Expression of Sam68 Associates with Neuronal Apoptosis and Reactive Astrocytes After Spinal Cord Injury

Src-associated in mitosis (Sam68; 68 kDa) is a novel RNA-binding protein that belongs to the signal transduction and activation of RNA family involved in various biological processes. However, the expression and roles of Sam68 in the central nervous system remain unknown. In the present study, we performed a spinal cord injury (SCI) model in adult rats and found a significant increase of Sam68 protein levels in this model, which reached a peak at day 3 and then gradually returned to normal levels at day 14 after SCI. We use immunohistochemistry analysis revealing a widespread distribution of Sam68 in the spinal cord. In addition, double-immunofluorescence staining showed that Sam68 immunoreactivity was found predominantly in neurons and astrocytes. Moreover, colocalization of Sam68/active caspase-3 has been respectively detected in neuronal nuclei, and colocalization of Sam68/PCNA has been detected in glial fibrillary acidic protein. In vitro, we found that depletion of Sam68 by short interfering RNA inhibits neuronal apoptosis and astrocyte proliferation and decreases cyclin D1 protein levels. In conclusion, this is the first study to find the Sam68 expression in SCI. Our results suggest that Sam68 might be illustrated in the apoptosis of neurons and proliferation of astrocytes after SCI. This research will provide new drug targets for clinical treatment of SCI.     

LIN28 Expression in Rat Spinal Cord After Injury

LIN28, an RNA-binding protein, is known to be involved in the regulation of many cellular processes, such as embryonic stem cell proliferation, cell fate succession, developmental timing, and oncogenesis. However, its expression and function in central nervous system still unclear. In this study, we performed an acute spinal cord contusion injury (SCI) model in adult rats and investigated the dynamic changes of LIN28 expression in spinal cord. Western blot and immunohistochemistry analysis revealed that LIN28 was present in normal spinal cord. It gradually increased, reached a peak at 3 day, and then nearly declined to the basal level at 14 days after SCI. Double immunofluorescence staining showed that LIN28 immunoreactivity was found in neurons, astrocytes and a handful of microglia. Interestingly, LIN28 expression was increased predominantly in astrocytes but not in neurons. Moreover, the colocalization of LIN28 and proliferating cell nuclear antigen was detected after injury. Western blot showed that LIN28 participated in lipopolysaccharide (LPS) induced astrocytes inflammatory responses by NF-κB signaling pathway. These results suggested that LIN28 may be involved in the pathologic process of SCI, and further research is needed to have a good understanding of its function and mechanism.     

Expression Profile and Role of EphrinA1 Ligand After Spinal Cord Injury

Spinal cord injury (SCI) triggers the re-expression of inhibitory molecules present in early stages of development, contributing to prevention of axonal regeneration. Upregulation of EphA receptor tyrosine kinases after injury suggest their involvement in the nervous system’s response to damage. However, the expression profile of their ephrinA ligands after SCI is unclear. In this study, we determined the expression of ephrinA ligands after contusive SCI. Adult Sprague-Dawley female rats were injured using the MASCIS impactor device at the T10 vertebrae, and levels of ephrinA mRNA and protein determined at different time points. Identification of the cell phenotype expressing the ephrin ligand and colocalization with Eph receptors was performed with immunohistochemistry and confocal microscopy. Behavioral studies were made, after blocking ephrinA1 expression with antisense (AS) oligonucleotides, to assess hindlimb locomotor activity. Real-time PCR demonstrated basal mRNA levels of ephrin (A1, A2, A3, and A5) in the adult spinal cord. Interestingly, ephrinA1 was the only ligand whose mRNA levels were significantly altered after SCI. Although ephrinA1 mRNA levels increased after 2 weeks and remain elevated, we did not observe this pattern at the protein level as revealed by western blot analysis. Immunohistochemical studies showed ephrinA1 expression in reactive astrocytes, axons, and neurons and also their colocalization with EphA4 and A7 receptors. Behavioral studies revealed worsening of locomotor activity when ephrinA1 expression was reduced. This study suggests that ephrinA1 ligands play a role in the pathophysiology of SCI.     

