Faculty Bibliography

It has become apparent that astrocytes may be important contributors to inflammatory immune responses within the brain in response to microbial challenges. To date, the mechanisms that underlie activation of this major glial cell type by such challenges have not been investigated. In the present study, we present evidence for members of a recently discovered family of receptors for highly conserved microbial components, the Toll-like receptors (TLRs), in isolated cultures of primary murine astrocytes. We describe the low-level constitutive expression of messenger RNA-encoding TLR2, TLR4, TLR5, and TLR9 in resting cultures of these cells. Importantly, the level of expression of messenger RNA for each of these receptors is markedly elevated following exposure to specific bacteria-derived ligands for these receptors. The functional expression of these receptor proteins is further supported by the ability of known ligands for each TLR to induce both message expression and protein secretion of the proinflammatory cytokine, interleukin-6. In addition, the recent availability of antibodies to TLR2 and TLR4 has enabled us to demonstrate directly the presence of these receptors on astrocytes by Western blot and immunofluorescence analysis, respectively. Furthermore, we have confirmed the sensitivity of such receptor expression to ligand stimulation. The present demonstration of Toll-like microbial pattern-recognition receptors on primary astrocytes provides a mechanistic link between bacterial challenge and inflammatory immune responses that may be an important component of the pathologies of bacterially induced inflammatory CNS disorders.

Nitric oxide acts as an important intracellular messenger in a variety of systems, including reproduction. Previous studies have shown the importance of nitric oxide in embryo development. NO is produced from l-arginine by the enzyme, nitric oxide synthase (NOS), which has three isoforms: endothelial (NOS3), neural (NOS1), and inducible (NOS2). We hypothesize that, because of the importance of NOS in development, at least two NOS isoforms are required in order for normal embryo development to occur. Through the generation of NOS3/NOS2, NOS3/NOS1, and NOS2/NOS1 double knockout mice, we found that while litter size remains unchanged, the expected number of generated double knockout mice varies significantly from what would be predicted by Mendelian genetics. Estrous cycles were similar for both DKO and the wild-type mice, and both groups were deemed fertile by their ability to mate with wild-type (CD-1) mice. Together, these results lead us to conclude that the lack of two NOS isoforms leads to a decreased viability in mice because of a developmental problem in the double knockout embryo.

Nitric oxide (NO) production plays an important role in regulating preimplantation embryo development. NO is produced from l-arginine by the enzyme nitric oxide synthase (NOS), which has three isoforms: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). It has been previously shown that inhibition of NO production by NG-nitro-l-arginine (l-NA) inhibits the development of two-cell embryos to the four-cell stage. However, excess NO also halts embryo development, possibly through the production of free radicals. We hypothesize that multiple NOS isoforms are expressed in order to ensure normal preimplantation embryo development and that, in this process, NO acts through the cGMP pathway. Using reverse transcription-polymerase chain reaction, mRNA for all three NOS isoforms was amplified from two-cell, four-cell, morula, and blastocyst embryos. However, blastocyst-stage embryos isolated midmorning on Day 4 of pregnancy expressed only nNOS and eNOS, whereas those isolated midafternoon again expressed all three NOS isoforms. Culture of one-cell embryos in various concentrations of Whitten (positive control), S-nitroso-N-acetylpenicillamine (SNP, a NO donor), l-NA, and/or 8-Br-cGMP demonstrated that NO is acting, at least in part, through cGMP in preimplantation embryo development. In addition, we determined that a critical concentration of NO and cGMP is required for normal embryo development and deviations from this concentration lead to developmental arrest and/or apoptosis of the embryo. This data provides support for a requirement of NO in preimplantation embryo development and one mechanism through which it regulates mitotic division in these embryos.