Exosomes originating from neural stem cells undergoing necroptosis participate in cellular communication by inducing TSC2 upregulation of recipient cells following spinal cord injury.

ABSTRACT: We previously demonstrated that inhibiting neural stem cells necroptosis enhances functional recovery after spinal cord injury. While exosomes are recognized as playing a pivotal role in neural stem cells exocrine function, their precise function in spinal cord injury remains unclear. To investigate the role of exosomes generated following neural stem cells necroptosis after spinal cord injury, we conducted single-cell RNA sequencing and validated that neural stem cells originate from ependymal cells and undergo necroptosis in response to spinal cord injury. Subsequently, we established an in vitro necroptosis model using neural stem cells isolated from embryonic mice aged 16-17 days and extracted exosomes. The results showed that necroptosis did not significantly impact the fundamental characteristics or number of exosomes. Transcriptome sequencing of exosomes in necroptosis group identified 108 differentially expressed messenger RNAs, 104 long non-coding RNAs, 720 circular RNAs, and 14 microRNAs compared with the control group. Construction of a competing endogenous RNA network identified the following hub genes: tuberous sclerosis 2 (Tsc2), solute carrier family 16 member 3 (Slc16a3), and forkhead box protein P1 (Foxpl). Notably, a significant elevation in TSC2 expression was observed in spinal cord tissues following spinal cord injury. TSC2-positive cells were localized around SRY-box transcription factor 2-positive cells within the injury zone. Furthermore, in vitro analysis revealed increased TSC2 expression in exosomal receptor cells compared with other cells. Further assessment of cellular communication following spinal cord injury showed that Tsc2 was involved in ependymal cellular communication at 1 and 3 days post-injury through the epidermal growth factor and midkine signaling pathways. In addition, Slc16a3 participated in cellular communication in ependymal cells at 7 days post-injury via the vascular endothelial growth factor and macrophage migration inhibitory factor signaling pathways. Collectively, these findings confirm that exosomes derived from neural stem cells undergoing necroptosis play an important role in cellular communication after spinal cord injury and induce TSC2 upregulation in recipient cells.

Extracellular vesicles released by transforming growth factor-beta 1-preconditional mesenchymal stem cells promote recovery in mice with spinal cord injury.

Spinal cord injury (SCI) causes neuroinflammation, neuronal death, and severe axonal connections. Alleviating neuroinflammation, protecting residual cells and promoting neuronal regeneration via endogenous neural stem cells (eNSCs) represent potential strategies for SCI treatment. Extracellular vesicles (EVs) released by mesenchymal stem cells have emerged as pathological mediators and alternatives to cell-based therapies following SCI. In the present study, EVs isolated from untreated (control, C-EVs) and TGF-ß1-treated (T-EVs) mesenchymal stem cells were injected into SCI mice to compare the therapeutic effects and explore the underlying mechanisms. Our study demonstrated for the first time that the application of T-EVs markedly enhanced the proliferation and antiapoptotic ability of NSCs in vitro. The infusion of T-EVs into SCI mice increased the shift from the M1 to M2 polarization of reactive microglia, alleviated neuroinflammation, and enhanced the neuroprotection of residual cells during the acute phase. Moreover, T-EVs increased the number of eNSCs around the epicenter. Consequently, T-EVs further promoted neurite outgrowth, increased axonal regrowth and remyelination, and facilitated locomotor recovery in the chronic stage. Furthermore, the use of T-EVs in Rictor-/- SCI mice (conditional knockout of Rictor in NSCs) showed that T-EVs failed to increase the activation of eNSCs and improve neurogenesis sufficiently, which suggested that T-EVs might induce the activation of eNSCs by targeting the mTORC2/Rictor pathway. Taken together, our findings indicate the prominent role of T-EVs in the treatment of SCI, and the therapeutic efficacy of T-EVs for SCI treatment might be optimized by enhancing the activation of eNSCs via the mTORC2/Rictor signaling pathway.

