Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease.

The immune cells of the central nervous system (CNS) comprise parenchymal microglia and at the CNS border regions meningeal, perivascular, and choroid plexus macrophages (collectively called CNS-associated macrophages, CAMs). While previous work has shown that microglial properties depend on environmental signals from the commensal microbiota, the effects of microbiota on CAMs are unknown. By combining several microbiota manipulation approaches, genetic mouse models, and single-cell RNA-sequencing, we have characterized CNS myeloid cell composition and function. Under steady-state conditions, the transcriptional profiles and numbers of choroid plexus macrophages were found to be tightly regulated by complex microbiota. In contrast, perivascular and meningeal macrophages were affected to a lesser extent. An acute perturbation through viral infection evoked an attenuated immune response of all CAMs in germ-free mice. We further assessed CAMs in a more chronic pathological state in 5xFAD mice, a model for Alzheimer's disease, and found enhanced amyloid beta uptake exclusively by perivascular macrophages in germ-free 5xFAD mice. Our results aid the understanding of distinct microbiota-CNS macrophage interactions during homeostasis and disease, which could potentially be targeted therapeutically.

Flow-cytometry-based protocol to analyze respiratory chain function in mouse microglia.

Most of the protocols to analyze metabolic features of cell populations from different tissues rely on in vitro cell culture conditions. Here, we present a flow-cytometry-based protocol for measuring the respiratory chain function in permeabilized mouse microglia ex vivo. We describe microglial cell isolation, followed by analyzing complex I and II using flow cytometry. This optimized protocol requires a low input of permeabilized cells and can be applied to other ex vivo isolated cells or cells derived from cell cultures. For complete details on the use and execution of this protocol, please refer to Erny et al. (2021).

Distinct Aß pathology in the olfactory bulb and olfactory deficits in a mouse model of Aß and α-syn co-pathology.

Several degenerative brain disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are characterized by the simultaneous appearance of amyloid-ß (Aß) and α-synuclein (α-syn) pathologies and symptoms that are similar, making it difficult to differentiate between these diseases. Until now, an accurate diagnosis can only be made by postmortem analysis. Furthermore, the role of α-syn in Aß aggregation and the arising characteristic olfactory impairments observed during the progression of these diseases is still not well understood. Therefore, we assessed Aß load in olfactory bulbs of APP-transgenic mice expressing APP695KM670/671NL and PSEN1L166P under the control of the neuron-specific Thy-1 promoter (referred to here as APPPS1) and APPPS1 mice co-expressing SNCAA30P (referred to here as APPPS1 × [A30P]aSYN). Furthermore, the olfactory capacity of these mice was evaluated in the buried food and olfactory avoidance test. Our results demonstrate an age-dependent increase in Aß load in the olfactory bulb of APP-transgenic mice that go along with exacerbated olfactory performance. Our study provides clear evidence that the presence of α-syn significantly diminished the endogenous and seed-induced Aß deposits and significantly ameliorated olfactory dysfunction in APPPS1 × [A30P]aSYN mice.

Gut microbiota drives age-related oxidative stress and mitochondrial damage in microglia via the metabolite N6-carboxymethyllysine.

Microglial function declines during aging. The interaction of microglia with the gut microbiota has been well characterized during development and adulthood but not in aging. Here, we compared microglial transcriptomes from young-adult and aged mice housed under germ-free and specific pathogen-free conditions and found that the microbiota influenced aging associated-changes in microglial gene expression. The absence of gut microbiota diminished oxidative stress and ameliorated mitochondrial dysfunction in microglia from the brains of aged mice. Unbiased metabolomic analyses of serum and brain tissue revealed the accumulation of N6-carboxymethyllysine (CML) in the microglia of the aging brain. CML mediated a burst of reactive oxygen species and impeded mitochondrial activity and ATP reservoirs in microglia. We validated the age-dependent rise in CML levels in the sera and brains of humans. Finally, a microbiota-dependent increase in intestinal permeability in aged mice mediated the elevated levels of CML. This study adds insight into how specific features of microglia from aged mice are regulated by the gut microbiota.

