MicroRNA339 Targeting PDXK Improves Motor Dysfunction and Promotes Neurite Growth in the Remote Cortex Subjected to Spinal Cord Transection.
ABSTRACT: Spinal cord injury (SCI) is a fatal disease that can cause severe disability. Cortical reorganization subserved the recovery of spontaneous function after SCI, although the potential molecular mechanism in this remote control is largely unknown. Therefore, using proteomics analysis, RNA interference/overexpression, and CRISPR/Cas9 in vivo and in vitro, we analyzed how the molecular network functions in neurological improvement, especially in the recovery of motor function after spinal cord transection (SCT) via the remote regulation of cerebral cortex. We discovered that the overexpression of pyridoxal kinase (PDXK) in the motor cortex enhanced neuronal growth and survival and improved locomotor function in the hindlimb. In addition, PDXK was confirmed as a target of miR-339 but not miR-124. MiR-339 knockout (KO) significantly increased the neurite outgrowth and decreased cell apoptosis in cortical neurons. Moreover, miR-339 KO rats exhibited functional recovery indicated by improved Basso, Beattie, and Bresnehan (BBB) score. Furthermore, bioinformatics prediction showed that PDXK was associated with GAP43, a crucial molecule related to neurite growth and functional improvement. The current research therefore confirmed that miR-339 targeting PDXK facilitated neurological recovery in the motor cortex of SCT rats, and the underlying mechanism was associated with regulating GAP43 in the remote cortex of rats subjected to SCT. These findings may uncover a new understanding of remoting cortex control following SCI and provide a new therapeutic strategy for the recovery of SCI in future clinical trials.
Project description:Acetylcholine receptors (AChRs) serve as connections between motor neurons and skeletal muscle and are essential for recovery from spinal cord transection (SCT). Recently, microRNAs have emerged as important potential biotherapeutics for several diseases; however, whether miRNAs operate in the modulation of AChRs remains unknown. We found increased AChRs numbers and function scores in rats with SCT; these increases were reduced following the injection of a eukaryotic translation initiation factor 5A1 (eIF5A1) shRNA lentivirus into the hindlimb muscle. Then, high-throughput screening for microRNAs targeting eIF5A1 was performed, and miR-434-3p was found to be robustly depleted in SCT rat skeletal muscle. Furthermore, a highly conserved miR-434-3p binding site was identified within the mRNA encoding eIF5A1 through bioinformatics analysis and dual-luciferase assay. Overexpression or knockdown of miR-434-3p in vivo demonstrated it was a negative post-transcriptional regulator of eIF5A1 expression and influenced AChRs expression. The microarray-enriched Gene Ontology (GO) terms regulated by miR-434-3p were muscle development terms. Using a lentivirus, one functional gene (map2k6) was confirmed to have a similar function to that of miR-434-3p in GO terms. Finally, HRM and MeDIP-PCR analyses revealed that DNA demethylation also up-regulated eIF5A1 after SCT. Consequently, miR-434-3p/eIF5A1 in muscle is a promising potential biotherapy for SCI repair.
Project description:Immunization with neural derived peptides (INDP) as well as scar removal-separately-have shown to induce morphological and functional improvement after spinal cord injury (SCI). In the present study, we compared the effect of INDP alone versus INDP with scar removal on motor recovery, regeneration-associated and cytokine gene expression, and axonal regeneration after chronic SCI. Scar removal was conducted through a single incision with a double-bladed scalpel along the stump, and scar renewal was halted by adding ?,?'-dipyridyl.During the chronic injury stage, two experiments were undertaken. The first experiment was aimed at testing the therapeutic effect of INDP combined with scar removal. Sixty days after therapeutic intervention, the expression of genes encoding for TNF?, IFN?, IL4, TGF?, BDNF, IGF1, and GAP43 was evaluated at the site of injury. Tyrosine hydroxylase and 5-hydroxytryptamine positive fibers were also studied. Locomotor evaluations showed a significant recovery in the group treated with scar removal + INDP. Moreover; this group presented a significant increase in IL4, TGF?, BDNF, IGF1, and GAP43 expression, but a decrease of TNF? and IFN?. Also, the spinal cord of animals receiving both treatments presented a significant increase of serotonergic and catecholaminergic fibers as compared to other the groups. The second experiment compared the results of the combined approach versus INDP alone. Rats receiving INDP likewise showed improved motor recovery, although on a lesser scale than those who received the combined treatment. An increase in inflammation and regeneration-associated gene expression, as well as in the percentage of serotonergic and catecholaminergic fibers was observed in INDP-treated rats to a lesser degree than those in the combined therapy group.These findings suggest that INDP, both alone and in combination with scar removal, could modify the non-permissive microenvironment prevailing at the chronic phase of SCI, providing the opportunity of improving motor recovery.
