Background: Many tuberous sclerosis patients develop subependymal nodules (SENs) -- small benign tumors that present in and around the V-SVZ, the largest stem cell niche in the pediatric and adult brain. Strikingly, although nodules may appear throughout this region and are mostly asymptomatic, larger, potentially lethal subependymal giant cell astrocytomas (SEGAs) preferentially present near the ventral V-SVZ and the foramen of Monro in both patients and preclinical models. It has been suggested that SEGAs arise ventrally due to the unique anatomical features or microenvironment of this area; however, this hypothesis has not been experimentally tested. It has recently been shown that V-SVZ neural progenitor cells are not homogeneous in their potential: they are organized into distinct subgroups that respond differently to developmental signals even when cultured or transplanted. My laboratory specializes in the precise targeting and single cell-level analysis of neural stem cell subpopulations in the neonatal mouse brain. I have previously identified developmental signaling pathways that are specifically activated in ventral stem cells. During the past 2 years, we have developed a model of SEN and SEGA development that dissects the contribution of dorsal and ventral progenitors to tumors. Our preliminary data suggest that ventral V-SVZ cells are the primary cell of origin for SEGAs. Concomitant with these studies, we have developed assays to measure per-cell activation of PI3K/Akt/mTOR signaling using phospho-specific flow cytometry. We have found that ventral neural progenitors exhibit higher levels of per-cell mTOR pathway activity even without genetic manipulation of Tsc1/2, and that this pattern persists (1) in Tsc2-heterozygous cells and (2) after exposure to rapamycin and other mTOR inhibitors. Therefore, the central hypothesis is that activating developmental signaling pathways driving a dorsal positional identity in ventral cells will exploit intrinsic developmental programming to inhibit SEGA growth. Our accompanying objective is to use the assays we have developed to identify novel SEGA-selective candidate therapeutics.Specific Aim 1: Test the ability of known dorsal-specific factors to reprogram ventral progenitors. We will use cultured neural progenitors derived from dorsal and ventral V-SVZ to test a series of drug-like molecules generated by our collaborators at Vanderbilt for their ability to reprogram the positional identity of ventral progenitors. We will use phospho-specific flow cytometry and confocal microscopy to measure the effects of these compounds on per-cell mTOR pathway activity, proliferation, apoptosis, the expression of location-specific transcripts, and the generation of dorsally or ventrally derived interneurons. If the intrinsic properties of ventral stem cells make them uniquely susceptible to "dorsalizing" factors, this would suggest a novel avenue for targeting SEGAs without affecting the entire germinal zone.Specific Aim 2: Identify novel compounds that uniquely inhibit proliferation, survival, or positional identity in ventral progenitors. Using the resources available through the Vanderbilt High-Throughput Screening shared resource, we will automate our microscopy and flow cytometry-based assays to test (1) curated libraries of compounds with a history of use in clinical trials or preclinical activity and (2) an unbiased library of compounds with a variety of biological activities. We will then identify candidate molecules that are preferentially cytostatic, cytotoxic, or "dorsalizing" in ventral neural progenitors. Promising candidates could then be tested in our mouse models of periventricular tumor development or patient-derived induced pluripotent stem cells.Innovation: The proposed studies are both conceptually and technically innovative. First, the idea that neural stem cell positional identity can be switched given the appropriate cues was only recently demonstrated, and to our knowledge, has not yet been applied to tuberous sclerosis complex (TSC) biology. Second, we have successfully developed a novel analytical technique, intracellular phospho-specific flow cytometry, to cultured neural progenitor cells and acutely dissociated V-SVZ tissue, and used this method to demonstrate differing levels of mTOR pathway activity in distinct, SEGA-relevant, subregions. Together with our Qualified Collaborator, Dr. Jonathan Irish, we are uniquely positioned to complete these studies.Impact: A critical barrier to research and treatment in TSC is that the true cell of origin and mechanism of ventral SEGA development remain to be elucidated. We hypothesize that ventral stem cells drive SEGA growth. Completion of the proposed studies will test whether altering a stem cell's positional identity is sufficient to alter the response of that cell to known inhibitors of the mTOR pathway, and will identify novel candidate therapeutics that may be used to block, slow, or reverse the growth of SEGAs -- a major area of concern for TSC patients.
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