UBA2 Activates Wnt/β-catenin Signaling Pathway during Protection of R28 Retinal Precursor Cells from Hypoxia by Extracellular Vesicles derived from Placental Mesenchymal Stem Cells

Abstract

<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background:</jats:bold> Stem cell transplantation has been proposed as an alternative treatment for intractable optic nerve disorders characterized by irrecoverable loss of cells. Mesenchymal stem cells, with varying tissue regeneration and recovery capabilities, are being considered for potential cell therapies. To overcome the limitations of cell therapy, we isolated exosomes from human placenta–derived mesenchymal stem cells (hPMSCs), and investigated their therapeutic effects in R28 cells (retinal precursor cells) exposed to CoCl<jats:sub>2</jats:sub>. <jats:bold>Method:</jats:bold> After nine hours of exposure to CoCl<jats:sub>2, </jats:sub>the hypoxic damaged R28 cells were divided into non treatment group (CoCl<jats:sub>2</jats:sub>+R28 cells) and treatment group (CoCl<jats:sub>2</jats:sub>+R28 cells treated with exosome). Immunoblot analysis was performed for Hif-1α, Vegf, Thy-1, Gap43, Ermn, Neuroflament, Wnt3a, β-catenin, phospo-GSK3β, Lef-1, UBA2, Skp1, βTrcp, and ubiquitin. The proteomes of each group were analyzed by liquid chromatography/tandem mass (LC-MS/MS) spectrometry. Differentially expressed proteins (DEPs) were detected by label-free quantification and the interactions of the proteins were examined through signal transduction pathway and gene ontology analysis. <jats:bold>Result:</jats:bold> Treatment group presented the decreased expression of Hif-1α protein (<jats:italic>P </jats:italic>&lt; 0.05) and increased expression of nerve regeneration–related factors such as Thy-1 and Neuroflament (<jats:italic>P </jats:italic>&lt; 0.05) compared with non treatment group. In total, 200 DEPs were identified in non treatment group and treatment group (fold change ≥ 2, <jats:italic>p</jats:italic> &lt; 0.05). Catenin and ubiquitin systems (UBA2, UBE2E3, UBE2I) were found in both the DEP lists of downregulated proteins from non treatment group and upregulated proteins from treatment group. The mRNA expressions of ubiquitin systems were significantly decreased under hypoxic condition. Moreover, UBA2 and Wnt/β-catenin protein were associated with rescue of the hypoxic damaged R28 cells. Using a siRNA system, we could find it out that hPMSC exosoms could not repair altered expressions of target proteins by CoCl<jats:sub>2</jats:sub> in lacking UBA2 R28 cells. <jats:bold>Conclusion: </jats:bold>This study reported that hypoxic damaged expression of regeneration markers in R28 cells were significantly recovered by hPMSC exosomes. We could also demonstrate that UBA2 played a key role in activating the Wnt/β-catenin signaling pathway during protection of hypoxic damaged R28 cells, induced by hPMSC exosomes.</jats:p>

Emerging Role of Exosomes in Retinal Diseases

“… Aires et al (2020) reported that exosomes produced by retinal microglia under high intraocular pressure can propagate the inflammatory signals and promote retinal degeneration. Another two groups found that exosome derived from human placental MSCs and human 293T cells which express brain-derived neurotrophic factor (BDNF) can protect R28 cells (retinal precursor cells that represent RGCs) from hypoxia injury ( Koh et al, 2020 ; Yan et al, 2020 ).…”