Microglia-synapse engulfment via PtdSer-TREM2 ameliorates neuronal hyperactivity in Alzheimer's disease models.

Neuronal hyperactivity is a key feature of early stages of Alzheimer's disease (AD). Genetic studies in AD support that microglia act as potential cellular drivers of disease risk, but the molecular determinants of microglia-synapse engulfment associated with neuronal hyperactivity in AD are unclear. Here, using super-resolution microscopy, 3D-live imaging of co-cultures, and in vivo imaging of lipids in genetic models, we found that spines become hyperactive upon Aß oligomer stimulation and externalize phosphatidylserine (ePtdSer), a canonical "eat-me" signal. These apoptotic-like spines are targeted by microglia for engulfment via TREM2 leading to amelioration of Aß oligomer-induced synaptic hyperactivity. We also show the in vivo relevance of ePtdSer-TREM2 signaling in microglia-synapse engulfment in the hAPP NL-F knock-in mouse model of AD. Higher levels of apoptotic-like synapses in mice as well as humans that carry TREM2 loss-of-function variants were also observed. Our work supports that microglia remove hyperactive ePtdSer+ synapses in Aß-relevant context and suggest a potential beneficial role for microglia in the earliest stages of AD.

Complement C3-Deficient Mice Fail to Display Age-Related Hippocampal Decline.

The complement system is part of the innate immune response responsible for removing pathogens and cellular debris, in addition to helping to refine CNS neuronal connections via microglia-mediated pruning of inappropriate synapses during brain development. However, less is known about the role of complement during normal aging. Here, we studied the role of the central complement component, C3, in synaptic health and aging. We examined behavior as well as electrophysiological, synaptic, and neuronal changes in the brains of C3-deficient male mice (C3 KO) compared with age-, strain-, and gender-matched C57BL/6J (wild-type, WT) control mice at postnatal day 30, 4 months, and 16 months of age. We found the following: (1) region-specific and age-dependent synapse loss in aged WT mice that was not observed in C3 KO mice; (2) age-dependent neuron loss in hippocampal CA3 (but not in CA1) that followed synapse loss in aged WT mice, neither of which were observed in aged C3 KO mice; and (3) significantly enhanced LTP and cognition and less anxiety in aged C3 KO mice compared with aged WT mice. Importantly, CA3 synaptic puncta were similar between WT and C3 KO mice at P30. Together, our results suggest a novel and prominent role for complement protein C3 in mediating aged-related and region-specific changes in synaptic function and plasticity in the aging brain. Significance statement: The complement cascade, part of the innate immune response to remove pathogens, also plays a role in synaptic refinement during brain development by the removal of weak synapses. We investigated whether complement C3, a central component, affects synapse loss during aging. Wild-type (WT) and C3 knock-out (C3 KO) mice were examined at different ages. The mice were similar at 1 month of age. However, with aging, WT mice lost synapses in specific brain regions, especially in hippocampus, an area important for memory, whereas C3 KO mice were protected. Aged C3 KO mice also performed better on learning and memory tests than aged WT mice. Our results suggest that complement C3, or its downstream signaling, is detrimental to synapses during aging.

PolyGR and polyPR knock-in mice reveal a conserved neuroprotective extracellular matrix signature in C9orf72 ALS/FTD neurons.

Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-ß1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-ß1 followed by COL6A1. Knockdown of TGF-ß1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-ß1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD.

New ELISAs with high specificity for soluble oligomers of amyloid ß-protein detect natural Aß oligomers in human brain but not CSF.

