Sparc Suppresses Microglial Neuroinflammation and Promotes Axonal Regeneration by Interacting With Uba52.

BACKGROUND

After spinal cord injury (SCI), pro-inflammatory microglia accumulate and impede axonal regeneration. We explored whether secreted protein acidic and rich in cysteine (Sparc) restrains microglial inflammation and fosters neurite outgrowth.

METHODS

Mouse microglial BV2 cells were polarized to a pro-inflammatory phenotype with lipopolysaccharides (LPSs). Sparc mRNA and protein were quantified by reverse transcription quantitative PCR (RT-qPCR). Sparc was overexpressed via plasmid transfection, then inflammatory cytokines, mitochondrial membrane potential (Δψm), reactive oxygen species (ROS), and oxidative-phosphorylation proteins, including voltage-dependent anion channel 1 (VDAC1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase α subunit (ATP5A), were assayed by Western blot, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. Immunoprecipitation plus mass spectrometry, co-immunoprecipitation, and immunofluorescence confirmed the interaction between Sparc and ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52). Effects of Sparc overexpression alone or combined with Uba52 small interfering RNA (si-Uba52) were compared in LPS-induced BV2 cells. Finally, BV2 cells and a mouse hippocampal neuron (HT-22) were co-cultured in the Transwell chamber, and the changes in proliferation, apoptosis, and III-tubulin content of the latter were detected.

RESULTS

In LPS-induced BV2 cells, the tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and ROS levels were elevated, while the IL-10 and transforming growth factor-β (TGF-β) levels, Δψm, and the proteins levels of the VDAC1, COX1, ATP5A, and Sparc decreased. Sparc overexpression reversed these changes. Mechanistically, Sparc bound Uba52 and upregulated its expression; Uba52 knockdown abolished the anti-inflammatory and mitochondrial-protective effects of Sparc. In co-culture, Sparc overexpression rescued HT-22 neurons apoptosis and enhanced axonal growth, but the effects were also reversed by Uba52 knockdown.

CONCLUSIONS

Sparc may maintain mitochondrial homeostasis by interacting with Uba52 to inhibit LPS-induced BV2 inflammatory response, thereby promoting neuronal axonal regeneration. This suggests that Sparc may play a potential role in SCI repair.