Microglia contribute to the propagation of Aß into unaffected brain tissue.

Microglia appear activated in the vicinity of amyloid beta (Aß) plaques, but whether microglia contribute to Aß propagation into unaffected brain regions remains unknown. Using transplantation of wild-type (WT) neurons, we show that Aß enters WT grafts, and that this is accompanied by microglia infiltration. Manipulation of microglia function reduced Aß deposition within grafts. Furthermore, in vivo imaging identified microglia as carriers of Aß pathology in previously unaffected tissue. Our data thus argue for a hitherto unexplored mechanism of Aß propagation.

Microbiota-derived acetate enables the metabolic fitness of the brain innate immune system during health and disease.

As tissue macrophages of the central nervous system (CNS), microglia constitute the pivotal immune cells of this organ. Microglial features are strongly dependent on environmental cues such as commensal microbiota. Gut bacteria are known to continuously modulate microglia maturation and function by the production of short-chain fatty acids (SCFAs). However, the precise mechanism of this crosstalk is unknown. Here we determined that the immature phenotype of microglia from germ-free (GF) mice is epigenetically imprinted by H3K4me3 and H3K9ac on metabolic genes associated with substantial functional alterations including increased mitochondrial mass and specific respiratory chain dysfunctions. We identified acetate as the essential microbiome-derived SCFA driving microglia maturation and regulating the homeostatic metabolic state, and further showed that it is able to modulate microglial phagocytosis and disease progression during neurodegeneration. These findings indicate that acetate is an essential bacteria-derived molecule driving metabolic pathways and functions of microglia during health and perturbation.

Different effects of constitutive and induced microbiota modulation on microglia in a mouse model of Alzheimer's disease.

It was recently revealed that gut microbiota promote amyloid-beta (Aß) burden in mouse models of Alzheimer's disease (AD). However, the underlying mechanisms when using either germ-free (GF) housing conditions or treatments with antibiotics (ABX) remained unknown. In this study, we show that GF and ABX-treated 5x familial AD (5xFAD) mice developed attenuated hippocampal Aß pathology and associated neuronal loss, and thereby delayed disease-related memory deficits. While Aß production remained unaffected in both GF and ABX-treated 5xFAD mice, we noticed in GF 5xFAD mice enhanced microglial Aß uptake at early stages of the disease compared to ABX-treated 5xFAD mice. Furthermore, RNA-sequencing of hippocampal microglia from SPF, GF and ABX-treated 5xFAD mice revealed distinct microbiota-dependent gene expression profiles associated with phagocytosis and altered microglial activation states. Taken together, we observed that constitutive or induced microbiota modulation in 5xFAD mice differentially controls microglial Aß clearance mechanisms preventing neurodegeneration and cognitive deficits.

Lithium and glutamine synthetase: Protective effects following stress.

Even though lithium is widely used as treatment for mood disorders, the exact mechanisms of lithium in the brain remain unknown. A potential mechanism affects the downstream target of the Wnt/ß-catenin signaling pathway, specifically glutamine synthetase (GS). Here, we investigate the effect of lithium on GS-promoter activity in the brain. Over seven days, B6C3H-Glultm(T2A-LacZ) mice that carry LacZ as a reporter gene fused to the GS-promotor received either daily intraperitoneal injections of lithium carbonate (25 mg/kg) or NaCl, or no treatment. Following histochemical staining of ß-galactosidase relative GS-promotor activity was measured by analyzing the intensity of the staining. Furthermore cell counts were conducted. GS-promotor activity was significantly decreased in female compared to male mice. Treatment group differences were only found in male hippocampi, with increased activity after NaCl treatment compared to both the lithium treatment and no treatment. Lithium treatment increased the overall number of cells in the CA1 region in males. Daily injections of NaCl might have been sufficient to induce stress-related GS-promotor activity changes in male mice; however, lithium was able to reverse the effect. Taken together, the current study indicates that lithium acts to prevent stress, rather affecting general GS-promoter activity.