Increased expression of nitric oxide synthase interacting protein (NOSIP) following traumatic spinal cord injury in rats

Previous studies indicated that nitric oxide (NO) is involved in secondary damage of spinal cord injury (SCI), which worsens the primary physical injury to the central nervous systems. Recently, nitric oxide synthase interacting protein (NOSIP) has been identified to interact with neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase by inhibiting the NO production. However, its expression and function after a central nervous system injury remains unclear. In this study, we examined the expression and cellular localization of NOSIP in the spinal cord of an adult rat. Western blot analysis indicated that NOSIP protein levels increased at day1 post-injury and peaked at day 14. Double immunofluorescence staining showed that NOSIP was primarily expressed in neurons and glial cells in the intact spinal cord. Interestingly, this study also showed that the expression of NOSIP significantly increased in astrocytes after injury. Furthermore, injury-induced expression of NOSIP was co-expressed with proliferating cell nuclear antigen (PCNA) positive astrocytes after injury. We also showed the NOSIP was co-localized with nNOS in gray matter and white matter after SCI. All these data taken together suggested that NOSIP may play an important roles in astrogliogenesis after a spinal cord injury.     

Spatiotemporal Pattern of RNA-Binding Motif Protein 3 Expression After Spinal Cord Injury in Rats

RNA-binding motif protein 3 (RBM3) belongs to a very small group of cold inducible proteins with anti-apoptotic and proliferative functions. To elucidate the expression and possible function of RBM3 in central nervous system (CNS) lesion and repair, we performed a spinal cord injury (SCI) model in adult rats. Western blot analysis revealed that RBM3 level significantly increased at 1 day after damage, and then declined during the following days. Immunohistochemistry further confirmed that RBM3 immunoactivity was expressed at low levels in gray and white matters in normal condition and increased at 1 day after SCI. Besides, double immunofluorescence staining showed RBM3 was primarily expressed in the neurons and a few of astrocytes in the normal group. While after injury, the expression of RBM3 increased both in neurons and astrocytes at 1 day. We also examined the expression profiles of proliferating cell nuclear antigen (PCNA) and active caspase-3 in injured spinal cords by western blot. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many RBM3-expressing cells and rare caspase-3 was observed in RBM3-expressing cells at 1 day after injury. Our data suggested that RBM3 might play important roles in CNS pathophysiology after SCI.     

Spatiotemporal Expression of Dexras1 After Spinal Cord Transection in Rats

Dexras1, a brain-enriched member of the Ras subfamily of GTPases, as a novel physiologic nitric oxide (NO) effector, anchor neuronal nitric oxide synthase (nNOS) that increased after spinal cord injury (SCI), to specific targets to enhance NO signaling, and is strongly and rapidly induced during treatment with dexamethasone. It is unknown how the central nervous system (CNS) trauma affects the expression of Dexras1. Here we used spinal cord transection (SCT) model to detect expression of Dexras1 at mRNA and protein level in spinal cord homogenates by real-time PCR and Western blot analysis. The results showed that Dexras1 mRNA upregulated at 3 day, 5 day, and 7 day significantly (P < 0.05) that was consistent with the protein level except at 7 day. Immunofluorescence revealed that both neurons and glial cells showed Dexras1 immunoreactivivty (IR) around SCT site, but the proportion is different. Importantly, injury-induced expression of Dexras1 was co-labeled by caspase-3 (apoptotic marker) and Tau-1 (marker for pathological oligodendrocyte). Furthermore, colocalization of Dexras1, carboxy-terminal PSD95/DLG/ZO-1 (PDZ) ligand of nNOS (CAPON) and nNOS was observed in neurons and glial cells, supporting the existence of ternary complexes in this model. Thus, the results that the transient high expression of Dexras1 which localized in apoptotic neurons and pathological oligodendrocytes might provide new insight into the secondary response after SCT. Xin Li, Chun Cheng, and Min Fei contributed equally to this work.     

Contribution of the delayed-rectifier potassium channel Kv2.1 to acute spinal cord injury in rats

Recent studies have reported that delayed-rectifier Kv channels regulate apoptosis in the nervous system. Herein, we investigated changes in the expression of the delayed-rectifier Kv channels Kv1.2, Kv2.1, and Kv3.1 after acute spinal cord injury (SCI) in rats. We performed RT-PCR analysis and found an increase in the level of Kv2.1 mRNA after SCI but no significant changes in the levels of Kv1.2 and Kv3.1 mRNA. Western blot analysis revealed that Kv2.1 protein levels rapidly decreased and then dramatically increased from 1 day, whereas Kv3.1b protein levels gradually and sharply decreased at 5 days. Kv1.2 protein levels did not change significantly. In addition, Kv2.1 clusters were disrupted in the plasma membranes of motor neurons after SCI. Interestingly, the expressional changes and translocation of Kv2.1 were consistent with the apoptotic changes on day 1. Therefore, these results suggest that Kv2.1 channels probably contribute to neuronal cell responses to SCI.     

