Proteomics of mouse brain endothelium uncovers dysregulation of vesicular transport pathways during aging.

Age-related decline in brain endothelial cell (BEC) function contributes critically to neurological disease. Comprehensive atlases of the BEC transcriptome have become available, but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting-based mouse BEC enrichment protocol compatible with proteomics and resolved the profiles of protein abundance changes during aging. Unsupervised cluster analysis revealed a segregation of age-related protein dynamics with biological functions, including a downregulation of vesicle-mediated transport. We found a dysregulation of key regulators of endocytosis and receptor recycling (most prominently Arf6), macropinocytosis and lysosomal degradation. In gene deletion and overexpression experiments, Arf6 affected endocytosis pathways in endothelial cells. Our approach uncovered changes not picked up by transcriptomic studies, such as accumulation of vesicle cargo and receptor ligands, including Apoe. Proteomic analysis of BECs from Apoe-deficient mice revealed a signature of accelerated aging. Our findings provide a resource for analysing BEC function during aging.

Microglia at sites of atrophy restrict the progression of retinal degeneration via galectin-3 and Trem2.

Outer retinal degenerations, including age-related macular degeneration (AMD), are characterized by photoreceptor and retinal pigment epithelium (RPE) atrophy. In these blinding diseases, macrophages accumulate at atrophic sites, but their ontogeny and niche specialization remain poorly understood, especially in humans. We uncovered a unique profile of microglia, marked by galectin-3 upregulation, at atrophic sites in mouse models of retinal degeneration and human AMD. In disease models, conditional deletion of galectin-3 in microglia led to phagocytosis defects and consequent augmented photoreceptor death, RPE damage, and vision loss, indicating protective roles. Mechanistically, Trem2 signaling orchestrated microglial migration to atrophic sites and induced galectin-3 expression. Moreover, pharmacologic Trem2 agonization led to heightened protection but in a galectin-3-dependent manner. In elderly human subjects, we identified this highly conserved microglial population that expressed galectin-3 and Trem2. This population was significantly enriched in the macular RPE-choroid of AMD subjects. Collectively, our findings reveal a neuroprotective population of microglia and a potential therapeutic target for mitigating retinal degeneration.

Association of Neurofilament Light Chain, [18F]PI-2620 Tau-PET, TSPO-PET, and Clinical Progression in Patients With ß-Amyloid-Negative CBS.

BACKGROUND AND OBJECTIVES: Corticobasal syndrome (CBS) with underlying 4-repeat tauopathy is a progressive neurodegenerative disease characterized by declining cognitive and motor functions. Biomarkers for assessing pathologic brain changes in CBS including tau-PET, 18 kDa translocator protein (TSPO)-PET, structural MRI, neurofilament light chain (NfL), or glial fibrillary acidic protein (GFAP) have recently been evaluated for differential diagnosis and disease staging, yet their association with disease trajectories remains unclear. Therefore, we performed a head-to-head comparison of neuroimaging (tau-PET, TSPO-PET, structural MRI) and plasma biomarkers (NfL, GFAP) as prognostic tools for longitudinal clinical trajectories in ß-amyloid (Aß)-negative CBS. METHODS: We included patients with clinically diagnosed Aß-negative CBS with clinical follow-up data who underwent baseline structural MRI and plasma-NfL analysis for assessing neurodegeneration, [18F]PI-2620-PET for assessing tau pathology, [18F]GE-180-PET for assessing microglia activation, and plasma-GFAP analysis for assessing astrocytosis. To quantify tau and microglia load, we assessed summary scores of whole-brain, cortical, and subcortical PET signal. For structural MRI analysis, we quantified subcortical and cortical gray matter volume. Plasma NfL and GFAP values were assessed using Simoa-based immunoassays. Symptom progression was determined using a battery of cognitive and motor tests (i.e., Progressive Supranuclear Palsy Rating Scale [PSPRS]). Using linear mixed models, we tested whether the assessed biomarkers at baseline were associated with faster symptom progression over time (i.e., time × biomarker interaction). RESULTS: Overall, 21 patients with Aß-negative CBS with ∼2-year clinical follow-up data were included. Patients with CBS with more widespread global tau-PET signal showed faster clinical progression (PSPRS: B/SE = 0.001/0.0005, p = 0.025), driven by cortical rather than subcortical tau-PET. By contrast, patients with higher global [18F]GE-180-PET readouts showed slower clinical progression (PSPRS: B/SE = -0.056/0.023, p = 0.019). No association was found between gray matter volume and clinical progression. Concerning fluid biomarkers, only higher plasma-NfL (PSPRS: B/SE = 0.176/0.046, p < 0.001) but not GFAP was associated with faster clinical deterioration. In a subsequent sensitivity analysis, we found that tau-PET, TSPO-PET, and plasma-NfL showed significant interaction effects with time on clinical trajectories when tested in the same model. DISCUSSION: [18F]PI-2620 tau-PET, [18F]GE-180 TSPO-PET, and plasma-NfL show prognostic potential for clinical progression in patients with Aß-negative CBS with probable 4-repeat tauopathy, which can be useful for clinical decision-making and stratifying patients in clinical trials.

