Patsch, C. | Search bioRxiv

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

A bottom-up approach identifies the antipsychotic and antineoplastic trifluoperazine and the ribose derivative deoxytubercidin as novel microglial phagocytosis inhibitors.

Phagocytosis is an indispensable function of microglia, the brain professional phagocytes. Microglia are particularly efficient phagocytosing cells that undergo programmed cell death (apoptosis) in physiological conditions. However, mounting evidence suggests microglial phagocytosis dysfunction in multiple brain disorders. These observations prompted us to search for phagocytosis modulators (enhancers or inhibitors) with therapeutic potential. We used a bottom-up strategy that consisted on the identification of phagocytosis modulators using phenotypic high throughput screenings (HTSs) in cell culture and validation in organotypic cultures and in vivo. We performed two complementary HTS campagnes: at Achucarro, we used primary cultures of mouse microglia and compounds of the Prestwick Chemical Library; at Roche, we used human iPSC derived macrophage-like cells and a proprietary chemo-genomic library with 2,200 compounds with known mechanism-of-action. Next, we validated the more robust compounds using hippocampal organotypic cultures and identified two hits: trifluoperazine, a dopaminergic and adrenergic antagonist used as an antipsychotic and antineoplastic; and deoxytubercidin, a ribose derivative. Finally, we tested whether these compounds were able to modulate phagocytosis of apoptotic newborn cells in the adult hippocampal neurogenic niche in vivo by administering them into the mouse hippocampus using osmotic minipumps. We confirmed that both trifluoperazine and deoxytubercidin have anti-phagocytic activity in vivo, and validated our bottom-up strategy to identify novel phagocytosis modulators. These results show that chemical libraries with anotated mechanism of action are an starting point for the pharmacological modulation of microglia in drug discovery projects aiming at the therapeutic manipulation of phagocytosis in brain diseases. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/599284v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@13ed994org.highwire.dtl.DTLVardef@13ea710org.highwire.dtl.DTLVardef@4a1c7dorg.highwire.dtl.DTLVardef@1945f00_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