Microglia maintain structural integrity during fetal brain morphogenesis.

Microglia (MG), the brain-resident macrophages, play major roles in health and disease via a diversity of cellular states. While embryonic MG display a large heterogeneity of cellular distribution and transcriptomic states, their functions remain poorly characterized. Here, we uncovered a role for MG in the maintenance of structural integrity at two fetal cortical boundaries. At these boundaries between structures that grow in distinct directions, embryonic MG accumulate, display a state resembling post-natal axon-tract-associated microglia (ATM) and prevent the progression of microcavities into large cavitary lesions, in part via a mechanism involving the ATM-factor Spp1. MG and Spp1 furthermore contribute to the rapid repair of lesions, collectively highlighting protective functions that preserve the fetal brain from physiological morphogenetic stress and injury. Our study thus highlights key major roles for embryonic MG and Spp1 in maintaining structural integrity during morphogenesis, with major implications for our understanding of MG functions and brain development.

Microglia Biology: One Century of Evolving Concepts.

Microglia were first recognized as a distinct cell population in the CNS one century ago. For a long time, they were primarily considered to be phagocytes responsible for removing debris during CNS development and disease. More recently, advances in imaging and genetics and the advent of single-cell technologies provided new insights into the much more complex and fascinating biology of microglia. The ontogeny of microglia was identified, and their functions in health and disease were better defined. Although many questions about microglia and their roles in human diseases remain unanswered, the prospect of targeting microglia for the treatment of neurological and psychiatric disorders is tantalizing.

Author Correction: Novel Hexb-based tools for studying microglia in the CNS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Novel Hexb-based tools for studying microglia in the CNS.

Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.

Microglia regulate central nervous system myelin growth and integrity.

Myelin is required for the function of neuronal axons in the central nervous system, but the mechanisms that support myelin health are unclear. Although macrophages in the central nervous system have been implicated in myelin health1, it is unknown which macrophage populations are involved and which aspects they influence. Here we show that resident microglia are crucial for the maintenance of myelin health in adulthood in both mice and humans. We demonstrate that microglia are dispensable for developmental myelin ensheathment. However, they are required for subsequent regulation of myelin growth and associated cognitive function, and for preservation of myelin integrity by preventing its degeneration. We show that loss of myelin health due to the absence of microglia is associated with the appearance of a myelinating oligodendrocyte state with altered lipid metabolism. Moreover, this mechanism is regulated through disruption of the TGFß1-TGFßR1 axis. Our findings highlight microglia as promising therapeutic targets for conditions in which myelin growth and integrity are dysregulated, such as in ageing and neurodegenerative disease2,3.

Origin, fate and dynamics of macrophages at central nervous system interfaces.

Perivascular, subdural meningeal and choroid plexus macrophages are non-parenchymal macrophages that mediate immune responses at brain boundaries. Although the origin of parenchymal microglia has recently been elucidated, much less is known about the precursors, the underlying transcriptional program and the dynamics of the other macrophages in the central nervous system (CNS). It was assumed that they have a high turnover from blood-borne monocytes. However, using parabiosis and fate-mapping approaches in mice, we found that CNS macrophages arose from hematopoietic precursors during embryonic development and established stable populations, with the notable exception of choroid plexus macrophages, which had dual origins and a shorter life span. The generation of CNS macrophages relied on the transcription factor PU.1, whereas the MYB, BATF3 and NR4A1 transcription factors were not required.

Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.

Genomic Characterization of Murine Monocytes Reveals C/EBPß Transcription Factor Dependence of Ly6C- Cells.

Monocytes are circulating, short-lived mononuclear phagocytes, which in mice and man comprise two main subpopulations. Murine Ly6C+ monocytes display developmental plasticity and are recruited to complement tissue-resident macrophages and dendritic cells on demand. Murine vascular Ly6C- monocytes patrol the endothelium, act as scavengers, and support vessel wall repair. Here we characterized population and single cell transcriptomes, as well as enhancer and promoter landscapes of the murine monocyte compartment. Single cell RNA-seq and transplantation experiments confirmed homeostatic default differentiation of Ly6C+ into Ly6C- monocytes. The main two subsets were homogeneous, but linked by a more heterogeneous differentiation intermediate. We show that monocyte differentiation occurred through de novo enhancer establishment and activation of pre-established (poised) enhancers. Generation of Ly6C- monocytes involved induction of the transcription factor C/EBPß and C/EBPß-deficient mice lacked Ly6C- monocytes. Mechanistically, C/EBPß bound the Nr4a1 promoter and controlled expression of this established monocyte survival factor.

