Single Nucleus Rna Sequencing
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Characterizing adipocyte heterogeneity in response to metabolic stress
Project Summary Adipose tissue is a central player in metabolism, storing energy healthily under normal conditions but becoming dysfunctional when overloaded. This can lead to the development of metabolic disease, most notably insulin resistance and type 2 diabetes (T2D). Understanding the contribution of adipose tissue to these complications requires knowledge of the individual cell types within adipose tissue and how they respond to different metabolic conditions. My previous work used single nucleus RNA sequencing to profile the cell types in adipose tissue and identified a number of subpopulations of white adipocytes that are differentially associated with clinical characteristics such as body mass index. In this grant, I now aim to better understand how a diverse array of stimuli influences adipocyte development and specification, the role that intra-individual variation plays in the response to these stimuli, and a better understanding of the relationship of adipocyte state to the development of metabolic disease. To do this, I propose using a model in which I can study human adipocyte development and function in mice to perform experiments such as high fat diet and cold exposure that are well-characterized in mice but not in humans. By performing experiments using cells from humans with a range of starting clinical characteristics, I can determine what changes will happen in response to a stimuli in all individuals verses those that only occur in specific populations. The experience that I have in characterizing adipocytes and adipose tissue both at the bench and computationally make me uniquely positioned to answer these questions. Taken together, these studies can test the behavior of adipocyte subpopulations from different people and under different conditions, ultimately leading to a better understanding of how subpopulations develop and, eventually, how we can target these populations to treat metabolic disease.
Pathogenic mechanisms of expanded ZFHX3 in SCA4 cerebellar organoids
Spinocerebellar ataxia type 4 (SCA4) is a disabling neurodegenerative disease characterized by progressive cerebellar ataxia, and the causative GGC-repeat expansion in ZFHX3 (ZHFX3-exp) was just discovered this year by our lab and others. Our research aims to understand how ZFHX3-exp causes SCA4 and to identify molecular therapeutic targets that can be quickly advanced into clinical trials. SCA4 is one of the four poly-glycine diseases that share the presence of neuronal intranuclear inclusion (NIIs) as a disease hallmark. In SCA4, NIIs are positive for ZFHX3, p62 and ubiquitin, indicating the loss of proteostasis as a mechanism of neurodegeneration. In addition, ZFHX3 RNA-gain-of-function may also contribute to neurodegeneration. Beyond this, knowledge of the disease mechanisms that underly SCA4 is extremely limited and there are currently no disease-modifying treatments for SCA4 or other polyG/NII diseases. There are no SCA4 mouse models and because of the high GC content in the repeat expansion complicates the production of SCA4 mouse models. We propose a novel approach to characterizing SCA4 Purkinje cell (PC) pathogenesis using human cerebellar organoids. Our approach allows for rapidly advancing the understanding of the pathogenesis and potential treatments of SCA4. Using cerebellar organoids will enable investigation on functional PCs, cerebellar neurodegeneration and the testing of potential therapeutic strategies. In aim 1, we will generate cerebellar organoids from five SCA4 patient-derived iPSC lines, and normal control iPSCs from individuals of the same family. These iPSC lines are already established in our laboratory. In aim 2, we will investigate PC viability, NII protein composition, proteostasis pathways, RNA gain-of-function and cell-type-specific dysregulated pathways by single nucleus RNA sequencing. In addition, we will study potential therapeutic targets by lentiviral knockdown and single nucleus RNA sequencing. SCA4 patient iPSCs express overabundant STAU1 and ATXN2. We will evaluate how lowering the abundance of these proteins modifies the PC molecular phenotype. Together, these experiments will establish a model to greatly enhance the understanding of human PC neurodegeneration, the pathological mechanisms of SCA4 and possible avenues of treatment.
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