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.

A microglial activity state biomarker panel differentiates FTD-granulin and Alzheimer's disease patients from controls.

BACKGROUND: With the emergence of microglia-modulating therapies there is an urgent need for reliable biomarkers to evaluate microglial activation states. METHODS: Using mouse models and human induced pluripotent stem cell-derived microglia (hiMGL), genetically modified to yield the most opposite homeostatic (TREM2-knockout) and disease-associated (GRN-knockout) states, we identified microglia activity-dependent markers. Non-targeted mass spectrometry was used to identify proteomic changes in microglia and cerebrospinal fluid (CSF) of Grn- and Trem2-knockout mice. Additionally, we analyzed the proteome of GRN- and TREM2-knockout hiMGL and their conditioned media. Candidate marker proteins were tested in two independent patient cohorts, the ALLFTD cohort (GRN mutation carriers versus non-carriers), as well as the proteomic data set available from the EMIF-AD MBD study. RESULTS: We identified proteomic changes between the opposite activation states in mouse microglia and CSF, as well as in hiMGL cell lysates and conditioned media. For further verification, we analyzed the CSF proteome of heterozygous GRN mutation carriers suffering from frontotemporal dementia (FTD). We identified a panel of six proteins (FABP3, MDH1, GDI1, CAPG, CD44, GPNMB) as potential indicators for microglial activation. Moreover, we confirmed three of these proteins (FABP3, GDI1, MDH1) to be significantly elevated in the CSF of Alzheimer's (AD) patients. Remarkably, each of these markers differentiated amyloid-positive cases with mild cognitive impairment (MCI) from amyloid-negative individuals. CONCLUSIONS: The identified candidate proteins reflect microglia activity and may be relevant for monitoring the microglial response in clinical practice and clinical trials modulating microglial activity and amyloid deposition. Moreover, the finding that three of these markers differentiate amyloid-positive from amyloid-negative MCI cases in the AD cohort suggests that these proteins associate with a very early immune response to seeded amyloid. This is consistent with our previous findings in the Dominantly Inherited Alzheimer's Disease Network (DIAN) cohort, where soluble TREM2 increases as early as 21 years before symptom onset. Moreover, in mouse models for amyloidogenesis, seeding of amyloid is limited by physiologically active microglia further supporting their early protective role. The biological functions of some of our main candidates (FABP3, CD44, GPNMB) also further emphasize that lipid dysmetabolism may be a common feature of neurodegenerative disorders.

Cerebrospinal fluid proteomics define the natural history of autosomal dominant Alzheimer's disease.

Alzheimer's disease (AD) pathology develops many years before the onset of cognitive symptoms. Two pathological processes-aggregation of the amyloid-ß (Aß) peptide into plaques and the microtubule protein tau into neurofibrillary tangles (NFTs)-are hallmarks of the disease. However, other pathological brain processes are thought to be key disease mediators of Aß plaque and NFT pathology. How these additional pathologies evolve over the course of the disease is currently unknown. Here we show that proteomic measurements in autosomal dominant AD cerebrospinal fluid (CSF) linked to brain protein coexpression can be used to characterize the evolution of AD pathology over a timescale spanning six decades. SMOC1 and SPON1 proteins associated with Aß plaques were elevated in AD CSF nearly 30 years before the onset of symptoms, followed by changes in synaptic proteins, metabolic proteins, axonal proteins, inflammatory proteins and finally decreases in neurosecretory proteins. The proteome discriminated mutation carriers from noncarriers before symptom onset as well or better than Aß and tau measures. Our results highlight the multifaceted landscape of AD pathophysiology and its temporal evolution. Such knowledge will be critical for developing precision therapeutic interventions and biomarkers for AD beyond those associated with Aß and tau.