Lifelong absence of microglia alters hippocampal glutamatergic networks but not synapse and spine density.

Microglia sculpt developing neural circuits by eliminating excess synapses in a process called synaptic pruning, by removing apoptotic neurons, and by promoting neuronal survival. To elucidate the role of microglia during embryonic and postnatal brain development, we used a mouse model deficient in microglia throughout life by deletion of the fms-intronic regulatory element (FIRE) in the Csf1r locus. Surprisingly, young adult Csf1rΔFIRE/ΔFIRE mice display no changes in excitatory and inhibitory synapse number and spine density of CA1 hippocampal neurons compared with Csf1r+/+ littermates. However, CA1 neurons are less excitable, receive less CA3 excitatory input and show altered synaptic properties, but this does not affect novel object recognition. Cytokine profiling indicates an anti-inflammatory state along with increases in ApoE levels and reactive astrocytes containing synaptic markers in Csf1rΔFIRE/ΔFIRE mice. Notably, these changes in Csf1rΔFIRE/ΔFIRE mice closely resemble the effects of acute microglial depletion in adult mice after normal development. Our findings suggest that microglia are not mandatory for synaptic pruning, and that in their absence pruning can be achieved by other mechanisms.

Mapping the origin and fate of myeloid cells in distinct compartments of the eye by single-cell profiling.

Similar to the brain, the eye is considered an immune-privileged organ where tissue-resident macrophages provide the major immune cell constituents. However, little is known about spatially restricted macrophage subsets within different eye compartments with regard to their origin, function, and fate during health and disease. Here, we combined single-cell analysis, fate mapping, parabiosis, and computational modeling to comprehensively examine myeloid subsets in distinct parts of the eye during homeostasis. This approach allowed us to identify myeloid subsets displaying diverse transcriptional states. During choroidal neovascularization, a typical hallmark of neovascular age-related macular degeneration (AMD), we recognized disease-specific macrophage subpopulations with distinct molecular signatures. Our results highlight the heterogeneity of myeloid subsets and their dynamics in the eye that provide new insights into the innate immune system in this organ which may offer new therapeutic targets for ophthalmological diseases.

Different inflammatory signatures based on CSF biomarkers relate to preserved or diminished brain structure and cognition.

Neuroinflammation is a hallmark of Alzheimer's disease (AD) and both positive and negative associations of individual inflammation-related markers with brain structure and cognitive function have been described. We aimed to identify inflammatory signatures of CSF immune-related markers that relate to changes of brain structure and cognition across the clinical spectrum ranging from normal aging to AD. A panel of 16 inflammatory markers, Aß42/40 and p-tau181 were measured in CSF at baseline in the DZNE DELCODE cohort (n = 295); a longitudinal observational study focusing on at-risk stages of AD. Volumetric maps of gray and white matter (GM/WM; n = 261) and white matter hyperintensities (WMHs, n = 249) were derived from baseline MRIs. Cognitive decline (n = 204) and the rate of change in GM volume was measured in subjects with at least 3 visits (n = 175). A principal component analysis on the CSF markers revealed four inflammatory components (PCs). Of these, the first component PC1 (highly loading on sTyro3, sAXL, sTREM2, YKL-40, and C1q) was associated with older age and higher p-tau levels, but with less pathological Aß when controlling for p-tau. PC2 (highly loading on CRP, IL-18, complement factor F/H and C4) was related to male gender, higher body mass index and greater vascular risk. PC1 levels, adjusted for AD markers, were related to higher GM and WM volumes, less WMHs, better baseline memory, and to slower atrophy rates in AD-related areas and less cognitive decline. In contrast, PC2 related to less GM and WM volumes and worse memory at baseline. Similar inflammatory signatures and associations were identified in the independent F.ACE cohort. Our data suggest that there are beneficial and detrimental signatures of inflammatory CSF biomarkers. While higher levels of TAM receptors (sTyro/sAXL) or sTREM2 might reflect a protective glia response to degeneration related to phagocytic clearance, other markers might rather reflect proinflammatory states that have detrimental impact on brain integrity.

Single-value brain activity scores reflect both severity and risk across the Alzheimer's continuum.

