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Sparc Suppresses Microglial Neuroinflammation and Promotes Axonal Regeneration by Interacting With Uba52
Hangyu Ji1,2, Kun Wang3, Yili Hu2
1Department of Spine Surgery, Zhongda Hospital Southeast University, 210009 Nanjing, Jiangsu, China.
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.
Insights
Secreted protein acidic and rich in cysteine (Sparc) restrains inflammation and promotes axonal regeneration after spinal cord injury by maintaining mitochondrial homeostasis. Sparc interacts with Uba52 to achieve these neuroprotective effects.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Spinal cord injury (SCI) leads to pro-inflammatory microglia accumulation, hindering axonal regeneration.
- The role of secreted protein acidic and rich in cysteine (Sparc) in modulating microglial inflammation and promoting neurite outgrowth remains unclear.
Purpose of the Study:
- To investigate whether Sparc can restrain microglial inflammation and foster axonal regeneration post-SCI.
- To elucidate the underlying molecular mechanisms involving Sparc and its interaction with Uba52.
Main Methods:
- Utilized LPS-induced BV2 microglial cells and HT-22 neurons for in vitro studies.
- Assessed inflammatory markers, mitochondrial function (Δψm, ROS), and protein expression via RT-qPCR, Western blot, ELISA, and flow cytometry.
- Confirmed Sparc-Uba52 interaction using co-immunoprecipitation and mass spectrometry.
Main Results:
- Sparc overexpression reduced pro-inflammatory cytokines (TNF-α, IL-6) and ROS, while restoring mitochondrial potential and key oxidative-phosphorylation proteins in LPS-treated BV2 cells.
- Sparc directly interacted with Uba52, and Uba52 knockdown abrogated Sparc's anti-inflammatory and mitochondrial protective effects.
- In co-cultures, Sparc overexpression reduced neuronal apoptosis and enhanced neurite outgrowth, effects dependent on Uba52.
Conclusions:
- Sparc maintains mitochondrial homeostasis by interacting with Uba52, inhibiting LPS-induced microglial inflammation.
- This mechanism promotes neuronal axonal regeneration, suggesting Sparc as a potential therapeutic target for SCI repair.