BACKGROUND: Soluble oligomers of amyloid ß-protein (Aß) have been increasingly linked to synaptic dysfunction, tau alteration, and neuritic dystrophy in Alzheimer's disease (AD) and mouse models. There is a great need for assays that quantify Aß oligomers with high specificity and sensitivity. METHODS: We designed and validated two oligomer-specific (o-) enzyme-linked immunoassays (ELISAs) using either an Aß aggregate-selective monoclonal for capture and a monoclonal to the free N-terminus for detection, or the latter antibody for both capture and detection. RESULTS: The o-ELISAs specifically quantified pure oligomers of synthetic Aß with sizes from dimers up to much larger assemblies and over a wide dynamic range of concentrations, whereas Aß monomers were undetectable. Natural Aß oligomers of similarly wide size and concentration ranges were measured in extracts of AD and control brains, revealing >1000-fold higher concentrations of Aß oligomers than monomers in the soluble fraction of AD cortex. The assays quantified the age-related rise in oligomers in hAPP transgenic mice. Unexpectedly, none of 90 human cerebrospinal fluid (CSF) samples gave a specific signal in either o-ELISA. CONCLUSIONS: These new o-ELISAs with rigorously confirmed specificity can quantify oligomer burden in human and mouse brains for diagnostic and mechanistic studies and for AD biomarker development. However, our data raise the likelihood that the hydrophobicity of Aß oligomers makes them very low in number or absent in aqueous CSF.

Stimulation of TREM2 with agonistic antibodies-an emerging therapeutic option for Alzheimer's disease.

Neurodegenerative disorders, including Alzheimer's disease, are associated with microgliosis. Microglia have long been considered to have detrimental roles in Alzheimer's disease. However, functional analyses of genes encoding risk factors that are linked to late-onset Alzheimer's disease, and that are enriched or exclusively expressed in microglia, have revealed unexpected protective functions. One of the major risk genes for Alzheimer's disease is TREM2. Risk variants of TREM2 are loss-of-function mutations affecting chemotaxis, phagocytosis, lipid and energy metabolism, and survival and proliferation. Agonistic anti-TREM2 antibodies have been developed to boost these protective functions in patients with intact TREM2 alleles. Several anti-TREM2 antibodies are in early clinical trials, and current efforts aim to achieve more efficient transport of these antibodies across the blood-brain barrier. PET imaging could be used to monitor target engagement. Data from animal models, and biomarker studies in patients, further support a rationale for boosting TREM2 functions during the preclinical stage of Alzheimer's disease.

Perivascular cells induce microglial phagocytic states and synaptic engulfment via SPP1 in mouse models of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-ß oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell-cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediated synaptic engulfment in mouse models of AD.

Dynamic analysis of amyloid ß-protein in behaving mice reveals opposing changes in ISF versus parenchymal Aß during age-related plaque formation.

Growing evidence supports the hypothesis that soluble, diffusible forms of the amyloid ß-peptide (Aß) are pathogenically important in Alzheimer's disease (AD) and thus have both diagnostic and therapeutic salience. To learn more about the dynamics of soluble Aß economy in vivo, we used microdialysis to sample the brain interstitial fluid (ISF), which contains the most soluble Aß species in brain at steady state, in >40 wake, behaving APP transgenic mice before and during the process of Aß plaque formation (age 3-28 months). Diffusible forms of Aß, especially Aß(42), declined significantly in ISF as mice underwent progressive parenchymal deposition of Aß. Moreover, radiolabeled Aß administered at physiological concentrations into ISF revealed a striking difference in the fate of soluble Aß in plaque-rich (vs plaque-free) mice: it clears more rapidly from the ISF and becomes more associated with the TBS-extractable pool, suggesting that cerebral amyloid deposits can rapidly sequester soluble Aß from the ISF. Likewise, acute γ-secretase inhibition in plaque-free mice showed a marked decline of Aß(38), Aß(40), and Aß(42), whereas in plaque-rich mice, Aß(42) declined significantly less. These results suggest that most of the Aß(42) that populates the ISF in plaque-rich mice is derived not from new Aß biosynthesis but rather from the large reservoir of less soluble Aß(42) in brain parenchyma. Together, these and other findings herein illuminate the in vivo dynamics of soluble Aß during the development of AD-type neuropathology and after γ-secretase inhibition and help explain the apparent paradox that CSF Aß(42) levels fall as humans develop AD.

Complement component C3 and complement receptor type 3 contribute to the phagocytosis and clearance of fibrillar Aß by microglia.

