Pyruvate kinase isoform M2 impairs cognition in systemic lupus erythematosus by promoting microglial synaptic pruning via the β-catenin signaling pathway

Abstract

Background Neuropsychiatric systemic lupus erythematosus (NPSLE) is a severe complication, which involves pathological damage to the brain and cognitive function. However, its exact mechanism of action still remains unclear. In this study, we explored the role of microglia in the cognitive dysfunction of NPSLE mice. We also analyzed and compared the metabolites in the hippocampal tissues of the lupus model and control mice. Methods MRL/MpJ-Faslpr (MRL/lpr) female mice were used as the NPSLE mouse model. Metabolomics was used to assess hippocampal glycolysis levels. Glucose, lactic acid, IL-6, and IL-1β of the hippocampus were detected by ELISA. Based on the glycolysis pathway, we found that pyruvate kinase isoform M2 (PKM2) in the hippocampus was significantly increased. Thus, the expression of PKM2 was detected by qRT-PCR and Western blotting, and the localization of PKM2 in microglia (IBA-1+) or neurons (NeuN+) was assessed by immunofluorescence staining. Flow cytometry was used to detect the number and phenotype of microglia; the changes in microglial phagocytosis and the β-catenin signaling pathway were detected in BV2 cells overexpressing PKM2. For in vivo experiments, MRL/lpr mice were treated with AAV9-shPKM2. After 2 months, Morris water maze and conditional fear tests were applied to investigate the cognitive ability of mice; H&E and immunofluorescence staining were used to evaluate brain damage; flow cytometry was used to detect the phenotype and function of microglia; neuronal synapse damage was monitored by qRT-PCR, Western blotting, and immunofluorescence staining. Results Glycolysis was elevated in the hippocampus of MRL/lpr lupus mice, accompanied by increased glucose consumption and lactate production. Furthermore, the activation of PKM2 in hippocampal microglia was observed in lupus mice. Cell experiments showed that PKM2 facilitated microglial activation and over-activated microglial phagocytosis via the β-catenin signaling pathway. In vivo, AAV9-shPKM2-treated mice showed decreased microglial activation and reduced neuronal synapses loss by blocking the β-catenin signaling pathway. Furthermore, the cognitive impairment and brain damage of MRL/lpr mice were significantly relieved after microglial PKM2 inhibition. Conclusion These data indicate that microglial PKM2 have potential to become a novel therapeutic target for treating lupus encephalopathy.

B cells in systemic lupus erythematosus

“…Inhibition of PKM2 improved splenomegaly in two lupus mouse models and reduced antidsDNA antibody titres and glomerular deposition of IgG and IgM [51]. Analogously, treatment of MRL/lpr mice with PKM2 inhibitor alleviated cognitive impairment and brain damage through decreased microglial activation [52].…”

sTREM-1 promotes the phagocytic function of microglia to induce hippocampus damage via the PI3K–AKT signaling pathway

“…Immunofluorescence staining was performed as previously described 62 , the paraffin sections were soaked in cold acetone for 15 min. After washing with PBS, pre-cooled methanol was added dropwise and treated at −20 °C for 30 min.…”

“…Half of the left and right hippocampus of four mice per group were used for western blot detection. Western blot was performed as previously described 62 . Briefly, hippocampus tissues or cells were lysed in ice-cold RIPA lysis buffer containing protease and phosphatase inhibitors.…”

“…Five mice per group were used for flow cytometric analysis. Perform sample preparation and flow cytometry according to the previous experimental description 62 . Briefly, after immunomagnetic beads sorting, single cells were pre-blocked with anti-CD16/CD32 Fc Blocker for 10 min, then stained with Zombie NIR™ Fixable Viability Kit (Biolegend, CA, USA), CD11b-FITC (clone number: M1/70, BD Biosciences, CA, USA), CD45-APC (clone number: RA3-6B2, Biolegend, CA, USA) antibodies for 30 min at 4 °C in the dark.…”

Glycolysis in Innate Immune Cells Contributes to Autoimmunity

“…PKM2 inhibitors suppress proline-rich tyrosine kinase 2, preventing TLR4/TLR7/TLR9 from activating the MAPK/NF-kB pathway (90). PKM2 and lactate levels were reported to be abnormally increased in the hippocampus of MRL/ lpr lupus mice (91). PKM2-b-catenin signaling leads to neuronal synapse loss by promoting microglial hyperactivation, hypersecretion of IL-6 and IL-1b, and hyperphagocytosis.…”