ABSTRACT
Haploinsufficiency of the progranulin (PGRN)-encoding gene (GRN) causes frontotemporal lobar degeneration (GRN-FTLD) and results in microglial hyperactivation, TREM2 activation, lysosomal dysfunction, and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology, we used genetic and pharmacological approaches to suppress TREM2-dependent transition of microglia from a homeostatic to a disease-associated state. Trem2 deficiency in Grn KO mice reduced microglia hyperactivation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies led to reduced TREM2 signaling due to its enhanced shedding. Furthermore, TREM2 antibody-treated PGRN-deficient microglia derived from human-induced pluripotent stem cells showed reduced microglial hyperactivation, TREM2 signaling, and phagocytic activity, but lysosomal dysfunction was not rescued. Similarly, lysosomal dysfunction, lipid dysregulation, and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Synaptic loss and neurofilament light-chain (NfL) levels, a biomarker for neurodegeneration, were further elevated in the Grn/Trem2 KO cerebrospinal fluid (CSF). These findings suggest that TREM2-dependent microglia hyperactivation in models of GRN deficiency does not promote neurotoxicity, but rather neuroprotection.
Subject(s)
Frontotemporal Lobar Degeneration/pathology , Membrane Glycoproteins/metabolism , Microglia/physiology , Monocytes/metabolism , Progranulins/deficiency , Receptors, Immunologic/metabolism , Animals , Antibodies/immunology , Antibodies/pharmacology , Brain/diagnostic imaging , Brain/physiopathology , Disease Models, Animal , Female , Frontotemporal Lobar Degeneration/metabolism , Humans , Lysosomes/metabolism , Lysosomes/pathology , Male , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Mice, Inbred C57BL , Mice, Knockout , Microglia/drug effects , Monocytes/drug effects , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , Syk Kinase/metabolismABSTRACT
Recovery after stroke is a multicellular process encompassing neurons, resident immune cells, and brain-invading cells. Stroke alters the gut microbiome, which in turn has considerable impact on stroke outcome. However, the mechanisms underlying gut-brain interaction and implications for long-term recovery are largely elusive. Here, we tested the hypothesis that short-chain fatty acids (SCFAs), key bioactive microbial metabolites, are the missing link along the gut-brain axis and might be able to modulate recovery after experimental stroke. SCFA supplementation in the drinking water of male mice significantly improved recovery of affected limb motor function. Using in vivo wide-field calcium imaging, we observed that SCFAs induced altered contralesional cortex connectivity. This was associated with SCFA-dependent changes in spine and synapse densities. RNA sequencing of the forebrain cortex indicated a potential involvement of microglial cells in contributing to the structural and functional remodeling. Further analyses confirmed a substantial impact of SCFAs on microglial activation, which depended on the recruitment of T cells to the infarcted brain. Our findings identified that microbiota-derived SCFAs modulate poststroke recovery via effects on systemic and brain resident immune cells.SIGNIFICANCE STATEMENT Previous studies have shown a bidirectional communication along the gut-brain axis after stroke. Stroke alters the gut microbiota composition, and in turn, microbiota dysbiosis has a substantial impact on stroke outcome by modulating the immune response. However, until now, the mediators derived from the gut microbiome affecting the gut-immune-brain axis and the molecular mechanisms involved in this process were unknown. Here, we demonstrate that short-chain fatty acids, fermentation products of the gut microbiome, are potent and proregenerative modulators of poststroke neuronal plasticity at various structural levels. We identified that this effect was mediated via circulating lymphocytes on microglial activation. These results identify short-chain fatty acids as a missing link along the gut-brain axis and as a potential therapeutic to improve recovery after stroke.
