Tommy L. Lewis Jr, Ph.D. - US grants

DESCRIPTION (provided by applicant): The development and maintenance of polarity is paramount to neuronal development and function. The loss of this cell state is realized in many neurodegenerative diseases and ultimately leads to cell death. This project will lead to a greater understanding of the mechanisms responsible for polarization and its maintenance. The goal of this project is to characterize the contribution that the recently discovered LKB1 kinase pathway plays in axon growth and branching through polarized transport of cargo. Specific Aim 1 will provide the necessary framework for understanding how LKB1 and NUAK1/2 affect the transport of mitochondria in the axon. Specific Aim 2 will characterize the mechanism by which transport is regulated by determining the downstream components of the kinase pathway. The interaction between LKB1/NUAK and the mitochondrial anchor protein syntaphilin will be the main focus of this aim. Specific Aim 3 will then attempt to link mitochondrial transport in the axon to proper axon branching and projection within layer 2/3 neurons of the cortex. This research will provide important new knowledge that will be useful in understanding the basic mechanisms at work in many neurodegenerative diseases including Alzheimer's, Huntington's and Parkinson's. PUBLIC HEALTH RELEVANCE: Neuron polarization is required for proper neuron function. Disruption of polarization and the mechanisms that underlie its maintenance are known to be major factors in many neurodegenerative diseases including ALS, Alzheimer's, Huntington's and Parkinson's.

Project Summary Mitochondria regulate a number of critical cellular pathways including energy homeostasis, calcium handling and lipid production. In a number of cell types, distinct populations of mitochondria are created and maintained within subcellular compartments driving unique responses to physiological challenges in different regions of the cell. While many of the molecular players that modulate mitochondrial shape, and therefore function, have been identified, complete understanding of their functions and interactions in establishing these subpopulations of mitochondria within cells remain difficult to define. The deficit in understanding subcellular mitochondrial shape and function is largely due to a limited ability to visualize, and manipulate, these dynamic organelles in a truly physiological environment at high spatial and temporal resolution. Our approaches are designed to address these gaps in knowledge by leveraging newly developed technologies enabling genetic labelling and manipulation, across multiple cell types, with high spatial and temporal imaging of mitochondrial morphology, dynamics and function in vivo. In project one, members of the laboratory will target the four known mammalian receptors (MFF, FIS1, MIEF1/2) of the dynamin-like protein one (DRP1), the main effector of mitochondrial fission, to test their roles in the creation and maintenance of different mitochondrial subpopulations in cortical neurons and skeletal myocytes in vivo. Through the use of loss of function experiments, CRISPR/Cas labeling and targeting-motif analysis coupled with high resolution imaging we will map the molecular mechanisms regulating subcellular mitochondrial fission dynamics across multiple mitochondrial subpopulations. In project two, members of the laboratory will implement methods for sparse, bright labeling of cortical neuron and skeletal myocyte mitochondria with fluorescent reporters for adenosine triphosphate, calcium, pH and reactive oxygen species, and couple it with 2-photon imaging in living mice to reveal how these mitochondrial subpopulations inform mitochondrial and cellular function in vivo. By manipulating different subpopulations and visualizing the effects on mitochondrial and cellular function in multiple cell types in vivo, we will provide a uniquely integrated approach to understanding the universal and cell-specific roles of mitochondrial subpopulations found within cells.