Targeting YAP1-regulated Glycolysis in Fibroblast-Like Synoviocytes Impairs Macrophage Infiltration to Ameliorate Diabetic Osteoarthritis Progression.

The interplay between immune cells/macrophages and fibroblast-like synoviocytes (FLSs) plays a pivotal role in initiating synovitis; however, their involvement in metabolic disorders, including diabetic osteoarthritis (DOA), is largely unknown. In this study, single-cell RNA sequencing (scRNA-seq) is employed to investigate the synovial cell composition of DOA. A significant enrichment of activated macrophages within eight distinct synovial cell clusters is found in DOA synovium. Moreover, it is demonstrated that increased glycolysis in FLSs is a key driver for DOA patients' synovial macrophage infiltration and polarization. In addition, the yes-associated protein 1 (YAP1)/thioredoxin-interacting protein (TXNIP) signaling axis is demonstrated to play a crucial role in regulating glucose transporter 1 (GLUT1)-dependent glycolysis in FLSs, thereby controlling the expression of a series of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) which may subsequently fine-tune the infiltration of M1-polarized synovial macrophages in DOA patients and db/db diabetic OA mice. For treatment, M1 macrophage membrane-camouflaged Verteporfin (Vt)-loaded PLGA nanoparticles (MVPs) are developed to ameliorate DOA progression by regulating the YAP1/TXNIP signaling axis, thus suppressing the synovial glycolysis and the infiltration of M1-polarized macrophages. The results provide several novel insights into the pathogenesis of DOA and offer a promising treatment approach for DOA.

Increased blood flow of spinal cord lesion after decompression improves neurological recovery of degenerative cervical myelopathy: an intraoperative ultrasonography-based prospective cohort study.

INTRODUCTION: Surgical decompression is a highly effective therapy for degenerative cervical myelopathy (DCM), but the mechanisms of neurological recovery following decompression remain unclear. This study aimed to evaluate the spinal cord blood flow status after sufficient decompression by intraoperative contrast-enhanced ultrasonography (CEUS) and to analyze the correlation between neurological recovery and postdecompressive spinal cord blood perfusion in DCM. MATERIALS AND METHODS: Patients with multilevel DCM were treated by ultrasound-guided modified French-door laminoplasty using a self-developed rongeur. Neurological function was evaluated using the modified Japanese Orthopaedic Association (mJOA) score preoperatively and at 12 months postoperatively. Spinal cord compression and cervical canal enlargement before and after surgery were assessed by magnetic resonance imaging and computerized tomography. The decompression status was evaluated in real time by intraoperative ultrasonography, while the spinal cord blood flow after sufficient decompression was assessed by CEUS. Patients were categorized as favourable (≥50%) or unfavourable (<50%) recovery according to the recovery rate of the mJOA score at 12 months postoperatively. RESULTS: Twenty-nine patients were included in the study. The mJOA scores were significantly improved in all patients from 11.2±2.1 preoperatively to 15.0±1.1 at 12 months postoperatively, with an average recovery rate of 64.9±16.2%. Computerized tomography and intraoperative ultrasonography confirmed adequate enlargement of the cervical canal and sufficient decompression of the spinal cord, respectively. CEUS revealed that patients with favourable neurological recovery had a greater increased blood flow signal in the compressive spinal cord segment after decompression. CONCLUSIONS: In DCM, intraoperative CEUS can clearly reflect spinal cord blood flow. Patients with increased blood perfusion of the spinal cord lesion immediately after surgical decompression tended to achieve greater neurological recovery.

Inhibiting tau protein improves the recovery of spinal cord injury in rats by alleviating neuroinflammation and oxidative stress.

