InFlamiN' the brain in Alzheimer's disease.

Although consistently implicated, the exact role of interferon (IFN) signaling in Alzheimer's disease remains largely unexplored. Roy et al. now demonstrate that type I IFNs may drive cognitive dysfunction through acting not only on microglia but, surprisingly, also on neurons.

Medin co-aggregates with vascular amyloid-ß in Alzheimer's disease.

Aggregates of medin amyloid (a fragment of the protein MFG-E8, also known as lactadherin) are found in the vasculature of almost all humans over 50 years of age1,2, making it the most common amyloid currently known. We recently reported that medin also aggregates in blood vessels of ageing wild-type mice, causing cerebrovascular dysfunction3. Here we demonstrate in amyloid-ß precursor protein (APP) transgenic mice and in patients with Alzheimer's disease that medin co-localizes with vascular amyloid-ß deposits, and that in mice, medin deficiency reduces vascular amyloid-ß deposition by half. Moreover, in both the mouse and human brain, MFG-E8 is highly enriched in the vasculature and both MFG-E8 and medin levels increase with the severity of vascular amyloid-ß burden. Additionally, analysing data from 566 individuals in the ROSMAP cohort, we find that patients with Alzheimer's disease have higher MFGE8 expression levels, which are attributable to vascular cells and are associated with increased measures of cognitive decline, independent of plaque and tau pathology. Mechanistically, we demonstrate that medin interacts directly with amyloid-ß to promote its aggregation, as medin forms heterologous fibrils with amyloid-ß, affects amyloid-ß fibril structure, and cross-seeds amyloid-ß aggregation both in vitro and in vivo. Thus, medin could be a therapeutic target for prevention of vascular damage and cognitive decline resulting from amyloid-ß deposition in the blood vessels of the brain.

Epigenetic control of microglial immune responses.

Microglia, the major population of brain-resident macrophages, are now recognized as a heterogeneous population comprising several cell subtypes with different (so far mostly supposed) functions in health and disease. A number of studies have performed molecular characterization of these different microglial activation states over the last years making use of "omics" technologies, that is transcriptomics, proteomics and, less frequently, epigenomics profiling. These approaches offer the possibility to identify disease mechanisms, discover novel diagnostic biomarkers, and develop new therapeutic strategies. Here, we focus on epigenetic profiling as a means to understand microglial immune responses beyond what other omics methods can offer, that is, revealing past and present molecular responses, gene regulatory networks and potential future response trajectories, and defining cell subtype-specific disease relevance through mapping non-coding genetic variants. We review the current knowledge in the field regarding epigenetic regulation of microglial identity and function, provide an exemplary analysis that demonstrates the advantages of performing joint transcriptomic and epigenomic profiling of single microglial cells and discuss how comprehensive epigenetic analyses may enhance our understanding of microglial pathophysiology.

Signatures of glial activity can be detected in the CSF proteome.

Single-cell transcriptomics has revealed specific glial activation states associated with the pathogenesis of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. While these findings may eventually lead to new therapeutic opportunities, little is known about how these glial responses are reflected by biomarker changes in bodily fluids. Such knowledge, however, appears crucial for patient stratification, as well as monitoring disease progression and treatment responses in clinical trials. Here, we took advantage of well-described mouse models of ß-amyloidosis and α-synucleinopathy to explore cerebrospinal fluid (CSF) proteome changes related to their respective proteopathic lesions. Nontargeted liquid chromatography-mass spectrometry revealed that the majority of proteins that undergo age-related changes in CSF of either mouse model were linked to microglia and astrocytes. Specifically, we identified a panel of more than 20 glial-derived proteins that were increased in CSF of aged ß-amyloid precursor protein- and α-synuclein-transgenic mice and largely overlap with previously described disease-associated glial genes identified by single-cell transcriptomics. Our results also show that enhanced shedding is responsible for the increase of several of the identified glial CSF proteins as exemplified for TREM2. Notably, the vast majority of these proteins can also be quantified in human CSF and reveal changes in Alzheimer's disease cohorts. The finding that cellular transcriptome changes translate into corresponding changes of CSF proteins is of clinical relevance, supporting efforts to identify fluid biomarkers that reflect the various functional states of glial responses in cerebral proteopathies, such as Alzheimer's and Parkinson's disease.

Innate immune memory in the brain shapes neurological disease hallmarks.

