NLRP3 inflammasome activation drives tau pathology.

Alzheimer's disease is characterized by the accumulation of amyloid-beta in plaques, aggregation of hyperphosphorylated tau in neurofibrillary tangles and neuroinflammation, together resulting in neurodegeneration and cognitive decline1. The NLRP3 inflammasome assembles inside of microglia on activation, leading to increased cleavage and activity of caspase-1 and downstream interleukin-1ß release2. Although the NLRP3 inflammasome has been shown to be essential for the development and progression of amyloid-beta pathology in mice3, the precise effect on tau pathology remains unknown. Here we show that loss of NLRP3 inflammasome function reduced tau hyperphosphorylation and aggregation by regulating tau kinases and phosphatases. Tau activated the NLRP3 inflammasome and intracerebral injection of fibrillar amyloid-beta-containing brain homogenates induced tau pathology in an NLRP3-dependent manner. These data identify an important role of microglia and NLRP3 inflammasome activation in the pathogenesis of tauopathies and support the amyloid-cascade hypothesis in Alzheimer's disease, demonstrating that neurofibrillary tangles develop downstream of amyloid-beta-induced microglial activation.

Systemic inflammation impairs microglial Aß clearance through NLRP3 inflammasome.

Alzheimer's disease is the most prevalent type of dementia and is caused by the deposition of extracellular amyloid-beta and abnormal tau phosphorylation. Neuroinflammation has emerged as an additional pathological component. Microglia, representing the brain's major innate immune cells, play an important role during Alzheimer's. Once activated, microglia show changes in their morphology, characterized by a retraction of cell processes. Systemic inflammation is known to increase the risk for cognitive decline in human neurogenerative diseases including Alzheimer's. Here, we assess for the first time microglial changes upon a peripheral immune challenge in the context of aging and Alzheimer's in vivo, using 2-photon laser scanning microscopy. Microglia were monitored at 2 and 10 days post-challenge by lipopolysaccharide. Microglia exhibited a reduction in the number of branches and the area covered at 2 days, a phenomenon that resolved at 10 days. Systemic inflammation reduced microglial clearance of amyloid-beta in APP/PS1 mice. NLRP3 inflammasome knockout blocked many of the observed microglial changes upon lipopolysaccharide, including alterations in microglial morphology and amyloid pathology. NLRP3 inhibition may thus represent a novel therapeutic target that may protect the brain from toxic peripheral inflammation during systemic infection.

Microglia-derived ASC specks cross-seed amyloid-ß in Alzheimer's disease.

The spreading of pathology within and between brain areas is a hallmark of neurodegenerative disorders. In patients with Alzheimer's disease, deposition of amyloid-ß is accompanied by activation of the innate immune system and involves inflammasome-dependent formation of ASC specks in microglia. ASC specks released by microglia bind rapidly to amyloid-ß and increase the formation of amyloid-ß oligomers and aggregates, acting as an inflammation-driven cross-seed for amyloid-ß pathology. Here we show that intrahippocampal injection of ASC specks resulted in spreading of amyloid-ß pathology in transgenic double-mutant APPSwePSEN1dE9 mice. By contrast, homogenates from brains of APPSwePSEN1dE9 mice failed to induce seeding and spreading of amyloid-ß pathology in ASC-deficient APPSwePSEN1dE9 mice. Moreover, co-application of an anti-ASC antibody blocked the increase in amyloid-ß pathology in APPSwePSEN1dE9 mice. These findings support the concept that inflammasome activation is connected to seeding and spreading of amyloid-ß pathology in patients with Alzheimer's disease.

Perinuclear positioning of endosomes can affect PS-ASO activities.

Phosphorothioate (PS) modified antisense oligonucleotide (ASO) drugs that act on cellular RNAs must enter cells and be released from endocytic organelles to elicit antisense activity. It has been shown that PS-ASOs are mainly released by late endosomes. However, it is unclear how endosome movement in cells contributes to PS-ASO activity. Here, we show that PS-ASOs in early endosomes display Brownian type motion and migrate only short distances, whereas PS-ASOs in late endosomes (LEs) move linearly along microtubules with substantial distances. In cells with normal microtubules and LE movement, PS-ASO-loaded LEs tend to congregate perinuclearly. Disruption of perinuclear positioning of LEs by reduction of dynein 1 decreased PS-ASO activity, without affecting PS-ASO cellular uptake. Similarly, disruption of perinuclear positioning of PS-ASO-LE foci by reduction of ER tethering proteins RNF26, SQSTM1 and UBE2J1, or by overexpression of P50 all decreased PS-ASO activity. However, enhancing perinuclear positioning through reduction of USP15 or over-expression of RNF26 modestly increased PS-ASO activity, indicating that LE perinuclear positioning is required for ensuring efficient PS-ASO release. Together, these observations suggest that LE movement along microtubules and perinuclear positioning affect PS-ASO productive release.