After spinal cord injury, the concentrations of total and hyperphosphorylated tau in cerebrospinal fluid increase, and levels of both correlate with injury severity. Tau inhibition is considered effective therapy for many central nervous system diseases, including traumatic brain injury and Alzheimer's disease. However, whether it can play a role in the treatment of spinal cord injury remains unclear. In this study, the therapeutic effects of tau inhibition were investigated in a rat model of transection spinal cord injury by injecting the rats with a lentivirus encoding tau siRNA that inhibits tau expression. We found that tau inhibition after spinal cord injury down-regulated the levels of inflammatory mediators, including tumor necrosis factor-α, interleukin-6 and interleukin-1ß. It also led to a shift of activated microglial polarization from the M1 pro-inflammatory phenotype to the M2 anti-inflammatory phenotype, and reduced the amount of reactive oxygen species in the acute phase. Furthermore, the survival of residual neural cells around the injury epicenter, and neuronal and axonal regeneration were also markedly enhanced, which promoted locomotor recovery in the model rats. Collectively, our findings support the conclusion that tau inhibition can attenuate neuroinflammation, alleviate oxidative stress, protect residual cells, facilitate neurogenesis, and improve the functional recovery after spinal cord injury, and thus suggest that tau could be a good molecular target for spinal cord injury therapy.

Inhibition of Neural Stem Cell Necroptosis Mediated by RIPK1/MLKL Promotes Functional Recovery After SCI.

Endogenous neural stem cells (eNSCs) are a new therapeutic strategy for the noninvasive repair of spinal cord injury (SCI). Necroptosis is a necrosome-dependent cell death process that serves as a significant regulatory mechanism in SCI. Current research shows that neurons, oligodendrocytes, and astrocytes all undergo necroptosis after SCI. However, it is unclear whether eNSCs are associated with necroptosis after SCI. By performing immunofluorescence analysis, we found that eNSCs undergo necroptosis during spinal cord injury repair in mice. Our present work demonstrates that receptor-interacting protein kinase 1 (RIPK1)/mixed lineage kinase domain-like protein (MLKL) are involved in necroptosis pathway in SCI mice. In vitro, the necroptosis induced by TNF-α/Smac-mimetic/Z-VAD-FMK (TSZ) treatment regulates phenotype of NSCs. In detail, the proliferative capacity of NSCs was significantly decreased in the presence of continual TSZ treatment, and the transcription of proinflammatory genes was upregulated, while the transcription of neurotrophic factors was inhibited. NSCs exhibited an obvious tendency to differentiate into glial cells under short-duration TSZ stimulation (6 h and 12 h); as the stimulus duration increased (24 h), the differentiation ability of the NSCs was significantly inhibited. These phenotypic changes are not conducive to neural cell survival and neural repair. Moreover, we examined the effect of necroptosis inhibitors on TSZ-treated NSCs. Necrostatin-1 and necrosulfonamide significantly reduced the necroptosis of NSCs after TSZ treatment and improved the phenotypic function of NSCs under TSZ stimulation. In additional in vivo experiments, after 2 weeks of administration, the necroptosis inhibitors reduced the necroptosis of NSCs and improved functional recovery in SCI mice. Taken together, these data indicate that the inhibition of NSC necroptosis with necroptosis inhibitors facilitates survival and phenotype maintenance in vitro and contributes to neuroprotection and repair in vivo. Our findings suggest that blocking necroptosis of eNSCs may be a potential therapeutic strategy for treating SCI.

Sequential targeting of YAP1 and p21 enhances the elimination of senescent cells induced by the BET inhibitor JQ1.

Chondrosarcoma (CHS) is the second most common bone malignancy with limited therapeutic approaches. Our previous study has found that Yes associated protein 1 (YAP1) is downregulated in CHS cells treated with bromodomain and extraterminal domain (BET) inhibitor JQ1. However, the precise role of YAP1 in CHS is largely unknown. Herein, we found that YAP1 expression was upregulated in CHS tissues, and positively correlated with its grading score. Loss of YAP1 inhibited CHS proliferation and induced cellular senescence, while expression of YAP1 mutants revealed YAP1/TEA domain family member (TEAD)-dependent negative regulation of p21 and subsequent cellular senescence. These results were validated by in vivo experiments using stable shYAP1 cell lines. Mechanistically, negative regulation of p21 by YAP1 occurred post-transcriptionally via Dicer-regulated miRNA networks, specifically, the miR-17 family. Furthermore, we demonstrated that sequential targeting of YAP1 and p21 enhanced the elimination of JQ1-induced senescent cells in a Bcl-2-like 1 (Bcl-XL)/Caspase-3 dependent manner. Altogether, we unveil a novel role of YAP1 signaling in mediating CHS cell senescence and propose a one-two punch approach that sequentially targets the YAP1/p21 axis to eliminate senescent cells.