Subject(s)
Brain/drug effects , Brain/immunology , Fatty Acids, Volatile/administration & dosage , Stroke/immunology , Animals , Brain/metabolism , Female , Lymphocytes/drug effects , Lymphocytes/immunology , Male , Mice, Inbred C57BL , Microglia/drug effects , Microglia/immunology , Recovery of Function/drug effects , Stroke/metabolism , Transcriptome/drug effectsABSTRACT
AIM: Understanding neuroinflammation after acute ischemic stroke is a crucial step on the way to an individualized post-stroke treatment. Microglia activation, an essential part of neuroinflammation, can be assessed using [18F]GE-180 18 kDa translocator protein positron emission tomography (TSPO-PET). However, the commonly used 60-90 min post-injection (p.i.) time window was not yet proven to be suitable for post-stroke neuroinflammation assessment. In this study, we compare semi-quantitative estimates derived from late time frames to quantitative estimates calculated using a full 0-90 min dynamic scan in a mouse photothrombotic stroke (PT) model. MATERIALS AND METHODS: Six mice after PT and six sham mice were included in the study. For a half of the mice, we acquired four serial 0-90 min scans per mouse (analysis cohort) and calculated standardized uptake value ratios (SUVRs; cerebellar reference) for the PT volume of interest (VOI) in five late 10 min time frames as well as distribution volume ratios (DVRs) for the same VOI. We compared late static 10 min SUVRs and the 60-90 min time frame of the analysis cohort to the corresponding DVRs by linear fitting. The other half of the animals received a static 60-90 min scan and was used as a validation cohort. We extrapolated DVRs by using the static 60-90 min p.i. time window, which were compared to the DVRs of the analysis cohort. RESULTS: We found high linear correlations between SUVRs and DVRs in the analysis cohort for all studied 10 min time frames, while the fits of the 60-70, 70-80, and 80-90 min p.i. time frames were the ones closest to the line of identity. For the 60-90 min time window, we observed an excellent linear correlation between SUVR and DVR regardless of the phenotype (PT vs. sham). The extrapolated DVRs of the validation cohort were not significantly different from the DVRs of the analysis group. CONCLUSION: Simplified quantification by a reference tissue ratio of the late 60-90 min p.i. [18F]GE-180 PET image can replace full quantification of a dynamic scan for assessment of microglial activation in the mouse PT model.
ABSTRACT
Neuroinflammation after stroke is characterized by the activation of resident microglia and the invasion of circulating leukocytes into the brain. Although lymphocytes infiltrate the brain in small number, they have been consistently demonstrated to be the most potent leukocyte subpopulation contributing to secondary inflammatory brain injury. However, the exact mechanism of how this minimal number of lymphocytes can profoundly affect stroke outcome is still largely elusive. Here, using a mouse model for ischemic stroke, we demonstrated that early activation of microglia in response to stroke is differentially regulated by distinct T cell subpopulations - with TH1 cells inducing a type I INF signaling in microglia and regulatory T cells (TREG) cells promoting microglial genes associated with chemotaxis. Acute treatment with engineered T cells overexpressing IL-10 administered into the cisterna magna after stroke induces a switch of microglial gene expression to a profile associated with pro-regenerative functions. Whereas microglia polarization by T cell subsets did not affect the acute development of the infarct volume, these findings substantiate the role of T cells in stroke by polarizing the microglial phenotype. Targeting T cell-microglia interactions can have direct translational relevance for further development of immune-targeted therapies for stroke and other neuroinflammatory conditions.
Subject(s)
Brain Ischemia , Stroke , Humans , Microglia/metabolism , Brain Ischemia/metabolism , Brain/metabolism , Signal Transduction/physiologyABSTRACT
Neuroinflammation is an emerging focus of translational stroke research. Preclinical studies have demonstrated a critical role for brain-invading lymphocytes in post-stroke pathophysiology. Reducing cerebral lymphocyte invasion by anti-CD49d antibodies consistently improves outcome in the acute phase after experimental stroke models. However, clinical trials testing this approach failed to show efficacy in stroke patients for the chronic outcome 3 mo after stroke. Here, we identify a potential mechanistic reason for this phenomenon by detecting chronic T cell accumulation-evading the systemic therapy-in the post-ischemic brain. We observed a persistent accumulation of T cells in mice and human autopsy samples for more than 1 mo after stroke. Cerebral T cell accumulation in the post-ischemic brain was driven by increased local T cell proliferation rather than by T cell invasion. This observation urges re-evaluation of current immunotherapeutic approaches, which target circulating lymphocytes for promoting recovery after stroke.
