Generation of new neurons in the aging adult brain: identifying mechanisms of adult neurogenesis by exploiting single cell spatial transcriptomics. - Project Summary: Discovery of glia-to-neuron identity conversion has opened the door for generation of new neurons to replace those lost to injury, aging or neurodegenerative disease including Huntington’s disease (HD) and Frontotemporal dementia (FTD). To this, I have used a therapeutically viable approach to successfully generate new neurons in the neurogenic niches of the aged adult mouse brain by transiently suppressing the RNA binding protein Polypyrimidine Tract Binding Protein-1 (PTBP1) using an antisense oligonucleotide (ASO) delivered by a single injection into cerebral spinal fluid. I further identified that Radial glial-like and subependymal-like cells (not astrocytes) convert into new neurons over a two-month period, acquire mature neuronal character, and functionally integrate into endogenous circuits that modify mouse behavior. Not yet established are the molecular events underlying glia-into-neuron identity conversion, including identification of the initiating glial cell(s), the events driving its conversion and subsequent maturation into a functional neuron, and application to HD and FTD. Additionally, the challenges pertaining to astrocyte-into-neuron conversion upon PTBP1 reduction have yet to be resolved. In this proposal, I will utilize combination of traditional single nuclear sequencing and immunofluorescence with a transformative single cell spatial transcriptomics technology, termed Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), to define the pathway(s) of generation of new neurons in aged neurogenic niches following a therapeutically viable injection to produce transient, ASO-mediated suppression of synthesis and accumulation of PTBP1. I will then extend the effort to test in mouse models of Frontotemporal dementia ( FTD) and Huntington’s disease (HD) whether glia-to- neuron conversion can generate functional replacement neurons. By leveraging my expertise with a network of collaborators I have assembled at UCSD and the surrounding research communities in San Diego, as well as strong collaboration with field leaders abroad, I will systematically identify the functionality, localization, cell origin and molecular pathways of glial cells undergoing identity conversion at multiple time points post conversion in healthy, FTD and HD contexts. During the mentored K99 phase, I will receive training under the guidance of Prof. Don Cleveland, who has trained more than 65 postdoctoral fellows, including 42 who at the end of their training obtained faculty positions. I have also assembled an outstanding team of collaborators including MERFISH pioneer Dr. Bogdan Bintu (UCSD), the Chief Scientific Officer of Ionis Pharmaceuticals Dr. C. Frank Bennett, neurogenesis expert Dr. Alysson Muotri (UCSD), and single nuclear sequencing expert Dr. Xin Jin (Scripps Research Institute) to assist my proposed research and provide me with additional scientific training and career support before, during, and after transitioning to an independent tenure-track faculty position. In the R00 phase, I will begin my long-term career goal of establishing a research program to understand adult neurogenesis and use mechanistic insights in reprograming for therapy development.
