CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death

Abstract

Accumulation of aggregated and misfolded proteins, leading to endoplasmic reticulum stress and activation of the unfolded protein response, is a hallmark of several neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. Genetic screens are powerful tools that are proving invaluable in identifying novel modulators of disease associated processes. Here, we performed a loss-of-function genetic screen using a human druggable genome library, followed by an arrayed-screen validation, in human iPSC-derived cortical neurons. We identified and genetically validated 13 genes, whose knockout was neuroprotective against Tunicamycin, a glycoprotein synthesis inhibitor widely used to induce endoplasmic reticulum stress. We also demonstrated that pharmacological inhibition of KAT2B, a lysine acetyltransferase identified by our genetic screens, by L-Moses, attenuates Tunicamycin-mediated neuronal cell death and activation of CHOP, a key pro-apoptotic member of the unfolded protein response in both cortical and dopaminergic neurons. Follow-up transcriptional analysis suggested that L-Moses provided neuroprotection by partly reversing the transcriptional changes caused by Tunicamycin. Finally, L-Moses treatment attenuated total protein levels affected by Tunicamycin, without affecting their acetylation profile. In summary, using an unbiased approach, we identified KAT2B and its inhibitor, L-Moses, as potential therapeutic targets for neurodegenerative diseases.

HNRNPH1 regulates the neuroprotective cold‐shock protein RBM3 expression through poison exon exclusion

“…For the fluorescence‐activated cell sorting (FACS)‐based whole‐genome CRISPR/Cas9 knockout screen, a fluorescence RBM3 reporter iPSC line was generated by Cas9‐mediated homology‐directed repair to insert GFP at the N‐terminus of the single copy of RBM3 on chromosome X in wild‐type (WT) iPSCs, which contain a doxycycline (dox)‐inducible neurogenin 2 expression cassette (Pawlowski et al , 2017 ) and express Cas9 driven by the GAPDH promoter (Cas9 WT) (Pavlou et al , 2023 ) (Fig EV1A ). The iPSCs can be differentiated into excitatory cortical neurons after continuous dox treatment for over 4 days (Pawlowski et al , 2017 ).…”

“…Human iPSC culture NGN2-OPTi-OX was generated by the Kotter laboratory at the University of Cambridge (Pawlowski et al, 2017), where the original iPSC line was sourced from the University of Cambridge (https:// hpscreg.eu/cell-line/CAMi014-A). iPSCs with Neurogenin-2 (NGN2) transgene stably integrated into a "safe-harbour" locus under doxycycline (Dox)-inducible promoter (Pavlou et al, 2023) were maintained under feeder-free conditions in TeSR-E8 medium in a 37°C, 5% CO 2 tissue culture incubator. They were cultured on vitronectin (3.3 lg/ml)-coated culture plates or glass-bottom dishes and fed every day with TeSR-E8 medium or every 2 days with StemFlex Medium.…”

Genome-wide CRISPR/Cas9 screen shows that loss of GET4 increases mitochondria-endoplasmic reticulum contact sites and is neuroprotective

“…To address this issue, we used two independent ER-Mito reporter clones that generated comparable results. Induced pluripotent stem cell-derived cortical neurons have recently been used to conduct both pooled and arrayed screens [ 53 ], and it would have been informative to determine whether a screen using cortical neurons can confirm our results.…”

Genome-wide CRISPR/Cas9 screen shows that loss of GET4 increases mitochondria-endoplasmic reticulum contact sites and is neuroprotective.

“…To address this issue, we used two independent ER-Mito reporter clones that generated comparable results. Induced pluripotent stem cell-derived cortical neurons have recently been used to conduct both pooled and arrayed screens 52, and it would have been informative to determine whether a screen using cortical neurons can con rm our results.…”