Microglia modulation through external vagus nerve stimulation in a murine model of Alzheimer's disease.

Chronically activated microglia contribute to the development of neurodegenerative diseases such as Alzheimer's disease (AD) by the release of pro-inflammatory mediators that compromise neuronal function and structure. Modulating microglia functions could be instrumental to interfere with disease pathogenesis. Previous studies have shown anti-inflammatory effects of acetylcholine (ACh) or norepinephrine (NE), which mainly activates the ß-receptors on microglial cells. Non-invasive vagus nerve stimulation (nVNS) is used in treatment of drug-resistant depression, which is a risk factor for developing AD. The vagus nerve projects to the brainstem's locus coeruleus from which noradrenergic fibers reach to the Nucleus Basalis of Meynert (NBM) and widely throughout the brain. Pilot studies showed first signs of cognitive-enhancing effects of nVNS in AD patients. In this study, the effects of nVNS on mouse microglia cell morphology were analyzed over a period of 280 min by 2-photon laser scanning in vivo microscopy. Total branch length, average branch order and number of branches, which are commonly used indicators for the microglial activation state were determined and compared between young and old wild-type and amyloid precursor protein/presenilin-1 (APP/PS1) transgenic mice. Overall, these experiments show strong morphological changes in microglia, from a neurodestructive to a neuroprotective phenotype, following a brief nVNS in aged animals, especially in APP/PS1 animals, whereas microglia from young animals were morphologically unaffected.

ARG1-expressing microglia show a distinct molecular signature and modulate postnatal development and function of the mouse brain.

Molecular diversity of microglia, the resident immune cells in the CNS, is reported. Whether microglial subsets characterized by the expression of specific proteins constitute subtypes with distinct functions has not been fully elucidated. Here we describe a microglial subtype expressing the enzyme arginase-1 (ARG1; that is, ARG1+ microglia) that is found predominantly in the basal forebrain and ventral striatum during early postnatal mouse development. ARG1+ microglia are enriched in phagocytic inclusions and exhibit a distinct molecular signature, including upregulation of genes such as Apoe, Clec7a, Igf1, Lgals3 and Mgl2, compared to ARG1- microglia. Microglial-specific knockdown of Arg1 results in deficient cholinergic innervation and impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, which in turn results in impaired long-term potentiation and cognitive behavioral deficiencies in female mice. Our results expand on microglia diversity and provide insights into microglia subtype-specific functions.

mTOR-dependent translation amplifies microglia priming in aging mice.

Microglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice had upregulated mTOR complex 1 signaling controlling translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual yet contrasting effect on microglia priming: it caused an NF-κB-dependent upregulation of priming genes at the mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation, and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating that upregulation of mTOR-dependent translation is an essential aspect of microglia priming in aging and neurodegeneration.

Human microglia states are conserved across experimental models and regulate neural stem cell responses in chimeric organoids.

Microglia are resident macrophages in the brain that emerge in early development and respond to the local environment by altering their molecular and phenotypic states. Fundamental questions about microglia diversity and function during development remain unanswered because we lack experimental strategies to interrogate their interactions with other cell types and responses to perturbations ex vivo. We compared human microglia states across culture models, including cultured primary and pluripotent stem cell-derived microglia. We developed a "report card" of gene expression signatures across these distinct models to facilitate characterization of their responses across experimental models, perturbations, and disease conditions. Xenotransplantation of human microglia into cerebral organoids allowed us to characterize key transcriptional programs of developing microglia in vitro and reveal that microglia induce transcriptional changes in neural stem cells and decrease interferon signaling response genes. Microglia additionally accelerate the emergence of synchronized oscillatory network activity in brain organoids by modulating synaptic density.

Microglia in Neurodegenerative Disorders.

Microglia are the brain's resident immune cells. Under physiological conditions, they participate in a myriad of processes mainly involved in housekeeping functions that promote tissue homeostasis. However, the triggering of an immune response is a common feature in neurodegenerative disorders. This shift in microglia cells toward a chronically activated phenotype contributing to neuronal dysfunction and cell death is of great interest nowadays. In this chapter, we review the implications of microglia activation in different neurodegenerative disorders.

