Dissecting Genetic Mechanisms of Differential Locomotion, Depression, and Allodynia after Spinal Cord Injury in Three Mouse Strains.

Strain differences have been reported for motor behaviors, and only a subset of spinal cord injury (SCI) patients develop neuropathic pain, implicating genetic or genomic contribution to this condition. Here, we evaluated neuropsychiatric behaviors in A/J, BALB/c, and C57BL/6 male mice and tested genetic or genomic alterations following SCI. A/J and BALB/c naive mice showed significantly less locomotor activity and greater anxiety-like behavior than C57BL/6 mice. Although SCI elicited locomotor dysfunction, C57BL/6 and A/J mice showed the best and the worst post-traumatic recovery, respectively. Mild (m)-SCI mice showed deficits in gait dynamics. All moderate/severe SCI mice exhibited similar degrees of anxiety/depression. mSCI in BALB/c and A/J mice resulted in depression, whereas C57BL/6 mice did not exhibit depression. mSCI mice had significantly lower mechanical thresholds than their controls, indicating high cutaneous hypersensitivity. C57BL/6, but not A/J and BLAB/c mice, showed significantly lower heat thresholds than their controls. C57BL/6 mice exhibited spontaneous pain. RNAseq showed that genes in immune responses and wound healing were upregulated, although A/J mice showed the largest increase. The cell cycle and the truncated isoform of trkB genes were robustly elevated in SCI mice. Thus, different genomics are associated with post-traumatic recovery, underscoring the likely importance of genetic factors in SCI.

Hspb1 and Lgals3 in spinal neurons are closely associated with autophagy following excitotoxicity based on machine learning algorithms.

Excitotoxicity represents the primary cause of neuronal death following spinal cord injury (SCI). While autophagy plays a critical and intricate role in SCI, the specific mechanism underlying the relationship between excitotoxicity and autophagy in SCI has been largely overlooked. In this study, we isolated primary spinal cord neurons from neonatal rats and induced excitotoxic neuronal injury by high concentrations of glutamic acid, mimicking an excitotoxic injury model. Subsequently, we performed transcriptome sequencing. Leveraging machine learning algorithms, including weighted correlation network analysis (WGCNA), random forest analysis (RF), and least absolute shrinkage and selection operator analysis (LASSO), we conducted a comprehensive investigation into key genes associated with spinal cord neuron injury. We also utilized protein-protein interaction network (PPI) analysis to identify pivotal proteins regulating key gene expression and analyzed key genes from public datasets (GSE2599, GSE20907, GSE45006, and GSE174549). Our findings revealed that six genes-Anxa2, S100a10, Ccng1, Timp1, Hspb1, and Lgals3-were significantly upregulated not only in vitro in neurons subjected to excitotoxic injury but also in rats with subacute SCI. Furthermore, Hspb1 and Lgals3 were closely linked to neuronal autophagy induced by excitotoxicity. Our findings contribute to a better understanding of excitotoxicity and autophagy, offering potential targets and a theoretical foundation for SCI diagnosis and treatment.

SP1 transcriptionally activates HTR2B to aggravate traumatic spinal cord injury by shifting microglial M1/M2 polarization.