Complement components and their receptors are found within and around amyloid ß (Aß) cerebral plaques in Alzheimer's disease (AD). Microglia defend against pathogens through phagocytosis via complement component C3 and/or engagement of C3 cleavage product iC3b with complement receptor type 3 (CR3, Mac-1). Here, we provide direct evidence that C3 and Mac-1 mediate, in part, phagocytosis and clearance of fibrillar amyloid-ß (fAß) by murine microglia in vitro and in vivo. Microglia took up not only synthetic fAß(42) but also amyloid cores from patients with AD, transporting them to lysosomes in vitro. Fibrillar Aß(42) uptake was significantly attenuated by the deficiency or knockdown of C3 or Mac-1 and scavenger receptor class A ligands. In addition, C3 or Mac-1 knockdown combined with a scavenger receptor ligand, fucoidan, further attenuated fibrillar Aß(42) uptake by N9 microglia. Fluorescent fibrillar Aß(42) microinjected cortically was significantly higher in C3 and Mac-1 knockout mice compared with wild-type mice 5 days after surgery, indicating reduced clearance in vivo. Together, these results demonstrate that C3 and Mac-1 are involved in phagocytosis and clearance of fAß by microglia, providing support for a potential beneficial role for microglia and the complement system in AD pathogenesis. © 2012 Wiley Periodicals, Inc.

Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain.

A key aspect of nearly all single-cell sequencing experiments is dissociation of intact tissues into single-cell suspensions. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression. Here, we demonstrate that use of enzymatic dissociation on brain tissue induces an aberrant ex vivo gene expression signature, most prominently in microglia, which is prevalent in published literature and can substantially confound downstream analyses. To address this issue, we present a rigorously validated protocol that preserves both in vivo transcriptional profiles and cell-type diversity and yield across tissue types and species. We also identify a similar signature in postmortem human brain single-nucleus RNA-sequencing datasets, and show that this signature is induced in freshly isolated human tissue by exposure to elevated temperatures ex vivo. Together, our results provide a methodological solution for preventing artifactual gene expression changes during fresh tissue digestion and a reference for future deeper analysis of the potential confounding states present in postmortem human samples.

Microglia states and nomenclature: A field at its crossroads.

Microglial research has advanced considerably in recent decades yet has been constrained by a rolling series of dichotomies such as "resting versus activated" and "M1 versus M2." This dualistic classification of good or bad microglia is inconsistent with the wide repertoire of microglial states and functions in development, plasticity, aging, and diseases that were elucidated in recent years. New designations continuously arising in an attempt to describe the different microglial states, notably defined using transcriptomics and proteomics, may easily lead to a misleading, although unintentional, coupling of categories and functions. To address these issues, we assembled a group of multidisciplinary experts to discuss our current understanding of microglial states as a dynamic concept and the importance of addressing microglial function. Here, we provide a conceptual framework and recommendations on the use of microglial nomenclature for researchers, reviewers, and editors, which will serve as the foundations for a future white paper.

Insight into the role of phosphatidylserine in complement-mediated synapse loss in Alzheimer's disease.

The innate immune system plays an integral role in the brain. Synaptic pruning, a fundamental process in developmental circuit refinement, is partially mediated by neuroimmune signalling at the synapse. In particular, microglia, the major tissue-resident macrophages of the brain, and the classical complement cascade, an innate immune pathway that aids in the clearance of unwanted material, have been implicated in mediating synapse elimination. Emerging data suggest that improper signalling of the innate immune pathway at the synapse leads to pathological synapse loss in age-related neurodegenerative diseases, including Alzheimer's disease. Now the key questions are whether synapses are targeted by complement and, if so, which synapses are vulnerable to elimination. Here, we review recent work implicating C1q, the initiator of the classical complement cascade, and surrounding glia as mediators of synapse loss. We examine how synapses could undergo apoptosis-like pathways in the Alzheimer brain, which may lead to the externalisation of phosphatidylserine on synapses. Finally, we discuss potential roles for microglia and astrocytes in this 'synaptic apoptosis'. Critical insight into neuroimmune regulatory pathways on synapses will be key to developing effective targets against pathological synapse loss in dementia.

Understanding microglial diversity and implications for neuronal function in health and disease.

