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Hypoxia is a critical pathological element in many human diseases, including ischemic stroke, myocardial infarction, and solid tumors. Of particular significance and interest of ours are the cellular and molecular mechanisms that underlie susceptibility or tolerance to low O2. Previous studies have demonstrated that Notch signaling pathway regulates hypoxia tolerance in both Drosophila melanogaster and humans. However, the mechanisms mediating Notch-conferred hypoxia tolerance are largely unknown. In this study, we delineate the evolutionarily conserved mechanisms underlying this hypoxia tolerant phenotype. We determined the role of a group of conserved genes that were obtained from a comparative genomic analysis of hypoxia-tolerant D.melanogaster populations and human highlanders living at the high-altitude regions of the world (Tibetans, Ethiopians, and Andeans). We developed a novel dual-UAS/Gal4 system that allows us to activate Notch signaling in the Eaat1-positive glial cells, which remarkably enhances hypoxia tolerance in D.melanogaster, and, simultaneously, knock down a candidate gene in the same set of glial cells. Using this system, we discovered that the interactions between Notch signaling and bnl (fibroblast growth factor), croc (forkhead transcription factor C), or Mkk4 (mitogen-activated protein kinase kinase 4) are important for hypoxia tolerance, at least in part, through regulating neuronal development and survival under hypoxic conditions. Becausethese genetic mechanisms are evolutionarily conserved, this group of genes may serve as novel targets for developing therapeutic strategies and have a strong potential to be translated to humans to treat/prevent hypoxia-related diseases.

FOXO3a, as a forkhead transcription factor, can control cell cycle through transcriptionally down-regulating p27(kip1) level, which is a key regulator of the mammalian cell cycle and a good candidate to regulate multiple aspects of neurogenesis. To elucidate their expression and function in nervous system lesion and repair, we performed an acute sciatic nerve crush model and studied differential expressions of Foxo3a and p27(kip1) in lumbar dorsal root ganglia. Temporally, Foxo3a protein level was reduced 1 day after injury, and following Foxo3a down-regulation, p27(kip1) mRNA and protein levels were also decreased after injury. Spatially, decreased levels of Foxo3a and p27(kip1) were predominant in neurons and glial cells, which were regenerating axons and largely proliferated after injury, respectively. Together with previous reports, we hypothesized decreased levels of Foxo3a and p27(kip1) in lumbar dorsal root ganglia were implicated in axonal regeneration and the proliferation of glial cells after sciatic nerve injury.

S-phase-associated kinase protein-2 (Skp2) is involved in ubiquitination and proteasome-mediated degradation of p27(kip1), which plays an important role in mammalian cell-cycle regulation and neurogenesis in the developing central nervous system. To investigate their expression and function in central nervous system injury and repair, we used a brain-penetrating injury model in adult rats. Western blot analysis showed a significant downregulation of p27(kip1) and a concomitant upregulation of Skp2 following brain injury, and their expression profiles were temporally correlative (r = -0.910, p = 0.037). Immunofluorescence double-labeling revealed that p27(kip1) was highly expressed in neurons (51%), astrocytes (72%), and microglia (76%) in the sham group, while its expression was decreased prominently in microglia (26%) and astrocytes (32%) at 3 days after injury. Meanwhile, Skp2 expression was very low in all cell types in the sham group; however, 3 days after injury, its expression was increased significantly in microglia (51%) and astrocytes (31%) (p0.001), and less significantly in neurons (8%) (p = 0.038), and the astrocytes and microglia had proliferated. We also examined the expression profiles of CDK2, threonine-187 phosphorylated p27(kip1), proliferating cell nuclear antigen (PCNA), and Ki67, and their changes correlated with the expression profiles of p27(kip1) and Skp2. Moreover, co-immunoprecipitation data suggested that the protein-protein interactions between p27(kip1) and Skp2 were enhanced after injury. Taken with results of previous reports, we hypothesize the Skp2 is related to the downregulation of p27(kip1) expression after brain injury, and such an event may be associated with glial proliferation, including that of astrocytes and microglia.

beta-1,4-Galactosyltransferase-I (beta-1,4-GalT-I) is one of the best studied glycosyltransferases. Previous studies demonstrated that beta-1,4-GalT-I was a major galactosyltransferase responsible for selectin-ligand biosynthesis and that inflammatory responses of beta-1,4-GalT-I deficient mice were impaired. Our previous study suggest that beta-1,4-GalT-I may play an important role in regulating immune cell migration into the inflammatory site. In this study, we investigate beta-1,4-GalT-I may play an important role in mediating microgliosis. The results of this study demonstrated that beta-1,4-GalT-I was strongly induced in the ventral midbrain by intranigral injection of LPS. Most galactose-containing glycans and beta-1,4-GalT-I were expressed in microglia. Moreover, an Ab against beta-1,4-GalT-I attenuated both LPS-induced microglial activation and phagocytosis. We therefore suggest that beta-1,4-GalT-I may play an important role in regulating immune cell migration into the inflammatory site and mediating microgliosis.

Annexin 7 (ANX7), also termed synexin, is a member of the annexin family of calcium-binding proteins. In the present study, we examined the distribution and cellular localization of ANX7-immunoreactivity in the rat hippocampus and its response to adrenalectomy (ADX). ANX7 was co-localized with OX42 in microglia distributed throughout the hippocampus of both control and ADX animals. ANX7-immunoreactivity was not detected in GFAP-positive astrocytes or in hippocampal neurons. At 1-week and 4-weeks following ADX, we observed a population of large, ameboid, ANX7-immunopositive microglia ("reactive microglia") which were largely confined to the granule cell layer of the dentate gyrus throughout its rostrocaudal extent. No reactive microglia were present in the hippocampus of sham-ADX or ADX + corticosterone treated animals. In 4-weeks ADX animals but not 1-week ADX, ANX7-immunostaining was significantly increased in the mossy fiber layer of CA3, due to the presence of many small, dark-staining "activated microglia". Our results show that ANX7 is abundantly expressed in the rat hippocampus by different microglial forms (e.g., ramified, activated and reactive microglia), suggesting an important role for this calcium-binding protein in microglial Ca2+-dependent processes.