EphA4 Regulates Hippocampal Neural Precursor Proliferation in the Adult Mouse Brain by d-Serine Modulation of N-Methyl-d-Aspartate Receptor Signaling.

The hippocampal dentate gyrus (DG) is a major region of the adult rodent brain in which neurogenesis occurs throughout life. The EphA4 receptor, which regulates neurogenesis and boundary formation in the developing brain, is also expressed in the adult DG, but whether it regulates adult hippocampal neurogenesis is not known. Here, we show that, in the adult mouse brain, EphA4 inhibits hippocampal precursor cell proliferation but does not affect precursor differentiation or survival. Genetic deletion or pharmacological inhibition of EphA4 significantly increased hippocampal precursor proliferation in vivo and in vitro, by blocking EphA4 forward signaling. EphA4 was expressed by mature hippocampal DG neurons but not neural precursor cells, and an EphA4 antagonist, EphA4-Fc, did not activate clonal cultures of precursors until they were co-cultured with non-precursor cells, indicating an indirect effect of EphA4 on the regulation of precursor activity. Supplementation with d-serine blocked the increased precursor proliferation induced by EphA4 inhibition, whereas blocking the interaction between d-serine and N-methyl-d-aspartate receptors (NMDARs) promoted precursor activity, even at the clonal level. Collectively, these findings demonstrate that EphA4 indirectly regulates adult hippocampal precursor proliferation and thus plays a role in neurogenesis via d-serine-regulated NMDAR signaling.

Complex expression patterns of Eph receptor tyrosine kinases and their ephrin ligands in colorectal carcinogenesis.

Aberrant expression of Eph and ephrin proteins in human cancers is extensively documented. However, data are frequently limited to one gene and therefore incomplete and in some instances conflicting. We analysed expression of all Eph and ephrin genes in colorectal cancer (CRC) cell lines and 153 clinical specimens, providing for the first time a comprehensive analysis of this system in CRC. Eph/ephrin mRNA expression was assessed by quantitative real-time PCR and correlated with protein expression (flow cytometry, Western blotting and immunocytochemistry). These data show that EphA1, EphA2, EphB2 and EphB4 were significantly over expressed in CRC. In all cases, at least one Eph gene was found in normal colon (EphA1, EphA2, EphB2, EphB4), where expression was observed at high levels in most CRCs. However, other Eph gene expression was lost in individual CRCs compared to the corresponding normal, EphA7 being a striking example. Loss of expression was more common in advanced disease and thus correlated with poor survival. This is consistent with the redundant functionality of Eph receptors, such that expression of a single Eph gene is sufficient for effector function. Overall, the data suggest a progressive loss of expression of individual Eph genes suggesting that individual CRCs need to be phenotyped to determine which Eph genes are highly expressed. Targeted therapies could then be selected from a group of specific antibodies, such as those developed for EphA1.

EphA4 blockers promote axonal regeneration and functional recovery following spinal cord injury in mice.

Upregulation and activation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family and their ligands, the ephrins, play a role in the inhibition of axonal regeneration following injury to the central nervous system. Previously we have demonstrated in a knockout model that axonal regeneration following spinal cord injury is promoted in the absence of the axon guidance protein EphA4. Antagonism of EphA4 was therefore proposed as a potential therapy to promote recovery from spinal cord injury. To further assess this potential, two soluble recombinant blockers of EphA4, unclustered ephrin-A5-Fc and EphA4-Fc, were examined for their ability to promote axonal regeneration and to improve functional outcome following spinal cord hemisection in wildtype mice. A 2-week administration of either of these blockers following spinal cord injury was sufficient to promote substantial axonal regeneration and functional recovery by 5 weeks following injury. Both inhibitors produced a moderate reduction in astrocytic gliosis, indicating that much of the effect of the blockers may be due to promotion of axon growth. These studies provide definitive evidence that soluble inhibitors of EphA4 function offer considerable therapeutic potential for the treatment of spinal cord injury and may have broader potential for the treatment of other central nervous system injuries.

ELK4 neutralization sensitizes glioblastoma to apoptosis through downregulation of the anti-apoptotic protein Mcl-1.