Innate immune memory is a vital mechanism of myeloid cell plasticity that occurs in response to environmental stimuli and alters subsequent immune responses. Two types of immunological imprinting can be distinguished-training and tolerance. These are epigenetically mediated and enhance or suppress subsequent inflammation, respectively. Whether immune memory occurs in tissue-resident macrophages in vivo and how it may affect pathology remains largely unknown. Here we demonstrate that peripherally applied inflammatory stimuli induce acute immune training and tolerance in the brain and lead to differential epigenetic reprogramming of brain-resident macrophages (microglia) that persists for at least six months. Strikingly, in a mouse model of Alzheimer's pathology, immune training exacerbates cerebral ß-amyloidosis and immune tolerance alleviates it; similarly, peripheral immune stimulation modifies pathological features after stroke. Our results identify immune memory in the brain as an important modifier of neuropathology.

Endogenous but not sensory-driven activity controls migration, morphogenesis and survival of adult-born juxtaglomerular neurons in the mouse olfactory bulb.

The development and survival of adult-born neurons are believed to be driven by sensory signaling. Here, in vivo analyses of motility, morphology and Ca2+ signaling, as well as transcriptome analyses of adult-born juxtaglomerular cells with reduced endogenous excitability (via cell-specific overexpression of either Kv1.2 or Kir2.1 K+ channels), revealed a pronounced impairment of migration, morphogenesis, survival, and functional integration of these cells into the mouse olfactory bulb, accompanied by a reduction in cytosolic Ca2+ fluctuations, phosphorylation of CREB and pCREB-mediated gene expression. Moreover, K+ channel overexpression strongly downregulated genes involved in neuronal migration, differentiation, and morphogenesis and upregulated apoptosis-related genes, thus locking adult-born cells in an immature and vulnerable state. Surprisingly, cells deprived of sensory-driven activity developed normally. Together, the data reveal signaling pathways connecting the endogenous intermittent neuronal activity/Ca2+ fluctuations as well as enhanced Kv1.2/Kir2.1 K+ channel function to migration, maturation, and survival of adult-born neurons.

Medin aggregation causes cerebrovascular dysfunction in aging wild-type mice.

Medin is the most common amyloid known in humans, as it can be found in blood vessels of the upper body in virtually everybody over 50 years of age. However, it remains unknown whether deposition of Medin plays a causal role in age-related vascular dysfunction. We now report that aggregates of Medin also develop in the aorta and brain vasculature of wild-type mice in an age-dependent manner. Strikingly, genetic deficiency of the Medin precursor protein, MFG-E8, eliminates not only vascular aggregates but also prevents age-associated decline of cerebrovascular function in mice. Given the prevalence of Medin aggregates in the general population and its role in vascular dysfunction with aging, targeting Medin may become a novel approach to sustain healthy aging.

Priming Microglia for Innate Immune Memory in the Brain.

Microglia, the resident macrophages of the brain, are highly plastic and well known to be pre-activated or 'primed' by active inflammatory processes, resulting in amplified responses to a second inflammatory insult. Furthermore, the capacity of microglia to develop 'innate immune memory' (IIM), that is, long-lasting molecular reprogramming, has recently been demonstrated. Depending on the initial stimulus, IIM can either enhance or suppress microglial responses to a delayed, secondary insult. Moreover, both priming and IIM can affect pathological hallmarks of neurological disease in mouse models, which may be consistent with certain clinical observations in patients. Here, we discuss the remarkable capacity of microglia to process inflammatory signals over short and long timeframes and propose new integrated nomenclature for these processes. We also highlight future research avenues, with implications for human brain disease.

Systemic inflammation induced the delayed reduction of excitatory synapses in the CA3 during ageing.