Subject(s)
Brain/immunology , Brain/pathology , Immunotherapy , Stroke/immunology , Stroke/therapy , T-Lymphocytes/immunology , Animals , Autopsy , Brain Ischemia/drug therapy , Brain Ischemia/immunology , Brain Ischemia/pathology , Cell Proliferation , Female , Humans , Integrin alpha4/immunology , Lymphocyte Count , Male , Mice, Inbred C57BL , Natalizumab/pharmacology , Natalizumab/therapeutic use , Neuronal Plasticity/drug effects , Recovery of Function/drug effects , Stroke/physiopathologyABSTRACT
The gut microbiome has been implicated as a key regulator of brain function in health and disease. But the impact of gut microbiota on functional brain connectivity is unknown. We used resting-state functional magnetic resonance imaging in germ-free and normally colonized mice under naive conditions and after ischemic stroke. We observed a strong, brain-wide increase of functional connectivity in germ-free animals. Graph theoretical analysis revealed significant higher values in germ-free animals, indicating a stronger and denser global network but with less structural organization. Breakdown of network function after stroke equally affected germ-free and colonized mice. Results from histological analyses showed changes in dendritic spine densities, as well as an immature microglial phenotype, indicating impaired microglia-neuron interaction in germ-free mice as potential cause of this phenomenon. These results demonstrate the substantial impact of bacterial colonization on brain-wide function and extend our so far mainly (sub) cellular understanding of the gut-brain axis.
ABSTRACT
Previous studies have identified a crucial role of the gut microbiome in modifying Alzheimer's disease (AD) progression. However, the mechanisms of microbiome-brain interaction in AD were so far unknown. Here, we identify microbiota-derived short chain fatty acids (SCFA) as microbial metabolites which promote Aß deposition. Germ-free (GF) AD mice exhibit a substantially reduced Aß plaque load and markedly reduced SCFA plasma concentrations; conversely, SCFA supplementation to GF AD mice increased the Aß plaque load to levels of conventionally colonized (specific pathogen-free [SPF]) animals and SCFA supplementation to SPF mice even further exacerbated plaque load. This was accompanied by the pronounced alterations in microglial transcriptomic profile, including upregulation of ApoE. Despite increased microglial recruitment to Aß plaques upon SCFA supplementation, microglia contained less intracellular Aß. Taken together, our results demonstrate that microbiota-derived SCFA are critical mediators along the gut-brain axis which promote Aß deposition likely via modulation of the microglial phenotype.
Subject(s)
Fatty Acids, Volatile/metabolism , Gastrointestinal Microbiome , Microglia/metabolism , Plaque, Amyloid/metabolism , Alzheimer Disease/metabolism , Animals , Female , Male , Mice , Specific Pathogen-Free OrganismsABSTRACT
The C9orf72 repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD). Non-canonical translation of the expanded repeat results in abundant poly-GA inclusion pathology throughout the CNS. (GA)149 -CFP expression in mice triggers motor deficits and neuroinflammation. Since poly-GA is transmitted between cells, we investigated the therapeutic potential of anti-GA antibodies by vaccinating (GA)149 -CFP mice. To overcome poor immunogenicity, we compared the antibody response of multivalent ovalbumin-(GA)10 conjugates and pre-aggregated carrier-free (GA)15 . Only ovalbumin-(GA)10 immunization induced a strong anti-GA response. The resulting antisera detected poly-GA aggregates in cell culture and patient tissue. Ovalbumin-(GA)10 immunization largely rescued the motor function in (GA)149 -CFP transgenic mice and reduced poly-GA inclusions. Transcriptome analysis showed less neuroinflammation in ovalbumin-(GA)10 -immunized poly-GA mice, which was corroborated by semiquantitative and morphological analysis of microglia/macrophages. Moreover, cytoplasmic TDP-43 mislocalization and levels of the neurofilament light chain in the CSF were reduced, suggesting neuroaxonal damage is reduced. Our data suggest that immunotherapy may be a viable primary prevention strategy for ALS/FTD in C9orf72 mutation carriers.