Single-value scores reflecting the deviation from (FADE score) or similarity with (SAME score) prototypical novelty-related and memory-related functional magnetic resonance imaging (fMRI) activation patterns in young adults have been proposed as imaging biomarkers of healthy neurocognitive aging. Here, we tested the utility of these scores as potential diagnostic and prognostic markers in Alzheimer's disease (AD) and risk states like mild cognitive impairment (MCI) or subjective cognitive decline (SCD). To this end, we analyzed subsequent memory fMRI data from individuals with SCD, MCI, and AD dementia as well as healthy controls (HC) and first-degree relatives of AD dementia patients (AD-rel) who participated in the multi-center DELCODE study (N = 468). Based on the individual participants' whole-brain fMRI novelty and subsequent memory responses, we calculated the FADE and SAME scores and assessed their association with AD risk stage, neuropsychological test scores, CSF amyloid positivity, and ApoE genotype. Memory-based FADE and SAME scores showed a considerably larger deviation from a reference sample of young adults in the MCI and AD dementia groups compared to HC, SCD and AD-rel. In addition, novelty-based scores significantly differed between the MCI and AD dementia groups. Across the entire sample, single-value scores correlated with neuropsychological test performance. The novelty-based SAME score further differed between Aß-positive and Aß-negative individuals in SCD and AD-rel, and between ApoE ε4 carriers and non-carriers in AD-rel. Hence, FADE and SAME scores are associated with both cognitive performance and individual risk factors for AD. Their potential utility as diagnostic and prognostic biomarkers warrants further exploration, particularly in individuals with SCD and healthy relatives of AD dementia patients.

A generalizable data-driven model of atrophy heterogeneity and progression in memory clinic settings.

Memory clinic patients are a heterogeneous population representing various aetiologies of pathological ageing. It is not known whether divergent spatiotemporal progression patterns of brain atrophy, as previously described in Alzheimer's disease patients, are prevalent and clinically meaningful in this group of older adults. To uncover distinct atrophy subtypes, we applied the Subtype and Stage Inference (SuStaIn) algorithm to baseline structural MRI data from 813 participants enrolled in the DELCODE cohort (mean ± standard deviation, age = 70.67 ± 6.07 years, 52% females). Participants were cognitively unimpaired (n = 285) or fulfilled diagnostic criteria for subjective cognitive decline (n = 342), mild cognitive impairment (n = 118) or dementia of the Alzheimer's type (n = 68). Atrophy subtypes were compared in baseline demographics, fluid Alzheimer's disease biomarker levels, the Preclinical Alzheimer Cognitive Composite (PACC-5) as well as episodic memory and executive functioning. PACC-5 trajectories over up to 240 weeks were examined. To test whether baseline atrophy subtype and stage predicted clinical trajectories before manifest cognitive impairment, we analysed PACC-5 trajectories and mild cognitive impairment conversion rates of cognitively unimpaired participants and those with subjective cognitive decline. Limbic-predominant and hippocampal-sparing atrophy subtypes were identified. Limbic-predominant atrophy initially affected the medial temporal lobes, followed by further temporal regions and, finally, the remaining cortical regions. At baseline, this subtype was related to older age, more pathological Alzheimer's disease biomarker levels, APOE ε4 carriership and an amnestic cognitive impairment. Hippocampal-sparing atrophy initially occurred outside the temporal lobe, with the medial temporal lobe spared up to advanced atrophy stages. This atrophy pattern also affected individuals with positive Alzheimer's disease biomarkers and was associated with more generalized cognitive impairment. Limbic-predominant atrophy, in all participants and in only unimpaired participants, was linked to more negative longitudinal PACC-5 slopes than observed in participants without or with hippocampal-sparing atrophy and increased the risk of mild cognitive impairment conversion. SuStaIn modelling was repeated in a sample from the Swedish BioFINDER-2 cohort. Highly similar atrophy progression patterns and associated cognitive profiles were identified. Cross-cohort model generalizability, at both the subject and the group level, was excellent, indicating reliable performance in previously unseen data. The proposed model is a promising tool for capturing heterogeneity among older adults at early at-risk states for Alzheimer's disease in applied settings. The implementation of atrophy subtype- and stage-specific end points might increase the statistical power of pharmacological trials targeting early Alzheimer's disease.

Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution.