Glioma is the most common adult primary brain tumor. Its most malignant form, glioblastoma multiforme (GBM), is almost invariably fatal, due in part to the intrinsic resistance of GBM to radiation- and chemotherapy-induced apoptosis. We analyzed B-cell leukemia-2 (Bcl-2) anti-apoptotic proteins in GBM and found myeloid cell leukemia-1 (Mcl-1) to be the highest expressed in the majority of malignant gliomas. Mcl-1 was functionally important, as neutralization of Mcl-1 induced apoptosis and increased chemotherapy-induced apoptosis. To determine how Mcl-1 was regulated in glioma, we analyzed the promoter and identified a novel functional single nucleotide polymorphism in an uncharacterized E26 transformation-specific (ETS) binding site. We identified the ETS transcription factor ELK4 as a critical regulator of Mcl-1 in glioma, since ELK4 downregulation was shown to reduce Mcl-1 and increase sensitivity to apoptosis. Importantly the presence of the single nucleotide polymorphism, which ablated ELK4 binding in gliomas, was associated with lower Mcl-1 levels and a greater dependence on Bcl-xL. Furthermore, in vivo, ELK4 downregulation reduced tumor formation in glioblastoma xenograft models. The critical role of ELK4 in Mcl-1 expression and protection from apoptosis in glioma defines ELK4 as a novel potential therapeutic target for GBM.

Activation of ephrin A proteins influences hematopoietic stem cell adhesion and trafficking patterns.

OBJECTIVE: To determine if Eph receptors and ephrins can modulate the homing of hematopoietic cells in a murine bone marrow transplantation model. MATERIALS AND METHODS: EphA and ephrin A gene expression by mouse hematopoietic stem cells and the progenitor cell line FDCP-1 was determined by real-time reverse transcription polymerase chain reaction and flow cytometry. The effect of ephrin A activation on adhesion of hematopoietic progenitors was determined by in vitro adhesion assays in which cells were exposed to fibronectin or vascular cell adhesion molecule-1 (VCAM-1) and an increasing gradient of immobilized EphA3-Fc. Adhesion to fibronectin and VCAM-1 was further investigated using soluble preclustered EphA3-Fc. We used soluble unclustered EphA3-Fc as an antagonist to block endogenous EphA-ephrin A interactions in vivo. The effect of injecting soluble EphA3-Fc on the mobilization of hematopoietic progenitor cells was examined. We determined the effect on short-term homing by pretreating bone marrow cells with EphA3-Fc or the control IgG before infusion into lethally irradiated mice. RESULTS: Preclustered and immobilized EphA3-Fc increased adhesion of progenitor cells and FDCP-1 to fibronectin and VCAM-1 (1.6- to 2-fold higher adhesion; p < 0.05) relative to control (0 µ/cm(2) EphA3-Fc extracellular molecule alone). Injection of the antagonist soluble EphA3-Fc increased progenitor cell and colony-forming unit-spleen cells in the peripheral blood (42% greater colony-forming unit in culture; p < 0.05, 3.8-fold higher colony-forming unit-spleen) relative to control. CONCLUSION: Treating bone marrow cells with EphA3-Fc resulted in a reduction by 31% in donor stem cells homing to the bone marrow and accumulation of donor cells in recipient spleens (50% greater than control) and greater recovery of donor stem cells from the peripheral blood.

Generation and characterization of EphA1 receptor tyrosine kinase reporter knockout mice.

Eph receptor tyrosine kinases (RTKs) are a highly conserved family of signaling proteins with functions in cellular migration, adhesion, apoptosis, and proliferation during both adult and embryonic life. Here, we describe a knock-in mouse in which EphA1 expression is disrupted via the insertion of an internal ribosome entry site (IRES)-human placental alkaline phosphatase (ALPP) reporter cassette into exon II of the EphA1 gene. This was shown to successfully knockout expression of endogenous EphA1 and enforce expression of the ALPP reporter by the EphA1 promoter. Staining for the ALPP reporter protein demonstrated an epithelially restricted expression pattern in mouse tissues. In EphA1 null mice, two separate phenotypes were identified: abnormal tail development manifesting as a kinky tail was found in approximately 80% of homozygous adults. A second, distinct abnormality present in approximately 18% of females was characterized by imperforate uterovaginal development with hydrometrocolpos and caused by a resistance of cells to apoptosis during reproductive tract canalization. These results indicate a possible role for EphA1 in tissue patterning and hormone-induced apoptotic processes.

Over-expression of Eph and ephrin genes in advanced ovarian cancer: ephrin gene expression correlates with shortened survival.

