposomes as a template for studying the interaction between Drp1 and its various membran (open full item for complete abstract)

Committee: Jason Mears (Advisor); Philip Kiser (Committee Chair); Rajesh Ramachandran (Committee Member); Derek Taylor (Committee Member); Edward Yu (Committee Member) Subjects: Biochemistry; Pharmacology

Doctor of Philosophy, Case Western Reserve University, 2017, Pharmacology

Mitochondria are dynamic organelles that continually undergo cycles of fission and fusion. Maintaining this fission/fusion balance is extremely important in sustaining cellular health. Mitochondrial fission serves to separate daughter mitochondria during division, segregate damaged mitochondria for autophagy and initiate apoptosis during disease conditions. In fact, in neurodegenerative, heart and liver disease, increased mitochondrial fission is observed, leading to cell death. The main mediator of mitochondrial fission is Drp1, a large GTPase of the dynamin superfamily. Drp1 can form oligomers capable of remodeling mitochondrial membranes, and this can lead to membrane partitioning. However, the mechanism of this process is not well understood. This work aimed to investigate the role of Drp1 in membrane constriction, the function of the uncharacterized variable domain (VD) of Drp1 and the structure of Drp1 on lipid membranes mimicking mitochondrial interactions. We assembled Drp1 on negatively charged membranes and induced constriction upon the addition of GTP. Moreover, we found that only GTP hydrolysis induces full constriction of the Drp1 lipid oligomer. Furthermore, the unstructured VD was found to keep Drp1 in a more active cytosolic conformation, as VD removal induced a hyperoligomeric state. We also found that alternatively spliced sequence insertion in the VD similarly reduces enzyme activity and narrows the diameters of Drp1-lipid polymers. In order to further investigate the function of these oligomers, we solved the 3D structure of Drp1 complexed on distinct lipid templates. Interestingly, we discovered that the mitochondrial specific lipid cardiolipin directly interacts with the VD and induces an activating conformational change to the rest of the Drp1 molecule. The helical assembly of Drp1 on CL templates is mediated exclusively through stalk and GTPase domain interaction sites. The VD has previously been termed a domain of unkn (open full item for complete abstract)

Committee: Jason Mears (Advisor) Subjects: Biochemistry; Pharmacology

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