Genetic data implicate microglia as central players in brain health and disease, urging the need to better understand what microglia do in the brain. Microglia are critical partners in neuronal wiring and function during development and disease. Emerging literature suggests that microglia have diverse functional roles, raising the intriguing question of which functions of microglia become impaired in disease to undermine proper neuronal function. It is also becoming increasingly clear that microglia exist in heterogeneous cell states. Microglial cell states appear context-dependent, that is, age, sex, location, and health of their microenvironment; these are further influenced by external signaling factors including gut microbiota and lipid metabolites. These data altogether suggest that microglia exist in functional clusters that impact, and are impacted by, surrounding neuronal microenvironment. However, we still lack understanding of how we can translate microglia cell states into function. Here, we summarize the state-of-the-art on the diverse functions of microglia in relation to neuronal health. Then, we discuss heterogeneity during developing, healthy adult and diseased brains, and whether this may be predetermined by origin and/or regulated by local milieu. Finally, we propose that it is critical to gain high-resolution functional discernment into microglia-neuron interactions while preserving the spatial architecture of the tissue. Such insight will reveal specific targets for biomarker and therapeutic development toward microglia-neuron crosstalk in disease.

Research priorities for neuroimmunology: identifying the key research questions to be addressed by 2030.

Neuroimmunology in the broadest sense is the study of interactions between the nervous and the immune systems. These interactions play important roles in health from supporting neural development, homeostasis and plasticity to modifying behaviour. Neuroimmunology is increasingly recognised as a field with the potential to deliver a significant positive impact on human health and treatment for neurological and psychiatric disorders. Yet, translation to the clinic is hindered by fundamental knowledge gaps on the underlying mechanisms of action or the optimal timing of an intervention, and a lack of appropriate tools to visualise and modulate both systems. Here we propose ten key disease-agnostic research questions that, if addressed, could lead to significant progress within neuroimmunology in the short to medium term. We also discuss four cross-cutting themes to be considered when addressing each question: i) bi-directionality of neuroimmune interactions; ii) the biological context in which the questions are addressed (e.g. health vs disease vs across the lifespan); iii) tools and technologies required to fully answer the questions; and iv) translation into the clinic. We acknowledge that these ten questions cannot represent the full breadth of gaps in our understanding; rather they focus on areas which, if addressed, may have the most broad and immediate impacts. By defining these neuroimmunology priorities, we hope to unite existing and future research teams, who can make meaningful progress through a collaborative and cross-disciplinary effort.

The Jekyll and Hyde of TREM2.

In a recent paper, Gratuze et al. demonstrated a putative neuroprotective role of a key Alzheimer risk variant, TREM2R47H, against tau-mediated neurodegeneration in a mouse model of tauopathy. This study highlights the context-dependent response of microglia, and proposes antagonistic roles of TREM2 in Aß- versus tau-mediated pathology.

Microglia modulate neurodegeneration in Alzheimer's and Parkinson's diseases.

Dementia is a rapidly rising global health crisis that silently disables families and ends lives and livelihoods around the world. To date, however, no early biomarkers or effective therapies exist. It is now clear that brain microglia are more than mere bystanders or amyloid phagocytes; they can act as governors of neuronal function and homeostasis in the adult brain. Here, we highlight the fundamental role of microglia as tissue-resident macrophages in neuronal health. Then, we suggest how chronic impairment in microglia-neuron cross-talk may secure the permanence of the failure of synaptic and neuronal function and health in Alzheimer's and Parkinson's diseases. Understanding how to assess and modulate microglia-neuron interactions critical for brain health will be key to developing effective therapies for dementia.

LRP promotes endocytosis and degradation, but not transcytosis, of the amyloid-beta peptide in a blood-brain barrier in vitro model.

