✍🏼 Anna Podlesny-Drabiniok, Gloriia Novikova, Yiyuan Liu, Josefine Dunst, Rose Temizer, Chiara Giannarelli, Samuele Marro, Taras Kreslavsky, Edoardo Marcora, Alison Mary Goate
🏠 Department of Genetics and Genomic Sciences, New York, NY, USA; Icahn School of Medicine at Mount Sinai, New York, NY, USA
📑 Nature Communications (2024)
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Abstract
Genetic and experimental evidence suggests that Alzheimer’s disease (AD) risk alleles and genes may influence disease susceptibility by altering the transcriptional and cellular responses of macrophages, including microglia, to damage of lipid-rich tissues like the brain. Recently, sc/nRNA sequencing studies identified similar transcriptional activation states in subpopulations of macrophages in aging and degenerating brains and in other diseased lipid-rich tissues. We collectively refer to these subpopulations of microglia and peripheral macrophages as DLAMs. Using macrophage sc/nRNA-seq data from healthy and diseased human and mouse lipid-rich tissues, we reconstructed gene regulatory networks and identified 11 strong candidate transcriptional regulators of the DLAM response across species. Loss or reduction of two of these transcription factors, BHLHE40/41, in iPSC-derived microglia and human THP-1 macrophages as well as loss of Bhlhe40/41 in mouse microglia, resulted in increased expression of DLAM genes involved in cholesterol clearance and lysosomal processing, increased cholesterol efflux and storage, and increased lysosomal mass and degradative capacity. These findings provide targets for therapeutic modulation of macrophage/microglial function in AD and other disorders affecting lipid-rich tissues.
How the WOLF was used in this study
The WOLF cell sorter was used during the generation of CRISPR/Cas9-edited human induced pluripotent stem cell (iPSC) lines. After electroporation to introduce CRISPR/Cas9 components and single-stranded donor templates targeting BHLHE40 and/or BHLHE41, individual human iPSCs were plated and then sorted into 96-well plates using the WOLF benchtop microfluidic cell sorter to isolate single viable cells. This step ensured clonal expansion from single cells carrying the intended edits, facilitating the derivation of homozygous knockout lines that were subsequently screened and validated for downstream transcriptional and functional analyses.
