✍🏼 Guang Lin, Burak Tepe, Geoff McGrane, Regine C Tipon, Gist Croft, Leena Panwala, Amanda Hope, Agnes JH Liang, Zhongyuan Zuo, Seul Kee Byeon, Lily Wang, Akhilesh Pandey, Hugo J Bellen
🏠 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX
📑 eLife (2023)
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Abstract
Infantile neuroaxonal dystrophy (INAD) is caused by recessive variants in PLA2G6 and is a lethal pediatric neurodegenerative disorder. Loss of the Drosophila homolog of PLA2G6, leads to ceramide accumulation, lysosome expansion, and mitochondrial defects. Here, we report that retromer function, ceramide metabolism, the endolysosomal pathway, and mitochondrial morphology are affected in INAD patient-derived neurons. We show that in INAD mouse models, the same features are affected in Purkinje cells, arguing that the neuropathological mechanisms are evolutionary conserved and that these features can be used as biomarkers. We tested 20 drugs that target these pathways and found that Ambroxol, Desipramine, Azoramide, and Genistein alleviate neurodegenerative phenotypes in INAD flies and INAD patient-derived neural progenitor cells. We also develop an AAV-based gene therapy approach that delays neurodegeneration and prolongs lifespan in an INAD mouse model.
How the WOLF was used in this study
The authors used the WOLF Cell Sorter as part of their CRISPR/Cas9 genome editing workflow to isolate individual induced pluripotent stem cells (iPSCs) after transfection. Following CRISPR-mediated editing of the PLA2G6 gene in patient-derived iPSCs, they single-cell sorted the transfected cells into 96-well plates using the WOLF sorter to obtain monoclonal populations. This gentle, low-pressure microfluidic sorting allowed efficient deposition of single cells with high viability and purity into individual wells, which were then expanded into colonies for genotyping and further analyses. The use of the WOLF sorter ensured reliable isolation of edited iPSC clones needed to generate isogenic cell lines for downstream characterization of disease phenotypes and therapeutic responses.