BACKGROUND: Spinal cord injury (SCI) can result in structural and functional damage to the spinal cord, which may lead to loss of limb movement and sensation, loss of bowel and bladder control, and other complications. Previous studies have revealed the critical influence of trans-acting transcription factor 1 (SP1) in neurological pathologies, however, its role and mechanism in SCI have not been fully studied. METHODS: The study was performed using mouse microglia BV2 stimulated using lipopolysaccharide (LPS) and male adult mice subjected to spinal hitting. Western blotting was performed to detect protein expression of SP1, 5-hydroxytryptamine (serotonin) receptor 2B (HTR2B), BCL2-associated x protein (Bax), B-cell lymphoma-2 (Bcl-2), inducible nitric oxide synthase (iNOS), clusters of differentiation 86 (CD86), Arginase 1 (Arg-1) and clusters of differentiation 206 (CD206). Cell viability and apoptosis were analyzed by MTT assay and TUNEL assay. mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-4 (IL-4) and tumor necrosis factor-ß (TNF-ß) were quantified by quantitative real-time polymerase chain reaction. The association of SP1 and HTR2B was identified by chromatin immunoprecipitation assay and dual-luciferase reporter assay. HE staining assay was performed to analyze the pathological conditions of spinal cord tissues. RESULTS: LPS treatment induced cell apoptosis and inhibited microglia polarization from M1 to M2 phenotype, accompanied by an increase of Bax protein expression and a decrease of Bcl-2 protein expression, however, these effects were relieved after SP1 silencing. Mechanism assays revealed that SP1 transcriptionally activated HTR2B in BV2 cells, and HTR2B knockdown rescued LPS-induced effects on BV2 cell apoptosis and microglial M1/M2 polarization. Moreover, SP1 absence inhibited BV2 cell apoptosis and promoted microglia polarization from M1 to M2 phenotype by decreasing HTR2B expression. SCI mouse model assay further showed that SP1 downregulation could attenuate spinal hitting-induced promoting effects on cell apoptosis of spinal cord tissues and microglial M1 polarization. CONCLUSION: SP1 transcriptionally activated HTR2B to aggravate traumatic SCI by shifting microglial M1/M2 polarization.

Small nucleolar RNA host gene 1 silence inhibits the lipopolysaccharide-induced microglial apoptosis and inflammation.

Microglia activation is an early mediator of neuroinflammation and a major contributor to spinal damage and motor dysfunction. This study was designed to investigate the role of small nucleolar RNA host gene 1 (SNHG1) on the apoptosis and inflammatory response of microglial cell BV-2 and its underlying molecular mechanism. The C5 lamina contusion-induced mouse model of spinal cord injury (SCI) was constructed. Mouse microglia BV2 was stimulated by lipopolysaccharide (LPS) to establish the in vitro model of SCI. The quantitative reverse transcription polymerase chain reaction method was used to quantify RNA expression levels. Enzyme-linked immunosorbent assays were used to quantify concentrations of inflammatory cytokines. Protein levels were assessed by western blotting, and apoptosis was assessed by flow cytometry. Dual luciferase reporter gene assay and RNA pull-down assay were conducted to investigate the binding relationships between molecules. Upregulation of SNHG1 and downregulation of miR-195-5p were observed in the spinal cords of SCI mouse model. LPS treatment led to elevation of SNHG1 expression in BV2 cells, as well as accelerated apoptosis and inflammation. Evident mitigation of LPS-induced BV2 cell damage was observed after SNHG1 knockdown. MiR-195-5p was identified as a target of SNHG1. Inhibition of miR-195-5p restored the impact of SNHG1 knockdown on cell damage of LPS-treated BV2 cells. Furthermore, miR-195-5p can target activating transcription factor-6 (ATF6). In summary, SNHG1 knockdown ameliorates LPS-induced microglial apoptosis and inflammatory response via the miR-195-5p/ATF6 axis, providing a novel direction for SCI treatment.

TP53INP2 knockdown inhibits inflammatory response and apoptosis after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a traumatic neurological disorder with limited therapeutic options. Tumor protein p53-inducible nuclear protein 2 (TP53INP2) is involved in the occurrence and development of various diseases, and it may play a role during SCI via affecting inflammation and neuronal apoptosis. This study investigated the associated roles and mechanisms of TP53INP2 in SCI. METHODS: Mouse and lipopolysaccharide (LPS)-induced SCI BV-2 cell models were constructed to explore the role of TP53INP2 in SCI and the associated mechanisms. Histopathological evaluation of spinal cord tissue was detected by hematoxylin and eosin staining. The Basso, Beattie, and Bresnahan score was used to measure the motor function of the mice, while the spinal cord water content was used to assess spinal cord edema. The expression of TP53INP2 was measured using RT-qPCR. In addition, inflammatory factors in the spinal cord tissue of SCI mice and LPS-treated BV-2 cells were measured using enzyme-linked immunosorbent assay. Apoptosis and related protein expression levels were detected by flow cytometry and western blot analysis, respectively. RESULTS: TP53INP2 levels increased in SCI mice and LPS-treated BV-2 cells. The results of in vivo and in vitro experiments showed that TP53INP2 knockdown inhibited the inflammatory response and neuronal apoptosis in mouse spinal cord tissue or LPS-induced BV-2 cells. CONCLUSIONS: After spinal cord injury, TP53INP2 was upregulated, and TP53INP2 knockdown inhibited the inflammatory response and apoptosis.

