Wun Chey Sin

Na+/H+ exchanger NHE1, a major determinant of intracellular pH (pH(i)) in mammalian central neurons, promotes neurite outgrowth under both basal and netrin-1-stimulated conditions. The small GTP binding proteins and their effectors have a dominant role in netrin-1-stimulated neurite outgrowth. Since NHE1 has been shown previously to work downstream of the Rho GTPases-mediated polarized membrane protrusion in non-neuronal cells, we examined whether NHE1 has a similar relationship with Cdc42, Rac1 and RhoA in neuronal morphogenesis. Interestingly, our results suggest the possibility that NHE1 acting upstream of Rho GTPases to promote neurite outgrowth induced by netrin-1. First, we found that netrin-1-induced increases in the activities of Rho GTPases using FRET (Forster Resonance Energy Transfer) analyses in individual growth cones; furthermore, their increased activities were abolished by cariporide, a specific NHE1 inhibitor. Second, NHE1 inhibition had no effect on neurite retraction induced by L-alpha-Lysophosphatidic acid (LPA), a potent RhoA activator. The regulation of Rho GTPases by NHE1 was further evidenced by reduced Rac1, Cdc42 and RhoA activities in NHE1-null neurons. Taken together, our findings suggest that NHE1-dependent neuronal morphogenesis involves the activation of Rho-family of small GTPases.

The environment that envelops the cancer cells intimately affects the malignancy of human cancers. In the case of glioma, an aggressive adult brain cancer, its high rate of recurrence after total resection is responsible for a poor prognosis. Connexin43 (Cx43) is a gap junction protein with a prominent presence in glioma-associated normal brain cells, specifically in the reactive astrocytes. We previously demonstrated that elimination of Cx43 in these astrocytes reduces glioma invasion in a syngeneic mouse model. To further our investigation in human glioma cells, we developed a scaffold-free 3D platform that takes into account both the tumor and its interaction with the surrounding tissue. Using cell-tracking dyes and 3D laser scanning confocal microscopy, we now report that the elimination of Cx43 protein in neural progenitor spheroids reduced the invasiveness of human brain tumor-initiating cells, confirming our earlier observation in an intact mouse brain. By investigating the glioma invasion in a defined multicellular system with a tumor boundary that mimics the intact brain environment, our findings strengthen Cx43 as a candidate target for glioma control.

Connexin43 (Cx43) gap junctions expressed in astrocytes can significantly impact neuronal survival in stroke. However, little is known regarding Cx43 spatial and temporal expression during the initial stages of brain ischemia. Using immunohistochemistry and Western blot analysis, we examined Cx43 spatial and temporal expression as a function of neuronal injury within the first 24 h after permanent middle cerebral artery occlusion (pMCAO). Western blot analysis showed a significant increase in Cx43 protein expression in the core ischemic area at 2 and 3 h after pMCAO. However, after 6 h of pMCAO Cx43 levels were significantly reduced. This reduction was due to cell death and concomitant Cx43 degradation in the expanding focal ischemic region, while the peri-infarct zone revealed intense Cx43 staining. The neuronal cell-death marker Fluoro-Jade C labeled injured neurons faintly at 1 h post-pMCAO with a time-dependent increase in both intensity and size of punctate staining. In addition, decreased microtubule-associated protein 2 (MAP2) immunoreactivity and thionin staining similarly indicated cell damage beginning at 1 h after pMCAO. Taken together, Cx43 expression is sensitive to neuronal injury and can be detected as early as 2 h post-pMCAO. These findings underscore Cx43 gap junction as a potential early target for therapeutic intervention in ischemic stroke.

Although genetic mutations are usually responsible for the initial tumor formation and progression, changes in the microenvironment also have a critical role in facilitating this process. Many ''untransformed'' cells infiltrate the tumors, and recent evidence suggests cancer cells can ‘reprogram’ normal cells by miRNAs, which are small, non-coding RNA molecules that regulate several protein targets. One prominent feature of glioma pathology is massive gliosis, an inflammatory response consisting of reactive astrocytes, in and around the tumor. We show that expression of Cx43, a major gap junction protein in astrocytes, is significantly enhanced in astrocytes at the tumor border. Using a mouse model consisting of syngeneic intracranial implantation of GL261 glioma cells into Nestin-Cre:Cx43fl/fl mice in which Cx43 is selectively eliminated in astrocytes, we demonstrate that reduction of astrocytic Cx43 decreases the dissemination of glioma cells from the tumor core. Similarly, knocking down Cx43 in astrocytes also reduces glioma invasion in a co-culture of glioma cells and astrocytes. By comparing the microRNA profiles of the astrocytes before and after co-culture with human glioma cells, we have identified a miR-5096 that appears to reprogram astrocytes to enhance the invasiveness of glioma cells. We are now examining whether we can prevent glioma cells from invading the brain and establishing recurrent secondary tumors by stopping the exchange of materials between glioma cells and astrocytes through eliminating Cx43 channel activity.

Reactive astrogliosis is associated with many pathologic processes in the central nervous system, including gliomas. The glycoprotein podoplanin (PDPN

Astrogliosis is a process that involves morphological and biochemical changes associated with astrocyte activation in response to cell damage in the brain. The upregulation of intermediate filament proteins including glial fibrillary acidic protein (GFAP), nestin and vimentin are often used as indicators for astrogliosis. Although connexin43 (Cx43), a channel protein widely expressed in adult astrocytes, exhibits enhanced immunoreactivity in the peri-lesion region, its role in astrogliosis is still unclear. Here, we correlated the temporal and spatial expression of Cx43 to the activation of astrocytes and microglia in response to an acute needle stab wound in vivo. We found large numbers of microglia devoid of Cx43 in the needle wound at 3 days post injury (dpi) while reactive astrocytes expressing Cx43 were present in the peripheral zone surrounding the injury site. A redistribution of Cx43 to the needle site, corresponding to the increased presence of GFAP-positive reactive astrocytes in the region, was only apparent from 6 dpi and sustained until at least 15 dpi. Interestingly, the extent of microglial activation and subsequent astrogliosis in the brain of Cx43 knockout mice was significantly larger than those of wild type, suggesting that Cx43 expression limits the degree of microgliosis. Although Cx43 is not essential for astrogliosis and microglial activation induced by a needle injury, our results demonstrate that Cx43 is a useful marker for injury induced astrogliosis due to its enhanced expression specifically within a small region of the lesion for an extended period. As a channel protein, Cx43 is a potential in vivo diagnostic tool of asymptomatic brain injury.