Shanquan Sun

Altered expression of long non-coding RNAs during genotoxic stress-induced cell death in human glioma cells.

Long non-coding RNAs (lncRNAs), a recently discovered class of non-coding genes, are transcribed throughout the genome. Emerging evidence suggests that lncRNAs may be involved in modulating various aspects of tumor biology, including regulating gene activity in response to external stimuli or DNA damage. No data are available regarding the expression of lncRNAs during genotoxic stress-induced apoptosis and/or necrosis in human glioma cells. In this study, we detected a change in the expression of specific candidate lncRNAs (neat1, GAS5, TUG1, BC200, Malat1, MEG3, MIR155HG, PAR5, and ST7OT1) during DNA damage-induced apoptosis in human glioma cell lines (U251 and U87) using doxorubicin (DOX) and resveratrol (RES). We also detected the expression pattern of these lncRNAs in human glioma cell lines under necrosis induced using an increased dose of DOX. Our results reveal that the lncRNA expression patterns are distinct between genotoxic stress-induced apoptosis and necrosis in human glioma cells. The sets of lncRNA expressed during genotoxic stress-induced apoptosis were DNA-damaging agent-specific. Generally, MEG3 and ST7OT1 are up-regulated in both cell lines under apoptosis induced using both agents. The induction of GAS5 is only clearly detected during DOX-induced apoptosis, whereas the up-regulation of neat1 and MIR155HG is only found during RES-induced apoptosis in both cell lines. However, TUG1, BC200 and MIR155HG are down regulated when necrosis is induced using a high dose of DOX in both cell lines. In conclusion, our findings suggest that the distinct regulation of lncRNAs may possibly involve in the process of cellular defense against genotoxic agents.

Immunolocalization of Aquaporins in Rat Brain

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Demyelination initiated by oligodendrocyte apoptosis through enhancing endoplasmic reticulum-mitochondria interactions and Id2 expression after compressed spinal cord injury in rats.

Demyelination is one of the most important pathological factors of spinal cord injury. Oligodendrocyte apoptosis is involved in triggering demyelination. However, fewer reports on pathological changes and mechanism of demyelination have been presented from compressed spinal cord injury (CSCI). The relative effect of oligodendrocyte apoptosis on CSCI‐induced demyelination and the mechanism of apoptosis remain unclear.

Aquaporin 9 in rat brain after severe traumatic brain injury

OBJECTIVE To reveal the expression and possible roles of aquaporin 9 (AQP9) in rat brain, after severe traumatic brain injury (TBI). METHODS Brain water content (BWC), tetrazolium chloride staining, Evans blue staining, immunohistochemistry (IHC), immunofluorescence (IF), western blot, and real-time polymerase chain reaction were used. RESULTS The BWC reached the first and second (highest) peaks at 6 and 72 hours, and the blood brain barrier (BBB) was severely destroyed at six hours after the TBI. The worst brain ischemia occurred at 72 hours after TBI. Widespread AQP9-positive astrocytes and neurons in the hypothalamus were detected by means of IHC and IF after TBI. The abundance of AQP9 and its mRNA increased after TBI and reached two peaks at 6 and 72 hours, respectively, after TBI. CONCLUSIONS Increased AQP9 might contribute to clearance of excess water and lactate in the early stage of TBI. Widespread AQP9-positive astrocytes might help lactate move into neurons and result in cellular brain edema in the later stage of TBI. AQP9-positive neurons suggest that AQP9 plays a role in energy balance after TBI.

Changes in lactate content and monocarboxylate transporter 2 expression in Aβ25-35-treated rat model of Alzheimer’s disease

Decreased brain energy metabolism is correlated with cognitive impairment in Alzheimer’s disease (AD). Accumulating evidence indicates that lactate and monocarboxylate transporters (MCTs) participate in brain energy metabolism. To date, changes in lactate level and expression of MCTs in AD remain unclear. This study was conducted to detect the changes in lactate content and expression of MCT2 in Aβ25-35-treated rat model of AD. Sprague–Dawley rats were randomly divided into control and model groups, which received bilateral intrahippocampal injections of saline and Aβ25-35, respectively. Cognitive functions were detected by Morris water-maze test. Lactate content in the cerebral cortex and hippocampus was measured by absorbance assay. The MCT2 level in the brain was examined by immunohistochemistry and Western blot. Morris water-maze test showed that the model group exhibited impaired learning and memory compared with the control group. Lactate content in the cerebral cortex and hippocampus was decreased in the model group compared with that in the control group. Immunohistochemistry and Western blot showed that the expression of MCT2 in the model group significantly decreased compared with that in the control group. Results indicate that decreased lactate content and downregulated MCT2 expression in the cerebral cortex and hippocampus reflected impaired energy metabolism in the brain, which may participate in the pathologic progression of AD.

Both endoplasmic reticulum and mitochondrial pathways are involved in oligodendrocyte apoptosis induced by capsular hemorrhage.

