Phosphorylated SOX9 drives neuroinflammatory astrocyte subtypes by boosting hexokinase 1 in neuropathic pain

Abstract

Neuropathic pain (NeP) is elicited by multiple etiologies, including injuries or diseases, casing spontaneous and persistent pain, which exerts adverse impacts on patients’ quality of life. Currently, there is no effective treatments. Detrimental activity of astrocytes in dorsal horn of the spinal cord (SDH) upon the nerve injury is thought to be one of the central mechanisms in driving long-lasting NeP. Exploring the molecular mechanisms underlying dynamic changes of astrocyte activities is critical for resolving the chronification of NeP. Due to heavily relying on glycolysis for energy production, glycolysis in astrocytes represents a central metabolic pathway in astrocytes under physiological conditions, and the regulation of glycolysis is essential for the conversion between neuroprotective and neurotoxic outcomes under pathological status. Here, by using scRNA-sequencing and perturbation studies in vivo and in vitro, we identified a pathologic astrocyte cluster that exhibited highly upregulated glycolytic genes, resulting in enhanced glycolytic flux during NeP chronification and the elevated secretion of pro-inflammatory modulators (e.g., cytokines and chemokines) for neuroinflammation. The emergency of this immunopathogenic astrocyte subtype was driven by the enrichment of histone H3K9 lactylation mediated by the excessive accumulation of lactate after nerve injury at the promoter region of multiple driving genes (e.g., C3, GFAP and CFB) and thus promoting their transcriptional activity. Importantly, it was found that Hexokinase I (HK1) in this astrocyte subtype, the first rate-limiting enzyme of glycolytic pathway, enhances glycolytic flux and contributes to the accumulation of lactate. Additionally, HK1 was regulated by the post-translational modification of SOX9, a transcriptional factor belonging to the SRY-like HMG-box family and uniquely expressed in astrocytes in central nerve system (CNS). SOX9 is a key regulator for astrocyte differentiation and is highly expressed in adult naïve astrocytes. Our results suggested that phosphorylation of SOX9 at the sites of S181 positively correlated with astrocytic immunopathological changes in SDH during pain development. Interestingly, genetic manipulation of SOX9 phosphorylation regulates its transcriptional activities by increasing the nucleus localization of SOX9 proteins. Phosphorylated SOX9 (p-SOX9) exhibits enhanced binding to the HK1 promoter, thereby trans-activating HK1 and reinforcing glycolytic flux in astrocytes.

In addition, elevated blood TNF- was detected in the NeP patients, which promotes the astrocytic pathological activities via enhancing the phosphorylation level of PAK1 (serine/threonine p21-activated kinase 1) at site Ser199/204 and thus upregulating the kinase enzyme activity of PAK1. Most importantly, PAK1, serving as the kinase enzyme, promotes SOX9 phosphorylation, contributing to the metabolic reprogramming and proinflammatory properties of astrocytes through upregulating p-SOX9/HK1 axis. Inhibition of PAK1 enzyme activity via administration of FDA approved drugs (e.g., Romedepsin and IPA3) decreases SOX9 phosphorylation level and reduces the HK1 enzyme activity, thereby impairs the glycolytic flux and neuroinflammation in astrocytes, attenuating the chronic neuropathic pain threshold. Interestingly, increased PAK1/p-PAK1 was also found in the plasma of NeP patients. This suggested that p-PAK1 cannot only be a target for developing therapies to treat NeP, but also be a potential biomarker for the diagnosis of NeP.