TRIM21-regulated Annexin A2 plasma membrane trafficking facilitates osteosarcoma cell differentiation through the TFEB-mediated autophagy.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, which is characterized by dysfunctional autophagy and poor differentiation. Our recent studies have suggested that the tripartite motif containing-21 (TRIM21) plays a crucial role in regulating OS cell senescence and proliferation via interactions with several proteins. Yet, its implication in autophagy and differentiation in OS is largely unknown. In the present study, we first showed that TRIM21 could promote OS cell autophagy, as determined by the accumulation of LC3-II, and the degradation of cargo receptor p62. Further, we were able to identify that Annexin A2 (ANXA2), as a novel interacting partner of TRIM21, was critical for TIRM21-induced OS cell autophagy. Although TRIM21 had a negligible effect on the mRNA and protein expressions of ANXA2, we did find that TRIM21 facilitated the translocation of ANXA2 toward plasma membrane (PM) in OS cells through a manner relying on TRIM21-mediated cell autophagy. This functional link has been confirmed by observing a nice co-expression of TRIM21 and ANXA2 (at the PM) in the OS tissues. Mechanistically, we demonstrated that TRIM21, via facilitating the ANXA2 trafficking at the PM, enabled to release the transcription factor EB (TFEB, a master regulator of autophagy) from the ANXA2-TFEB complex, which in turn entered into the nucleus for the regulation of OS cell autophagy. In accord with previous findings that autophagy plays a critical role in the control of differentiation, we also demonstrated that autophagy inhibited OS cell differentiation, and that the TRIM21/ANXA2/TFEB axis is implicated in OS cell differentiation through the coordination with autophagy. Taken together, our results suggest that the TRIM21/ANXA2/TFEB axis is involved in OS cell autophagy and subsequent differentiation, indicating that targeting this signaling axis might lead to a new clue for OS treatment.

PRMT5-TRIM21 interaction regulates the senescence of osteosarcoma cells by targeting the TXNIP/p21 axis.

Osteosarcoma (OS) is the most common bone malignancy in adolescents and has poor clinical outcomes. Protein arginine methyltransferase 5 (PRMT5) has recently been shown to be aberrantly expressed in various cancers, yet its role in OS remains elusive. Here, we found that PRMT5 was overexpressed in OS and its overexpression predicted poor clinical outcomes. PRMT5 knockdown significantly triggered pronounced senescence in OS cells, as evidenced by the increase in senescence-associated ß-galactosidase (SA-ß-gal)-stained cells, induction of p21 expression, and upregulation of senescence-associated secretory phenotype (SASP) gene expression. In addition, we found that PRMT5 plays a key role in regulating DNA damaging agents-induced OS cell senescence, possibly, via affecting the repair of DNA damage. Furthermore, we found that TXNIP acts as a key factor mediating PRMT5 depletion-induced DNA damage and cellular senescence. Mechanistically, TRIM21, which interacts with PRMT5, was essential for the regulation of TXNIP/p21 expression. In summary, we propose a model in which PRMT5, by interaction with TRIM21, plays a key role in regulating the TXNIP/p21 axis during senescence in OS cells. The present findings suggest that PRMT5 overexpression in OS cells might confer resistance to chemotherapy and that targeting the PRMT5/TRIM21/TXNIP signaling may enhance the therapeutic efficacy in OS.