Identification of hub genes and key pathways in spinal cord injury via bioinformatics analysis.

This study aimed to explore the hub genes and related key pathways in Spinal Cord Injury (SCI) based on the bioinformatics analysis. Two microarray datasets (GSE45006, GSE45550) were obtained from the GEO database and were merged and batch-corrected. The differentially expressed genes (DEGs) in SCI were explored with the Limma, and the weighted gene co-expression network analysis (WGCNA) was conducted to explore the module genes. Functional enrichment analysis and Gene set variation analysis (GSVA) were used to investigate the biological functions and key pathways of the key genes related to SCI. Then the protein-protein interaction (PPI) network was generated using the STING online tool, and the hub genes in SCI were identified. Receiver operating characteristic (ROC) curves were applied to assess the diagnostic value of the selected hub genes. We identified 554 DEGs in SCI, and 1236 key genes in SCI were selected via WGCNA. Totally 111 key genes related to SCI were discovered. Furthermore, the functional enrichment analysis showed that these key mRNAs were primarily enriched in the extracellular matrix (ECM)-related pathways and processes associated with wound healing and cell growth. The PPI network further filtered six hub genes (Cd44, Timp1, Loxl1, Col6a1, Col3a1, Col5a1) ranked by the degree, and the diagnostic value of the six hub genes was confirmed by the ROC curves. Six hub genes including Cd44, Timp1, Loxl1, Col6a1, Col3a1, and Col5a1 were identified in SCI, with differential expression and excellent diagnostic value, which might provide insight into the targeted therapy of SCI.

Conditioned medium-enriched umbilical cord mesenchymal stem cells: a potential therapeutic strategy for spinal cord injury, unveiling transcriptomic and secretomic insights.

INTRODUCTION: Spinal cord injury (SCI) leads to significant destruction of nerve tissue, causing the degeneration of axons and the formation of cystic cavities. This study aimed to examine the characteristics of human umbilical cord-derived mesenchymal stem cells (HUCMSCs) cultured in a serum-free conditioned medium (CM) and assess their effectiveness in a well-established hemitransection SCI model. MATERIALS AND METHODS: In this study, HUCMSCs cultured medium was collected and characterized by measuring IL-10 and identifying proteomics using mass spectroscopy. This collected serum-free CM was further used in the experiments to culture and characterize the HUMSCs. Later, neuronal cells derived from CM-enriched HUCMSC were tested sequentially using an injectable caffeic acid-bioconjugated gelatin (CBG), which was further transplanted in a hemitransection SCI model. In vitro, characterization of CM-enriched HUCMSCs and differentiated neuronal cells was performed using flow cytometry, immunofluorescence, electron microscopy, and post-transplant analysis using immunohistology analysis, qPCR, in vivo bioluminescence imaging, and behavioral analysis using an infrared actimeter. RESULTS: The cells that were cultured in the conditioned media produced a pro-inflammatory cytokine called IL-10. Upon examining the secretome of the conditioned media, the Kruppel-like family of KRAB and zinc-finger proteins (C2H2 and C4) were found to be activated. Transcriptome analysis also revealed an increased expression of ELK-1, HOXD8, OTX2, YY1, STAT1, ETV7, and PATZ1 in the conditioned media. Furthermore, the expression of Human Stem-101 confirmed proliferation during the first 3 weeks after transplantation, along with the migration of CBG-UCNSC cells within the transplanted area. The gene analysis showed increased expression of Nestin, NeuN, Calb-2, Msi1, and Msi2. The group that received CBG-UCNSC therapy showed a smooth recovery by the end of week 2, with most rats regaining their walking abilities similar to those before the spinal cord injury by week 5. CONCLUSIONS: In conclusion, the CBG-UCNSC method effectively preserved the integrity of the transplanted neuronal-like cells and improved locomotor function. Thus, CM-enriched cells can potentially reduce biosafety risks associated with animal content, making them a promising option for clinical applications in treating spinal cord injuries.

ATF3 is a neuron-specific biomarker for spinal cord injury and ischaemic stroke.

