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A multiplexed siRNA screen identifies key kinase signaling networks of brain glia.

ABSTRACT: The dynamic behaviors of brain glial cells in various neuroinflammatory conditions and neurological disorders have been reported; however, little is known about the underlying intracellular signaling pathways. Here, we developed a multiplexed kinome-wide siRNA screen to identify the kinases regulating several inflammatory phenotypes of mouse glial cells in culture, including inflammatory activation, migration, and phagocytosis of glia. Subsequent proof-of-concept experiments involving genetic and pharmacological inhibitions indicated the importance of T-cell receptor signaling components in microglial activation and a metabolic shift from glycolysis to oxidative phosphorylation in astrocyte migration. This time- and cost-effective multiplexed kinome siRNA screen efficiently provides exploitable drug targets and novel insight into the mechanisms underlying the phenotypic regulation of glial cells and neuroinflammation. Moreover, the kinases identified in this screen may be relevant in other inflammatory diseases and cancer, wherein kinases play a critical role in disease signaling pathways.

SUBMITTER: Kim JH

PROVIDER: S-EPMC9990460 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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A multiplexed siRNA screen identifies key kinase signaling networks of brain glia.

Kim Jong-Heon JH Han Jin J Afridi Ruqayya R Kim Jae-Hong JH Rahman Md Habibur MH Park Dong Ho DH Lee Won Suk WS Song Gyun Jee GJ Suk Kyoungho K

Life science alliance 20230306 5

The dynamic behaviors of brain glial cells in various neuroinflammatory conditions and neurological disorders have been reported; however, little is known about the underlying intracellular signaling pathways. Here, we developed a multiplexed kinome-wide siRNA screen to identify the kinases regulating several inflammatory phenotypes of mouse glial cells in culture, including inflammatory activation, migration, and phagocytosis of glia. Subsequent proof-of-concept experiments involving genetic an ...[more]

PMID: 36878638

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Project description:The strain designated Chlamydia trachomatis serovar L2 that was used for experiments in this paper is Chlamydia muridarum, a species closely related to C. trachomatis (and formerly termed the Mouse Pneumonitis strain of C. trachomatis). This conclusion was verified by deep sequencing and by PCR using species-specific primers. All data presented in the results section that refer to C. trachomatis should be interpreted as referring to C. muridarum. Since C. muridarum TARP lacks the consensus tyrosine repeats present in C. trachomatis TARP, we cannot make any conclusions about the role of TARP phosphorylation and C. muridarum entry. However, the conclusion that C. trachomatis L2 TARP is a target of Abl kinase is still valid as these experiments were performed with C. trachomatis L2 TARP [corrected]. To elucidate the mechanisms involved in early events in Chlamydia trachomatis infection, we conducted a large scale unbiased RNA interference screen in Drosophila melanogaster S2 cells. This allowed identification of candidate host factors in a simple non-redundant, genetically tractable system. From a library of 7,216 double stranded RNAs (dsRNA), we identified approximately 226 host genes, including two tyrosine kinases, Abelson (Abl) kinase and PDGF- and VEGF-receptor related (Pvr), a homolog of the Platelet-derived growth factor receptor (PDGFR). We further examined the role of these two kinases in C. trachomatis binding and internalization into mammalian cells. Both kinases are phosphorylated upon infection and recruited to the site of bacterial attachment, but their roles in the infectious process are distinct. We provide evidence that PDGFRbeta may function as a receptor, as inhibition of PDGFRbeta by RNA interference or by PDGFRbeta neutralizing antibodies significantly reduces bacterial binding, whereas depletion of Abl kinase has no effect on binding. Bacterial internalization can occur through activation of PDGFRbeta or through independent activation of Abl kinase, culminating in phosphorylation of the Rac guanine nucleotide exchange factor (GEF), Vav2, and two actin nucleators, WAVE2 and Cortactin. Finally, we show that TARP, a bacterial type III secreted actin nucleator implicated in entry, is a target of Abl kinase. Together, our results demonstrate that PDGFRbeta and Abl kinases function redundantly to promote efficient uptake of this obligate intracellular parasite.

Dataset Information

A multiplexed siRNA screen identifies key kinase signaling networks of brain glia.

Publications

A multiplexed siRNA screen identifies key kinase signaling networks of brain glia.

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