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Cuprizone-induced demyelination is a mouse model of multiple sclerosis (MS) as cuprizone-fed mice exhibit neuroin-flammation and demyelination in the brain. Upon removal of cuprizone from the diet, inflammation is resolved and reparative remyelination occurs. In an Affymetrix Gene-Chip analysis, the stress-associated gene p8 was strongly upregulated (>10×) during cuprizone-induced demyelination but not remyelination. We verified this upregulation (>15×) of p8 in the CNS during demyelination by real-time polymerase chain reaction (PCR). This upregulation is brain-specific, as p8 is not elevated in the liver, lung, kidney, spleen, and heart of cuprizone-treated mice. We also localized the cellular source of p8 during cuprizone treatment, and further found elevated expression during embryogenesis but not in normal adult brain. Compared with wild-type controls, the death of oligodendrocytes in p8−/− mice is delayed, as is microglial recruitment to areas of demyelination. The corpus callosum of p8−/− mice demyelinates at a slower rate than wild-type mice, suggesting that p8 exacerbates CNS inflammation and demyelination. Enhanced expression of p8 is also observed in the spinal cords of mice with acute experimental autoimmune encephalomyelitis (EAE) induced by PLP139–151 peptide (10×). Increased expression is detected during disease onset and expression wanes during the remission phase. Finally, p8 is found upregulated (8×) in post-mortem tissue from MS patients and is higher in the plaque tissue compared with adjacent normal-appearing white and gray matter. Thus, p8 is an excellent candidate as a novel biomarker of demyelination.

Owing to the difficulties of isolating adequate numbers of microglia from adult tissue, much of our understanding of their function is based on characterizations of microglia that develop in mixed glial cultures. To learn more about the nature of these cells in vivo, we have compared the phenotypes of murine microglia isolated from adults, neonates, and from mixed glial cultures with spleen cells from fetuses, neonates, and adults. In the adult CNS, the only resident population of cells that express CD45, a protein tyrosine phosphatase, are the F4/80+ and FcR+ cells: the microglia. In contrast to all other differentiated cells of hemopoietic origin, microglial CD45 levels fail to increase from the neonatal period through adulthood. Rather, their levels are indistinguishable from the low levels found on a small population of embryonic day 16 liver cells. Conversely, we find that the F4/80 values of microglia are elevated as compared to splenic macrophages. Strikingly, microglia that develop in mixed glial cultures display a more activated phenotype, with low F4/80 values, weak MHC class II expression, and the appearance of a subset of cells positive for the dendritic cell marker, NLDC145. Additionally, CD45 values are elevated to a level intermediate between that of adult microglia and adult spleen, a level similar to that found on microglia activated in vivo. Consistent with this activated phenotype, indomethacin revealed the ability of mixed glial culture microglia to present a peptide antigen to naive T-cells expressing a defined T-cell receptor. Although adult microglia did express costimulatory molecules, B7.2, ICAM-1, and CD40, and could be induced to express MHC class II, they failed to present antigen in the same assay. Interestingly, these same cells could stimulate T-cell proliferation in a mixed lymphocyte reaction but not in an allogeneic specific manner. Taken together these data suggest that adult microglia remain in a relatively immature and unactivated state of differentiation as compared to other tissue macrophages.

The mRNA for the 5-hydroxytryptamine receptor 5-HT5A was detected at embryonic day 18 in the rat central nervous system and peaked by postnatal day 20. At all time points examined, 5-HT5A immunoreactivity observed on astrocyte cell bodies and in the stellate processes not only colocalized with the astrocyte-specific marker glial fibrillary acidic protein (GFAP) but was coordinately regulated with GFAP, increasing during development and during gliosis. Transfection of 5-HT5A into glioma cells prevented the 5-HT-induced increase in cAMP observed in untransfected cells and decreased the relative forskolin response by approximately 20%, suggesting that the 5-HT5A receptor couples negatively to adenylyl cyclase in astrocytes. Together, these results indicate a neuron-to-astrocyte serotonergic signaling pathway mediating cAMP concentrations, which could provide a neuronally driven mechanism for regulating astrocyte physiology with relevance to gliosis.