Title : Knockdown of YTHDF2 mitigates OGD-induced microglial inflammation by preventing m6A-dependent PARP14 degradation
Abstract:
Neuroinflammation, a pathological process caused by immune responses in the central nervous system（CNS）, plays a pivotal role in the onset and progression of various neurological disorders, including neurodegenerative diseases, ischemic stroke, and neuropathic pain. Reader proteins, functioning as the “effectors” of m⁶A methylation, influence physiological functions like development, immune response, and neural repair by recognizing and modulating m⁶A-modified RNA. Although studies have demonstrated that the m⁶A reader protein YTHDF2 plays a critical regulatory role in the progression of ischemic stroke, its role and mechanisms in neuroinflammation associated with ischemic brain diseases remain unclear. This study employed an oxygen-glucose deprivation (OGD) model to simulate ischemic conditions in the brain. And through YTHDF2 knockdown, we investigated its effect on OGD-induced neuroinflammation. The results revealed that YTHDF2 are involved in neuroinflammation induced by OGD. Decreasing YTHDF2 levels facilitates the transition of microglia from M1 to M2 phenotype following OGD, thereby exerting an anti-inflammatory effect. Mechanistically, qPCR results was employed to identify that Nos2 and Il1b, which serve as specific transcripts for M1 microglia, were significantly elevated in BV2 cells following OGD. However, YTHDF2 knockdown not only suppressed the expression of these M1-related markers but also upregulated the expression of M2-related anti-inflammatory markers such as Arg, Tgfb, Mrc1, Socs3 and Il10. Previous research indicated that Poly (ADP-ribose) polymerase family, member 14 (PARP14) had a crucial role in regulating the inflammatory response to cerebral ischemic injury and in modulating the M1/M2 phenotype transition of microglia following ischemia. Therefore, this study further investigated whether YTHDF2 regulated PARP14 expression in BV2 cells after OGD through m⁶A methylation. qPCR and Western blotting analyses demonstrated an increase in PARP14 expression in BV2 cells following OGD, and YTHDF2 knockdown further elevating PARP14 levels. Using CatRAPID and SRAMP, we predicted three high-confidence m⁶A modification sites on PARP14 mRNA and identified potential binding affinity between PARP14 and YTHDF2. Subsequently, Western blotting and ELISA were conducted, revealing that inflammatory markers (including iNOS, TNF-α, IL-1β, and IL-6) were significantly upregulated in the group with dual knockdown of YTHDF2 and PARP14. Moreover, the expression of M2-related anti-inflammatory markers was suppressed by PARP14 knockdown. Our findings demonstrate that YTHDF2 knockdown increased the expression of PARP14 and promoted a PARP14-driven phenotypic switch in microglia, resulting in diminished inflammation. This discovery underscores the significance of PARP14 in the inflammatory response and indicates that the YTHDF2-PARP14 axis could serve as a promising target for developing new therapeutic strategies to counteract inflammation in ischemic brain injury.
