Fei Zhuo

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

With 3 figures and 2 tables

Hyperosmotic induction of aquaporin expression in rat astrocytes through a different MAPK pathway

Water homeostasis of the nervous system is important during neural signal transduction. Astrocytes are crucial in water transport in the central nervous system under both physiological and pathological conditions. To date, five aquaporins (AQP) have been found in rat brain astrocytes. Most studies have focused on AQP4 and AQP9, however, little is known about the expression of AQP3, ‐5, and ‐8 as well as their regulating mechanism in astrocytes. The expression patterns of AQP3, ‐5, and ‐8 in astrocytes exposed to hyperosmotic solutions were examined to clarify the roles of AQP3, ‐5, and ‐8 in astrocyte water movement. The expression of AQP4 and AQP9 under the same hyperosmotic conditions was also investigated. The AQP4 and AQP9 expressions continuously increased until 12 h after hyperosmotic solution exposure, whereas the AQP3, ‐5, and ‐8 expressions continued to increase until 6 h after hyperosmotic solution exposure. The different AQPs decreased at corresponding time points (24 h for AQP4 andAQP9; 12 h for AQP3, ‐5, and ‐8 after hyperosmotic solution exposure). The ERK inhibitor can attenuate the expression of AQP3, ‐5, and ‐8 after hyperosmotic solution exposure. The p38 inhibitor can inhibit the AQP4 and AQP9 expressions in cultured astrocytes. AQP expression is directly related to the extracellular hyperosmotic stimuli. Moreover, different AQPs can be regulated by a distinct MAPK signal transduction pathway. J. Cell. Biochem. 114: 111–119, 2012.

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.

The internalization and lysosomal degradation of brain AQP4 after ischemic injury.

The membrane-bound water channel aquaporin-4 (AQP4) plays a significant role in maintaining brain water homeostasis. In ischemic brain, changes in the expression level of AQP4 have been reported. Previous studies suggest that the internalization of several membrane-bound proteins, including AQP4, may occur with or without lysosomal degradation. In this study, the internalization of AQP4 was detected in the ischemic rat brain via double immunofluorescence labeling. Specifically, AQP4 and early endosome antigen-1 (EEA1) co-localized after 1 h post-ischemic injury. Moreover, the co-expression of AQP4 and lysosomal-associated membrane protein-1 (LAMP1) was observed after 3 h post-ischemia. These findings suggest that AQP4 is internalized and the lysosome is involved in degrading the internalized AQP4 in the ischemic brain. AQP4 is known to be downregulated by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) in vivo and in vitro. The results in this study displayed that PMA infusion could decrease brain edema accompanied by AQP4 downregulation in ischemic brain. However, compared with vehicle infusion, PKC activator infusion did not increase the ratio of internalized or lysosomal degraded AQP4. That is, we have not found out evidence to prove protein kinase C activator PMA can promote the internalization or lysosomal degradation of AQP4 in the ischemic brain.

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.

Lost Polarization of Aquaporin4 and Dystroglycan in the Core Lesion after Traumatic Brain Injury Suggests Functional Divergence in Evolution.

Objective. To understand how aquaporin4 (AQP4) and dystroglycan (DG) polarized distribution change and their roles in brain edema formation after traumatic brain injury (TBI). Methods. Brain water content, Evans blue detection, real-time PCR, western blot, and immunofluorescence were used. Results. At an early stage of TBI, AQP4 and DG maintained vessel-like pattern in perivascular endfeet; M1, M23, and M1/M23 were increased in the core lesion. At a later stage of TBI, DG expression was lost in perivascular area, accompanied with similar but delayed change of AQP4 expression; expression of M1, M23, and DG and the ratio of M1/M2 were increased. Conclusion. At an early stage, AQP4 and DG maintained the polarized distribution. Upregulated M1 and M23 could retard the cytotoxic edema formation. At a later stage AQP4 and DG polarized expression were lost from perivascular endfeet and induced the worst cytotoxic brain edema. The alteration of DG expression could regulate that of AQP4 expression after TBI.

Upregulation and lysosomal degradation of AQP4 in rat brains with bacterial meningitis

Brain edema is among the major complications in children with bacterial meningitis. Aquaporins are integral membrane pore proteins that form channels to regulate cellular water content. Aquaporin-4 (AQP4), which is enriched in parts of astrocytic membranes that are apposed to pial or perivascular basal laminae, is the predominant aquaporin in the central nervous system. Dystroglycan is among the proteins that are responsible for the site-specific anchorage of AQP4. To elucidate the role of AQP4 in the development of brain edema induced by meningitis, a model of bacterial meningitis was established by injecting group B β-hemolytic Streptococci into the cerebrospinal fluid of three-week-old rats. The brain water content increased in this model compared with that in the control group. The expression of AQP4 and dystroglycan was examined by Western blot and the degradation route of AQP4 was investigated by double immunofluorescence labeling. Western blot results showed that the expression of AQP4 and dystroglycan in rat brain increased in the meningitis model. Meanwhile, AQP4 was co-localized with the marker of lysosome in this model, indicating that the lysosome is involved in AQP4 degradation.