The pathogenesis of Alzheimer's disease is characterized by aggregation of the amyloid-beta protein (Abeta) into neurotoxic plaques. Recent in vivo studies have suggested the non-proteolytic clearance of Abeta via receptor-mediated transport across the blood-brain barrier (BBB). The aim of this study was to investigate the role of P-glycoprotein (Pgp) and the low-density lipoprotein receptor-related protein (LRP) in Abeta efflux across the BBB. We developed an in vitro BBB-like model using Madin-Darby Canine Kidney (MDCK) cells seeded on filters separating apical (blood) and basolateral (brain) compartments. MDCK cells were stably transfected with Pgp or mLRP4, an LRP mini-receptor. When compared to empty vector-transfected cells, MDCK-Pgp cells did not transcytose radiolabeled Abeta in the basolateral-to-apical direction. MDCK-mLRP4 cells were found to endocytose and degrade, but not to trasncytose intact radiolabeled Abeta. These results implicate LRP as a mediator of Abeta degradation, but indicate that overexpression of LRP or Pgp alone is insufficient for non-proteolytic transcytosis of intact Abeta.

TREM2: Keeping Microglia Fit during Good Times and Bad.

Microglia are the macrophages of the brain and play an important role in Alzheimer's disease (AD). In Cell, Ulland et al. (2017) recently reported that mutations in TREM2, a protein implicated in AD, disrupt microglial energy state and function, thus sabotaging the microglia's ability to defend the brain against amyloid plaques.

Structured Illumination Microscopy for the Investigation of Synaptic Structure and Function.

The neuronal synapse is a primary building block of the nervous system to which alterations in structure or function can result in numerous pathologies. Studying its formation and elimination is the key to understanding how brains are wired during development, maintained throughout adulthood plasticity, and disrupted during disease. However, due to its diffraction-limited size, investigations of the synaptic junction at the structural level have primarily relied on labor-intensive electron microscopy or ultra-thin section array tomography. Recent advances in the field of super-resolution light microscopy now allow researchers to image synapses and associated molecules with high-spatial resolution, while taking advantage of the key characteristics of light microscopy, such as easy sample preparation and the ability to detect multiple targets with molecular specificity. One such super-resolution technique, Structured Illumination Microscopy (SIM), has emerged as an attractive method to examine synapse structure and function. SIM requires little change in standard light microscopy sample preparation steps, but results in a twofold improvement in both lateral and axial resolutions compared to widefield microscopy. The following protocol outlines a method for imaging synaptic structures at resolutions capable of resolving the intricacies of these neuronal connections.

Complement C3 deficiency protects against neurodegeneration in aged plaque-rich APP/PS1 mice.

The complement cascade not only is an innate immune response that enables removal of pathogens but also plays an important role in microglia-mediated synaptic refinement during brain development. Complement C3 is elevated in Alzheimer's disease (AD), colocalizing with neuritic plaques, and appears to contribute to clearance of Aß by microglia in the brain. Previously, we reported that C3-deficient C57BL/6 mice were protected against age-related and region-specific loss of hippocampal synapses and cognitive decline during normal aging. Furthermore, blocking complement and downstream iC3b/CR3 signaling rescued synapses from Aß-induced loss in young AD mice before amyloid plaques had accumulated. We assessed the effects of C3 deficiency in aged, plaque-rich APPswe/PS1dE9 transgenic mice (APP/PS1;C3 KO). We examined the effects of C3 deficiency on cognition, Aß plaque deposition, and plaque-related neuropathology at later AD stages in these mice. We found that 16-month-old APP/PS1;C3 KO mice performed better on a learning and memory task than did APP/PS1 mice, despite having more cerebral Aß plaques. Aged APP/PS1;C3 KO mice also had fewer microglia and astrocytes localized within the center of hippocampal Aß plaques compared to APP/PS1 mice. Several proinflammatory cytokines in the brain were reduced in APP/PS1;C3 KO mice, consistent with an altered microglial phenotype. C3 deficiency also protected APP/PS1 mice against age-dependent loss of synapses and neurons. Our study suggests that complement C3 or downstream complement activation fragments may play an important role in Aß plaque pathology, glial responses to plaques, and neuronal dysfunction in the brains of APP/PS1 mice.

Microglia: Phagocytosing to Clear, Sculpt, and Eliminate.

Microglia are the primary phagocytes of the central nervous system. They eliminate excess functional connections between neurons to sculpt neuronal circuits during development and throughout adulthood. Understanding how microglia recognize and prune synapses during development is providing insight into synapse loss and dysfunction in disease.