Abstract
Alzheimer’s disease (AD) is characterized by a progressive cognitive decline and believed to be driven by the self-aggregation of amyloid-β (Aβ) peptide into oligomers and fibrils that accumulate as senile plaques. It is widely accepted that microglia-mediated inflammation is a significant contributor to disease pathogenesis; however, different microglia phenotypes were identified along AD progression and excessive Aβ production was shown to dysregulate cell function. As so, the contribution of microglia to AD pathogenesis remains to be elucidated. In this study, we wondered if isolated microglia cultured for 16 days in vitro (DIV) would react differentially from the 2 DIV cells upon treatment with 1000 nM Aβ1–42 for 24 h. No changes in cell viability were observed and morphometric alterations associated to microglia activation, such as volume increase and process shortening, were obvious in 2 DIV microglia, but less evident in 16 DIV cells. These cells showed lower phagocytic, migration and autophagic properties after Aβ treatment than the 2 DIV cultured microglia. Reduced phagocytosis may derive from increased CD33 expression, reduced triggering receptor expressed on myeloid cells 2 (TREM2) and milk fat globule-EGF factor 8 protein (MFG-E8) levels, which were mainly observed in 16 DIV cells. Activation of inflammatory mediators, such as high mobility group box 1 (HMGB1) and pro-inflammatory cytokines, as well as increased expression of Toll-like receptor 2 (TLR2), TLR4 and fractalkine/CX3C chemokine receptor 1 (CX3CR1) cell surface receptors were prominent in 2 DIV microglia, while elevation of matrix metalloproteinase 9 (MMP9) was marked in 16 DIV cells. Increased senescence-associated β-galactosidase (SA-β-gal) and upregulated miR-146a expression that were observed in 16 DIV cells showed to increase by Aβ in 2 DIV microglia. Additionally, Aβ downregulated miR-155 and miR-124, and reduced the CD11b+ subpopulation in 2 DIV microglia, while increased the number of CD86+ cells in 16 DIV microglia. Simultaneous M1 and M2 markers were found after Aβ treatment, but at lower expression in the in vitro aged microglia. Data show key-aging associated responses by microglia when incubated with Aβ, with a loss of reactivity from the 2 DIV to the 16 DIV cells, which course with a reduced phagocytosis, migration and lower expression of inflammatory miRNAs. These findings help to improve our understanding on the heterogeneous responses that microglia can have along the progression of AD disease and imply that therapeutic approaches may differ from early to late stages.
Highlights
Alzheimer’s disease (AD) is the most common dementing disorder in the elderly affecting around 35.6 million people worldwide and is expected to duplicate in the years to come (Prince et al, 2013)
In the present work we used our established model of reactive and aged-like microglia phenotypes, in which the microglial cells are separated from mixed cultures with astrocytes and maintained for 2 days in vitro (DIV) and 16 DIV in culture, respectively (Caldeira et al, 2014)
We decided to incubate the cells with a mixture of Aβ oligomers and fibrils, as previously used in the N9 microglial cell line (Falcão et al, 2017), to recapitulate the neuropathology of AD associated to the different activation processes of microglia by such species (Sondag et al, 2009)
Summary
Alzheimer’s disease (AD) is the most common dementing disorder in the elderly affecting around 35.6 million people worldwide and is expected to duplicate in the years to come (Prince et al, 2013). Deposition of Aβ was shown to trigger the activation of both astrocytes and microglia leading to the production of pro-inflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, among other inflammatory mediators (Wyss-Coray, 2006), generating neuroinflammation and contributing to AD progression and severity (Heneka et al, 2015). It was shown that fenamate NSAIDs decreased microglia activation and IL-1β processing in rodent models, which led the authors to suggest that they may be repurposed as inhibitors of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome (Daniels et al, 2016). The underlying molecular mechanisms of microglia failure in AD pathogenesis remain to be identified
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