Project description:Incomplete spinal cord injury (SCI) elicits structural plasticity of the spared motor system, including the motor cortex, which may underlie some of the spontaneous recovery of motor function seen after injury. Promoting structural plasticity may become an important component of future strategies to improve functional outcomes. We have recently observed dynamic changes in the density and morphology of dendritic spines in the motor cortex following SCI. The present study sought to test whether SCI-induced changes in spine density and morphology could be modulated by potential strategies to enhance functional recovery. We examined the effects of enriched environment, transplants, and neurotrophin-3 on the plasticity of synaptic structures in the motor cortex following SCI. Housing rats in an enriched environment increased spine density in the motor cortex regardless of injury. SCI led to a more slender and elongated spine morphology. Enriched housing mitigated the SCI-induced morphological alterations, suggesting that the environmental modification facilitates maturation of synaptic structures. Transplantation of embryonic spinal cord tissue and delivery of neurotrophin-3 at the injury site further increased spine density when combined with enriched housing. This combinatorial treatment completely abolished the injury-induced changes, restoring a preinjury pattern of spine morphology. These results demonstrated that remodeling of dendritic spines in the motor cortex after SCI can be modulated by enriched housing, and the combinatorial treatment with embryonic transplants and neurotrophin-3 can potentiate the effects of enriched housing. We suggest that synaptic remodeling processes in the motor cortex can be targeted for an intervention to enhance functional recovery after SCI.
Project description:We present a new method for inducing a circumscribed subcortical capsular infarct (SCI), which imposes a persistent motor impairment in rats. Photothrombotic destruction of the internal capsule (IC) was conducted in Sprague Dawley rats (male; n=38). The motor performance of all animals was assessed using forelimb placing, forelimb use asymmetry, and the single pellet reaching test. On the basis of the degree of motor recovery, rats were subdivided into either the poor recovery group (PRG) or the moderate recovery group (MRG). Imaging assessment of the impact of SCI on brain metabolism was performed using 2-deoxy-2-[(18)F]-fluoro-D-glucose ([(18)F]-FDG) microPET (positron emission tomography). Photothrombotic lesioning using low light energy selectively disrupted circumscribed capsular fibers. The MRG showed recovery of motor performance after 1 week, but the PRG showed a persistent motor impairment for >3 weeks. Damage to the posterior limb of the IC (PLIC) is more effective for producing a severe motor deficit. Analysis of PET data revealed decreased regional glucose metabolism in the ipsilesional motor and bilateral sensory cortex and increased metabolism in the contralesional motor cortex and bilateral hippocampus during the early recovery period after SCI. Behavioral, histologic, and functional imaging findings support the usefulness of this novel SCI rat model for investigating motor recovery.