Sepsis-associated encephalopathy (SAE) represents diverse cerebral dysfunctions in response to pathogen-induced systemic inflammation. Peripheral exposure to lipopolysaccharide (LPS), a component of the gram-negative bacterial cell wall, has been extensively used to model systemic inflammation. Our previous studies suggested that LPS led to hippocampal neuron death and synaptic destruction in vivo. However, the underlying roles of activated microglia in these neuronal changes remained unclear. Here, LPS from two different bacterial strains (Salmonella enterica or E. coli) were compared and injected in 14- to 16-month-old mice and evaluated for neuroinflammation and neuronal integrity in the hippocampus at 7 or 63 days post-injection (dpi). LPS injection resulted in persistent neuroinflammation lasting for seven days and a subsequent normalisation by 63 dpi. Of note, increases in proinflammatory cytokines, microglial morphology and microglial mean lysosome volume were more pronounced after E. coli LPS injection than Salmonella LPS at 7 dpi. While inhibitory synaptic puncta density remained normal, excitatory synaptic puncta were locally reduced in the CA3 region of the hippocampus at 63 dpi. Finally, we provide evidence that excitatory synapses coated with complement factor 3 (C3) decreased between 7 dpi and 63 dpi. Although we did not find an increase of synaptic pruning by microglia, it is plausible that microglia recognised and eliminated these C3-tagged synapses between the two time points of investigation. Since a region-specific decline of CA3 synapses has previously been reported during normal ageing, we postulate that systemic inflammation may have accelerated or worsened the CA3 synaptic changes in the ageing brain.

Inflammatory mechanisms in neurodegeneration.

This review discusses the profound connection between microglia, neuroinflammation, and Alzheimer's disease (AD). Theories have been postulated, tested, and modified over several decades. The findings have further bolstered the belief that microglia-mediated inflammation is both a product and contributor to AD pathology and progression. Distinct microglia phenotypes and their function, microglial recognition and response to protein aggregates in AD, and the overall role of microglia in AD are areas that have received considerable research attention and yielded significant results. The following article provides a historical perspective of microglia, a detailed discussion of multiple microglia phenotypes including dark microglia, and a review of a number of areas where microglia intersect with AD and other pathological neurological processes. The overall breadth of important discoveries achieved in these areas significantly strengthens the hypothesis that neuroinflammation plays a key role in AD. Future determination of the exact mechanisms by which microglia respond to, and attempt to mitigate, protein aggregation in AD may lead to new therapeutic strategies.

Microglial phagocytosis of live neurons.

Microglia, the brain's professional phagocytes, can remove dead and dying neurons as well as synapses and the processes of live neurons. However, we and others have recently shown that microglia can also execute neuronal death by phagocytosing stressed-but-viable neurons - a process that we have termed phagoptosis. In this Progress article, we discuss evidence suggesting that phagoptosis may contribute to neuronal loss during brain development, inflammation, ischaemia and neurodegeneration.

Infiltrating monocytes promote brain inflammation and exacerbate neuronal damage after status epilepticus.

The generalized seizures of status epilepticus (SE) trigger a series of molecular and cellular events that produce cognitive deficits and can culminate in the development of epilepsy. Known early events include opening of the blood-brain barrier (BBB) and astrocytosis accompanied by activation of brain microglia. Whereas circulating monocytes do not infiltrate the healthy CNS, monocytes can enter the brain in response to injury and contribute to the immune response. We examined the cellular components of innate immune inflammation in the days following SE by discriminating microglia vs. brain-infiltrating monocytes. Chemokine receptor 2 (CCR2(+)) monocytes invade the hippocampus between 1 and 3 d after SE. In contrast, only an occasional CD3(+) T lymphocyte was encountered 3 d after SE. The initial cellular sources of the chemokine CCL2, a ligand for CCR2, included perivascular macrophages and microglia. The induction of the proinflammatory cytokine IL-1ß was greater in FACS-isolated microglia than in brain-invading monocytes. However, Ccr2 knockout mice displayed greatly reduced monocyte recruitment into brain and reduced levels of the proinflammatory cytokine IL-1ß in hippocampus after SE, which was explained by higher expression of the cytokine in circulating and brain monocytes in wild-type mice. Importantly, preventing monocyte recruitment accelerated weight regain, reduced BBB degradation, and attenuated neuronal damage. Our findings identify brain-infiltrating monocytes as a myeloid-cell subclass that contributes to neuroinflammation and morbidity after SE. Inhibiting brain invasion of CCR2(+) monocytes could represent a viable method for alleviating the deleterious consequences of SE.

Conversion of Synthetic Aß to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.