Subject(s)
Amyotrophic Lateral Sclerosis , C9orf72 Protein/genetics , Frontotemporal Dementia , Microglia , Amyotrophic Lateral Sclerosis/therapy , Animals , Humans , Mice , Mice, Inbred C57BL , VaccinationABSTRACT
Microglia are the main immune cells in the brain and have roles in brain homeostasis and neurological diseases. Mechanisms underlying microglia-neuron communication remain elusive. Here, we identified an interaction site between neuronal cell bodies and microglial processes in mouse and human brain. Somatic microglia-neuron junctions have a specialized nanoarchitecture optimized for purinergic signaling. Activity of neuronal mitochondria was linked with microglial junction formation, which was induced rapidly in response to neuronal activation and blocked by inhibition of P2Y12 receptors. Brain injury-induced changes at somatic junctions triggered P2Y12 receptor-dependent microglial neuroprotection, regulating neuronal calcium load and functional connectivity. Thus, microglial processes at these junctions could potentially monitor and protect neuronal functions.
Subject(s)
Brain Injuries/immunology , Brain/immunology , Intercellular Junctions/immunology , Microglia/immunology , Neurons/immunology , Receptors, Purinergic P2Y12/physiology , Animals , Brain/ultrastructure , Brain Injuries/pathology , Calcium , Cell Communication/immunology , HEK293 Cells , Humans , Mice , Mitochondria/immunology , Shab Potassium Channels/genetics , Shab Potassium Channels/physiology , Signal TransductionABSTRACT
Microglia are the resident immune cells of the brain and react quickly to changes in their environment with transcriptional regulation and morphological changes. Brain tissue injury such as ischemic stroke induces a local inflammatory response encompassing microglial activation. The change in activation status of a microglia is reflected in its gradual morphological transformation from a highly ramified into a less ramified or amoeboid cell shape. For this reason, the morphological changes of microglia are widely utilized to quantify microglial activation and studying their involvement in virtually all brain diseases. However, the currently available methods, which are mainly based on manual rating of immunofluorescent microscopic images, are often inaccurate, rater biased, and highly time consuming. To address these issues, we created a fully automated image analysis tool, which enables the analysis of microglia morphology from a confocal Z-stack and providing up to 59 morphological features. We developed the algorithm on an exploratory dataset of microglial cells from a stroke mouse model and validated the findings on an independent data set. In both datasets, we could demonstrate the ability of the algorithm to sensitively discriminate between the microglia morphology in the peri-infarct and the contralateral, unaffected cortex. Dimensionality reduction by principal component analysis allowed to generate a highly sensitive compound score for microglial shape analysis. Finally, we tested for concordance of results between the novel automated analysis tool and the conventional manual analysis and found a high degree of correlation. In conclusion, our novel method for the fully automatized analysis of microglia morphology shows excellent accuracy and time efficacy compared to traditional analysis methods. This tool, which we make openly available, could find application to study microglia morphology using fluorescence imaging in a wide range of brain disease models.
ABSTRACT
Microbiome alterations have been shown to affect stroke outcome. However, to what extent the presence of a gut microbiome per se is affecting post-stroke neuroinflammation has not been tested. By comparing germfree mice with recolonized (Ex-GF) and conventional SPF mice, we were able to demonstrate that bacterial colonization reduces stroke volumes. Bacterial colonization increased cerebral expression of cytokines as well as microglia/macrophage cell counts in contrast to improved stroke outcome. Interestingly, the microbiome-mediated brain protection was absent in lymphocyte-deficient mice. These findings support the concept of lymphocyte-driven protective neuroinflammation after stroke under control of the microbiome.