Project description:Spinal cord injury (SCI) is frequently accompanied by a degree of spontaneous functional recovery. The underlying mechanisms through which such recovery is generated remain elusive. In this study, we observed a significant spontaneous motor function recovery 14 to 28 days after spinal cord transection (SCT) in rats. Using a comparative proteomics approach, caudal to the injury, we detected difference in 20 proteins. Two of these proteins, are eukaryotic translation initiation factor 5A1 (eIF5A1) that is involved in cell survival and proliferation, and Rho GDP dissociation inhibitor alpha (RhoGDI?), a member of Rho GDI family that is involved in cytoskeletal reorganization. After confirming the changes in expression levels of these two proteins following SCT, we showed that in vivo eIF5A1 up-regulation and down-regulation significantly increased and decreased, respectively, motor function recovery. In vitro, eIF5A1 overexpression in primary neurons increased cell survival and elongated neurite length while eIF5A1 knockdown reversed these results. We found that RhoGDI? up-regulation and down-regulation rescues the effect of eIF5A1 down-regulation and up-regulation both in vivo and in vitro. Therefore, we have identified eIF5A1/RhoGDI? pathway as a new therapeutic target for treatment of spinal cord injured patients.
Project description:Recent evidence has demonstrated that remote responses in the brain, as well as local responses in the injured spinal cord, can be induced after spinal cord injury (SCI). Intravenous infusion of mesenchymal stem cells (MSCs) has been shown to provide functional improvements in SCI through local therapeutic mechanisms that provide neuroprotection, stabilization of the blood-spinal cord barrier, remyelination, and axonal sprouting. In the present study, we examined the brain response that might be associated with the functional improvements induced by the infused MSCs after SCI. Genome-wide RNA profiling was performed in the motor cortex of SCI rats at 3 days post-MSC or vehicle infusion. Then, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) data revealed that the "behaviorally-associated differentially expressed genes (DEGs)" were identified by the Pearson's correlation analysis with the behavioral function, suggesting that the "behaviorally-associated DEGs" may be related to the functional recovery after systemic infusion of MSCs in SCI. These results suggested that the infused MSCs alter the gene expression signature in the brain and that these expression changes may contribute to the improved function in SCI.
Project description:Axonal regeneration after spinal cord injury (SCI) is difficult to achieve, and no fundamental treatment can be applied in clinical settings. DNA methylation has been suggested to play a role in regeneration capacity and neuronal growth after SCI by controlling the expression of regeneration-associated genes (RAGs). The aim of this study was to examine changes in neuronal DNA methylation status after SCI and to determine whether modulation of DNA methylation with ascorbic acid can enhance neuronal regeneration or functional restoration after SCI. Changes in epigenetic marks (5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC)); the expression of Ten-eleven translocation (Tet) family genes; and the expression of genes related to inflammation, regeneration, and degeneration in the brain motor cortex were determined following SCI. The 5hmC level within the brain was increased after SCI, especially in the acute and subacute stages, and the mRNA levels of Tet gene family members (Tet1, Tet2, and Tet3) were also increased. Administration of ascorbic acid (100 mg/kg) to SCI rats enhanced 5hmC levels; increased the expression of the Tet1, Tet2, and Tet3 genes within the brain motor cortex; promoted axonal sprouting within the lesion cavity of the spinal cord; and enhanced recovery of locomotor function until 12 weeks. In conclusion, we found that epigenetic status in the brain motor cortex is changed after SCI and that epigenetic modulation using ascorbic acid may contribute to functional recovery after SCI.
Project description:Functional and neural tissue recovery has been reported in many animal studies conducted with stem cells. However, the combined effect of cytokines and stem cells has not yet been adequately researched. Here, we analyzed the additive effects of granulocyte colony-stimulating factor (GCSF) on adipose-derived stem cells (ADSCs) infusion in the treatment of acute spinal cord injury (SCI) in rats.Four days after intrathecal infusion tubes implantation in Sprague-Dawley rats, SCI was induced with an infinite horizon impactor. In the Sham group (n=5), phosphate-buffered saline was injected 3, 7, and 14 days after SCI. GCSF, ADSCs, and ADSCs with GCSF were injected at the same time in the GCSF (n=8), ADSC (n=8), and ADSC+GCSF groups (n=7), respectively.The ADSC and ADSC+GCSF groups, but not the GCSF group, showed significantly higher Basso-Beattie-Bresnahan scores than the Sham group during 8 weeks (p<0.01), but no significant difference between the ADSC and ADSC+GCSF groups. In the ladder rung test, all four groups were significantly different from each other, with the ADSC+GCSF group showing the best improvement (p<0.01). On immunofluorescent staining (GAP43, MAP2), western blotting (GAP43), and reverse transcription polymerase chain reaction (GAP43, nerve growth factor), the ADSC and ADSC+GCSF groups showed higher levels than the Sham and GCSF groups.Our analyses suggest that the combination of GCSF and ADSCs infusions in acute SCI in the rat does not have a significant additive effect. Hence, when combination agents for SCI stem cell therapy are considered, molecules other than GCSF, or modifications to the methodology, should be investigated.
