Deubiquitinase UCHL1 promotes angiogenesis and blood-spinal cord barrier function recovery after spinal cord injury by stabilizing Sox17.

Improving the function of the blood-spinal cord barrier (BSCB) benefits the functional recovery of mice following spinal cord injury (SCI). The death of endothelial cells and disruption of the BSCB at the injury site contribute to secondary damage, and the ubiquitin-proteasome system is involved in regulating protein function. However, little is known about the regulation of deubiquitinated enzymes in endothelial cells and their effect on BSCB function after SCI. We observed that Sox17 is predominantly localized in endothelial cells and is significantly upregulated after SCI and in LPS-treated brain microvascular endothelial cells. In vitro Sox17 knockdown attenuated endothelial cell proliferation, migration, and tube formation, while in vivo Sox17 knockdown inhibited endothelial regeneration and barrier recovery, leading to poor functional recovery after SCI. Conversely, in vivo overexpression of Sox17 promoted angiogenesis and functional recovery after injury. Additionally, immunoprecipitation-mass spectrometry revealed the interaction between the deubiquitinase UCHL1 and Sox17, which stabilized Sox17 and influenced angiogenesis and BSCB repair following injury. By generating UCHL1 conditional knockout mice and conducting rescue experiments, we further validated that the deubiquitinase UCHL1 promotes angiogenesis and restoration of BSCB function after injury by stabilizing Sox17. Collectively, our findings present a novel therapeutic target for treating SCI by revealing a potential mechanism for endothelial cell regeneration and BSCB repair after SCI.

Assessing the utility of MRI-based vertebral bone quality (VBQ) for predicting lumbar pedicle screw loosening.

STUDY DESIGN: Retrospective cohort. OBJECTIVE: The purpose of this study is to assess the potential of utilizing the MRI-based vertebral bone quality (VBQ) score as a predictive tool for pedicle screw loosening (PSL) in patients who have undergone pedicle screw fixation and to identify risk factors associated with VBQ scores. METHODS: One hundred and sixteen patients who had undergone pedicle screw fixation between December 2019 and January 2021 and had more than a year of follow-up were divided into two groups of PSL and non-PSL. The radiological and clinical parameters investigated were age, gender, body mass index, the VBQ score, length of fusion and the DXA T-score. RESULTS: Of the 116 patients included in the study, 22 patients developed pedicle screw loosening after surgery (18.97%). VBQ score of PSL group was higher than the non-PSL group (3.61 ± 0.63 vs. 2. 86 ± 0.43, p < 0.001). According to logistic regression, PSL was independently linked with a higher VBQ score (OR = 3.555, 95% confidence interval [1.620-7.802], p < 0.005). The AUC of predicting screw loosening was 0.774 (p < 0.001) for VBQ score, and the best threshold was 3.055 (sensitivity, 81.8%; specificity, 71.3%). High VBQ score was associated with age (r (114) = 0.29, p = 0.002), while it was not negatively correlated with T-scores of each part. CONCLUSION: VBQ score is an independent predictor of pedicle screw loosening, with higher scores indicating a greater risk. Our results showed that older patients and women had higher VBQ scores.

Do Cervical Parameters Increase the Risk of Thoracic Spinal Stenosis in Patients with Cervical Spinal Stenosis?

OBJECTIVES: The diagnosis and treatment of tandem stenosis have been widely discussed. However, studies on concurrent cervical and thoracic spinal stenosis are rare in the literature. This study was aimed to investigate the risk factors for thoracic spinal stenosis (TSS) in patients with cervical spinal stenosis (CSS). METHODS: This retrospective cohort study assessed the risk factors for TSS in 162 patients who were diagnosed with CSS. Patients were divided into TSS (n = 45) and non-TSS (n = 117) groups. We retrospectively analyzed clinical characteristics and radiographic parameters including age, gender, body mass index, ossification of the cervical posterior longitudinal ligament (OPLLc), hypertrophy of cervical ligamentum flavum (HLFc), cervical stenosis segments, and cervical sagittal parameters. Cervical sagittal parameters were measured on T2-weighted magnetic resonance imaging including C2-7 Cobb angle, cervical tilt, T1 slope, thoracic inlet angle (TIA), C2-C7 sagittal vertical axis (C2-C7), and cervical curvature. RESULTS: Two groups showed significant differences in ossification of the cervical posterior longitudinal ligament, HLFc, cervical stenosis segments, and TIA. The receiver operating characteristic curves demonstrated that the optimal threshold for TIA was 68.25. In multivariate logistic regression analysis, OPLLc (odds ratio [OR] = 4.403, 95% confidence interval [CI] = 1.782-10.880, P = 0.001), HLFc (OR = 4.849, 95% CI = 1.995-11.782, P < 0.001), and TIA >68.25 degrees (OR = 2.555, 95% CI = 1.044-6.251, P = 0.040) were independent risk factors for TSS. Moreover, the multiindex receiver operating characteristic curve demonstrated that the area under the curve for predicted probability was 0.799 (P < 0.001). CONCLUSIONS: Routine thoracic magnetic resonance imaging assessment is strongly recommended in CSS patients with OPLLc, HLFc, and enlarged TIA to screen for neglected but vital thoracic disease.