BACKGROUND: Although many molecules have been investigated as biomarkers for spinal cord injury (SCI) or ischemic stroke, none of them are specifically induced in central nervous system (CNS) neurons following injuries with low baseline expression. However, neuronal injury constitutes a major pathology associated with SCI or stroke and strongly correlates with neurological outcomes. Biomarkers characterized by low baseline expression and specific induction in neurons post-injury are likely to better correlate with injury severity and recovery, demonstrating higher sensitivity and specificity for CNS injuries compared to non-neuronal markers or pan-neuronal markers with constitutive expressions. METHODS: In animal studies, young adult wildtype and global Atf3 knockout mice underwent unilateral cervical 5 (C5) SCI or permanent distal middle cerebral artery occlusion (pMCAO). Gene expression was assessed using RNA-sequencing and qRT-PCR, while protein expression was detected through immunostaining. Serum ATF3 levels in animal models and clinical human samples were measured using commercially available enzyme-linked immune-sorbent assay (ELISA) kits. RESULTS: Activating transcription factor 3 (ATF3), a molecular marker for injured dorsal root ganglion sensory neurons in the peripheral nervous system, was not expressed in spinal cord or cortex of naïve mice but was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Additionally, ATF3 protein levels in mouse blood significantly increased 1 day after SCI or ischemic stroke. Importantly, ATF3 protein levels in human serum were elevated in clinical patients within 24 hours after SCI or ischemic stroke. Moreover, Atf3 knockout mice, compared to the wildtype mice, exhibited worse neurological outcomes and larger damage regions after SCI or ischemic stroke, indicating that ATF3 has a neuroprotective function. CONCLUSIONS: ATF3 is an easily measurable, neuron-specific biomarker for clinical SCI and ischemic stroke, with neuroprotective properties. HIGHLIGHTS: ATF3 was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Serum ATF3 protein levels are elevated in clinical patients within 24 hours after SCI or ischemic stroke. ATF3 exhibits neuroprotective properties, as evidenced by the worse neurological outcomes and larger damage regions observed in Atf3 knockout mice compared to wildtype mice following SCI or ischemic stroke.

Long non-coding RNA H19 mediates osteogenic differentiation of bone marrow mesenchymal stem cells through the miR-29b-3p/DKK1 axis.

Single immobilization theory cannot fully account for the extensive bone loss observed after spinal cord injury (SCI). Bone marrow mesenchymal stem cells (BMSCs) are crucial in bone homeostasis because they possess self-renewal capabilities and various types of differentiation potential. This study aimed to explore the molecular mechanism of long non-coding RNA H19 in osteoporosis after SCI and provide new research directions for existing prevention strategies. We used small interfering RNA to knockdown H19 expression and regulated miR-29b-2p expression using miR-29b-3p mimetics and inhibitors. Western blotting, real-time fluorescence quantitative PCR, Alizarin red staining, alkaline phosphatase staining and double-luciferase reporter gene assays were used to assess gene expression, osteogenic ability and binding sites. lncRNA H19 was upregulated in BMSCs from the osteoporosis group, whereas miR-29b-3p was downregulated. We identified the binding sites between miR-29b-3p and lncRNAs H19 and DKK1. H19 knockdown promoted BMSCs' osteogenic differentiation, whereas miR-29b-3p inhibition attenuated this effect. We discovered potential binding sites for miR-29b-3p in lncRNAs H19 and DKK1. Our findings suggest that long non-coding RNA H19 mediates BMSCs' osteogenic differentiation in osteoporosis after SCI through the miR-29b-3p/DKK1 axis and by directly inhibiting the ß-catenin signalling pathway.

CircRNA CDR1as affects functional repair after spinal cord injury and regulates fibrosis through the SMAD pathway.

Spinal cord injury (SCI) is a complex problem in modern medicine. Fibroblast activation and fibroscarring after SCI impede nerve recovery. Non-coding RNA plays an important role in the progression of many diseases, but the study of its role in the progression of spinal fibrosis is still emerging. Here, we investigated the function of circular RNAs, specifically antisense to the cerebellar degeneration-related protein 1 (CDR1as), in spinal fibrosis and characterized its molecular mechanism and pathophysiology. The presence of CDR1as in the spinal cord was verified by sequencing and RNA expression assays. The effects of inhibition of CDR1as on scar formation, inflammation and nerve regeneration after spinal cord injury were investigated in vivo and in vitro. Further, gene expression of miR-7a-5p and protein expression of transforming Growth Factor Beta Receptor II (TGF-ßR2) were measured to evaluate their predicted interactions with CDR1as. The regulatory effects and activation pathways were subsequently verified by miR-7a-5p inhibitor and siCDR1as. These results indicate that CDR1as/miR-7a-5p/TGF-ßR2 interactions may exert scars and nerves functions and suggest potential therapeutic targets for treating spinal fibrotic diseases.