A MICROGLIAL ACTIVITY STATE BIOMARKER PANEL DIFFERENTIATES FTD-GRANULIN AND ALZHEIMER'S DISEASE PATIENTS FROM CONTROLS.

Background: With the emergence of microglia-modulating therapies there is an urgent need for reliable biomarkers to evaluate microglial activation states. Methods: Using mouse models and human induced pluripotent stem cell-derived microglia (hiMGL), which were genetically modified to yield the most opposite homeostatic ( TREM2- knockout) and disease-associated ( GRN -knockout) states, we identified microglia activity-dependent markers. Non-targeted mass spectrometry was used to identify changes in microglial and cerebrospinal (CSF) proteome of Grn - and Trem2 -knockout mice. Additionally, we analyzed the proteome of GRN - and TREM2 -knockout hiMGL and their conditioned media. Candidate marker proteins were tested in two independent patient cohorts, the ALLFTD cohort with 11 GRN mutation carriers and 12 non-carriers, as well as the proteomic data set available from the European Medical Information Framework Alzheimer's Disease Multimodal Biomarker Discovery (EMIF-AD MBD). Findings: We identified proteomic changes between the opposite activation states in mouse microglia and cerebrospinal fluid (CSF), as well as in hiMGL cell lysates and conditioned media. For further verification, we analyzed the CSF proteome of heterozygous GRN mutation carriers suffering from frontotemporal dementia (FTD). We identified a panel of six proteins (FABP3, MDH1, GDI1, CAPG, CD44, GPNMB) as potential indicators for microglial activation. Moreover, we confirmed three of these proteins (FABP3, GDI1, MDH1) to be significantly elevated in the CSF of AD patients. In AD, these markers differentiated amyloid-positive cases with mild cognitive impairment (MCI) from amyloid-negative individuals. Interpretation: The identified candidate proteins reflect microglia activity and may be relevant for monitoring the microglial response in clinical practice and clinical trials modulating microglial activity and amyloid deposition. Moreover, the finding that three of these markers differentiate amyloid-positive from amyloid-negative MCI cases in the AD cohort suggests that these marker proteins associate with a very early immune response to seeded amyloid. This is consistent with our previous findings in the DIAN (Dominantly Inherited Alzheimer's Disease Network) cohort, where soluble TREM2 increases as early as 21 years before symptom onset. Moreover, in mouse models for amyloidogenesis, seeding of amyloid is limited by physiologically active microglia further supporting their early protective role. The biological functions of some of our main candidates (FABP3, CD44, GPNMB) also further emphasize that lipid dysmetabolism may be a common feature of neurodegenerative disorders. Funding: This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy - ID 390857198 to CH, SFL and DP) and a Koselleck Project HA1737/16-1 (to CH).

Microglia at Sites of Atrophy Restrict the Progression of Retinal Degeneration via Galectin-3 and Trem2 Interactions.

Degenerative diseases of the outer retina, including age-related macular degeneration (AMD), are characterized by atrophy of photoreceptors and retinal pigment epithelium (RPE). In these blinding diseases, macrophages are known to accumulate ectopically at sites of atrophy, but their ontogeny and functional specialization within this atrophic niche remain poorly understood, especially in the human context. Here, we uncovered a transcriptionally unique profile of microglia, marked by galectin-3 upregulation, at atrophic sites in mouse models of retinal degeneration and in human AMD. Using disease models, we found that conditional deletion of galectin-3 in microglia led to defects in phagocytosis and consequent augmented photoreceptor death, RPE damage and vision loss, suggestive of a protective role. Mechanistically, Trem2 signaling orchestrated the migration of microglial cells to sites of atrophy, and there, induced galectin-3 expression. Moreover, pharmacologic Trem2 agonization led to heightened protection, but only in a galectin-3-dependent manner, further signifying the functional interdependence of these two molecules. Likewise in elderly human subjects, we identified a highly conserved population of microglia at the transcriptomic, protein and spatial levels, and this population was enriched in the macular region of postmortem AMD subjects. Collectively, our findings reveal an atrophy-associated specialization of microglia that restricts the progression of retinal degeneration in mice and further suggest that these protective microglia are conserved in AMD.