Up-regulation of TRAF2 Suppresses Neuronal Apoptosis after Rat Spinal Cord Injury

Tumor necrosis factor receptor-associated factor 2 (TRAF2) for signal transduction of the cell death receptor is well established. However, the role of TRAF2 in spinal cord injury (SCI) remains unclear. In this study, we detected the dynamic change patterns of TRAF2 expression using an acute spinal cord contusion (SCC) model in adult rats. Western blot analysis and immunohistochemistry identified significant upregulation of TRAF2 after SCI. Double-immunofluorescent staining demonstrated that the upregulated TRAF2 was found predominantly in neurons. Moreover, colocalization of TRAF2 with active caspase-3/-8 was detected in NeuN-positive cells. In vitro, we analyzed the association of TRAF2 with active caspase-3/8 on PC12 cells by western blot analysis, which paralleled the in vivo data. Knockdown ofTRAF2 with siRNA demonstrated its probable anti-apoptotic role in the process of neuronal apoptosis after SCI. To summarize, we have revealed for the first time the temporal and spatial expression profile of TRAF2 in SCI. Our data suggest that upregulation of TRAF2 triggered by trauma plays an important role in suppressing neuronal apoptosis after SCI.     

Upregulation of miRNA-223-3p ameliorates RIP3-mediated necroptosis and inflammatory responses via targeting RIP3 after spinal cord injury

Spinal cord injury (SCI) has been a major burden on the society because of the high rate of disability. Receptor-interacting protein 3 (RIP3)-mediated necroptosis is a newly discovered pathway of programmed cell death and is involved in multiple pathologies of various human diseases. Micro RNAs (miRNAs) have been shown to be a potential target for therapeutic interventions after SCI. The aim of the present study is to explore the potential role of miR-223-3p and possible mechanism in SCI. We found that miR-223-3p was significantly downregulated in spinal neurons after H₂O ₂-induced damage, while RIP3-mediated necroptosis was elevated. Accordingly, RIP3-mediated necroptosis and the inflammatory factor secretion could be significantly inhibited by Nec-1 treatment. In adittion, overexpression of miR-223-3p in spinal neurons protected against H ₂O ₂-induced necroptosis, and ablation of miR-223-3p exhibited the opposite effect. We found that miR-223-3p bound to the 3′-untranslated region of RIP3 mRNA to negatively regulate the expression of RIP3. Moreover, the activated RIP3 reversed the inhibition of RIP3 and MLKL expression and the levels of TNF-α, IL-1β, and lactate dehydrogenase, which were induced by transfection with miR-223-3p in a H ₂O ₂-induced model. Finally, these results indicate that miR-223-3p negatively regulates the RIP3 necroptotic signaling cascades and inflammatory factor secretion, which significantly relieves injury of spinal neurons. The miR-223-3p/RIP3 pathway offers a novel therapeutic target for the protection of spinal neurons after SCI.     

The Expression of VHL (Von Hippel-Lindau) After Traumatic Spinal Cord Injury and Its Role in Neuronal Apoptosis

The VHL (Von Hippel-Lindau) gene is a tumor suppressor gene, which is best known as an E3 ubiquitin ligase that negatively regulates the hypoxia inducible factor. The inactivation of VHL gene could result in the abnormal synthesis of VHL protein, which is in contact with the development and occurrence of renal clear cell carcinoma. However, the expression and possible function of VHL in central nervous system (CNS) is still unclear. To examine the function of VHL in CNS injury and repair, we used an acute spinal cord injury (SCI) model in adult rats. Western blot analysis showed an important upregulation of VHL protein, reaching a peak at day 3 and then declined during the following days. Double immunofluorescence staining showed that VHL was co-expressed with neurons, but not with astrocytes and microglia. Moreover, we detected that active caspase-3 had co-localized with VHL in neurons after SCI. Additionally in vitro, VHL depletion, by short interfering RNA, significantly reduced neuronal apoptosis. In conclusion, these data suggested that the change of VHL protein expression was related to neuronal apoptosis after SCI.

     

Stem Cells Downregulate the Elevated Levels of Tissue Plasminogen Activator in Rats After Spinal Cord Injury

We investigated the involvement of tPA after SCI in rats and effect of treatment with human umbilical cord blood derived stem cells. tPA expression and activity were determined in vivo after SCI in rats and in vitro in rat embryonic spinal neurons in response to injury with staurosporine, hydrogen peroxide and glutamate. The activity and/or expression of tPA increased after SCI and reached peak levels on day 21 post-SCI. Notably, the tPA mRNA activity was upregulated by 310-fold compared to controls on day 21 post-SCI. As expected, MBP expression is minimal at the time of peak tPA activity and vice versa. Implantation of hUCB after SCI resulted in the downregulation of elevated tPA activity/expression in vivo in rats as well as in vitro in spinal neurons. Our results demonstrated the involvement of tPA in the secondary pathogenesis after SCI as well as the therapeutic potential of hUCB.     

