Cultured normal adult human astrocytes as a model to investigate patho-physiological mechanisms in chronic inflammatory brain disorders

When the brain is injured by stroke, head trauma or by the Alzheimer and Parkinson disease processes, astrocytes respond to the build-up of proinflammatory cytokines, such as IL-1ß, TNF-α, and IFN-γ from activated microglia by making among other things the short-lived, weakly oxidizing nitric oxide (NO), which is converted to the long-lived, cytotoxic, strongly oxidizing peroxynitrite radical (ONOO_) that can harm and kill the neurons functionally and physically associated with the astrocytes. This NO can be made by any one of the three main types of NOS (NO synthase)—neuronal NOS-1 (or nNOS), the inducible and the most productive NOS-2 (or iNOS), and endothelial cell NOS-3 (eNOS or ecNOS). Since NO injures and kills neurons in variously damaged brains, finding out which nitric oxide synthase induces it and which are the signalling pathways that modulate the enzymatic activity in the human adult astrocytes, should help to design effective therapeutics. In fact, for example, it has been demonstrated that inhibiting the expression of the NOS-2 gene or the activation of the NOS-2 protein significantly reduces the damage from a head blow or focal ischemia in rat and mouse brains, respectively. For this reason, the aims of my study have been (1) to find out which NOS enzyme is involved in NO-driven killing cascade, and (2) to explicate the mechanisms responsible for the build-up and the regulation of NOS in freshly isolated proliferatively quiescent, phenotypically stable, adult human cerebral astrocytes exposed to the three proinflammatory cytokines (IL-1ß, IFN-γ, TNF-αCM trio) that are most often found in injured or diseased brains. Finally, I have also investigated (3) the effects of a soluble Aß, the Aß (1-40) peptide driving the development of AD , as well as the myelin basic protein (MBP) that may be produced by neuronal degeneration and oligodendroglial cell damage, on the NO production by the normal adult human astrocytes equivalent to those found in a normal adult brain. I found that when these astrocytes were exposed to three proinflammatory cytokines (IL-1ß, IFN-γ, TNF-α), they somehow triggered the expression of the NOS-2 gene by MEK–ERK activity-dependent events. The critical MEK1/MEK2-dependent process had been available for starting NOS-2 activation only between 24 and 24.5 h after CM trio addition because it could not start again and drive NO production for at least 6.5 days after the treatment with the MEK1/MEK2 inhibitor U0126. The second novel finding was that the U0126-sensitive process in the CM trio-stimulated astrocytes was the synthesis of the NOS-2 cofactor tetrahydrobiopterin (BH4), first because the U0126 prevented the build up of BH4 that accompanies the surge of NO production, and second because adding BH4 to the culture medium enabled the U0126- treated cells to generate nearly normal amounts of NO. The BH4 accumulating along with the new NOS-2 proteins in CM trio-treated cells would have operated by binding with very high affinity to single NOS-2 pre-enzyme proteins and promoting their dimerization into functional enzymes. I also found that Ca2+-sensing receptors (CASR) signaling was needed to activate the NOS-2 because exposing the cells to NPS 89636 prevented NO production without affecting NOS-2 protein expression. It appeared that NPS 89636 could mimic U0126 by preventing ERK activation, and this inhibition by NPS 89636 could be completely overridden by adding BH4 to the medium. I then focused my efforts on finding out how CASR signals and MEK-ERK kinases control the production of BH4 by GTP-cyclohydrolase-I (GTP-CH-I) in human astrocytes activated by pro-inflammatory cytokines. In this study I demonstrated that GTP-CH-I is not constitutively expressed and was coinduced in human astrocytes stimulated by CM trio to produce NO. It was my intention to investigate which proteins could be implicated in the activation and post-translational modifications of GTP-CH-I and by mean of proteomic analysis I found that proteins involved probably as adaptors in the signalling transduction of a G-protein-coupled receptor, like CASR were part of the immunocomplexes isolated using an anti-GTP-CH-I antibody from CM trio-treated astrocytes. Some of them could be also involved in the modulation of the intracellular GTP level. GTP is the substrate of GTP-CH-I and modulates GTP-CH-I activity by cooperative binding and thereby changing the enzyme kinetics. Finally, from my results, I have also learned that a soluble Aß, the Aß (1-40) peptide, as well as the MBP that may be produced by neuronal degeneration and oligodendroglial cell damage, could stimulate non-proliferating phenol typically normal adult human astrocytes equivalent to those found in a normal adult brain to start expressing NOS-2 and manufacturing NO. The mechanism(s) by which Aß (1-40) and MBP might have stimulated NO overproduction in the astrocytes is unknown. Clearly the next steps will be to find out whether Aßs and MBP induce normal mature adult human astrocytes to express GTP-CH-I and NOS-2 and make NO using the same MEK/ERK-dependent mechanism as the INF-γ+IL-1ß+TNF-α cytokine triad. Every clarification of the steps leading to the coinduction of GTP-CH-I and NOS-2 activity in the human astrocytes may be an important finding that help the understanding of the molecular events taking place in the neuro inflammation and in the neuronal damage, as manifested in AD brain.