Microglia phenotypes are associated with subregional patterns of concomitant tau, amyloid-β and α-synuclein pathologies in the hippocampus of patients with Alzheimer’s disease and dementia with Lewy bodies
Sonja FixemerCorrado AmeliGaël P. HammerLuis SalamancaOihane Uriarte HuarteChantal SchwartzJean‐Jacques GérardyNaguib MechawarAlexander SkupinMichel MittelbronnDavid Bouvier
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Abstract The cellular alterations of the hippocampus lead to memory decline, a shared symptom between Alzheimer’s disease (AD) and dementia with Lewy Bodies (DLB) patients. However, the subregional deterioration pattern of the hippocampus differs between AD and DLB with the CA1 subfield being more severely affected in AD. The activation of microglia, the brain immune cells, could play a role in its selective volume loss. How subregional microglia populations vary within AD or DLB and across these conditions remains poorly understood. Furthermore, how the nature of the hippocampal local pathological imprint is associated with microglia responses needs to be elucidated. To this purpose, we employed an automated pipeline for analysis of 3D confocal microscopy images to assess CA1, CA3 and DG/CA4 subfields microglia responses in post-mortem hippocampal samples from late-onset AD ( n = 10), DLB ( n = 8) and age-matched control (CTL) ( n = 11) individuals. In parallel, we performed volumetric analyses of hyperphosphorylated tau (pTau), amyloid-β (Aβ) and phosphorylated α-synuclein (pSyn) loads. For each of the 32,447 extracted microglia, 16 morphological features were measured to classify them into seven distinct morphological clusters. Our results show similar alterations of microglial morphological features and clusters in AD and DLB, but with more prominent changes in AD. We identified two distinct microglia clusters enriched in disease conditions and particularly increased in CA1 and DG/CA4 of AD and CA3 of DLB. Our study confirms frequent concomitance of pTau, Aβ and pSyn loads across AD and DLB but reveals a specific subregional pattern for each type of pathology, along with a generally increased severity in AD. Furthermore, pTau and pSyn loads were highly correlated across subregions and conditions. We uncovered tight associations between microglial changes and the subfield pathological imprint. Our findings suggest that combinations and severity of subregional pTau, Aβ and pSyn pathologies transform local microglia phenotypic composition in the hippocampus. The high burdens of pTau and pSyn associated with increased microglial alterations could be a factor in CA1 vulnerability in AD.Keywords:
Amyloid (mycology)
Despite the significant role microglia play in the pathology of multiple sclerosis (MS), medications that act within the central nervous system (CNS) to inhibit microglia have not yet been identified as treatment options.We screened 1040 compounds with the aim of identifying inhibitors of microglia to reduce neuroinflammation.The NINDs collection of 1040 compounds, where most are therapeutic medications, was tested at 10 µM final concentration on lipopolysaccharide (LPS)-activated human microglia. An ELISA was run on the media to measure the level of TNF-α as an indicator of microglia activity. For compounds that reduce LPS-activated TNF-α levels by over 50%, considered as a potential inhibitor of interest, toxicity tests were conducted to exclude non-specific cytotoxicity. Promising compounds were subjected to further analyses, including toxicity to other CNS cell types, and multiplex assays.Of 1040 compounds tested, 123 reduced TNF-α levels of LPS-activated microglia by over 50%. However, most of these were cytotoxic to microglia at the concentration tested while 54 were assessed to be non-toxic. Of the latter, spironolactone was selected for further analyses. Spironolactone reduced TNF-α levels of activated microglia by 50-60% at 10 µM, and this concentration did not kill microglia, neurons or astrocytes. In multiplex assays, spironolactone reduced several molecules in activated microglia. Finally, during the screening, we identified 9 compounds that elevated further the TNF-α levels in LPS-activated microglia.Many of the non-toxic compounds identified in this screen as inhibitors of microglia, including spironolactone, may be explored as viable therapeutic options in MS.
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Microglia, the major inflammatory cells of the brain, play a pivotal role in the initiation and progression of Alzheimer's Disease (AD) by either phagozytosing amyloid‐β deposits or by releasing cytotoxic and pro‐inflammatory substances in response to activation by amyloid‐β aggregates, including amyloid‐β oligomers (AβO). We here propose microglial Kv1.3 channels as a novel target for curbing the harmful effects of Aβ‐induced microglia activation. Microglia isolated from the brains of adult 5xFAD mice expressed higher levels of Kv1.3 than microglia from age‐matched control mice. We further observed strong Kv1.3 immunoreactivities in microglia associated with amyloid plaques in brains of 5xFAD mice. Proof for the functional importance of Kv1.3 in microglia comes from our observations that the Kv1.3 blocker PAP‐1 inhibits AβO‐stimulated NO production as well as microglia‐mediated neurotoxicity in dissociated cultures and organotypic brain slices. A 6‐week course of daily PAP‐1 injections also reduced the degree of microglia activation in 5xFAD mice. In contrast, Kv1.3 blockade with PAP‐1 does not affect phagocytosis of Aβ aggregates by microglia. These observations suggest that Kv1.3 blockers might preferentially inhibit microglia mediated neuronal killing without affecting beneficial functions such as scavenging of debris. Supported by NIH and Alzheimer's Association
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Amyloid (mycology)
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Abstract Brain iron accumulation has been found to accelerate disease progression in amyloid-β(Aβ) positive Alzheimer patients, though the mechanism is still unknown. Microglia have been identified as key players in the disease pathogenesis, and are highly reactive cells responding to aberrations such as increased iron levels. Therefore, using histological methods, multispectral immunofluorescence and an automated in-house developed microglia segmentation and analysis pipeline, we studied the occurrence of iron-accumulating microglia and the effect on its activation state in human Alzheimer brains. We identified a subset of microglia with increased expression of the iron storage protein ferritin light chain (FTL), together with increased Iba1 expression, decreased TMEM119 and P2RY12 expression. This activated microglia subset represented iron-accumulating microglia and appeared morphologically dystrophic. Multispectral immunofluorescence allowed for spatial analysis of FTL + Iba1 + -microglia, which were found to be the predominant Aβ-plaque infiltrating microglia. Finally, an increase of FTL + Iba1 + -microglia was seen in patients with high Aβ load and Tau load. These findings suggest iron to be taken up by microglia and to influence the functional phenotype of these cells, especially in conjunction with Aβ.