UNLABELLED: The aggregation of amyloid-ß peptide (Aß) in brain is an early event and hallmark of Alzheimer's disease (AD). We combined the advantages of in vitro and in vivo approaches to study cerebral ß-amyloidosis by establishing a long-term hippocampal slice culture (HSC) model. While no Aß deposition was noted in untreated HSCs of postnatal Aß precursor protein transgenic (APP tg) mice, Aß deposition emerged in HSCs when cultures were treated once with brain extract from aged APP tg mice and the culture medium was continuously supplemented with synthetic Aß. Seeded Aß deposition was also observed under the same conditions in HSCs derived from wild-type or App-null mice but in no comparable way when HSCs were fixed before cultivation. Both the nature of the brain extract and the synthetic Aß species determined the conformational characteristics of HSC Aß deposition. HSC Aß deposits induced a microglia response, spine loss, and neuritic dystrophy but no obvious neuron loss. Remarkably, in contrast to in vitro aggregated synthetic Aß, homogenates of Aß deposits containing HSCs induced cerebral ß-amyloidosis upon intracerebral inoculation into young APP tg mice. Our results demonstrate that a living cellular environment promotes the seeded conversion of synthetic Aß into a potent in vivo seeding-active form. SIGNIFICANCE STATEMENT: In this study, we report the seeded induction of Aß aggregation and deposition in long-term hippocampal slice cultures. Remarkably, we find that the biological activities of the largely synthetic Aß aggregates in the culture are very similar to those observed in vivo This observation is the first to show that potent in vivo seeding-active Aß aggregates can be obtained by seeded conversion of synthetic Aß in a living (wild-type) cellular environment.

Eaten alive! Cell death by primary phagocytosis: 'phagoptosis'.

Phagoptosis, also called primary phagocytosis, is a recently recognised form of cell death caused by phagocytosis of viable cells, resulting in their destruction. It is provoked by exposure of 'eat-me' signals and/or loss of 'don't-eat-me' signals by viable cells, causing their phagocytosis by phagocytes. Phagoptosis mediates turnover of erythrocytes, neutrophils and other cells, and thus is quantitatively one of the main forms of cell death in the body. It defends against pathogens and regulates inflammation and immunity. However, recent results indicate that inflamed microglia eat viable brain neurons in models of neurodegeneration, and cancer cells can evade phagocytosis by expressing a 'don't-eat-me' signal, suggesting that too much or too little phagoptosis can contribute to pathology. This review provides an overview of the molecular signals that regulate phagoptosis and the physiological and pathological circumstances in which it has been observed.

Phagocytosis executes delayed neuronal death after focal brain ischemia.

Delayed neuronal loss and brain atrophy after cerebral ischemia contribute to stroke and dementia pathology, but the mechanisms are poorly understood. Phagocytic removal of neurons is generally assumed to be beneficial and to occur only after neuronal death. However, we report herein that inhibition of phagocytosis can prevent delayed loss and death of functional neurons after transient brain ischemia. Two phagocytic proteins, Mer receptor tyrosine kinase (MerTK) and Milk fat globule EGF-like factor 8 (MFG-E8), were transiently up-regulated by macrophages/microglia after focal brain ischemia in vivo. Strikingly, deficiency in either protein completely prevented long-term functional motor deficits after cerebral ischemia and strongly reduced brain atrophy as a result of inhibiting phagocytosis of neurons. Correspondingly, in vitro glutamate-stressed neurons reversibly exposed the "eat-me" signal phosphatidylserine, leading to their phagocytosis by microglia; this neuronal loss was prevented in the absence of microglia and reduced if microglia were genetically deficient in MerTK or MFG-E8, both of which mediate phosphatidylserine-recognition. Thus, phagocytosis of viable neurons contributes to brain pathology and, surprisingly, blocking this process is strongly beneficial. Therefore, inhibition of specific phagocytic pathways may present therapeutic targets for preventing delayed neuronal loss after transient cerebral ischemia.

Inhibition of UDP/P2Y6 purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo.