Exosomal USP13 derived from microvascular endothelial cells regulates immune microenvironment and improves functional recovery after spinal cord injury by stabilizing IκBα.

Spinal cord injury (SCI) can result in irreversible sensory and motor disability with no effective treatment currently. After SCI, infiltrated macrophages accumulate in epicenter through destructed blood-spinal cord barrier (BSCB). Further, great majority of macrophages are preferentially polarized to M1 phenotype, with only a few transient M2 phenotype. The purpose of this study was to explore roles of vascular endothelial cells in microglia/macrophages polarization and the underlying mechanism. Lipopolysaccharide (LPS) was used to pretreat BV2 microglia and RAW264.7 macrophages followed by administration of conditioned medium from microvascular endothelial cell line bEnd.3 cells (ECM). Analyses were then performed to determine the effects of exosomes on microglia/macrophages polarization and mitochondrial function. The findings demonstrated that administration of ECM shifted microglia/macrophages towards M2 polarization, ameliorated mitochondrial impairment, and reduced reactive oxygen species (ROS) production in vitro. Notably, administration of GW4869, an exosomal secretion inhibitor, significantly reversed these observed benefits. Further results revealed that exosomes derived from bEnd.3 cells (Exos) promote motor rehabilitation and M2 polarization of microglia/macrophages in vivo. Ubiquitin-specific protease 13 (USP13) was shown to be significantly enriched in BV2 microglia treated with Exos. USP13 binds to, deubiquitinates and stabilizes the NF-κB inhibitor alpha (IκBα), thus regulating microglia/macrophages polarization. Administration of the selective IκBα inhibitor betulinic acid (BA) inhibited the beneficial effect of Exos in vivo. These findings uncovered the potential mechanism underlying the communications between vascular endothelial cells and microglia/macrophages after SCI. In addition, this study indicates exosomes might be a promising therapeutic strategy for SCI treatment.

USP1/UAF1-Stabilized METTL3 Promotes Reactive Astrogliosis and Improves Functional Recovery after Spinal Cord Injury through m6A Modification of YAP1 mRNA.

RNA N6-methyladenosine (m6A) modification is involved in diverse biological processes. However, its role in spinal cord injury (SCI) is poorly understood. The m6A level increases in injured spinal cord, and METTL3, which is the core subunit of methyltransferase complex, is upregulated in reactive astrocytes and further stabilized by the USP1/UAF1 complex after SCI. The USP1/UAF1 complex specifically binds to and subsequently removes K48-linked ubiquitination of the METTL3 protein to maintain its stability after SCI. Moreover, conditional knockout of astrocytic METTL3 in both sexes of mice significantly suppressed reactive astrogliosis after SCI, thus resulting in widespread infiltration of inflammatory cells, aggravated neuronal loss, hampered axonal regeneration, and impaired functional recovery. Mechanistically, the YAP1 transcript was identified as a potential target of METTL3 in astrocytes. METTL3 could selectively methylate the 3'-UTR region of the YAP1 transcript, which subsequently maintains its stability in an IGF2BP2-dependent manner. In vivo, YAP1 overexpression by adeno-associated virus injection remarkably contributed to reactive astrogliosis and partly reversed the detrimental effects of METTL3 knockout on functional recovery after SCI. Furthermore, we found that the methyltransferase activity of METTL3 plays an essential role in reactive astrogliosis and motor repair, whereas METTL3 mutant without methyltransferase function failed to promote functional recovery after SCI. Our study reveals the previously unreported role of METTL3-mediated m6A modification in SCI and might provide a potential therapy for SCI.SIGNIFICANCE STATEMENT Spinal cord injury is a devastating trauma of the CNS involving motor and sensory impairments. However, epigenetic modification in spinal cord injury is still unclear. Here, we propose an m6A regulation effect of astrocytic METTL3 following spinal cord injury, and we further characterize its underlying mechanism, which might provide promising strategies for spinal cord injury treatment.

Engineered extracellular vesicles for delivery of siRNA promoting targeted repair of traumatic spinal cord injury.