Exosomes derived from vMIP-II-Lamp2b gene-modified M2 cells provide neuroprotection by targeting the injured spinal cord, inhibiting chemokine signals and modulating microglia/macrophage polarization in mice.

Inflammation is one of the key injury factors for spinal cord injury (SCI). Exosomes (Exos) derived from M2 macrophages have been shown to inhibit inflammation and be beneficial in SCI animal models. However, lacking targetability restricts their application prospects. Considering that chemokine receptors increase dramatically after SCI, viral macrophage inflammatory protein II (vMIP-II) is a broad-spectrum chemokine receptor binding peptide, and lysosomal associated membrane protein 2b (Lamp2b) is the key membrane component of Exos, we speculated that vMIP-II-Lamp2b gene-modified M2 macrophage-derived Exos (vMIP-II-Lamp2b-M2-Exo) not only have anti-inflammatory properties, but also can target the injured area by vMIP-II. In this study, using a murine contusive SCI model, we revealed that vMIP-II-Lamp2b-M2-Exo could target the chemokine receptors which highly expressed in the injured spinal cords, inhibit some key chemokine receptor signaling pathways (such as MAPK and Akt), further inhibit proinflammatory factors (such as IL-1ß, IL-6, IL-17, IL-18, TNF-α, and iNOS), and promote anti-inflammatory factors (such as IL-4 and Arg1) productions, and the transformation of microglia/macrophages from M1 into M2. Moreover, the improved histological and functional recoveries were also found. Collectively, our results suggest that vMIP-II-Lamp2b-M2-Exo may provide neuroprotection by targeting the injured spinal cord, inhibiting some chemokine signals, reducing proinflammatory factor production and modulating microglia/macrophage polarization.

PTPRO inhibition ameliorates spinal cord injury through shifting microglial M1/M2 polarization via the NF-κB/STAT6 signaling pathway.

Spinal cord injury (SCI) induces severe neuroinflammation, and subsequently neurological dysfunction. Activated microglia are critical for modulation of neuroinflammation. Protein tyrosine phosphatase receptor type O (PTPRO), a member of protein tyrosine phosphatases (PTPs), exerts a pro-inflammatory role in multiple human diseases; however, its role in SCI remains unclarified. Here, a T7 spinal cord compression injury model was established in Sprague-Dawley (SD) rats, and PTPRO expression was upregulated in injured spinal cord and microglia after SCI. Microglia M1 and M2 polarization in vitro were induced using LPS/IFN-γ and IL-4, respectively. PTPRO expression was elevated in M1-polarized microglia, and PTPRO downregulation mediated by PTPRO shRNA (shPTPRO) decreased CD86+ cell proportion, iNOS, TNF-α, IL-1ß, and IL-6 levels, and p65 phosphorylation. PTPRO was downregulated in M2 microglia, and PTPRO upregulation by PTPRO overexpression plasmid (OE-PTPRO) reduced CD206+ cell percentage, Arg-1, IL-10, and TGF-ß1 levels and STAT6 phosphorylation. Mechanistically, the transcription factor SOX4 elevated PTPRO expression and its promoter activity. SOX4 overexpression enhanced M1 polarization and p65 phosphorylation, while its knockdown promoted M2 polarization and STAT6 phosphorylation. PTPRO might mediate the function of SOX4 in BV2 microglia polarization. Furthermore, lentivirus-mediated downregulation of PTPRO following SCI improved locomotor functional recovery, demonstrated by elevated BBB scores, incline angle, consistent hindlimb coordination, and reduced lesion area and neuronal apoptosis. PTPRO downregulation promoted microglia M2 polarization, NF-κB inactivation and STAT6 activation after injury. In conclusion, PTPRO inhibition improves spinal cord injury through facilitating M2 microglia polarization via the NF-κB/STAT6 signaling pathway, which is probably controlled by SOX4.

