Extracellular vesicles derived from melatonin‐preconditioned mesenchymal stem cells containing USP29 repair traumatic spinal cord injury by stabilizing NRF2

Abstract

Spinal cord injury (SCI) is a devastating trauma that leads to irreversible motor and sensory dysfunction and is, so far, without effective treatment. Recently, however, nano-sized extracellular vesicles derived from preconditioned mesenchymal stem cells (MSCs) have shown great promise in treating various diseases, including SCI. In this study, we investigated whether extracellular vesicles (MEVs) derived from MSCs pretreated with melatonin (MT), which is well recognized to be useful in treating diseases, including Alzheimer's disease, non-small cell lung cancer, acute ischemia-reperfusion liver injury, chronic kidney disease, and SCI, are better able to promote functional recovery in mice after SCI than extracellular vesicles derived from MSCs without preconditioning (EVs). MEVs were found to facilitate motor behavioral recovery more than EVs and to increase microglia/macrophages polarization from M1-like to M2like in mice. Experiments in BV2 microglia and RAW264.7 macrophages confirmed that MEVs facilitate M2-like polarization and also showed that they reduce the production of reactive oxygen species (ROS) and regulate mitochondrial function. Proteomics analysis revealed that ubiquitin-specific protease 29 (USP29) was markedly increased in MEVs, and knockdown of USP29 in MEVs (shUSP29-MEVs) abolished MEVs-mediated benefits in vitro and in vivo. We then showed that USP29 interacts with, deubiquitinates and therefore stabilizes nuclear factor-like 2 (NRF2), thereby regulating microglia/macrophages polarization. In NRF2 knockout mice, MEVs failed to promote functional recovery and M2-like microglia/macrophages polarization. We also showed that MT reduced global N6-methyladenosine (m 6 A) modification and levels of the m 6 A "writer" methyltransferase-like 3 (METTL3). The stability of USP29 mRNA in MSCs was enhanced by treatment with MT, but inhibited by overexpression of METTL3.This study describes a very promising extracellular vesicle-based approach for treating SCI.

Melatonin and the Programming of Stem Cells

“…The findings concerning microglia are, again, in good agreement with corresponding actions of directly administered melatonin [59]. The microvesicles collected from melatonin-pretreated cells were also shown to be enriched with USP29 (ubiquitin-specific protease 29, systematic name: ubiquitin carboxyl-terminal hydrolase 29) [227], a deubiquitinase that prevents the proteasomal decay of Nrf2 (nuclear factor erythroid 2-related factor 2), a key transcription factor of anti-inflammatory and antioxidant signaling. This study also revealed another novel aspect of MSC function concerning the stability of Usp29 mRNA, which was enhanced by melatonin, but strongly decreased by overexpression of METTL3 (methyltransferase-like 3), an RNA methylating enzyme, also referred to as the "writer" of adenosine 6-methylation.…”

“…This is explained by the PTEN-catalyzed dephosphorylation of PIP3 (phosphatidylinositol 3,4,5-tris-phosphate) to PIP2, a known inhibitor of Akt signaling. In a study on spinal cord injury, the melatoninpromoted M1-to-M2 shift was corroborated for both macrophages and microglia when treated with microvesicles from melatonin-preconditioned MSCs [227]. The findings concerning microglia are, again, in good agreement with corresponding actions of directly administered melatonin [59].…”

“…This study also revealed another novel aspect of MSC function concerning the stability of Usp29 mRNA, which was enhanced by melatonin, but strongly decreased by overexpression of METTL3 (methyltransferase-like 3), an RNA methylating enzyme, also referred to as the "writer" of adenosine 6-methylation. At first glance, these opposite effects of melatonin and METTL3 might appear to be independent, but it can be concluded that they interact or compete, since melatonin was shown to decrease m 6 A (6-methyladenosine) in overall RNA by reducing METTL3 expression [227]. This finding is worth strongly emphasizing, because it represents an entirely novel, unprecedented action mode of melatonin, which takes place at the level of regulation by RNA modification.…”

Neuroinflammation and Scarring After Spinal Cord Injury: Therapeutic Roles of MSCs on Inflammation and Glial Scar

“…During the process of skin and muscle wounds healing, macrophages will shift from M1 to M2 phenotype in response to the change of microenvironmental stimulus signals at the lesion site (50,51). Whereas, an analogous M1 to M2 conversion was not observed in the process of spinal cord tissue repair, and the persistent presence of M1 macrophages would exacerbate secondary injury (52)(53)(54). Therefore, inducing macrophages switch towards M2 phenotype following acute inflammatory response may contribute to anatomical and functional recovery post-SCI.…”

Melatonin Prevents Cartilage Degradation in Early-Stage Osteoarthritis Through Activation of miR-146a/NRF2/HO-1 Axis

“…Melatonin‐stimulated extracellular vesicles (MEVs) upregulated ubiquitin‐specific protease 29 (USP29) to deubiquitinate and stabilize NRF2, thereby regulating microglia/macrophages polarization and mitochondrial function. ( 44 )…”