Modulation of cerebral β-amyloidosis by myeloid cells

Abstract

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder and the most common form of dementia. Thereby, the abnormal deposition of the amyloid-β (Aβ) peptide into plaques is considered to be the primary neuropathological insult in AD. For a small proportion of all AD cases it is well known that rare genetic mutations are causative for very early Aβ deposition (familial Alzheimer’s disease). However, the vast majority of all AD cases manifest at later ages (late-onset Alzheimer’s disease (LOAD)) and are most likely caused by an interplay of multiple genetic variants and the environment. During the last ten years, genome-wide association studies revealed several risk loci that increase the susceptibility for LOAD, and interestingly, many of these genetic variants were found to be associated with innate immune functions of which the resident tissue macrophages of the brain – the microglia – are prime regulators. In general, the innate immune response mediated by the resident tissue macrophages is considered protective as it induces the production of inflammatory modulators and enables phagocytosis and killing of pathogens to prevent further tissue damage. However in the AD brain, the progressive accumulation of Aβ deposits leads to a chronic exposure of microglia to Aβ aggregates and induces an excessive neuro-inflammatory response that is thought to promote disease progression. Interestingly, microglia display a highly plastic phenotype and studies from peripheral tissue macrophages reported that a variety of environmental stimuli can determine but also reprogram their functional phenotype. To this end, this thesis summarizes three different approaches, which aimed to understand but also modulate the myeloid cell immune function during AD with regard to their effects on the pathology of cerebral β-amyloidosis. To begin with, we examined whether peripheral monocytes, which were previously shown to adopt a microglia-like phenotype in the healthy brain, can replace dysfunctional microglia in brains of two different mouse models of cerebral β-amyloidosis and may then restrict Aβ accumulation. For this purpose, we depleted microglia in APPPS1 and APP23 transgenic (tg) mice that expressed the herpes simplex virus thymidine kinase (HSVTK) under the myeloid-cell specific CD11b promoter; the application of the thymidine kinase substrate ganciclovir (GCV), which is converted into a cytotoxic product, then induced microglial death. After a two-week ganciclovir treatment, application was discontinued from two weeks up to six months to allow the peripheral monocytes to repopulate the brain. Interestingly, during the first weeks of repopulation the number of infiltrated monocytes were twice the number of resident microglia in control mice, but the engrafted monocytes failed to cluster around Aβ plaques. Consequently, we did not observe alterations in plaque pathology. Also, a pro-longed incubation for up to six months did not change Aβ load. However, long-term monocyte engraftment for five months induced in pre-depositing APP23 mice enabled the infiltrated monocytes to behave most similar to resident microglia: they began clustering around Aβ depositions, the cell number was virtually equal to control mice and plaque-associated monocytes were TREM2-positive. However, these cells also failed to alter Aβ plaque load. This work indicates that the tissue environment in the brain dominates over myeloid cell origin and thus reprograms myeloid cells to match the resident microglia population, however without prevention of Aβ pathology. Recent studies provide evidence that cells of the innate immune system can, similar to the adaptive immune cells, acquire immunological memory. In particular, a distinct set of primary immune stimuli can either enhance or suppress a subsequent immune response, which is referred to as “training” and “tolerance”, respectively. In a second study, we tested the applicability of the immune memory concept to microglia and examined if the induction of innate immune memory can induce long-lasting changes in the brain’s immune response and thereby alter pathology of neurological diseases. To this end, we injected two different doses of the endotoxin lipopolysaccharide (LPS) into pre-depositing APP23 mice. Whereas a single LPS injection was identified to induce acute training effects, consecutive injections for four days induced tolerance effects in microglia. Accordingly, in the brain, we acutely measured initially enhanced concentrations of inflammatory cytokines which decreased with further LPS injections. When we examined the long-lasting effects of the induced immune memory on Aβ pathology and cortical ischemia at the later time points, the initial training stimulus increased while the tolerance stimulus reduced pathology, which was reflected by changes in Aβ plaque load and neuronal damage, respectively. Immune memory in macrophages was previously shown to be mediated by epigenetic changes in enhancer regions that either stimulate or prevent gene transcription. In accordance, we performed chromatin immunoprecipitation sequencing for histone modifications in isolated microglia to determine changes in their enhancer landscape. Notably, we identified the active enhancer repertoire for hypoxia-inducible factor 1α (HIF-1α), a key modulator for macrophage inflammatory responses, to be enriched in microglia after the induction of trained immune memory (1xLPS). In contrast, pathways related to phagocytic functions showed an increase in active enhancers in the 4xLPS treatment group. Importantly, these epigenetic alterations were reflected by expression changes in the respective genes in the isolated microglia population. By this study, we provide first evidence for long-lasting innate immune memory in the brain that can shape neurological disease outcome and is driven by epigenetic modifications of the microglial enhancer landscape. In a last study, we focused on the microglial phagocytic capacity as an important factor for the modulation of Aβ plaque pathology, as in vitro experiments have reported that microglia can bind to, and engulf Aβ fibrils. However, so far, in vivo studies have not convincingly confirmed these results. Therefore, we investigated the role of the soluble milk fat globuleepidermal growth-factor 8 (MFG-E8) protein, that was recently hypothesized to mediate Aβ phagocytosis in AD pathology. To test the in vivo function of MFG-E8, we crossed mice expressing a functional knockout variant of Mfge8 (Mfge8-/-) with the APPPS1 and APP23 tg mouse models of cerebral β-amyloidosis. In contrast to previous reports, our results indicated that the depletion of MFG-E8 has no impact on Aβ uptake by microglia or subsequent Aβ degradation processes. However, contrary to our expectations, MFG-E8 deficiency reduced Aβ plaque load and Aβ levels in both mouse models without affecting amyloid precursor protein (APP) processing. When we immunohistochemically analyzed MFG-E8 distribution in the brain we observed a strong accumulation of MFG-E8 with congophilic Aβ deposits and co-staining of MFG-E8 with Aβ even showed a partial co-localization of both proteins at the sites of Aβ plaques. While the mechanism of these effects requires further studies, our results suggests that a direct interaction between MFG-E8 and Aβ promotes amyloid aggregation. Taken together, these studies examined different ways of modulating the microglial immune response during AD pathology. Interestingly, the replacement of dysfunctional microglia by peripheral monocytes in the diseased brain did not modify Aβ deposition although the infiltrated monocytes adopted features of plaque-associated microglia. However, when we applied the concept of innate immune memory to the brain through the remodeling of the innate immune response by epigenetic reprogramming of the microglial enhancer repertoire, we identified a promising approach to modify Aβ pathology. Especially the induction of a microglial tolerance state had beneficial long-term effects on the pathology of cerebral β- amyloidosis while training aggravated disease outcome. These results provide, for the first time, evidence that long-lasting modulation of the innate immune reaction may occur due to immunological priming – a mechanism that introduces new targets for dampening Aβ pathology in Alzheimer’s disease. However, in contrast, a direct modification of microglial Aβ phagocytosis through the knockout of Mfge8 is most likely not sufficient to modulate microglia function in AD.