Alexander Kreymerman, PhD - US grants

DESCRIPTION (provided by applicant): The role of mitochondrial fission/fusion in CNS axon regeneration Central nervous system (CNS) disease or injury is often accompanied by progressive axon degeneration, leading to lost sensory, motor, or cognitive abilities, with little o no regenerative response. In search of signaling factors to restore degenerated CNS axons, we identified a group of developmentally regulated transcription factors, the Kruppel-like transcription factors (KLFs), which differentially suppress or enhance hippocampal, corticospinal neuron, and retinal ganglion cell (RGC) axon growth. However, the downstream mechanisms by which KLFs regulate axon growth are unknown. Evidence suggests one downstream effector may be mitochondrial (Mt) fission/fusion dynamics. We recently showed that suppressing fission (increasing fusion) leads to a loss in axon growth inhibition by chondroitin sulfate proteoglycans, supporting a hypothesis in which CNS axon growth and guidance is regulated by Mt fission-fusion dynamics. These data also suggest suppressing Mt fission is a potential therapeutic strategy for improving axon regeneration after CNS trauma or disease. To identify whether Mt fission/fusion mechanisms also underlie the axon suppressing/enhancing activity of KLFs, we are investigating the potential ability for KLFs to critically regulate Mt genes for axon growth. Pertinent to our previous findings, we found that axon growth-suppressing KLF9 increased and growth- promoting KLF7 decreased the genetic expression of mitochondrial fission process 1,18 kDa (MTP18), a positive regulator of Mt fission, supporting the hypothesis that increased fission is inhibitory for axon growth in CNS neurons. Furthermore, our recent data analyzing exome sequencing of familial axonopathies also pointed to a disease association with a number of mitochondrial proteins thought to act on fission/fusion dynamics, including MTP18. Therefore, we hypothesize that KLF7/9-mediated regulation of the mitochondrial fission enhancer MTP18 regulates intrinsic axon growth ability in CNS neurons. To address this hypothesis, we will express/knockdown MTP18 in combination with or independent of KLF7/9 expression/knockdown in RGCs both in vitro and in vivo, identifying the neuronal role of MTP18 in regulating Mt fission/fusion dynamics, CNS axon growth and guidance, and KLF7/9-mediated axon regeneration. The overall goal is to improve our understanding of how Mt fission/fusion regulates axon regeneration and identify strategies for restoring axon growth after CNS injury or disease.

The role of mitochondrial DNA mutations in chemotherapy induced cardiomyopathy. Doxorubicin(DOX), a broadly applied cancer therapeutic, is a significant contributor to irreversible cardiomyopathy. Evidence suggests that DOX treatment-induced cardiomyopathy is associated with elevated levels of mitochondrial DNA (mtDNA) mutations. Mitochondria (mt) are critical for cardiac myocyte physiology, and it is not surprising, therefore, that mtDNA mutations effect cardiac myocyte function and are associated with cardiomyopathy. However, it is unclear if or how the mtDNA mutation load contributes to cardiomyopathy. This question is confounded by the fact that disease presentation can be dependent on the heterogeneity of ?diseased? vs ?healthy? mitochondria in a given cell or cell population, known as mitochondrial heteroplasmy. Here, I will use patient genetics and iPSC-derived cardiomyocytes to resolve how mtDNA gene variants or mutations affect DOX-induced cardiomyopathy. The overarching goal of this proposal is to resolve whether specific mtDNA mutations and/or the proportion of mutated vs non-mutated mtDNA should be treated as a risk factor or interpreted as causative in the development of cardiomyopathy after DOX treatment. I hypothesize that mtDNA mutations are induced or selected for in patients treated for cancer and that mtDNA mutations and that their heteroplasmic load plays a causative role in the cardiomyopathy developed after cancer treatments such as DOX. We will test this hypothesis through patient-based association studies: 1) mining the 100,000 Genomes Project database to identify risk factors between cancer treatments, the development of cardiomyopathy, and the presence/heteroplasmic load of specific mtDNA mutations. In addition, I will perform functional studies using phenotypic and genetic high throughput screening approaches in iPSC-derived cardiomyocytes to determine 2) whether DOX induces de novo versus selects for pre-existing mtDNA mutations, and 3) whether induced or selected mt mutations directly cause myopathy. The results of this investigation should resolve whether DOX causes mutations or selects for pre-existing variants, and should help inform clinical decision making by answering the key question of ?Should patients that are heteroplasmic for certain mtDNA variants be monitored carefully after DOX treatment?? Moreover, it should lay the groundwork for my future research career, in which I would like to develop therapeutic strategies to potentially alter mtDNA heteroplasmy in patients at risk for developing cardiomyopathy after cancer treatment. Thus, it is important to identify the selection events in a patient?s life that could contribute to creating or increasing the presence of mtDNA mutations, especially since there are no current treatments for mt disease.