Upregulated expression of ubiquitin ligase TRIM21 promotes PKM2 nuclear translocation and astrocyte activation in experimental autoimmune encephalomyelitis

Reactive astrocytes play critical roles in the occurrence of various neurological diseases such as multiple sclerosis. Activation of astrocytes is often accompanied by a glycolysis-dominant metabolic switch. However, the role and molecular mechanism of metabolic reprogramming in activation of astrocytes have not been clarified. Here, we found that PKM2, a rate-limiting enzyme of glycolysis, displayed nuclear translocation in astrocytes of EAE (experimental autoimmune encephalomyelitis) mice, an animal model of multiple sclerosis. Prevention of PKM2 nuclear import by DASA-58 significantly reduced the activation of mice primary astrocytes, which was observed by decreased proliferation, glycolysis and secretion of inflammatory cytokines. Most importantly, we identified the ubiquitination-mediated regulation of PKM2 nuclear import by ubiquitin ligase TRIM21. TRIM21 interacted with PKM2, promoted its nuclear translocation and stimulated its nuclear activity to phosphorylate STAT3, NF-κB and interact with c-myc. Further single-cell RNA sequencing and immunofluorescence staining demonstrated that TRIM21 expression was upregulated in astrocytes of EAE. TRIM21 overexpressing in mice primary astrocytes enhanced PKM2-dependent glycolysis and proliferation, which could be reversed by DASA-58. Moreover, intracerebroventricular injection of a lentiviral vector to knockdown TRIM21 in astrocytes or intraperitoneal injection of TEPP-46, which inhibit the nuclear translocation of PKM2, effectively decreased disease severity, CNS inflammation and demyelination in EAE. Collectively, our study provides novel insights into the pathological function of nuclear glycolytic enzyme PKM2 and ubiquitination-mediated regulatory mechanism that are involved in astrocyte activation. Targeting this axis may be a potential therapeutic strategy for the treatment of astrocyte-involved neurological disease.