Circular RNA METTL9 contributes to neuroinflammation following traumatic brain injury by complexing with astrocytic SND1

Abstract Background Circular RNAs (circRNAs) are highly enriched in the central nervous system and have been implicated in neurodegenerative diseases. However, whether and how circRNAs contribute to the pathological processes induced by traumatic brain injury (TBI) has not been fully elucidated. Methods We conducted a high-throughput RNA sequencing screen for well-conserved, differentially expressed circRNAs in the cortex of rats subjected to experimental TBI. Circular RNA METTL9 (circMETTL9) was ultimately identified as upregulated post-TBI and further characterized by RT-PCR and agarose gel electrophoresis, Sanger sequencing, and RNase R treatment. To examine potential involvement of circMETTL9 in neurodegeneration and loss of function following TBI, circMETTL9 expression in cortex was knocked-down by microinjection of a shcircMETTL9 adeno-associated virus. Neurological functions were evaluated in control, TBI, and TBI-KD rats using a modified neurological severity score, cognitive function using the Morris water maze test, and nerve cell apoptosis rate by TUNEL staining. Pull-down assays and mass spectrometry were conducted to identify circMETTL9-binding proteins. Co-localization of circMETTL9 and SND1 in astrocytes was examined by fluorescence in situ hybridization and immunofluorescence double staining. Changes in the expression levels of chemokines and SND1 were estimated by quantitative PCR and western blotting. Results CircMETTL9 was significantly upregulated and peaked at 7 d in the cerebral cortex of TBI model rats, and it was abundantly expressed in astrocytes. We found that circMETTL9 knockdown significantly attenuated neurological dysfunction, cognitive impairment, and nerve cell apoptosis induced by TBI. CircMETTL9 directly bound to and increased the expression of SND1 in astrocytes, leading to the upregulation of CCL2, CXCL1, CCL3, CXCL3, and CXCL10, and ultimately to enhanced neuroinflammation. Conclusion Altogether, we are the first to propose that circMETTL9 is a master regulator of neuroinflammation following TBI, and thus a major contributor to neurodegeneration and neurological dysfunction. Graphical Abstract