Spinal cord injury (SCI) is a severe disease of the nervous system that causes irreparable damage and loss of function, for which no effective treatments are available to date. Engineered extracellular vesicles (EVs) carrying therapeutic molecules hold promise as an alternative SCI therapy depending on the specific functionalized EVs and the appropriate engineering strategy. In this study, we demonstrated the design of a drug delivery system of peptide CAQK-modified, siRNA-loaded EVs (C-EVs-siRNA) for SCI-targeted therapy. The peptide CAQK was anchored through a chemical modification to the membranes of EVs isolated from induced neural stem cells (iNSCs). CCL2-siRNA was then loaded into the EVs through electroporation. The modified EVs still maintained the basic properties of EVs and showed favorable targeting and therapeutic effects in vitro and in vivo. C-EVs-siRNA specifically delivered siRNA to the SCI region and was taken up by target cells. C-EVs-siRNA used the inherent anti-inflammatory and neuroreparative functions of iNSCs-derived EVs in synergy with the loaded siRNA, thus enhancing the therapeutic effect against SCI. The combination of targeted modified EVs and siRNA effectively regulated the microenvironmental disturbance after SCI, promoted the transformation of microglia/macrophages from M1 to M2 and limited the negative effects of the inflammatory response and neuronal injury on functional recovery in mice after SCI. Thus, engineered EVs are a potentially feasible and efficacious treatment for SCI, and may also be used to develop targeted treatments for other diseases.

TSG-6 released from adipose stem cells-derived small extracellular vesicle protects against spinal cord ischemia reperfusion injury by inhibiting endoplasmic reticulum stress.

BACKGROUND: Spinal cord ischemia reperfusion injury (SCIRI) is a complication of aortic aneurysm repair or spinal cord surgery that is associated with permanent neurological deficits. Mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) have been shown to be potential therapeutic options for improving motor functions after SCIRI. Due to their easy access and multi-directional differentiation potential, adipose-derived stem cells (ADSCs) are preferable for this application. However, the effects of ADSC-derived sEVs (ADSC-sEVs) on SCIRI have not been reported. RESULTS: We found that ADSC-sEVs inhibited SCIRI-induced neuronal apoptosis, degradation of tight junction proteins and suppressed endoplasmic reticulum (ER) stress. However, in the presence of the ER stress inducer, tunicamycin, its anti-apoptotic and blood-spinal cord barrier (BSCB) protective effects were significantly reversed. We found that ADSC-sEVs contain tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) whose overexpression inhibited ER stress in vivo by modulating the PI3K/AKT pathway. CONCLUSIONS: ADSC-sEVs inhibit neuronal apoptosis and BSCB disruption in SCIRI by transmitting TSG-6, which suppresses ER stress by modulating the PI3K/AKT pathway.

Radiographic predictors for recurrence of lumbar symptoms after prioritized cervical surgery in patients with tandem spinal stenosis.

OBJECTIVES: The purpose of the current study was to explore radiographic predictors for recurrence of lumbar symptoms after prioritized cervical surgery in patients with tandem spinal stenosis (TSS). METHODS: The current retrospective cohort study included 74 patients with TSS, who underwent prioritized cervical surgery. Based on presence or absence of improvement in lower limb symptoms, patients were grouped into improved and non-improved groups. Medical records and radiological parameters including age, sex, body mass index, cervical and lumbar parameters were analyzed. In improved group, patients were divided into relapsed and non-relapsed groups based on recurrence in lower limb symptoms. RESULTS: Lumbar symptoms improved in 70.1% (n = 52) of patients. Comparison between the improved and non-improved group showed that there were no statistically significant differences in cervical parameters while comparisons between the relapsed and non-relapsed groups showed significant differences in redundant nerve roots (RNRs) (p = 0.029), narrow segment (p = 0.042) and lumbar stenosis index (LSI) (p = 0.003). In multivariate logistic regression analysis, LSI > 10 (p = 0.016) was independently associated with recurrence of lumbar symptoms. CONCLUSIONS: Finding of the current study indicated that LSI > 10 was associated with recurrence of lumbar symptoms in TSS patients following cervical surgery.

TRIM22 inhibits osteosarcoma progression through destabilizing NRF2 and thus activation of ROS/AMPK/mTOR/autophagy signaling.

