Previous studies suggest that neuronal activity may guide the development of synaptic connections in the central nervous system through mechanisms involving glutamate receptors and GTPase-dependent modulation of the actin cytoskeleton. Here we demonstrate by in vivo time-lapse imaging of optic tectal cells in Xenopus laevis tadpoles that enhanced visual activity driven by a light stimulus promotes dendritic arbor growth. The stimulus-induced dendritic arbor growth requires glutamate-receptor-mediated synaptic transmission, decreased RhoA activity and increased Rac and Cdc42 activity. The results delineate a role for Rho GTPases in the structural plasticity driven by visual stimulation in vivo.
Reactive astrocytes are integral to the glioma microenvironment. Connexin43 (Cx43) is a major gap junction protein in astrocytes and its expression is enhanced significantly in glioma-associated astrocytes, especially at the peri-tumoral region. Although downregulation of Cx43-mediated intercellular communication is associated with increased malignancy in tumor cells, the role of Cx43 in stromal cells in glioma progression is not defined. Using a mouse model consisting of syngeneic intracranial implantation of GL261 glioma cells into Nestin-Cre:Cx43(fl/fl) mice where Cx43 was eliminated in astrocytes, we demonstrate a role of astrocytic Cx43 in the dissemination of glioma cells from the tumor core. To determine whether heterocellular communication between astrocytes and glioma cells is essential for reduced invasion in the absence of astrocytic Cx43, we abolished channel formation between glioma cells and astrocytes by either knocking down Cx43 in glioma cells with short hairpin RNA (shRNA) or overexpressing a dominant-negative channel-defective Cx43-T154A mutant in these cells. Although Cx43shRNA in glioma cells reduced invasion, expression of Cx43-T154A had no effect on glioma invasion, suggesting tumoral Cx43 may influence motility independently from its channel function. Alteration in astrocytic Cx43 function, such as by replacing the wild-type allele with a C-terminal truncated Cx43 mutant exhibiting reduced intercellular coupling, is sufficient to reduce glioma spreading into the brain parenchyma. Our results reveal a novel role of astrocytic Cx43 in the formation of an invasive niche and raise the possibility to control glioma progression by manipulating the microenvironment.
The invasiveness of high-grade glioma is the primary reason for poor survival following treatment. Interaction between glioma cells and surrounding astrocytes are crucial to invasion. We investigated the role of gap junction mediated miRNA transfer in this context. By manipulating gap junctions with a gap junction inhibitor, siRNAs, and a dominant negative connexin mutant, we showed that functional glioma-glioma gap junctions suppress glioma invasion while glioma-astrocyte and astrocyte-astrocyte gap junctions promote it in an in vitro transwell invasion assay. After demonstrating that glioma-astrocyte gap junctions are permeable to microRNA, we compared the microRNA profiles of astrocytes before and after co-culture with glioma cells, identifying specific microRNAs as candidates for transfer through gap junctions from glioma cells to astrocytes. Further analysis showed that transfer of miR-5096 from glioma cells to astrocytes is through gap junctions; this transfer is responsible, in part, for the pro-invasive effect. Our results establish a role for glioma-astrocyte gap junction mediated microRNA signaling in modulation of glioma invasive behavior, and that gap junction coupling among astrocytes magnifies the pro-invasive signaling. Our findings reveal the potential for therapeutic interventions based on abolishing alteration of stromal cells by tumor cells via manipulation of microRNA and gap junction channel activity.
The GPR4 subfamily consists of four G protein-coupled receptors that share significant sequence homology. In addition to GPR4, this subfamily includes OGR1, TDAG8 and G2A. G2A has previously been shown to be a potent transforming oncogene for murine 3T3 cells. Here we show that GPR4 also malignantly transforms NIH3T3 cells and that TDAG8 malignantly transforms the normal mammary epithelial cell line NMuMG. Overexpression of GPR4 or TDAG8 in HEK293 cells led to transcriptional activation from SRE-and CREdriven promoters, independent of exogenously added ligand. TDAG8 and GPR4 are also overexpressed in a range of human cancer tissues. Our results suggest that GPR4 and TDAG8 overexpression in human tumors plays a role in driving or maintaining tumor formation.