BACKGROUND: Increased expression of Eph receptor tyrosine kinases and their ephrin ligands has been implicated in tumor progression in a number of malignancies. This report describes aberrant expression of these genes in ovarian cancer, the commonest cause of death amongst gynaecological malignancies. METHODS: Eph and ephrin expression was determined using quantitative real time RT-PCR. Correlation of gene expression was measured using Spearman's rho statistic. Survival was analysed using log-rank analysis and (was visualised by) Kaplan-Meier survival curves. RESULTS: Greater than 10 fold over-expression of EphA1 and a more modest over-expression of EphA2 were observed in partially overlapping subsets of tumors. Over-expression of EphA1 strongly correlated (r = 0.801; p < 0.01) with the high affinity ligand ephrin A1. A similar trend was observed between EphA2 and ephrin A1 (r = 0.387; p = 0.06). A striking correlation of both ephrin A1 and ephrin A5 expression with poor survival (r = -0.470; p = 0.02 and r = -0.562; p < 0.01) was observed. Intriguingly, there was no correlation between survival and other clinical parameters or Eph expression. CONCLUSION: These data imply that increased levels of ephrins A1 and A5 in the presence of high expression of Ephs A1 and A2 lead to a more aggressive tumor phenotype. The known functions of Eph/ephrin signalling in cell de-adhesion and movement may explain the observed correlation of ephrin expression with poor prognosis.

The phylogenetic diversity of Thermus and Meiothermus from microbial mats of an Australian subsurface aquifer runoff channel.

Spectral analysis of the cell free extracts of four mat samples colonizing a Great Artesian Bain (GAB) aquifer bore runoff channel suggested that Thermus was present in the 75 degrees C grey mat, Meiothermus was present in the 66 degrees C red mat, a mixed population of Meiothermus/Thermus and photosynthetic microbes were present in the 57 degrees C green mat and photosynthetic microbes were present in the 52 degrees C brown mat. Enumeration studies indicated that Thermus dominated the grey mats and Meiothermus dominated the red mat but both were absent in samples collected at the bore source (89 degrees C) and below the bore source (88 degrees C). Culture-dependent studies followed by 16S rRNA gene sequence analysis indicated that 13 of the 14 Thermus isolates clustered closely with each other and to T. igniterrae, with the remaining strain clustering with Thermus strain SRI-96. The two Meiothermus isolates were closely related to Meiothermus ruber. A culture-independent study with 367 16S rRNA gene clones concurred that Thermus dominated the grey mat, but to a lesser extent in the red mat and the green mats and its complete absence in the brown mat. Of the four Thermus phylogroups identified one phylogroup dominated the cloned library and was related to the cluster represented by T. scotoductus. The second most dominant phylogroup was related to the cluster represented by T. igniterrae and the third and fourth phylogroups, which were the least dominant, were related to cluster represented by Thermus strain SRI-248 and T. oshima respectively. Meiothermus was only represented in the 16S rRNA gene libraries of the red, green and brown mats and formed two phylogroups, of which the most dominant was associated with the red mat and phylogenetically related to M. ruber while the second phylogroup was found only in the green mat gene library and was related to M. cerberus.

Thermaerobacter subterraneus sp. nov., a novel aerobic bacterium from the Great Artesian Basin of Australia, and emendation of the genus Thermaerobacter.

A strictly aerobic, thermophilic, gram-positive, spore-producing, rod-shaped bacterium (2.0-10.0 x 0.3 microm), designated isolate C21T, was isolated from a sample collected from an open drain run-off channel of a bore in the Great Artesian Basin of Australia (New Lorne Bore, registered number 17263). Isolate C21T grew optimally at 70 degrees C (temperature range for growth was 55-80 degrees C) and pH 8.5 (pH range for growth was 6.0-10.5), with a generation time of 90 min. The isolate was strictly heterotrophic and grew on yeast extract and/or tryptone as carbon and energy sources. An increase in growth was not observed with carbohydrates (sucrose, cellobiose, glucose, dextrin, amylopectin, chitin, carboxymethylcellulose, xylan, inositol, arabinose, mannose, fructose, gelatin, starch, amylose, galactose, dextrose, xylose, maltose, L-sorbose or raffinose), organic acids (lactic acid, pyruvic acid or benzoic acid) or Casamino acids as sole carbon sources or in the presence of yeast extract and/or tryptone. The G+C content of the chromosomal DNA, as measured by the thermal denaturation method, was 71 mol%. Phylogenetic analysis of the 16S rRNA gene of isolate C21T placed it as a member of the phylum Firmicutes, with Thermaerobacter marianensis as the closest relative (similarity value of 98%). However, isolate C21T and T. marianensis differed in a number of key physiological and phenotypic properties and also had a DNA-DNA hybridization value of less than 5%. Based on this evidence, it is proposed that strain C21T be designated Thermaerobacter subterraneus sp. nov. (type strain C21T = ATCC BAA-137T = DSM 13965T).