MSC-derived exosomes deliver ZBTB4 to mediate transcriptional repression of ITIH3 in astrocytes in spinal cord injury.

BACKGROUND: BMSC-secreted exosomes (BMSC-Exos) have shown potential for promoting behavioral recovery following spinal cord injury (SCI). However, its role in blocking astrocyte activation remains unclear. Thus, this study aimed to determine whether BMSC-Exos impair the function of astrocytes following SCI in mice and to seek the mechanism. METHODS: BMSC-Exos were collected by ultracentrifugation and identified. The SCI mice were developed by laminectomy combined with spinal cord shock, followed by BMSC-Exos or nerve growth factor (positive control) treatment. HE staining, Nissl staining, and TUNEL were conducted to analyze the pathological structural damage and neuronal damage in the mouse spinal cord. Bioinformatics was used to screen altered molecules under the BMSC-Exos treatment. Effects of BMSC-Exos and changes in ZBTB4 and ITIH3 expression on neuronal damage induced by activated astrocytes in the co-culture system were analyzed by CCK-8 and flow cytometry. RESULTS: Nerve growth factor and BMSC-Exos promoted motor function recovery, alleviated nerve injury, and reduced apoptosis in mice with SCI. ZBTB4 was enriched in BMSC-Exos and lowly expressed in SCI. Downregulation of ZBTB4 diminished the therapeutic effects of BMSC-Exos against SCI. ITIH3 was a downstream target of ZBTB4. Neurotoxic activation of astrocytes induced neuronal injury, which was alleviated by BMSC-Exos. However, ZBTB4 knockdown overturned the effects of BMSC-Exos in vitro and combined ITIH3 knockdown alleviated the accentuating effects of ZBTB4 knockdown on neuronal injury. CONCLUSION: BMSC-Exos protected against astrocyte-induced neuronal injury by delivering ZBTB4 to repress ITIH3, ultimately improving motor function in mice with SCI.

Oligodendrocyte-selective deletion of the eIF2α kinase Perk/Eif2ak3 limits functional recovery after spinal cord injury.

After spinal cord injury (SCI), re-establishing cellular homeostasis is critical to optimize functional recovery. Central to that response is PERK signaling, which ultimately initiates a pro-apoptotic response if cellular homeostasis cannot be restored. Oligodendrocyte (OL) loss and white matter damage drive functional consequences and determine recovery potential after thoracic contusive SCI. We examined acute (<48 h post-SCI) and chronic (6 weeks post-SCI) effects of conditionally deleting Perk from OLs prior to SCI. While Perk transcript is expressed in many types of cells in the adult spinal cord, its levels are disproportionately high in OL lineage cells. Deletion of OL-Perk prior to SCI resulted in: (1) enhanced acute phosphorylation of eIF2α, a major PERK substrate and the critical mediator of the integrated stress response (ISR), (2) enhanced acute expression of the downstream ISR genes Atf4, Ddit3/Chop, and Tnfrsf10b/Dr5, (3) reduced acute OL lineage-specific Olig2 mRNA, but not neuronal or astrocytic mRNAs, (4) chronically decreased OL content in the spared white matter at the injury epicenter, (5) impaired hindlimb locomotor recovery, and (6) reduced chronic epicenter white matter sparing. Cultured primary OL precursor cells with reduced PERK expression and activated ER stress response showed: (1) unaffected phosphorylation of eIF2α, (2) enhanced ISR gene induction, and (3) increased cytotoxicity. Therefore, OL-Perk deficiency exacerbates ISR signaling and potentiates white matter damage after SCI. The latter effect is likely mediated by increased loss of Perk-/- OLs.

Transplantation of dorsal root ganglia overexpressing the NaChBac sodium channel improves locomotion after complete SCI.

