Single-cell RNA sequencing integrated with bulk RNA sequencing analysis reveals the protective effects of lactate-mediated lactylation of microglia-related proteins on spinal cord injury.

Abstract

Spinal cord injury (SCI) results in significant neurological deficits, and microglia play the critical role in regulating the immune microenvironment and neurological recovery. Protein lactylation has been found to modulate the function of immune cells. Therefore, this study aimed to elucidate the effects of glycolysis-derived lactate on microglial function and its potential neuroprotective mechanisms via lactylation after SCI. Single-cell RNA sequencing (scRNA-seq) data were obtained from figshare to analyze cellular and molecular alterations within the spinal cord post-SCI, further focusing on the expression of microglia-related genes for cell sub-clustering, trajectory analysis, and glycolysis function analysis. We also evaluated the expression of lactylation-related genes in microglia between day 7 after SCI and sham group. Additionally, we established the mice SCI model and performed the bulk RNA sequencing in a time-dependent manner. The expression of glycolysis- and lactylation-related genes was evaluated, as well as the immune infiltration analysis based on the lactylation-related genes. Then, we investigated the bio-effects of lactate on the inflammation and polarization phenotype of microglia. Finally, adult male C57BL/6 mice were subjected to exercise first to increase lactate level, before SCI surgery, aiming to evaluate the protective effects of lactate-mediated lactylation of microglia-related proteins on SCI. scRNA-seq identified a subcluster of microglia, recombinant chemokine C-X3-C-motif receptor 1+ (CX3CR1+) microglia, which is featured by M1-like phenotype and increased after SCI. KEGG analysis revealed the dysfunctional glycolysis in microglia after SCI surgery, and AUCell analysis suggested that the decreased glycolysis an increased oxidative phosphorylation in CX3CR1+ microglia. Differential gene analysis suggested that several lactylation-related genes (Fabp5, Lgals1, Vim, and Nefl) were downregulated in CX3CR1+ microglia at day 7 after SCI, further validated by the results from bulk RNA sequencing. Immunofluorescence staining indicated the expression of lactate dehydrogenase A (LDHA) in CX3CR1+ microglia also decreased at day 7 after SCI. Cellular experiments demonstrated that the administration of lactate could increase the lactylation level and inhibit the pro-inflammatory phenotype in microglia. Functionally, exercise-mediated lactate production resulted in improved locomotor recovery and decreased inflammatory markers in SCI mice compared to SCI alone. In the subacute phase of SCI, metabolic remodeling in microglia may be key therapeutic targets to promote nerve regeneration, and lactate contributed to neuroprotection after SCI by influencing microglial lactylation and inflammatory phenotype, which offered a novel approach for therapeutic intervention.