In Vivo Phagocytosis Analysis of Amyloid Beta.

Alzheimer's disease (AD) pathology is characterized by the presence of extracellular amyloid beta (Aß), tau hyperphosphorylation, and neuroinflammation. One striking feature in the disease is the clustering of microglia around Aß plaques. These cells exhibit a highly and chronically activated phenotype, performing a variety of functions, phagocytosis being one of them. Since Aß phagocytosis by microglia could represent a key aspect in the pathogenesis and progression of the disease, robust and comprehensive methods to evaluate this process are needed. Here, we describe a detailed flow cytometry-based protocol for the analysis of Aß phagocytosis in vivo.

Cannabinoid 1 Receptor Signaling on Hippocampal GABAergic Neurons Influences Microglial Activity.

Microglia, the resident immune cells of the brain, play important roles in defending the brain against pathogens and supporting neuronal circuit plasticity. Chronic or excessive pro-inflammatory responses of microglia damage neurons, therefore their activity is tightly regulated. Pharmacological and genetic studies revealed that cannabinoid type 1 (CB1) receptor activity influences microglial activity, although microglial CB1 receptor expression is very low and activity-dependent. The CB1 receptor is mainly expressed on neurons in the central nervous system (CNS)-with an especially high level on GABAergic interneurons. Here, we determined whether CB1 signaling on this neuronal cell type plays a role in regulating microglial activity. We compared microglia density, morphology and cytokine expression in wild-type (WT) and GABAergic neuron-specific CB1 knockout mice (GABA/CB1-/-) under control conditions (saline-treatment) and after 3 h, 24 h or repeated lipopolysaccharide (LPS)-treatment. Our results revealed that hippocampal microglia from saline-treated GABA/CB1-/- mice resembled those of LPS-treated WT mice: enhanced density and larger cell bodies, while the size and complexity of their processes was reduced. No further reduction in the size or complexity of microglia branching was detected after LPS-treatment in GABA/CB1-/- mice, suggesting that microglia in naïve GABA/CB1-/- mice were already in an activated state. This result was further supported by correlating the level of microglial tumor necrosis factor α (TNFα) with their size. Acute LPS-treatment elicited in both genotypes similar changes in the expression of pro-inflammatory cytokines (TNFα, interleukin-6 (IL-6) and interleukin 1ß (IL-1ß)). However, TNFα expression was still significantly elevated after repeated LPS-treatment in WT, but not in GABA/CB1-/- mice, indicating a faster development of tolerance to LPS. We also tested the possibility that the altered microglia activity in GABA/CB1-/- mice was due to an altered expression of neuron-glia interaction proteins. Indeed, the level of fractalkine (CX3CL1), a neuronal protein involved in the regulation of microglia, was reduced in hippocampal GABAergic neurons in GABA/CB1-/- mice, suggesting a disturbed neuronal control of microglial activity. Our result suggests that CB1 receptor agonists can modulate microglial activity indirectly, through CB1 receptors on GABAergic neurons. Altogether, we demonstrated that GABAergic neurons, despite their relatively low density in the hippocampus, have a specific role in the regulation of microglial activity and cannabinoid signaling plays an important role in this arrangement.

United Again: STING and the Police.

Neuroinflammation is a common feature of aging and neurodegeneration. In this issue of Neuron, Mathur et al. (2017) report that the antiviral drug Ganciclovir induces an interferon type I response in microglia through activation of the STING pathway, inhibiting inflammation and leading to protection in a model of multiple sclerosis.

Microglia in Alzheimer's disease: the good, the bad and the ugly.

Traditionally the brain has been viewed as being an immune-privileged organ. However, endogenous stimuli such as the presence of misfolded or aggregated proteins, as well as systemic inflammatory events may lead to the activation of microglial cells, the brain's innate immune system, and, subsequently, to neuroinflammation. Alzheimer's disease, the leading cause of dementia, is characterized by amyloid beta deposition and tau hyperphosphorylation. Neuroinflammation in Alzheimer's disease has been identified as major contributor to disease pathogenesis. Once activated, microglia release several pro and anti-inflammatory mediators of which several affect the function and structure of the brain. Modulation of this microglial activation in Alzheimer's disease might open new therapeutic avenues.