Spinal cord injury (SCI) is a debilitating condition currently lacking treatment. Severe SCI causes the loss of most supraspinal inputs and neuronal activity caudal to the injury, which, coupled with the limited endogenous capacity for spontaneous regeneration, can lead to complete functional loss even in anatomically incomplete lesions. We hypothesized that transplantation of mature dorsal root ganglia (DRGs) genetically modified to express the NaChBac sodium channel could serve as a therapeutic option for functionally complete SCI. We found that NaChBac expression increased the intrinsic excitability of DRG neurons and promoted cell survival and neurotrophic factor secretion in vitro. Transplantation of NaChBac-expressing dissociated DRGs improved voluntary locomotion 7 weeks after injury compared to control groups. Animals transplanted with NaChBac-expressing DRGs also possessed higher tubulin-positive neuronal fiber and myelin preservation, although serotonergic descending fibers remained unaffected. We observed early preservation of the corticospinal tract 14 days after injury and transplantation, which was lost 7 weeks after injury. Nevertheless, transplantation of NaChBac-expressing DRGs increased the neuronal excitatory input by an increased number of VGLUT2 contacts immediately caudal to the injury. Our work suggests that the transplantation of NaChBac-expressing dissociated DRGs can rescue significant motor function, retaining an excitatory neuronal relay activity immediately caudal to injury.

P2X7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury.

In contrast to mammals, zebrafish undergo successful neural regeneration following spinal cord injury. Spinal cord ependymo-radial glia (ERG) undergo injury-induced proliferation and neuronal differentiation to replace damaged cells and restore motor function. However, the molecular cues driving these processes remain elusive. Here, we demonstrate that the evolutionarily conserved P2X7 receptors are widely distributed on neurons and ERG within the zebrafish spinal cord. At the protein level, the P2X7 receptor expressed in zebrafish is a truncated splice variant of the full-length variant found in mammals. The protein expression of this 50 kDa isoform was significantly downregulated at 7 days post-injury (dpi) but returned to basal levels at 14 dpi when compared to naïve controls. Pharmacological activation of P2X7 following SCI resulted in a greater number of proliferating cells around the central canal by 7 dpi but did not affect neuronal differentiation at 14 dpi. Our findings suggest that unlike in mammals, P2X7 signaling may not play a maladaptive role following SCI in adult zebrafish and may also work to curb the proliferative response of ERG following injury.

Kdm6a-CNN1 axis orchestrates epigenetic control of trauma-induced spinal cord microvascular endothelial cell senescence to balance neuroinflammation for improved neurological repair.

Cellular senescence assumes pivotal roles in various diseases through the secretion of proinflammatory factors. Despite extensive investigations into vascular senescence associated with aging and degenerative diseases, the molecular mechanisms governing microvascular endothelial cell senescence induced by traumatic stress, particularly its involvement in senescence-induced inflammation, remain insufficiently elucidated. In this study, we present a comprehensive demonstration and characterization of microvascular endothelial cell senescence induced by spinal cord injury (SCI). Lysine demethylase 6A (Kdm6a), commonly known as UTX, emerges as a crucial regulator of cell senescence in injured spinal cord microvascular endothelial cells (SCMECs). Upregulation of UTX induces senescence in SCMECs, leading to an amplified release of proinflammatory factors, specifically the senescence-associated secretory phenotype (SASP) components, thereby modulating the inflammatory microenvironment. Conversely, the deletion of UTX in endothelial cells shields SCMECs against senescence, mitigates the release of proinflammatory SASP factors, and promotes neurological functional recovery after SCI. UTX forms an epigenetic regulatory axis by binding to calponin 1 (CNN1), orchestrating trauma-induced SCMECs senescence and SASP secretion, thereby influencing neuroinflammation and neurological functional repair. Furthermore, local delivery of a senolytic drug reduces senescent SCMECs and suppresses proinflammatory SASP secretion, reinstating a local regenerative microenvironment and enhancing functional repair after SCI. In conclusion, targeting the UTX-CNN1 epigenetic axis to prevent trauma-induced SCMECs senescence holds the potential to inhibit SASP secretion, alleviate neuroinflammation, and provide a novel treatment strategy for SCI repair.

Identification of key autophagy-related genes and pathways in spinal cord injury.

