Functional properties of microglia in mouse models of Alzheimer’s disease

Abstract

Microglia, resident macrophages of the CNS, execute various functions: they participate in oligodendrogenesis, neurogenesis, learning and behavior, phagocytose harmful material as well as tissue debris and mount crucial innate immune responses upon CNS infection and damage (Hanisch & Kettenmann, 2007). Ageing and associated neurodegenerative processes can impair these functions. In Alzheimer´s disease (AD), microglia are incapable to clear the toxic amyloid β peptide (Aβ). This may lead to a massive accumulation and deposition of the peptide. Additionally, in an AD environment, microglia seem to be activated, leading to excessive production of inflammatory mediators, such as pro-inflammatory cytokines and chemokines, which can further damage the vulnerable CNS circuitry. The main focus of this study was to investigate if these changes in microglia properties are reversible in a healthy environment. Furthermore, microglial priming (described as their exaggerated response to an inflammatory stimulus compared with stimulus-naïve microglia; Norden & Godbout, 2013) was studied in mouse models of AD. Using 3, 6 and 9 months old 5XFAD mice, as an animal model of AD we could mainly show that the activity of microglia to phagocytose or produce pro-inflammatory factors does not differ from microglia derived from wild-type (WT) mice ex vivo. However, we observed a dramatic age-dependent decrease in both of these activities independent of the genotype. These data demonstrate that microglial alteration in AD environments -described in former studies- are reversible, depending on the environment. In addition, we studied the hyper-sensitivity of microglia in the vicinity of Aβ plaques. Characterization of these microglia in APP23, APPswePS1dE9 and 5XFAD mice revealed expression of microglial activation/priming markers such as Mac-2, CD68 and MHC II. Isolation of MHC II positive and -negative microglia from whole brains of 9 months old 5XFAD and WT mice also showed significant changes towards pro-inflammatory characterisitcs in MHC II positive microglia compared to the MHC II negatives. In addition, immunohistochemical analysis of systemic LPS-induced inflammation in 5XFAD mice led to overexpression of Mac-2, CD68, MHC II and IL-1β exclusively in the vicinity of Aβ plaques. In contrast, LPS-induced priming and inflammation was absent in plaque free regions. These data indicate that microglia in the vicinity of Aβ depositions are primed. In the third part we determined how the activation of beta 2 adrenergic receptors (β2ARs) in LPS-stimulated microglia influences the pro-inflammatory response of microglia. This investigation was based on previously observed anti-inflammatory effects of the adrenergic system on macrophages including microglia and its positive effects on AD. In the investigations, ex vivo or in vivo, microglia were treated with LPS and salbutamol (β2AR agonist) simultaneously and the subsequent microglial production of pro-inflammatory cyto-/ chemokines and microglia-induced infiltration of immune cells from the periphery was analyzed. We observed that the production of some but not all pro-inflammatory proteins are inhibited by salbutamol. For instance, the production of TNFα is almost completely inhibited. In contrast, the production of CCL5 is almost not inhibited. Previous studies on microglia lacking the mediator protein TRIF suggested that TLR4 signalling through the TRIF pathway is a supporting path to escape from the inhibitory effects of salbutamol. Our current data concerning involvement of specific TRIF dependent genes and also data from mRNA sequencing experiments in microglia treated with LPS alone or combined with salbutamol proved involvement of the TRIF pathway as an escaping route. Moreover, flow cytometry analyses of mice treated with LPS alone or combined with salbutamol revealed significant decreases in infiltration of immune cells in the brain. Using immunohistochemistry we additionally showed that expression of Iba-1 and GFAP on microglia and astrocytes are not affected by salbutamol. These data clearly show selective effects of the adrenergic system on pro-inflammatory factors in microglia. Reduced recruitment of immune cells from the periphery by activation of the adrenergic system is possibly an important factor in improving AD inflammation.