Individual regional associations between Aß-, tau- and neurodegeneration (ATN) with microglial activation in patients with primary and secondary tauopathies.

ß-amyloid (Aß) and tau aggregation as well as neuronal injury and atrophy (ATN) are the major hallmarks of Alzheimer's disease (AD), and biomarkers for these hallmarks have been linked to neuroinflammation. However, the detailed regional associations of these biomarkers with microglial activation in individual patients remain to be elucidated. We investigated a cohort of 55 patients with AD and primary tauopathies and 10 healthy controls that underwent TSPO-, Aß-, tau-, and perfusion-surrogate-PET, as well as structural MRI. Z-score deviations for 246 brain regions were calculated and biomarker contributions of Aß (A), tau (T), perfusion (N1), and gray matter atrophy (N2) to microglial activation (TSPO, I) were calculated for each individual subject. Individual ATN-related microglial activation was correlated with clinical performance and CSF soluble TREM2 (sTREM2) concentrations. In typical and atypical AD, regional tau was stronger and more frequently associated with microglial activation when compared to regional Aß (AD: ßT = 0.412 ± 0.196 vs. ßA = 0.142 ± 0.123, p < 0.001; AD-CBS: ßT = 0.385 ± 0.176 vs. ßA = 0.131 ± 0.186, p = 0.031). The strong association between regional tau and microglia reproduced well in primary tauopathies (ßT = 0.418 ± 0.154). Stronger individual associations between tau and microglial activation were associated with poorer clinical performance. In patients with 4RT, sTREM2 levels showed a positive association with tau-related microglial activation. Tau pathology has strong regional associations with microglial activation in primary and secondary tauopathies. Tau and Aß related microglial response indices may serve as a two-dimensional in vivo assessment of neuroinflammation in neurodegenerative diseases.

Loss of TDP-43 causes ectopic endothelial sprouting and migration defects through increased fibronectin, vcam 1 and integrin α4/ß1.

Aggregation of the Tar DNA-binding protein of 43 kDa (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia and likely contributes to disease by loss of nuclear function. Analysis of TDP-43 function in knockout zebrafish identified an endothelial directional migration and hypersprouting phenotype during development prior lethality. In human umbilical vein cells (HUVEC) the loss of TDP-43 leads to hyperbranching. We identified elevated expression of FIBRONECTIN 1 (FN1), the VASCULAR CELL ADHESION MOLECULE 1 (VCAM1), as well as their receptor INTEGRIN α4ß1 (ITGA4B1) in HUVEC cells. Importantly, reducing the levels of ITGA4, FN1, and VCAM1 homologues in the TDP-43 loss-of-function zebrafish rescues the angiogenic defects indicating the conservation of human and zebrafish TDP-43 function during angiogenesis. Our study identifies a novel pathway regulated by TDP-43 important for angiogenesis during development.

Myelin dysfunction drives amyloid-ß deposition in models of Alzheimer's disease.

The incidence of Alzheimer's disease (AD), the leading cause of dementia, increases rapidly with age, but why age constitutes the main risk factor is still poorly understood. Brain ageing affects oligodendrocytes and the structural integrity of myelin sheaths1, the latter of which is associated with secondary neuroinflammation2,3. As oligodendrocytes support axonal energy metabolism and neuronal health4-7, we hypothesized that loss of myelin integrity could be an upstream risk factor for neuronal amyloid-ß (Aß) deposition, the central neuropathological hallmark of AD. Here we identify genetic pathways of myelin dysfunction and demyelinating injuries as potent drivers of amyloid deposition in mouse models of AD. Mechanistically, myelin dysfunction causes the accumulation of the Aß-producing machinery within axonal swellings and increases the cleavage of cortical amyloid precursor protein. Suprisingly, AD mice with dysfunctional myelin lack plaque-corralling microglia despite an overall increase in their numbers. Bulk and single-cell transcriptomics of AD mouse models with myelin defects show that there is a concomitant induction of highly similar but distinct disease-associated microglia signatures specific to myelin damage and amyloid plaques, respectively. Despite successful induction, amyloid disease-associated microglia (DAM) that usually clear amyloid plaques are apparently distracted to nearby myelin damage. Our data suggest a working model whereby age-dependent structural defects of myelin promote Aß plaque formation directly and indirectly and are therefore an upstream AD risk factor. Improving oligodendrocyte health and myelin integrity could be a promising target to delay development and slow progression of AD.