Analysis of transient expression of estrogen receptor-alpha in newborn rat primary auditory cortex

In the primary temporal cortex (Te1) of newborn rats, we detected transient expression of alpha-type estrogen receptor (ER alpha). Since they have a pyramidal-like shape, they are considered neurons. By immunohistochemistry we found that they were absolutely devoid of glial fibrillary acidic protein but some of them contained calretinin, a calcium binding protein. It is already known that neurons in layer V of the Te1 extend their projections to the contralateral side of the Te1, the ipsilateral inferior colliculus, or the ipsilateral medial geniculate nucleus. Thus, we applied a retrograde track tracer into those regions of newborn rats and examined the possible colocalization of ER alpha signals and the tracer in the same cells. So far no clear colocalization of both signals has been detected in cells in the Te1. Thus, the cells expressing ER alpha transiently are not projecting to the assumed regions, at least at the newborn age examined in the present experiment. The possibility exists that (1) they are not projection neurons but local interneurons, (2) even though they are projection neurons, they did not have any synaptic contacts with their target region(s), (3) they may die after they are attached to the target neurons. Further analyses are needed to clarify the biological roles of ER alpha expressed transiently in these neurons. On the other hand, no ER beta cells were detected in the same region of the brain under the same condition. Thus, this finding was limited to the ER alpha.     

Receptor for Advanced Glycation End-Products (RAGE) Blockade Do Damage to Neuronal Survival via Disrupting Wnt/β-Catenin Signaling in Spinal Cord Injury

Wnt signaling are recognized key factors in neuronal development, cell proliferation and axonal guidance. However, RAGE effect on wnt signaling after spinal cord injury (SCI) are poorly understood. Our study aims to explore RAGE blockade effect on wnt signaling after SCI. We constructed Allen SCI model and micro-injected with RAGE neutralizing antibody or IgG after injury. We determined β-catenin, wnt3a and its receptor frizzled-5 via Western blot. We determined β-catenin/NeuN expression at 2 weeks after SCI via immunofluorescence (IF). We found that β-catenin, wnt3a and wnt receptor frizzled5 expression were activated after SCI at 3 days after injury. However, RAGE blockade inhibit β-catenin, wnt3a and frizzled5 expression. We found that β-catenin accumulation in NeuN cells were activated after SCI via IF, however, RAGE blockade reduced β-catenin and NeuN positive cells. RAGE blockade attenuated number of survived neurons and decreased area of spared white matter around the epicenter. RAGE signaling may involved in disrupting wnt signaling to aids neuronal recovery after SCI.     

Restoration of ER proteostasis attenuates remote apoptotic cell death after spinal cord injury by reducing autophagosome overload

The pathogenic mechanisms that underlie the progression of remote degeneration after spinal cord injury (SCI) are not fully understood. In this study, we examined the relationship between endoplasmic reticulum (ER) stress and macroautophagy, hereafter autophagy, and its contribution to the secondary damage and outcomes that are associated with remote degeneration after SCI. Using a rat model of spinal cord hemisection at the cervical level, we measured ER stress and autophagy markers in the axotomized neurons of the red nucleus (RN). In SCI animals, mRNA and protein levels of markers of ER stress, such as GRP78, CHOP, and GADD34, increased 1 day after the injury, peaking on Day 5. Notably, in SCI animals, the increase of ER stress markers correlated with a blockade in autophagic flux, as evidenced by the increase in microtubule-associated protein 2 light chain 3 (LC3-II) and p62/SQSTM1 (p62) and the decline in LAMP1 and LAMP2 levels. After injury, treatment with guanabenz protected neurons from UPR failure and increased lysosomes biogenesis, unblocking autophagic flux. These effects correlated with greater activation of TFEB and improved neuronal survival and functional recovery—effects that persisted after suspension of the treatment. Collectively, our results demonstrate that in remote secondary damage, impairments in autophagic flux are intertwined with ER stress, an association that contributes to the apoptotic cell death and functional damage that are observed after SCI.Subject terms: Cell death in the nervous system, Neurodegeneration, Molecular neuroscience