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Cellular signaling of human microglia in response to [beta]-amyloid 1-40

University of British Columbia

Microglia are resident immune cells of the brain that are activated in response to trauma and inflammation. Activated microglia exhibit characteristics similar to peripheral macrophages, such as the expression of irnmunomolecules, secretion of proinflammatory substances and phagocytic activity. Like macrophages, microglia exhibit these characteristics in order to defend the brain from infection and aid in the repair of damaged tissue. However, in Alzheimer's Disease (AD) microglia can become overactivated resulting in the release of substances that escalate Marnmation and ultimately cause neuronal death. A protein implicated in the progression of AD is β-amyloid (Aβ). Aβ production is increased in AD and deposits of Aβ form throughout the brain, which are correlated to the activation of microglia and neuronal death. Studies have shown that Aβ can activate microglia and cause changes in the cellular functions of these cells. For example, in microglia Aβ has been shown to cause increases in the production of pro-inflarnrnatory cytokines and reactive oxygen species. The objective of this work was to characterize the actions of Aβ 40, a commonly expressed form of Aβ, on the mobilization of intracellular calcium ([Ca²⁺]0 in human microglia. The rational was that subsequent pharmacological modulation of the calcium signals induced by Aβ 40 could then be used to alter the cellular functions of microglia, such as the secretion of neurotoxic factors. The first study used calcium sensitive microfluorescence to examine Aβ 40 actions on [Ca²], mediated signaling pathways. Aβ 40 application (4 and 10 μM) to microglia caused a rise in [Ca²j to a plateau level which was sustained following the removal of the peptide. Calcium-free external solution (Ca- free PSS) was used to show that the primary contribution to the [Ca²]; rise came from the influx of extracellular calcium. A small amount of intracellular calcium release is also possible since Ca-free PSS did not totally inhibit the Aβ 40-induced [Ca²][sub i] response. The Aβ 40 mediated calcium influx was sensitive to depolarization since low chloride solution applied extracellularly inhibited the influx of calcium. Additional experiments suggested that a store-operated channel (SOC) did not mediate the Aβ 40-induced calcium influx since an inhibitor of this pathway, SKF96365, had no effect on the [Ca²][sub i] rise. At present, a specific modifier of the Aβ 40-induced influx pathway has not been identified. The next study examined the actions of Aβ 40 on COX-2 expression. COX-2 is an enzyme responsible for prostaglandin synthesis and free radical formation that is overexpressed in AD . Microglia cultures were treated with Aβ 40 for 24 hrs and the expression of COX-2 was determined through RT-PCR analysis. The results show that Aβ 40 significantly increased COX-2 expression. However, it is not know if the enhancement of COX-2 is linked to the Aβ 40-induced increase in [Ca²][sub i]. The third study examined the potential of Aβ 40 to induce the production of neurotoxic substances by human microglia. Neuroblastoma cells were treated with supernatant from microglia exposed to Aβ 40 and the neurotoxic effects were evaluated by assessing cell viability. The results indicate that supernatant from Aβ 40 treated microglia decreased neuroblastoma viability, however the decrease was not significantly different from Aβ 40 applied directly to neuroblastoma cells. This result suggests that a larger number of human microglia are required to record the effects of neurotoxic substances in the cell viability assays used.

Thesis/Dissertation

application/pdf

2009-08-05

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http://hdl.handle.net/2429/11750

Neuroscience

University of British Columbia

UBCV

DSpace