Osteosarcoma (OS) is a malignant bone tumor that mainly occurs in adolescents. It is accompanied by a high rate of lung metastasis, and high mortality. Recent studies have suggested the important roles of tripartite motif-containing (TRIM) family proteins in regulating various substrates and signaling pathways in different tumors. However, the detailed functional role of TRIM family proteins in the progression of OS is still unknown and requires further investigations. In this study, we found that tripartite motif-containing 22 (TRIM22) was downregulated in OS tissues and was hence associated with better prognosis. In vitro and in vivo functional analysis demonstrated that TRIM22 inhibits proliferation and metastasis of OS cells. Nuclear factor erythroid 2-related factor 2 (NRF2), a redox regulator, was identified as a novel target for TRIM22. TRIM22 interacts with and accelerates the degradation of NRF2 by inducing its ubiquitination dependent on its E3 ligase activity but independent of Kelch-like ECH-associated protein 1 (KEAP1). Further, a series of gain- and loss-of-function experiments showed that knockdown or overexpression of NRF2 reversed the functions of knockdown or overexpression of TRIM22 in OS. Mechanistically, TRIM22 inhibited OS progression through NRF2-mediated intracellular reactive oxygen species (ROS) imbalance. ROS production was significantly promoted and mitochondrial potential was remarkably inhibited when overexpressing TRIM22, thus activating AMPK/mTOR signaling. Moreover, TRIM22 was also found to inhibit Warburg effect in OS cells. Autophagy activation was found in OS cells which were overexpressed TRIM22, thus leading to autophagic cell death. Treatment with N-Acetylcysteine (NAC), a ROS scavenger or the autophagy inhibitor 3-Methyladenine (3-MA) abolished the decreased malignant phenotypes in TRIM22 overexpressing OS cells. In conclusion, our study indicated that TRIM22 inhibits OS progression by promoting proteasomal degradation of NRF2 independent of KEAP1, thereby activating ROS/AMPK/mTOR/Autophagy signaling that leads to autophagic cell death in OS. Therefore, our findings indicated that targeting TRIM22/NRF2 could be a promising therapeutic target for treating OS.

Analysis of Functional Outcomes and Risk Factors for Facet Joint Distraction During Anterior Cervical Discectomy and Fusion for Cervical Spondylotic Myelopathy.

OBJECTIVE: This study aimed to clarify functional outcomes of facet joint distraction (FJD) and identify specific risk factors for excessive FJD during single-level anterior cervical discectomy and fusion (ACDF) for cervical spondylotic myelopathy (CSM). METHODS: This study retrospectively analyzed 100 patients who underwent single-level ACDF for CSM from January 2016 to May 2020. Anteroposterior and lateral radiographs were obtained before surgery and 12 months after surgery. Radiographic parameters including anterior intervertebral height (AIH), posterior intervertebral height, facet joint gap, cage posterior depth (CPD), upper vertebral length, cervical segmental Cobb angle (CSCA), C2-C7 Cobb angle, and C2-C7 sagittal vertical axis were analyzed. Functional outcomes were evaluated using the modified Japanese Orthopedic Association Score, visual analog scale (VAS), and Neck Disability Index (NDI). RESULTS: Comparison between the appropriate FJD and excessive FJD groups showed statistically significant differences in the NDI, VAS, CPD, and ΔAIH (P < 0.05). Multivariate logistic regression analysis showed that independent factors associated with excessive FJD were as follows: a ΔAIH > 2.28 mm (odds ratio [OR] = 6.792, 95% confidence interval [CI] = 1.885-24.470, P = 0.003), CPD > 12.45 mm (OR = 5.876, 95% CI = 1.828-18.895, P = 0.003), and post-CSCA < 0° (OR = 6.251, 95% CI = 1.275-30.633, P = 0.024). Furthermore, receiver operating characteristic curve analysis for the multilevel logistic regression model produced an area under the curve of 0.783 (P < 0.001). CONCLUSION: Patients with an FJD of >0.905 mm had worse NDI and VAS pain scores, but not a poorer modified Japanese Orthopedic Association Score recovery rate. Our findings suggested that a ΔAIH > 2.28 mm, CPD > 12.45 mm, and post-CSCA < 0° were independent risk factors for excessive FJD after single-level ACDF for CSM.

USP11 regulates autophagy-dependent ferroptosis after spinal cord ischemia-reperfusion injury by deubiquitinating Beclin 1.

