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Beta-Amyloid₁₋₄₂- induced intracellular signaling pathways, functional responses and modulation by 4-aminopyridine in microglia Franciosi, Sonia


Alzheimer's Disease (AD) is a progressive, neurodegenerative disease characterized by gradual cognitive decline and memory loss. Although research has focused on elucidating the risk factors, pathophysiologic abnormalities associated with AD and on mechanisms of impeding disease progression, results indicate that a variety of factors may contribute to AD which makes treating this disease difficult. The neuropathological hallmarks of AD include senile plaques which are composed of extracellular deposits of amyloid beta (Aβ) peptide as well as neurofibrillary tangles, neuronal loss and inflammation. Microglia, the immune cells of the CNS, are abundantly found in the vicinity of neuritic plaques. It is believed that microglia become activated in response to Aβ leading to an inflammatory response and subsequent neuronal loss associated with AD pathogenesis. Modulation of the Aβ-induced intracellular signaling and functional responses of microglia could serve as a therapeutic strategy for AD. Full length amyloid beta, Aβ₁₋₄₂, induced distinct intracellular signaling pathways in human microglia. Electophysiological studies indicated that Aβ₁₋₄₂ acutely applied to human microglia upregulated the expression of a novel outward K⁺ current, sensitive to the nonselective K⁺ channel blocker 4-aminopyridine (4-AP). A similar outward K⁺ current was activated by intracellular application of GTPγS which suggests that Aβ₁₋₄₂ induces an outward K⁺ current in microglia via a G protein. Molecular biology studies indicated that the K⁺ channel upregulated by Aβ₁₋₄₂ was likely due to Kv3.1. Aβ₁₋₄₂ also caused a transient depolarization of microglia and increased the expression of the FcγII receptor. The FcγII receptor mediated this depolarization since antibody inhibition of the FcγII receptor inhibited the Aβ₁₋₄₂-induced depolarization. In addition to its ability to block the outward K⁺ current upregulated by Aβ₁₋₄₂, several in vitro and in vivo assays indicated that 4-AP modulates Aβ₁₋₄₂-induced intracellular signaling and functional responses of microglia including neurotoxicity. Calcium spectrofluorometric studies indicated that Aβ₁₋₄₂ activated a calcium entry pathway which was blocked by 4-AP. Chronic exposure of microglia to Aβ₁₋₄₂ led to increased p38 MAP kinase expression and NFκB activation; in the presence of 4-AP, both factors were inhibited. Stimulation with Aβ₁₋₄₂ also led to the expression and production of pro-inflammatory mediators; 4-AP was effective in reducing the expression and production of these factors. Furthermore, 4-AP attenuated neurotoxicity induced by conditioned medium from Aβ₁₋₄₂ stimulated microglia. In vivo, injection of Aβ₁₋₄₂ into rat hippocampus caused neuronal damage and increased microglial activation. Daily administration of 4-AP was found to suppress microglial activation and exhibited neuroprotection. These results suggest that 4-AP modulation of Aβ₁₋₄₂-induced intracellular signaling pathways and functional responses in human microglia including microglial-mediated neurotoxicity serves as a potential therapeutic strategy in AD pathology. The chemokine CXCL8 (IL-8) appears to potentiate Aβ₁₋₄₂ responses in human microglia. RT-PCR and ELISA studies indicated that CXCL8 potentiated Aβ₁₋₄₂-induced expression and production of pro-inflammatory mediators; the expression of antiinflammatory cytokines IL-10 and TGFβ₁ remained unchanged from basal levels despite treatment with stimuli. Stimulation with CXCL8 itself was effective in increasing microglial expression of pro-inflammatory mediators however, had no effect on protein levels of all these factors. CXCL8 potentiation of Aβ₁₋₄₂-induced inflammatory mediators may have particular relevance to AD brain which exhibits elevated levels of the chemokine.

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