PROJECT SUMMARY Zellweger spectrum disorders (ZSDs), which affect 1:50,000 individuals in the U.S., are characterized by dysfunction in any one of 13 peroxisomal biogenesis proteins (known as peroxins) and result in severe neurological phenotypes including seizures, developmental delay, and abnormal white matter growth in the brain. Life expectancy for patients rarely surpasses a few years. The peroxisomal biogenesis factor 11 beta (PEX11b), which is essential for peroxisomal fission, is among the peroxins mutated in ZSDs. However, the exact mechanisms by which peroxisomal morphology may contribute to disease pathophysiology remain unclear. This proposal aims to directly manipulate peroxisomal morphology during neurodevelopment by genetically deleting PEX11b and characterizing how PEX11b deficiency affects peroxisomal morphology, peroxisome-mediated metabolic functions, and early human neurogenesis. We aim to approach these questions by using PEX11b knockout (KO) human induced pluripotent stem cells (hiPSCs), which I have generated using CRISPR/Cas9. We will first assess the effects of PEX11b deficiency on peroxisomal morphology using super-resolution microscopy and rigorous, automated analysis of peroxisomal fission events, and peroxisomal length and volume from acquired images. To evaluate neurodevelopmental effects of knocking out PEX11b, we will evaluate self- renewal and multipotent differentiation potential in hiPSC-derived neural progenitor cells, as well as self-renewal of the neural progenitor pool and cortical layer formation in hiPSC-derived forebrain organoids. To assess the metabolic consequences of knocking out PEX11b, we will leverage metabolomics and imaging-mass spectrometry, among other cutting-edge metabolic profiling techniques, to evaluate whether downregulation of PEX11b alters cellular levels of long-chain, branched-chain, and very-long-chain-fatty acids. Successful completion of these aims would help establish the contributions of peroxisomal morphology during neurodevelopment. Additionally, it would help determine whether disruption of peroxisomal dynamics underlies ZSDs.