Investigations into Alzheimer's disease pathogenetic mechanisms using normal adult human astrocytes in culture

Alterations in neurovascular mechanisms and blood-brain barrier (BBB) disorders have been related to late-onset Alzheimer’s disease (AD). An increased expression of vascular endhotelial growth factor (VEGF) has seen in cluster of reactive astrocytes in AD brains and into rat hippocampus Aβ1-42 injected. We found that the Aβ25-35, an amyloid β1-42 active fragment, and proinflammatory cytokines (CM-trio, IL-1β + IFN-γ + TNF-α) treatments has induced an increased protein steady state levels of the hypoxia-inducible factor transcriptional complex (HIF-1α•HIF-1β) in normal adult human astrocytes (NAHAs). This transcription factor is the main regulator of the VEGF expression, but also the PGC-1α cofactor seems to be related to its upregulation in AD. We analyzed the mRNA expression of both these factors, but only the complex HIF-1α•HIF-1β was implicated in the VEGF-A expression. Indeed, the enhanced HIF-1α•HIF-1β nuclear translocation and its binding at the hypoxia response element (HRE) sequence in the VEGF-A promoter, found after both treatments, were correlated with the increased VEGF-A mRNA splice variants (121, 165, and 189) expression. The CM-trio was the most powerful stimulus inducing the VEGF-A secretion in NAHAs culture medium, also induced, less than CM-trio, by the paired cytokines IFN-γ + TNF-α, Aβ25-35 and CM-trio + Aβ25-35 treatments. Hence the astrocytes in an AD brain can produce a raised amount of VEGF-A in response to Aβ peptide and the proinflammatory cytokines promoting the neoangiogenic process. We found that the HIF-1α•HIF-1β complex was furthermore responsible for the β-secretase (BACE-1) mRNA and protein up-regulation in our experimental model treated with Aβ25-35. After the same treatment we found moreover an enhancement of the β and γ-secretase activities, the two secretases involved in the amyloid precursor protein (APP) processing. The immunofluorescence and immunoblotting assays confirmed the Aβ production in NAHAs, suggesting the existence of a positive feed-back loop for the Aβ synthesis in astrocytes. This findings support the theory that astrocytes, as well as neurones, are involved in the AD decline of cognitive functions. Moreover, the immunofluorescence assay shows the Aβ25-35 uptake, defining a possible role of astocytes as Aβ peptides scavengers.