Spinal cord injury (SCI) can cause a range of functional impairments, and patients with SCI have limited potential for functional recovery. Previous studies have demonstrated that autophagy plays a role in the pathological process of SCI, but the specific mechanism of autophagy in this context remains unclear. Therefore, we explored the role of autophagy in SCI by identifying key autophagy-related genes and pathways. This study utilized the GSE132242 expression profile dataset, which consists of four control samples and four SCI samples; autophagy-related genes were sourced from GeneCards. R software was used to screen differentially expressed genes (DEGs) in the GSE132242 dataset, which were then intersected with autophagy-related genes to identify autophagy-related DEGs in SCI. Subsequently, the expression levels of these genes were confirmed and analyzed with gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). A protein-protein interaction (PPI) analysis was conducted to identify interaction genes, and the resulting network was visualized with Cytoscape. The MCODE plug-in was used to build gene cluster modules, and the cytoHubba plug-in was applied to screen for hub genes. Finally, the GSE5296 dataset was used to verify the reliability of the hub genes. We screened 129 autophagy-related DEGs, including 126 up-regulated and 3 down-regulated genes. GO and KEGG pathway enrichment analysis showed that these 129 genes were mainly involved in the process of cell apoptosis, angiogenesis, IL-1 production, and inflammatory reactions, the TNF signaling pathway and the p53 signaling pathway. PPI identified 10 hub genes, including CCL2, TGFB1, PTGS2, FN1, HGF, MYC, IGF1, CD44, CXCR4, and SERPINEL1. The GSE5296 dataset revealed that the control group exhibited lower expression levels than the SCI group, although only CD44 and TGFB1 showed significant differences. This study identified 129 autophagy-related genes that might play a role in SCI. CD44 and TGFB1 were identified as potentially important genes in the autophagy process after SCI. These findings provide new targets for future research and offer new perspectives on the pathogenesis of SCI.

Necroptosis pathway emerged as potential diagnosis markers in spinal cord injury.

The present research focused on identifying necroptosis-related differentially expressed genes (NRDEGs) in spinal cord injury (SCI) to highlight potential therapeutic and prognostic target genes in clinical SCI. Three SCI-related datasets were downloaded, including GSE151371, GSE5296 and GSE47681. MSigDB and KEGG datasets were searched for necroptosis-related genes (NRGs). Differentially expressed genes (DEGs) and NRGs were intersected to obtain NRDEGs. The MCC algorithm was employed to select the first 10 genes as hub genes. A protein-protein interaction (PPI) network related to NRDEGs was developed utilizing STRING. Several databases were searched to predict interactions between hub genes and miRNAs, transcription factors, potential drugs, and small molecules. Immunoassays were performed to identify DEGs using CIBERSORTx. Additionally, qRT-PCR was carried out to verify NRDEGs in an animal model of SCI. Combined analysis of all datasets identified 15 co-expressed DEGs and NRGs. GO and KEGG pathway analyses highlighted DEGs mostly belonged to pathways associated with necroptosis and apoptosis. Hub gene expression analysis showed high accuracy in SCI diagnosis was associated with the expression of CHMP7 and FADD. A total of two hub genes, i.e. CHMP7, FADD, were considered potential targets for SCI therapy.

Epigenetic regulation and factors that influence the effect of iPSCs-derived neural stem/progenitor cells (NS/PCs) in the treatment of spinal cord injury.

Spinal cord injury (SCI) is a severe neurological disorder that causes neurological impairment and disability. Neural stem/progenitor cells (NS/PCs) derived from induced pluripotent stem cells (iPSCs) represent a promising cell therapy strategy for spinal cord regeneration and repair. However, iPSC-derived NS/PCs face many challenges and issues in SCI therapy; one of the most significant challenges is epigenetic regulation and that factors that influence this mechanism. Epigenetics refers to the regulation of gene expression and function by DNA methylation, histone modification, and chromatin structure without changing the DNA sequence. Previous research has shown that epigenetics plays a crucial role in the generation, differentiation, and transplantation of iPSCs, and can influence the quality, safety, and outcome of transplanted cells. In this study, we review the effects of epigenetic regulation and various influencing factors on the role of iPSC-derived NS/PCs in SCI therapy at multiple levels, including epigenetic reprogramming, regulation, and the adaptation of iPSCs during generation, differentiation, and transplantation, as well as the impact of other therapeutic tools (e.g., drugs, electrical stimulation, and scaffolds) on the epigenetic status of transplanted cells. We summarize our main findings and insights in this field and identify future challenges and directions that need to be addressed and explored.