Expression And Cell Distribution Of SENP3 In Brain Tissue After Traumatic Brain Injury In Mice

Background:Traumatic brain injury (TBI) occurs when an exterior force traumatically injures the brain, which induces focal and/or diffuse pathologies. Increasing evidencethat neuronal cell death neuron apoptisis response contributes to secondary brain injury following traumatic brain injury. TBI is usually divided into two stages:the primary injury and secondary injury. It is postulated that primary brain injury cannot be prevented without avoiding the injury itself, however, secondary brain injury is amenable to therapeutic interventions. Multiple pathogenic factors have been attested involved in this period, such as reactive oxygen species (ROS), calcium release, neuron apoptisis, glutamate toxicity, mitochondrial dysfunction, and inflammation.The treatment of secondary brain injury represents an unmet medical need, as no effective treatment currently exists. The development of such a treatment requires a fundamental understanding of the pathophysiological mechanisms that underpin the sequelae resulting from TBI, particularly the ensuing neuron apoptisis and cognitive impairments. Small ubiquitin-like modifier (SUMO) conjugation is a posttranslational protein modification whereby SUMOs are conjugated to proteins to modify such cellular pathways as nuclear transport, transcription, chromosome segregation and DNA repair. SUMO conjugation can alter the activity, stability, and function of target proteins, and thereby modulate almost all major cellular processes. SUMO can enhance the SUMOylation of inhibitor of nuclear factor-kappa B (IκB) and Caspase, leading to an inhibition of nuclear factor-kappa B (NF-κB) transcriptional activity and Caspase cleaving. Inflammation and apoptosis are two important parameters involved in the secondary damage following TBI and can aggravate this damage. Sentrin/SUMO-specific proteases (SENPs) regulate the covalent modification of proteins by SUMO reversibly. Six SENPs have been acknowledged in humans thus far. The balance between SUMO conjugation and SENP-mediated SUMO removal determines the SUMOylation status of substrate proteins. Among the three types of SUMOs in mammals, SUMO-2/3-conjugated targets are more successfully deconjugated by SENP3 than SUMO-1-containing species and SUMO-2/3 conjugation while not SUMO-1 is an endogenous neuroprotective mechanism. SENP3 is critical for maintaining the level of SUMOylated and de-SUMOylated substrates required for normal physiology owing to its isopeptidase activity, whereby it cleaves the isopeptide bond between SUMO and target proteins, as a result, SENP3 may weaken the neuroprotective effect of SUMO-2/3. Dynamic changes in SENP3 stability is interrelated to apoptosis and inflammation. Previous studies have demonstrated that up-regulation of SENP3 is implicated in neuronal apoptosis and declines in SENP3 could lead to a suppression of apoptosis both in cerebral ischemia and spinal cord injury.Objective:The aim of the current study is to explore the cerebral ccortical expression patten of SENP3 and its potential role in neuron apoptisis following TBI.Methods:The present study was conducted with adult male ICR mice (6-8 weeks post-natal) weighing 29-34 g. A total of 84 male ICR mice were randomly divided into seven groups:a sham group (n=12) and a TBI group that was divided into six subgroups (n =12 per group), namely, groups examined 3 h,6 h,12 h,24 h,3 days,5 days after TBI. The animals were subjected to experimental TBI and were sacrificed at the aforementioned six time-points after injury. Sham animals were sacrificed 1 day after the sham operation. The mouse model of TBI was induced by free-falling method, the operation of the sham group was identical with that of the other groups except the mice in the sham group were not subjected to the impact of the dropped weight. The sample cortex surrounding the contused area was collected and stored immediately in liquid nitrogen for Western blot and real-time PCR analysis. The spatial and temporal expression patterns of SENP3 and cysteinyl aspartate specific proteinase 3 (Caspase-3) in the cerebral cortex were analyzed by Western blot. Reverse transcription polymerase chain reaction (RT-PCR) assay were used to test the mRNA levels of SENP3 in the pericontusional cortex. Immunohistochemistry and immunofluorescence study were performed to evaluate the expression and localization of SENP3 in the brain after TBI. Results:The mRNA level of SENP3 increased significantly as early as 6 h following TBI, peaking at 12 h, and lasted at least 5 days. Consistent the mRNA level, the protein level of SENP3 increased as early as 12 h following TBI, peaking at 24 h, and also lasted at least 5 days. To provide the molecular evidence of apoptosis following TBI, Western blot of cleaved caspase-3 was performed. Significant increase of cleaved caspase-3 protein level was examined at 24 h following TBI. An IHC analysis was performed to assess the expression of SENP3. SENP3-positive cells were observed both in the sham and 24 h post-TBI groups. However, robust expression of SENP3 was seldom observed in the sham group, while it was obviously enhanced in the 24 h post-TBI group. Notably, nuclear expression of SENP3 was significantly increased in the 24 h post-TBI group.To identify the type of brain cells in which SENP3 was predominantly expressed, DI staining was performed for SENP3 and neural cell specific proteins (NeuN for neurons, GFAP for astrocytes, and Ibal for microglia). These results indicated that the expression of SENP3 increased notably at 24 h following TBI and increased SENP3 mainly localized in neurons and astrocytes. Furthermore, DI staining was used to investigate the relationship between SENP3-positive and cleaves caspase-3-positive or apoptotic cells.24 h following TBI, SENP3-positive cells were mainly the cleaved caspase-3-positive cells, indicating that SENP3 might be involved in the apoptosis following TBI.Conclusion:In this study, we reveal that both mRNA and protein levels of SENP3 were upregulated in the cerebral cortex following experimental TBI in mice. The SENP3-positive cells were mainly neurons and astrocytes. Moreover, co-localization of SENP3 /caspase-3 was detected in the cerebral cortex. Collectively, these findings seem to indicate that TBI enhances the expression of SENP3 in neurons and SENP3 might be involved in the secondary injury following TBI.