Project description:INTRODUCTION: A growing number of studies have highlighted the potential of stem cell and more-differentiated neural cell transplantation as intriguing therapeutic approaches for neural repair after spinal cord injury (SCI). METHODS: A conditionally immortalized neural stem cell line derived from human fetal spinal cord tissue (SPC-01) was used to treat a balloon-induced SCI. SPC-01 cells were implanted into the lesion 1 week after SCI. To determine the feasibility of tracking transplanted stem cells, a portion of the SPC-01 cells was labeled with poly-L-lysine-coated superparamagnetic iron-oxide nanoparticles, and the animals grafted with labeled cells underwent magnetic resonance imaging. Functional recovery was evaluated by using the BBB and plantar tests, and lesion morphology, endogenous axonal sprouting and graft survival, and differentiation were analyzed. Quantitative polymerase chain reaction (qPCR) was used to evaluate the effect of transplanted SPC-01 cells on endogenous regenerative processes. RESULTS: Transplanted animals displayed significant motor and sensory improvement 2 months after SCI, when the cells robustly survived in the lesion and partially filled the lesion cavity. qPCR revealed the increased expression of rat and human neurotrophin and motor neuron genes. The grafted cells were immunohistologically positive for glial fibrillary acidic protein (GFAP); however, we found 25% of the cells to be positive for Nkx6.1, an early motor neuron marker. Spared white matter and the robust sprouting of growth-associated protein 43 (GAP43)(+)?axons were found in the host tissue. Four months after SCI, the grafted cells matured into Islet2(+) and choline acetyltransferase (ChAT)(+) neurons, and the graft was grown through with endogenous neurons. Grafted cells labeled with poly-L-lysine-coated superparamagnetic nanoparticles before transplantation were detected in the lesion on T2-weighted images as hypointense spots that correlated with histologic staining for iron and the human mitochondrial marker MTCO2. CONCLUSIONS: The transplantation of SPC-01 cells produced significant early functional improvement after SCI, suggesting an early neurotrophic action associated with long-term restoration of the host tissue, making the cells a promising candidate for future cell therapy in patients with SCI.
Project description:The rapid decline of injury-induced neuronal circuit remodelling after birth is paralleled by the accumulation of chondroitin sulphate proteoglycans (CSPGs) in the extracellular matrix, culminating with the appearance of perineuronal nets (PNNs) around parvalbumin-expressing GABAergic interneurons. We used a spinal cord injury (SCI) model to study the interplay between integrity of PNN CSPGs in the sensorimotor cortex, anatomical remodelling of the corticospinal tract (CST) and motor recovery in adult mice. We showed that thoracic SCI resulted in an atrophy of GABAergic interneurons in the axotomized hindlimb cortex, as well as in a more widespread downregulation of parvalbumin expression. In parallel, spontaneous changes in the integrity of CSPG glycosaminoglycan (GAG) chains associated with PNNs occurred at the boundary between motor forelimb and sensorimotor hindlimb cortex, a region previously showed to undergo reorganization after thoracic SCI. Surprisingly, full digestion of CSPG GAG chains by intracortical chondroitinase ABC injection resulted in an aggravation of motor deficits and reduced sprouting of the axotomized CST above the lesion. Altogether, our data show that changes in the expression pattern of GABAergic markers and PNNs occur in regions of the sensorimotor cortex undergoing spontaneous reorganization after SCI, but suggest that these changes have to be tightly controlled to be of functional benefit.