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Microglia are central nervous system (CNS) resident macrophages. They play a prominent role in virtually all neurodegenerative and traumatic brain injuries. Visualizing microglia using label-free methodologies will allow a better understanding of how microglia participate in CNS disorders in the absence of perturbations from external fluorescent dyes. Fluorescence Lifetime Imaging (FLIM) of NADH is an effective tool for monitoring cell intrinsic metabolic changes, and can imply metabolic state based on free/bound NADH lifetime quantification. Recently, FLIM based NADH lifetime quantification was used to characterize macrophages and other immune cell types. Here, we use a lifetime-based, label-free method to characterize microglia in vitro. We have found that there is a unique and statistically significant difference (p<0.05, n=5) in the NADH lifetime and free/bound NADH levels in microglia compared to other CNS cell types. Activated (i.e. inflammatory) microglia play a particularly important role in CNS diseases compared to quiescent microglia, and to our knowledge, there are no markers that can uniquely identify activated microglia, and distinguish them from related immune cell types. Thus, we have extended our NADH FLIM-based approach to differentiate quiescent microglia from activated microglia and found that there is a statistically significant difference (p<0.05,n=5) in NADH mean lifetime in the activated cells. Identifying microglia in a mixed population of CNS cells, and distinguishing activated from quiescent microglia will pave the way to better understanding their roles in the healthy and diseased brain.
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Abstract Age-associated microglial dysfunction contributes to the accumulation of amyloid-β (Aβ) plaques in Alzheimer’s disease. Although several studies have shown age-related declines in the phagocytic capacity of myeloid cells, relatively few have examined phagocytosis of normally aged microglia. Furthermore, much of the existing data on aging microglial function have been generated in accelerated genetic models of Alzheimer’s disease. Here we found that naturally aged microglia phagocytosed less Aβ over time. To gain a better understanding of such dysfunction, we assessed differences in gene expression between young and old microglia that either did or did not phagocytose Aβ. Young microglia had both phagocytic and neuronal maintenance signatures indicative of normal microglial responses, whereas, old microglia, regardless of phagocytic status, exhibit signs of broad dysfunction reflective of underlying neurologic disease states. We also found downregulation of many phagocytic receptors on old microglia, including TREM2, an Aβ phagocytic receptor. TREM2 protein expression was diminished in old microglia and loss of TREM2 + microglia was correlated with impaired Aβ uptake, suggesting a mechanism for phagocytic dysfunction in old microglia. Combined, our work reveals that normally aged microglia have broad changes in gene expression, including defects in Aβ phagocytosis that likely underlies the progression to neurologic disease.
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Author(s): Rice, Rachel Anne | Advisor(s): Green, Kim N | Abstract: Microglia are the immune competent cells of the central nervous system (CNS). During development, microglia play critical roles in pruning synapses and refining neuronal connectivity. In the adult brain, microglia constantly survey the parenchyma for cellular damage or invading pathogens. Upon detection of such events, microglia become activated and shift to a phagocytic phenotype, secreting pro-inflammatory molecules and adopting an amoeboid morphology. As part of the resolution/repair process, microglia return to a surveillant state and produce anti-inflammatory molecules. Unfortunately, with severe insults, such as traumatic brain injury or chronic neurodegeneration, microglia remain activated and contribute to an inflammatory process that is never, or poorly, resolved. In this way, we hypothesize that microglia contribute deleteriously to functional outcomes.The goal of my dissertation is to determine the contributions of microglia to neuronal health and cognition in both the healthy and injured brain. The direct assessment of microglia-specific contributions is possible due to the discovery by our lab that microglia are dependent upon signaling through the colony-stimulating factor 1 receptor (CSF1R) for their survival. Treatment with a small-molecule CSF1R inhibitor eliminates g99% of microglia from the adult mouse brain. Critically, microglia fully repopulate the CNS upon withdrawal of the CSF1R inhibitor, effectively renewing this cellular compartment. Using a genetic model of inducible neuronal loss, I have determined that the elimination of microglia during a lesion is detrimental to cellular health, while the elimination of microglia following a lesion results in the reversal of many lesion-induced deficits. Importantly, this research suggests that the microglia-mediated immune response is beneficial during insult or injury, but deleterious after such an event. Moreover, repopulation of the brain with new microglia following neuronal lesioning largely resets the inflammatory milieu and confers functional benefits.Finally, long-term elimination of microglia was employed in order to determine if these cells shape the synaptic landscape in the healthy adult brain, as they do during development. Indeed, I found that microglial elimination in healthy adult mice results in brain-wide and robust increases in dendritic spine numbers and excitatory neuronal connectivity, indicating that microglia modulate synaptic function throughout the course of the lifetime.
Synaptic Pruning
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