Biology subjects

Roudnicky, F.

Publications and source records attributed to Roudnicky, F..

3 recordsLinked to original sources

bioRxiv2024

A CRISPR/Cas9 screen reveals proteins at the endosome-Golgi interface that modulate cellular ASO activity

Anti-sense oligonucleotides (ASOs) are modified synthetic single-stranded molecules with enhanced stability, activity, and bioavailability. They associate with RNA through sequence complementarity and can reduce or alter mRNA expression upon binding of splice site positions. To target RNA in the nucleus or cytoplasm, ASOs must cross membranes, a poorly understood process. We have performed an unbiased CRISPR/Cas9 knockout screen with a genetic splice reporter to identify genes that can increase or decrease ASOs activity, resulting in the most comprehensive catalog of ASOs-activity modifier genes. Distinct targets were uncovered, including AP1M1 and TBC1D23, linking ASOs activity to transport of cargo between the Golgi and endosomes. AP1M1 absence strongly increased ASO activity by delaying endosome-to-lysosome transport in vitro and in vivo. Prolonged ASOs residence time in the endosomal system may increase the likelihood of ASOs escape from this organelle before they reach lysosomes. This insight into AP1M1 role in ASOs trafficking suggests a way for enhancing the therapeutic efficacy of ASOs by manipulating the endolysosomal pathways.

cell biology↗

bioRxiv2024

An arrayed CRISPR/Cas9 screen identifies mTORC1 as a regulator of lipid droplet accumulation in APOE E3 and APOE KO iPSC-derived microglia

Variants of the Apolipoprotein E (APOE) gene, particularly the E4 allele, are significantly associated with an increased risk of Alzheimers Disease and have been implicated in neuroinflammatory processes due to disrupted lipid metabolism. Lipid alterations can manifest in glial cells as an excessive buildup of lipids, potentially contributing to neuroinflammation. In this study, we observed a heightened lipid load in APOE-deficient human induced pluripotent stem cell (iPSC)-derived microglia relative to cells with other APOE isoforms. To explore the mechanisms governing lipid handling within microglia, we established a technique for the nucleofection of CRISPR/Cas9 ribonucleoprotein complexes into iPSC-derived myeloid cells. Utilizing this method, we performed a targeted screen to identify key upstream modifiers in lipid droplet formation. Our findings highlight the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway as a pivotal influence on lipid storage in microglia with both APOE3 and APOE knockout genotypes, underscoring its role in lipid dysregulation associated with Alzheimers Disease and neuroinflammation.

neuroscience↗

bioRxiv2019

Decoding neuronal diversity by single-cell Convert-seq

The conversion of cell fates is controlled by hierarchical gene regulatory networks (GRNs) that induce remarkable alterations in cellular and transcriptome states. The identification of key regulators within these networks from myriad of candidate genes, however, poses a major research challenge. Here we present Convert-seq, combining single-cell RNA sequencing (scRNA-seq) and pooled ectopic gene expression with a new strategy to discriminate sequencing reads derived from exogenous and endogenous transcripts. We demonstrate Convert-seq by associating hundreds of single cells during reprogramming of human fibroblasts to induced neurons (iN) with exogenous and endogenous transcriptional signatures. Convert-seq identified GRNs modulating the emergence of developmental trajectories and predicted combinatorial activation of exogenous transcription factors controlling iN subtype specification. Functional validation of iN subtypes generated by novel combinations of exogenous transcription factors establish Convert-seq as a broadly applicable workflow to rapidly identify key transcription factors and GRNs orchestrating the reprogramming of virtually any cell type.

neuroscience↗