OBJECTIVE The white matter injury caused by intracerebral hemorrhage (ICH) includes demyelination and axonal injury. Oligodendrocyte apoptosis is reported to be involved in triggering demyelination. Experimental observations indicate that both endoplasmic reticulum and mitochondrial pathways could mediate cell apoptosis. The purpose of this study was to investigate the demyelination and the possible mechanisms in an autologous blood-injected rat model of internal capsule hemorrhage. METHODS Transmission electron microscope was applied to examine the pathological changes of myelinated nerve fibers in internal capsule. Western blotting was used to detect the myelin basic protein (MBP) which was an important component of myelin sheath. Double immunofluorescence and Western blotting were used to determine the apoptosis and apoptotic pathways. The levels of caspase-12 (a representative protein of endoplasmic reticulum stress) and cytochrome c (an apoptosis factor released from mitochondria) were assessed in this study. RESULTS Demyelination occurred on day 1, 3, and 7 after ICH onset. Myelin sheaths of internal capsule nerve fibers were swollen and broken down in ICH groups. MBP expression showed a downregulation after ICH with its minimum value occurred on day 7 post-ICH. Besides, neuron and oligodendrocyte apoptosis were observed at different time intervals post-ICH accompanied with an upregulated caspase-12 expression and enhanced cytochrome c release. CONCLUSIONS These results suggested that oligodendrocyte and neuron apoptosis may contribute to the demyelination induced by internal capsule hemorrhage and oligodendrocyte apoptosis is positively mediated through both endoplasmic reticulum and mitochondrial pathways.

Loss of AQP4 polarized localization with loss of β-dystroglycan immunoreactivity may induce brain edema following intracerebral hemorrhage.

The aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular and subpial membrane domains of astrocytes where it is concentrated. These membranes form the interface between the neuropil and the extracellular liquid spaces. The brain-selective deletion of the dystroglycan (DG) gene causes a disorganization of AQP4 on the astroglial endfeet. First, we analyzed the expression of AQP4, β-DG in the brain following intracerebral hemorrhage (ICH) and correlated AQP4 expression with the expression pattern of the β-DG, which is a component of dystrophin-dystroglycan complex (DDC). Besides, the vessels ultrastructure and brain water content were investigated at different time points post-ICH (day 1, day 3, day 7). We found that AQP4 polarity was disturbed in parallel with the loss of β-DG in the perihematomal area post-ICH. At day 1 post-ICH, brain edema was obvious and the damage of vascular ultrastructure was the most severe. These results suggest a role for β-DG in targeting and stabilizing AQP4 channel in astrocytic cells, which may be critical for water homeostasis in brain.

Internalization of Aquaporin-4 After Collagenase-Induced Intracerebral Hemorrhage

Brain edema formation following intracerebral hemorrhage (ICH) appears to be related with aquaporin‐4 (AQP4), which is critically involved in brain volume homeostasis and water balance. Despite its importance, the regulation of AQP4 expression involved in transmembrane water movements still remains rudimentary. Many studies suggest that the internalization of several membrane‐bound proteins, including AQP4, may occur with or without lysosomal degradation. Previously, we investigated the internalization of AQP4 in retinal ischemic‐reperfusion model. Here, we test the hypothesis that AQP4 is internalized post‐ICH and then degraded in the lysosome. The results demonstrated that both AQP4 and the mannose‐6‐phosphate receptor (MPR) co‐localized in perihematomal region at 6 hr post‐ICH. In addition, AQP4 and lysosomal‐associated membrane protein 1 (LAMP1) also co‐localized in perihematomal region, with co‐expression increasing followed by a gradual decrease at different time windows post‐ICH (6, 12, 24, 48, and 72 hr). After ICH, the Evans blue leakage happened very early at 1 hr and the brain swelling occurred at 3 hr. Moreover, we also found the AQP4 mRNA and AQP4 protein were increased post‐ICH. These results suggest that AQP4 is internalized and the lysosome is involved in degrading the internalized AQP4 post‐ICH. Both the AQP4 internalization and lysosomal degradation may provide biophysical insights regarding the potential of new treatments for brain edema. Anat Rec, 298:554–561, 2015.

Long noncoding RNA GAS5 regulates the proliferation, migration, and invasion of glioma cells by negatively regulating miR-18a-5p: LIU et al.

Long noncoding RNAs, transcribed from a recently discovered class of noncoding genes, may play a critical role in regulating cellular processes, such as cell proliferation and apoptosis, as well as in cancer progression and metastasis. We previously detected the induction of growth arrest–specific 5 (GAS5) during glioma cell death. However, the function and underlying mechanism of GAS5 in human gliomas remain to be elucidated.

p53-Mediated oligodendrocyte apoptosis initiates demyelination after compressed spinal cord injury by enhancing ER-mitochondria interaction and E2F1 expression

Oligodendrocyte apoptosis mediated demyelination is a pathological change characteristic of compressed spinal cord injury (CSCI). However, the mechanism of demyelination due to oligodendrocyte apoptosis is not known. In this study, after successfully establishing a rat CSCI model using a custom-made compressor, we investigated the pathological changes, MBP expression, as well as apoptosis-related protein (p53, active caspase-3) expression to determine whether or not apoptosis and demyelination occurred after injury. To understand the possible mechanism of oligodendrocyte apoptosis, caspase-12 and cytochrome C were analyzed to explore the relationship between oligodendrocyte apoptosis and endoplasmic reticulum(ER)-mitochondria interaction. The transcription factor, E2F1, was also detected by immunofluorescence and Western blot assays. The results showed that CSCI increased the expression levels of p53, E2F1 and active caspase-3 followed by the swelling and breakdown of myelin sheaths. The number of myelinated nerve fibers also decreased with down-regulated expression of MBP. Expression levels of caspase-12 and cytochrome C were enhanced along with a reduction in the number of oligodendrocytes. After treatment of CSCI in rats with Pifithrin-μ(PFT-μ), a specific inhibitor of p53, pathomorphological changes of myelin sheath improved significantly. Expression levels of E2F1, active caspase-3, caspase-12 and cytochrome C were down-regulated, consistent with reduced the number of apoptotic oligodendrocytes. These results demonstrated that over-expression of p53 could mediate oligodendrocyte apoptosis thus resulting in demyelination in two ways; by enhancing ER-mitochondria interaction and by triggering the E2F1 mediated apoptosis pathway.