Microglia have critical roles not only in neural development and homeostasis, but also in neurodegenerative and neuroinflammatory diseases of the central nervous system1-4. These highly diverse and specialized functions may be executed by subsets of microglia that already exist in situ, or by specific subsets of microglia that develop from a homogeneous pool of cells on demand. However, little is known about the presence of spatially and temporally restricted subclasses of microglia in the central nervous system during development or disease. Here we combine massively parallel single-cell analysis, single-molecule fluorescence in situ hybridization, advanced immunohistochemistry and computational modelling to comprehensively characterize subclasses of microglia in multiple regions of the central nervous system during development and disease. Single-cell analysis of tissues of the central nervous system during homeostasis in mice revealed specific time- and region-dependent subtypes of microglia. Demyelinating and neurodegenerative diseases evoked context-dependent subtypes of microglia with distinct molecular hallmarks and diverse cellular kinetics. Corresponding clusters of microglia were also identified in healthy human brains, and the brains of patients with multiple sclerosis. Our data provide insights into the endogenous immune system of the central nervous system during development, homeostasis and disease, and may also provide new targets for the treatment of neurodegenerative and neuroinflammatory pathologies.

Author Correction: Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution.

In this Letter, Dominic Grün and Sagar have been added to the author list (affiliated with Max-Planck-Institute of Immunology and Epigenetics (MPI-IE), Freiburg, Germany). The author list, 'Author contribution' and 'Acknowledgements' sections have been corrected online. See accompanying Amendment.

Consistently lower volumes across thalamus nuclei in very premature-born adults.

Lasting thalamus volume reduction after preterm birth is a prominent finding. However, whether thalamic nuclei volumes are affected differentially by preterm birth and whether nuclei aberrations are relevant for cognitive functioning remains unknown. Using T1-weighted MR-images of 83 adults born very preterm (≤ 32 weeks' gestation; VP) and/or with very low body weight (≤ 1,500 g; VLBW) as well as of 92 full-term born (≥ 37 weeks' gestation) controls, we compared thalamic nuclei volumes of six subregions (anterior, lateral, ventral, intralaminar, medial, and pulvinar) across groups at the age of 26 years. To characterize the functional relevance of volume aberrations, cognitive performance was assessed by full-scale intelligence quotient using the Wechsler Adult Intelligence Scale and linked to volume reductions using multiple linear regression analyses. Thalamic volumes were significantly lower across all examined nuclei in VP/VLBW adults compared to controls, suggesting an overall rather than focal impairment. Lower nuclei volumes were linked to higher intensity of neonatal treatment, indicating vulnerability to stress exposure after birth. Furthermore, we found that single results for lateral, medial, and pulvinar nuclei volumes were associated with full-scale intelligence quotient in preterm adults, albeit not surviving correction for multiple hypotheses testing. These findings provide evidence that lower thalamic volume in preterm adults is observable across all subregions rather than focused on single nuclei. Data suggest the same mechanisms of aberrant thalamus development across all nuclei after premature birth.

Negative symptoms, striatal dopamine and model-free reward decision-making in schizophrenia.

Negative symptoms, such as lack of motivation or social withdrawal, are highly prevalent and debilitating in patients with schizophrenia. Underlying mechanisms of negative symptoms are incompletely understood, thereby preventing the development of targeted treatments. We hypothesized that in patients with schizophrenia during psychotic remission, impaired influences of both model-based and model-free reward predictions on decision-making ('reward prediction influence', RPI) underlie negative symptoms. We focused on psychotic remission, because psychotic symptoms might confound reward-based decision-making. Moreover, we hypothesized that impaired model-based/model-free RPIs depend on alterations of both associative striatum dopamine synthesis and storage (DSS) and executive functioning. Both factors influence RPI in healthy subjects and are typically impaired in schizophrenia. Twenty-five patients with schizophrenia with pronounced negative symptoms during psychotic remission and 24 healthy controls were included in the study. Negative symptom severity was measured by the Positive and Negative Syndrome Scale negative subscale, model-based/model-free RPI by the two-stage decision task, associative striatum DSS by 18F-DOPA positron emission tomography and executive functioning by the symbol coding task. Model-free RPI was selectively reduced in patients and associated with negative symptom severity as well as with reduced associative striatum DSS (in patients only) and executive functions (both in patients and controls). In contrast, model-based RPI was not altered in patients. Results provide evidence for impaired model-free reward prediction influence as a mechanism for negative symptoms in schizophrenia as well as for reduced associative striatum dopamine and executive dysfunction as relevant factors. Data suggest potential treatment targets for patients with schizophrenia and pronounced negative symptoms.

