Cytoplasmic dynein and one of its regulators, LIS1, are essential for the proliferation of neural progenitor (stem) cells, generation of neural and glial precursor cells, and their subsequent migration to destinations within the brain. Nuclei within the neural progenitor cells undergo a very unique oscillatory movement which is critical to their growth and proliferation. Interestingly, knockdown of cytoplasmic dynein or LIS1 completely blocks the oscillatory movement of these nuclei. Results from our laboratory indicate the possibility that cytoplasmic dynein associated with the nuclear envelope in neural progenitor cells may be responsible for nuclear oscillations, suggesting the exciting possibility that the nucleus behaves as a dynein cargo. A primary focus of this proposal will be to test whether dynein, indeed, functions in this capacity and to identify mechanisms by which dynein is recruited to the nuclear envelope. The focus of this application is, therefore, to identify proteins that may be involved in recruiting dynein to the nuclear envelope in neural progenitor cells as well as to study their role in this process by RNAi, live imaging, and localization analysis in neural progenitor cells. Many dynein adaptor proteins have already been identified in non-neuronal cells, and these will be tested in embryonic rat brains for recruitment of dynein to the nuclear envelope. Preliminary evidence suggests that depletion of the candidate adaptor proteins acutely affects neuronal distribution, but their exact function is unclear. The first specific aim will analyze the affect of knockdown of these proteins in nuclear oscillations by extensive live imaging of brains electroporated with RNAi constructs against each protein. The second specific aim will support the first by identifying the localization pattern of these proteins within the neural progenitor cells by immunofluoresence. The direction of oscillatory movement is tightly correlated with the cell cycle stage of the nucleus, and is likely regulated by the cell cycle. The localization of these proteins will be correlated with the cell cycle stage by use of cell cycle markers to identify how the cell cycle might regulate dynein attachment to the nuclear envelope. In the long run, this project aims to identify new genes that are involved in brain development. Preliminary results already indicate an obvious role for these proteins in neuronal redistribution. This project will also generate a wealth of data on the other roles of these proteins in the embryonic brain, and shed light on the poorly understood regulation of nuclear oscillations by the cell cycle, which has the potential of a long-term project. The neural progenitor cells studied in this proposal are immensely important as till date, they are the only known brain stem cells to persist into adulthood. Understanding their proliferation and differentiation is therefore critical to developing technology for regeneration of nerves following nerve injury, often caused by strokes. Additionally, these new genes will provide tools for understanding and manipulating neural stem cell behavior, an issue of critical importance for the production, preservation, and therapeutic use of stem cells.
The neural progenitor cells studied in this proposal are the only known brain stem cells to persist into adulthood. Understanding their proliferation and differentiation is therefore critical to developing technology for regeneration of nerves following nerve injury, often caused by strokes.