Spinal cord ischemia-reperfusion injury (SCIRI) is a serious trauma that can lead to loss of sensory and motor function. Ferroptosis is a new form of regulatory cell death characterized by iron-dependent accumulation of lipid peroxides. Ferroptosis has been studied in various diseases; however, the exact function and molecular mechanism of ferroptosis in SCIRI remain unknown. In this study, we demonstrated that ferroptosis is involved in the pathological mechanism of SCIRI. Inhibition of ferroptosis could promote the recovery of motor function in mice after SCIRI. In addition, we found that ubiquitin-specific protease 11 (USP11) was significantly upregulated in neuronal cells after hypoxia-reoxygenation and in the spinal cord in mice with I/R injury. Knockdown of USP11 in vitro and KO of USP11 in vivo (USP11-/Y) significantly decreased neuronal cell ferroptosis. In mice, this promotes functional recovery after SCIRI. In contrast, in vitro, USP11 overexpression leads to classic ferroptosis events. Overexpression of USP11 in mice resulted in increased ferroptosis and poor functional recovery after SCIRI. Interestingly, upregulating the expression of USP11 also appeared to increase the production of autophagosomes and to cause substantial autophagic flux, a potential mechanism through which USP11 may enhance ferroptosis. The decreased autophagy markedly weakened the ferroptosis mediated by USP11 and autophagy induction had a synergistic effect with USP11. Importantly, USP11 promotes autophagy activation by stabilizing Beclin 1, thereby leading to ferroptosis. In conclusion, this study shows that ferroptosis is closely associated with SCIRI, and that USP11 plays a key role in regulating ferroptosis and additionally identifies USP11-mediated autophagy-dependent ferroptosis as a promising target for the treatment of SCIRI.

Small extracellular vesicles encapsulating CCL2 from activated astrocytes induce microglial activation and neuronal apoptosis after traumatic spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a severe traumatic disease which causes high disability and mortality rates. The molecular pathological features after spinal cord injury mainly involve the inflammatory response, microglial and neuronal apoptosis, abnormal proliferation of astrocytes, and the formation of glial scars. However, the microenvironmental changes after spinal cord injury are complex, and the interactions between glial cells and nerve cells remain unclear. Small extracellular vesicles (sEVs) may play a key role in cell communication by transporting RNA, proteins, and bioactive lipids between cells. Few studies have examined the intercellular communication of astrocytes through sEVs after SCI. The inflammatory signal released from astrocytes is known to initiate microglial activation, but its effects on neurons after SCI remain to be further clarified. METHODS: Electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting were applied to characterize sEVs. We examined microglial activation and neuronal apoptosis mediated by astrocyte activation in an experimental model of acute spinal cord injury and in cell culture in vitro. RESULTS: Our results indicated that astrocytes activated after spinal cord injury release CCL2, act on microglia and neuronal cells through the sEV pathway, and promote neuronal apoptosis and microglial activation after binding the CCR2. Subsequently, the activated microglia release IL-1ß, which acts on neuronal cells, thereby further aggravating their apoptosis. CONCLUSION: This study elucidates that astrocytes interact with microglia and neurons through the sEV pathway after SCI, enriching the mechanism of CCL2 in neuroinflammation and spinal neurodegeneration, and providing a new theoretical basis of CCL2 as a therapeutic target for SCI.

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

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 M2-like 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 (m6 A) modification and levels of the m6 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.

Prevalence and Predictive Factors of Asymptomatic Spondylotic Cervical Spinal Stenosis in Patients with Symptomatic Lumbar Spinal Stenosis.

OBJECTIVE: We performed a retrospective cohort study to investigate the prevalence of and risk factors for asymptomatic spondylotic cervical spinal stenosis (ASCSS) in the setting of lumbar spinal stenosis (LSS). METHODS: A total of 114 patients with a diagnosis of LSS without cervical myelopathy and radiculopathy were grouped into ASCSS and non-ASCSS groups. The medical data and radiological parameters, including age, sex, body mass index, Charlson comorbidity index, symptom duration, redundant nerve roots, dural sac cross-sectional area (DCSA), facet joint angle, lumbar lordosis angle (LLA), pelvic incidence (PI), Torg-Pavlov ratio, and lumbosacral transitional vertebrae, were analyzed. The lumbar stenosis index and cervical stenosis index of the 114 patients were also analyzed. RESULTS: ASCSS occurred in 70 of the 114 patients with LSS (61.4%). The two groups showed significant differences in symptom duration, redundant nerve roots, LLA, DCSA, and PI. On multivariate logistic regression analysis, an LLA >35.85° (P < 0.001) and a DCSA <84.50 mm2 (P = 0.003) were independently associated with ASCSS. The multi-index receiver operating characteristic curve showed that the area under the curve for predicted probability was 0.805 (P < 0.001). Linear regression analysis revealed that cervical stenosis index significantly and positively correlated with the lumbar stenosis index (r = 0.430; P < 0.001). CONCLUSIONS: Our findings suggest that an LLA >35.85° and a DCSA <84.50 mm2 are risk factors for the development of ASCSS. For LSS patients with an enlarged LLA and reduced DSCA, a whole spinal magnetic resonance imaging examination should be performed.