APOE and immunity: Research highlights.

INTRODUCTION: At the Alzheimer's Association's APOE and Immunity virtual conference, held in October 2021, leading neuroscience experts shared recent research advances on and inspiring insights into the various roles that both the apolipoprotein E gene (APOE) and facets of immunity play in neurodegenerative diseases, including Alzheimer's disease and other dementias. METHODS: The meeting brought together more than 1200 registered attendees from 62 different countries, representing the realms of academia and industry. RESULTS: During the 4-day meeting, presenters illuminated aspects of the cross-talk between APOE and immunity, with a focus on the roles of microglia, triggering receptor expressed on myeloid cells 2 (TREM2), and components of inflammation (e.g., tumor necrosis factor α [TNFα]). DISCUSSION: This manuscript emphasizes the importance of diversity in current and future research and presents an integrated view of innate immune functions in Alzheimer's disease as well as related promising directions in drug development.

Depletion and activation of microglia impact metabolic connectivity of the mouse brain.

AIM: We aimed to investigate the impact of microglial activity and microglial FDG uptake on metabolic connectivity, since microglial activation states determine FDG-PET alterations. Metabolic connectivity refers to a concept of interacting metabolic brain regions and receives growing interest in approaching complex cerebral metabolic networks in neurodegenerative diseases. However, underlying sources of metabolic connectivity remain to be elucidated. MATERIALS AND METHODS: We analyzed metabolic networks measured by interregional correlation coefficients (ICCs) of FDG-PET scans in WT mice and in mice with mutations in progranulin (Grn) or triggering receptor expressed on myeloid cells 2 (Trem2) knockouts (-/-) as well as in double mutant Grn-/-/Trem2-/- mice. We selected those rodent models as they represent opposite microglial signatures with disease associated microglia in Grn-/- mice and microglia locked in a homeostatic state in Trem2-/- mice; however, both resulting in lower glucose uptake of the brain. The direct influence of microglia on metabolic networks was further determined by microglia depletion using a CSF1R inhibitor in WT mice at two different ages. Within maps of global mean scaled regional FDG uptake, 24 pre-established volumes of interest were applied and assigned to either cortical or subcortical networks. ICCs of all region pairs were calculated and z-transformed prior to group comparisons. FDG uptake of neurons, microglia, and astrocytes was determined in Grn-/- and WT mice via assessment of single cell tracer uptake (scRadiotracing). RESULTS: Microglia depletion by CSF1R inhibition resulted in a strong decrease of metabolic connectivity defined by decrease of mean cortical ICCs in WT mice at both ages studied (6-7 m; p = 0.0148, 9-10 m; p = 0.0191), when compared to vehicle-treated age-matched WT mice. Grn-/-, Trem2-/- and Grn-/-/Trem2-/- mice all displayed reduced FDG-PET signals when compared to WT mice. However, when analyzing metabolic networks, a distinct increase of ICCs was observed in Grn-/- mice when compared to WT mice in cortical (p < 0.0001) and hippocampal (p < 0.0001) networks. In contrast, Trem2-/- mice did not show significant alterations in metabolic connectivity when compared to WT. Furthermore, the increased metabolic connectivity in Grn-/- mice was completely suppressed in Grn-/-/Trem2-/- mice. Grn-/- mice exhibited a severe loss of neuronal FDG uptake (- 61%, p < 0.0001) which shifted allocation of cellular brain FDG uptake to microglia (42% in Grn-/- vs. 22% in WT). CONCLUSIONS: Presence, absence, and activation of microglia have a strong impact on metabolic connectivity of the mouse brain. Enhanced metabolic connectivity is associated with increased microglial FDG allocation.

sTREM2 is associated with amyloid-related p-tau increases and glucose hypermetabolism in Alzheimer's disease.