n-Chimerin (al-chimerin) is a brain GTPase-activating protein (GAP) for the ras-related p2l'. We now report the occurrence of another form of chimerin, termed a2-chimerin. This is the product of an alternately spliced transcript of the human n-chimerin gene encoding an N-terminal SH2 (src homology 2) domain in addition to the phorbol ester receptor and GAP domains. ad-and ca2-chimerin mRNAs were expressed differently. In the rat brain, only ael-chimerin mRNA was expressed in cerebellar Purkinje cells, although both al-and a2-chimerin mRNAs occurred in neurons in the cerebral cortex, hippocampus, and thalamus. Only ca2-chimerin RNA was expressed in rat testes, in early pachytene spermatocytes. A 45-kDa SH2-containing chimerin corresponding to the a2 form was purified from rat brain. As with Escherichia coli 45-kDa recombinant cv2-chimerin, purified brain ca2-chimerin exhibited racGAP activity which was stimulated by phosphatidylserine. The recombinant SH2 domain bound several 32P-labelled phosphoproteins of PC12 cells, whose phosphorylation increased in response to trophic factors, including nerve growth factor. To examine the relationships of cl-and at2-chimerin transcripts, human genomic DNA clones were characterized. In a2-chimerin mRNA, a 3' splice acceptor site within exon 1 of acl-chimerin mRNA was used, replacing its 5' untranslated region and N-terminal coding sequence. The single human n-chimerin gene was mapped to chromosome 2q31-q32.1, colocalizing with the CRE-BP1 transcription factor gene (2q32). It contained several splice junctions conserved with the sequence-related protein kinase C and ber genes. af2-Chimerin is only the second SH2-containing GAP and the first example of an SH2 domain generated by alternate splicing.The ras superfamily of GTP-binding proteins has diverse functions in cell growth, differentiation, and secretion. These proteins bind GTP and are down-regulated by GTPase-activating proteins (GAPs), which stimulate their intrinsic GTPase activity (8). n-Chimerin is one of a new family of GAPs which regulate members of the rho subfamily (16,24,25), which is involved in cytoskeletal organization (23). rho and rac play a role in the formation of actin stress fibers and focal contacts and in membrane ruffling in response to growth factors (60, 61).n-Chimerin, Bcr, rhoGAP (16), and the rasGAP-associated protein p190 (66) . p190 is a GAP for rho, rac, and cdc42Hs (65). In contrast, n-chimerin acts selectively on rac and is restricted to neurons (40,42). It is enriched in brain regions involved in learning and memory and may have a specialized rac-mediated role in neuronal signalling. A second chimerin, 3-chimerin, is restricted to spermatocytes in late stages of maturation (36). The cysteine-rich phorbol ester-binding and GAP sequences are conserved in 1-chimerin (97 and 72% amino acid identity, respectively) (36). Both domains are present in the putative product of the Drosophila rotund gene, which is expressed in imaginal discs and the developing testis (1). rasGAP both regulates GTPase...
GJA1 (connexin43) has been predicted as the top key driver of an astrocyte enriched subnetwork associated with Alzheimer’s disease (AD). In this study, we comprehensively examined GJA1 expression across 29 transcriptomic and proteomic datasets from post-mortem AD and normal control brains. We demonstrated that GJA1 was strongly associated with AD amyloid and tau pathologies and cognitive functions. RNA sequencing analysis of Gja1−/− astrocytes validated that Gja1 regulated the subnetwork identified in AD, and many genes involved in Aβ metabolism. Astrocytes lacking Gja1 showed reduced Apoe protein levels as well as impaired Aβ phagocytosis. Consistent with this, wildtype neurons co-cultured with Gja1−/− astrocytes contained higher levels of Aβ species than those with wildtype astrocytes. Moreover, Gja1−/− astrocytes was more neuroprotective under Aβ stress. Our results underscore the importance of GJA1 in AD pathogenesis and its potential for further investigation as a promising pharmacological target in AD.Electronic supplementary materialThe online version of this article (10.1186/s40478-018-0642-x) contains supplementary material, which is available to authorized users.
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