Examining the Impact of Peroxisomal Fission on Cell Fate Decisions During Neurodevelopment

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.

Grant Number: 5F31HD114431-02
NIH Institute/Center: NIH
Principal Investigator: Caroline Bodnya