Microglial activation occurs early in Alzheimer's disease (AD) and previous studies reported both detrimental and protective effects of microglia on AD progression. Here, we used CSF sTREM2 to investigate disease stage-dependent drivers of microglial activation and to determine downstream consequences on AD progression. We included 402 patients with measures of earliest beta-amyloid (CSF Aß1-42 ) and late-stage fibrillary Aß pathology (amyloid-PET centiloid), as well as sTREM2, p-tau181 , and FDG-PET. To determine disease stage, we stratified participants into early Aß-accumulators (Aß CSF+/PET-; n = 70) or late Aß-accumulators (Aß CSF+/PET+; n = 201) plus 131 controls. In early Aß-accumulators, higher centiloid was associated with cross-sectional/longitudinal sTREM2 and p-tau181 increases. Further, higher sTREM2 mediated the association between centiloid and cross-sectional/longitudinal p-tau181 increases and higher sTREM2 was associated with FDG-PET hypermetabolism. In late Aß-accumulators, we found no association between centiloid and sTREM2 but a cross-sectional association between higher sTREM2, higher p-tau181 and glucose hypometabolism. Our findings suggest that a TREM2-related microglial response follows earliest Aß fibrillization, manifests in inflammatory glucose hypermetabolism and may facilitate subsequent p-tau181 increases in earliest AD.

Dynamics of monocyte-derived macrophage diversity in experimental myocardial infarction.

AIMS: Macrophages have a critical and dual role in post-ischaemic cardiac repair, as they can foster both tissue healing and damage. Multiple subsets of tissue resident and monocyte-derived macrophages coexist in the infarcted heart, but their precise identity, temporal dynamics, and the mechanisms regulating their acquisition of discrete states are not fully understood. To address this, we used multi-modal single-cell immune profiling, combined with targeted cell depletion and macrophage fate mapping, to precisely map monocyte/macrophage transitions after experimental myocardial infarction. METHODS AND RESULTS: We performed single-cell transcriptomic and cell-surface marker profiling of circulating and cardiac immune cells in mice challenged with acute myocardial infarction, and integrated single-cell transcriptomes obtained before and at 1, 3, 5, 7, and 11 days after infarction. Using complementary strategies of CCR2+ monocyte depletion and fate mapping of tissue resident macrophages, we determined the origin of cardiac macrophage populations. The macrophage landscape of the infarcted heart was dominated by monocyte-derived cells comprising two pro-inflammatory populations defined as Isg15hi and MHCII+Il1b+, alongside non-inflammatory Trem2hi cells. Trem2hi macrophages were observed in the ischaemic area, but not in the remote viable myocardium, and comprised two subpopulations sequentially populating the heart defined as Trem2hiSpp1hi monocyte-to-macrophage intermediates, and fully differentiated Trem2hiGdf15hi macrophages. Cardiac Trem2hi macrophages showed similarities to 'lipid-associated macrophages' found in mouse models of metabolic diseases and were observed in the human heart, indicating conserved features of this macrophage state across diseases and species. Ischaemic injury induced a shift of circulating Ly6Chi monocytes towards a Chil3hi state with granulocyte-like features, but the acquisition of the Trem2hi macrophage signature occurred in the ischaemic tissue. In vitro, macrophages acquired features of the Trem2hi signature following apoptotic-cell efferocytosis. CONCLUSION: Our work provides a comprehensive map of monocyte/macrophage transitions in the ischaemic heart, constituting a valuable resource for further investigating how these cells may be harnessed and modulated to promote post-ischaemic heart repair.

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.

Response to Comment on "Microglial activation states drive glucose uptake and FDG-PET alterations in neurodegenerative diseases".

Microglial FDG uptake alterations are the source of FDG-PET changes in models of neurodegenerative diseases.

If amyloid drives Alzheimer disease, why have anti-amyloid therapies not yet slowed cognitive decline?

Strong genetic evidence supports an imbalance between production and clearance of amyloid ß-protein (Aß) in people with Alzheimer disease (AD). Microglia that are potentially involved in alternative mechanisms are actually integral to the amyloid cascade. Fluid biomarkers and brain imaging place accumulation of Aß at the beginning of molecular and clinical changes in the disease. So why have clinical trials of anti-amyloid therapies not provided clear-cut benefits to patients with AD? Can anti-amyloid therapies robustly decrease Aß in the human brain, and if so, could this lowering be too little, too late? These central questions in research on AD are being urgently addressed.

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.