Microglia activated through Toll-like receptor (TLR)-2 or -4 can cause neuronal death by phagocytosing otherwise-viable neurons-a form of cell death called "phagoptosis." UDP release from neurons has been shown to provoke microglial phagocytosis of neurons via microglial P2Y6 receptors, but whether inhibition of this process affects neuronal survival is unknown. We tested here whether inhibition of P2Y6 signaling could prevent neuronal death in inflammatory conditions, and whether UDP signaling can induce phagoptosis of stressed but viable neurons. We find that delayed neuronal loss and death in mixed neuronal/glial cultures induced by the TLR ligands lipopolysaccharide (LPS) or lipoteichoic acid was prevented by: apyrase (to degrade nucleotides), Reactive Blue 2 (to inhibit purinergic signaling), or MRS2578 (to specifically block P2Y6 receptors). In each case, inflammatory activation of microglia was not affected, and the rescued neurons remained viable for at least 7 days. Blocking P2Y6 receptors with MRS2578 also prevented phagoptosis of neurons induced by 250 nM amyloid beta 1-42, 5 µM peroxynitrite, or 50 µM 3-morpholinosydnonimine (which releases reactive oxygen and nitrogen species). Furthermore, the P2Y6 receptor agonist UDP by itself was sufficient to stimulate microglial phagocytosis and to induce rapid neuronal loss that was prevented by eliminating microglia or inhibiting phagocytosis. In vivo, injection of LPS into rat striatum induced microglial activation and delayed neuronal loss and blocking P2Y6 receptors with MRS2578 prevented this neuronal loss. Thus, blocking UDP/P2Y6 signaling is sufficient to prevent neuronal loss and death induced by a wide range of stimuli that activate microglial phagocytosis of neurons.

Combined Analysis of mRNA Expression and Open Chromatin in Microglia.

The advance of single-cell RNA-sequencing technologies in the past years has enabled unprecedented insights into the complexity and heterogeneity of microglial cell states in the homeostatic and diseased brain. This includes rather complex proteomic, metabolomic, morphological, transcriptomic, and epigenetic adaptations to external stimuli and challenges resulting in a novel concept of core microglia properties and functions. To uncover the regulatory programs facilitating the rapid transcriptomic adaptation in response to changes in the local microenvironment, the accessibility of gene bodies and gene regulatory elements can be assessed. Here, we describe the application of a previously published method for simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq) on microglia nuclei isolated from frozen mouse brain tissue.

MFG-E8 mediates primary phagocytosis of viable neurons during neuroinflammation.

Milk-fat globule EGF factor-8 (MFG-E8, SED1, lactadherin) is known to mediate the phagocytic removal of apoptotic cells by bridging phosphatidylserine (PS)-exposing cells and the vitronectin receptor (VR) on phagocytes. However, we show here that MFG-E8 can mediate phagocytosis of viable neurons during neuroinflammation induced by lipopolysaccharide (LPS), thereby causing neuronal death. In vitro, inflammatory neuronal loss is independent of apoptotic pathways, and is inhibited by blocking the PS/MFG-E8/VR pathway (by adding PS blocking antibodies, annexin V, mutant MFG-E8 unable to bind VR, or VR antagonist). Neuronal loss is absent in Mfge8 knock-out cultures, but restored by adding recombinant MFG-E8, without affecting inflammation. In vivo, LPS-induced neuronal loss is reduced in the striatum of Mfge8 knock-out mice or by coinjection of an MFG-E8 receptor (VR) inhibitor into the rat striatum. Our data show that blocking MFG-E8-dependent phagocytosis preserves live neurons, implying that phagocytosis actively contributes to neuronal death during brain inflammation.

Inhibition of microglial phagocytosis is sufficient to prevent inflammatory neuronal death.

It is well-known that dead and dying neurons are quickly removed through phagocytosis by the brain's macrophages, the microglia. Therefore, neuronal loss during brain inflammation has always been assumed to be due to phagocytosis of neurons subsequent to their apoptotic or necrotic death. However, we report in this article that under inflammatory conditions in primary rat cultures of neurons and glia, phagocytosis actively induces neuronal death. Specifically, two inflammatory bacterial ligands, lipoteichoic acid or LPS (agonists of glial TLR2 and TLR4, respectively), stimulated microglial proliferation, phagocytic activity, and engulfment of ∼30% of neurons within 3 d. Phagocytosis of neurons was dependent on the microglial release of soluble mediators (and peroxynitrite in particular), which induced neuronal exposure of the eat-me signal phosphatidylserine (PS). Surprisingly, however, eat-me signaling was reversible, so that blocking any step in a phagocytic pathway consisting of PS exposure, the PS-binding protein milk fat globule epidermal growth factor-8, and its microglial vitronectin receptor was sufficient to rescue up to 90% of neurons without reducing inflammation. Hence, our data indicate a novel form of inflammatory neurodegeneration, where inflammation can cause eat-me signal exposure by otherwise viable neurons, leading to their death through phagocytosis. Thus, blocking phagocytosis may prevent some forms of inflammatory neurodegeneration, and therefore might be beneficial during brain infection, trauma, ischemia, neurodegeneration, and aging.