Organelle dynamics profoundly affect the physiology of the cell and are regulated by interactions with the cytoskeleton. Alterations in organelle dynamics (i.e., inter-organelle and cytoskeletal contacts, fission, and mobility) are associated with a variety of human diseases, particularly neuropathies. Charcot-Marie-Tooth (CMT) disease is the most commonly inherited neuropathy and is caused by mutations in at least eighty different genes. Although the mutations that cause CMT are often in genes linked to altered organelle dynamics, questions remain regarding the pathogenic mechanism. Mitochondrial fission is mediated by the polymerization of actin at ER-mitochondria contact sites via the ER-anchored, actin polymerizing protein INF2. Dominant activating mutations in INF2 cause increased mitochondrial fission and excessive actin accumulation on mitochondria, which reduces mitochondrial mobility. Similar mutations in INF2 also cause CMT. Preliminary data show that 1) actin accumulates at fission sites of other organelles including endosomes, lysosomes, peroxisomes, and the Golgi, and 2) that CMT mutations in INF2 cause a reduction in endosome and lysosome mobility. This leads to the central hypothesis of this proposal: there is a conserved molecular mechanism regulating organelle fission and mobility mediated by actin cytoskeletal proteins at ER-organelle contact sites. Furthermore, it is proposed that reduction in organelle mobility specifically affects peripheral neurons due to the extreme length of these cells and hence may be a general pathogenic feature of CMT.
The Specific Aims of this project are as follows:
Aim 1 focuses on the role of INF2 in mitochondrial, endosomal, and lysosomal fission and how these processes are altered by mutations in INF2 that cause CMT. These studies will be carried out in primary human fibroblasts via live-cell imaging, including the use of a novel, innovative probe that specifically labels ER-associated actin. Experiments to assess organelle functions and to directly implicate actin polymerization in the phenotypes observed will also be performed.
Aim 2 will be carried out in cultured primary mouse neurons in order to properly assess how alterations in organelle dynamics and mobility affect neuronal health. Deep learning-based image restoration will be used to achieve high spatiotemporal resolution imaging of organelle mobility.
Aim 3 will include organelle and neuronal health assays in the context of disease-relevant models of CMT. Specifically, neurons derived from CMT patient fibroblasts and neurons from mice injected with AAVs directing expression of CMT- mutant INF2, MFN2, or RAB7A will be analyzed. Completion of these aims will provide mechanistic insight into the role of actin in organelle fission and mobility, how these processes are coupled, and test the novel hypothesis that CMT involves global disruption of mobility of multiple organelles. This will further our understanding of the pathogenic mechanism of CMT and perhaps other neurodegenerative disorders. The project will also enhance my scientific training by providing me with invaluable training in neurobiology and neurodegeneration, designing novel imaging probes, advanced imaging techniques, stem-cell based reprogramming, and mouse models.
Alterations in organelles (e.g. mitochondria) are linked to various neurodegenerative diseases, such as Charcot- Marie-Tooth (CMT) disease; however, our understanding of how organelle dynamics are regulated is still not complete. While actin has a known role in ER-mediated mitochondrial fission through the activities of the actin- regulatory protein INF2, our preliminary results suggest a common mechanism at play in other organelles. This proposal focuses on ER- and actin-mediated organelle fission, how this process impacts organelle motility, and the consequences of CMT mutations that disrupt these processes in nerve cells in culture and in mice.
