IL-6 promotes astrogliosis79 and activates microglia.80 Increased IL-6 found in the AD brain could come from either microglia or astrocytes, or both. As we have shown, β-AP induces β-AP secretion by microglia,82 so local levels of this stimulus would also increase, leading to further microglia secretion of IL-1, and to additional neuronal M-CSF expression. In this way, a self-perpetuating pathophysiologic

cascade is initiated. It is important, that the augmenting effect of M-CSF is specific. Our results show that costimulation of BV-2 cells with β-AP 1-40 and GM-CSF, another colony-stimulating factor Inhibitors,research,lifescience,medical produced by astrocytes that activates microglia,54 does not augment IL-1 or NO (nitrite) production. Many features of this model could be tested. In our current experiments, we are focusing on microglial production of NO, IL-1, IL-6, and ROS in response to β-AP,

IL-1, and M-CSF stimulation, and on how these Inhibitors,research,lifescience,medical events affect neurons in Selisistat Organotypic hippocampal cultures. Organotypic hippocampal cultures contain the full complement of cerebral cell types including neurons, astrocytes, and microglia. Hence, they more closely model the intact, brain than do monotypic cultures of neurons or glia.83 Using the reverse transcriptase polymerase chain reaction Inhibitors,research,lifescience,medical (RT-PCR), we have found that treatment of organotypic hippocampal cultures with β-AP (22 µM, 24 hours’ treatment.) and M-CSF results in a larger increase in the mRNA for IL-1 and iNOS than either agent, alone. M-CSF augmentation of β-AP-induced

IL-1 expression can also be detected in conditioned media from organotypic cultures using enzyme-linked immunosorbent, assay (ELISA). Note that there is no toxicity after 24 hours’ treatment, Inhibitors,research,lifescience,medical as assessed by lactic dehydrogenase (LDH) in conditioned media. We are currently using immunohistochemical techniques with organotypic cultures to identify the cell type(s) that show increased synthesis of IL-1 and NO after treatment with β-AP and M-CSF. Inhibitors,research,lifescience,medical Organotypic cultures may also be useful in modeling inflammation-mediated neurotoxicity in AD. β-AP at a dose of 47 µM induces a significant increase in LDH in slice culture medium after 72 hours of treatment. M-CSF synergistically augments this toxicity (Figure 2). Figure 2. M-CSF augments β-AP-induced Oxygenase toxicity in hippocampal slice cultures. Rat organotypic hippocampal cultures were treated for 72 hours with medium, β-AP 40-1 (inactive control peptide), β-AP 1-40, M-CSF 50 ng/mL, or β-AP 1-40 … We are also examining expression of M-CSF and its receptor in transgenic animal models for AD. In these models, mutant human beta-amyloid precursor protein transgenes result in deposition of P-AP in the brain, and a robust glial reaction surrounding these deposits.84,85 Our hypothesis is that increased β-AP deposition in these animals should lead to increased expression of M-CSF and possibly its receptor.