Cholinergic white matter pathways along the Alzheimer's disease continuum.

Previous studies have shown that the cholinergic nucleus basalis of Meynert and its white matter projections are affected in Alzheimer's disease dementia and mild cognitive impairment. However, it is still unknown whether these alterations can be found in individuals with subjective cognitive decline, and whether they are more pronounced than changes found in conventional brain volumetric measurements. To address these questions, we investigated microstructural alterations of two major cholinergic pathways in individuals along the Alzheimer's disease continuum using an in vivo model of the human cholinergic system based on neuroimaging. We included 402 participants (52 Alzheimer's disease, 66 mild cognitive impairment, 172 subjective cognitive decline and 112 healthy controls) from the Deutsches Zentrum für Neurodegenerative Erkrankungen Longitudinal Cognitive Impairment and Dementia Study. We modelled the cholinergic white matter pathways with an enhanced diffusion neuroimaging pipeline that included probabilistic fibre-tracking methods and prior anatomical knowledge. The integrity of the cholinergic white matter pathways was compared between stages of the Alzheimer's disease continuum, in the whole cohort and in a CSF amyloid-beta stratified subsample. The discriminative power of the integrity of the pathways was compared to the conventional volumetric measures of hippocampus and nucleus basalis of Meynert, using a receiver operating characteristics analysis. A multivariate model was used to investigate the role of these pathways in relation to cognitive performance. We found that the integrity of the cholinergic white matter pathways was significantly reduced in all stages of the Alzheimer's disease continuum, including individuals with subjective cognitive decline. The differences involved posterior cholinergic white matter in the subjective cognitive decline stage and extended to anterior frontal white matter in mild cognitive impairment and Alzheimer's disease dementia stages. Both cholinergic pathways and conventional volumetric measures showed higher predictive power in the more advanced stages of the disease, i.e. mild cognitive impairment and Alzheimer's disease dementia. In contrast, the integrity of cholinergic pathways was more informative in distinguishing subjective cognitive decline from healthy controls, as compared with the volumetric measures. The multivariate model revealed a moderate contribution of the cholinergic white matter pathways but not of volumetric measures towards memory tests in the subjective cognitive decline and mild cognitive impairment stages. In conclusion, we demonstrated that cholinergic white matter pathways are altered already in subjective cognitive decline individuals, preceding the more widespread alterations found in mild cognitive impairment and Alzheimer's disease. The integrity of the cholinergic pathways identified the early stages of Alzheimer's disease better than conventional volumetric measures such as hippocampal volume or volume of cholinergic nucleus basalis of Meynert.

The ReCoDe addiction research consortium: Losing and regaining control over drug intake-Findings and future perspectives.

Substance use disorders (SUDs) are seen as a continuum ranging from goal-directed and hedonic drug use to loss of control over drug intake with aversive consequences for mental and physical health and social functioning. The main goals of our interdisciplinary German collaborative research centre on Losing and Regaining Control over Drug Intake (ReCoDe) are (i) to study triggers (drug cues, stressors, drug priming) and modifying factors (age, gender, physical activity, cognitive functions, childhood adversity, social factors, such as loneliness and social contact/interaction) that longitudinally modulate the trajectories of losing and regaining control over drug consumption under real-life conditions. (ii) To study underlying behavioural, cognitive and neurobiological mechanisms of disease trajectories and drug-related behaviours and (iii) to provide non-invasive mechanism-based interventions. These goals are achieved by: (A) using innovative mHealth (mobile health) tools to longitudinally monitor the effects of triggers and modifying factors on drug consumption patterns in real life in a cohort of 900 patients with alcohol use disorder. This approach will be complemented by animal models of addiction with 24/7 automated behavioural monitoring across an entire disease trajectory; i.e. from a naïve state to a drug-taking state to an addiction or resilience-like state. (B) The identification and, if applicable, computational modelling of key molecular, neurobiological and psychological mechanisms (e.g., reduced cognitive flexibility) mediating the effects of such triggers and modifying factors on disease trajectories. (C) Developing and testing non-invasive interventions (e.g., Just-In-Time-Adaptive-Interventions (JITAIs), various non-invasive brain stimulations (NIBS), individualized physical activity) that specifically target the underlying mechanisms for regaining control over drug intake. Here, we will report on the most important results of the first funding period and outline our future research strategy.