Identification of clinical disease trajectories in neurodegenerative disorders with natural language processing.

Neurodegenerative disorders exhibit considerable clinical heterogeneity and are frequently misdiagnosed. This heterogeneity is often neglected and difficult to study. Therefore, innovative data-driven approaches utilizing substantial autopsy cohorts are needed to address this complexity and improve diagnosis, prognosis and fundamental research. We present clinical disease trajectories from 3,042 Netherlands Brain Bank donors, encompassing 84 neuropsychiatric signs and symptoms identified through natural language processing. This unique resource provides valuable new insights into neurodegenerative disorder symptomatology. To illustrate, we identified signs and symptoms that differed between frequently misdiagnosed disorders. In addition, we performed predictive modeling and identified clinical subtypes of various brain disorders, indicative of neural substructures being differently affected. Finally, integrating clinical diagnosis information revealed a substantial proportion of inaccurately diagnosed donors that masquerade as another disorder. The unique datasets allow researchers to study the clinical manifestation of signs and symptoms across neurodegenerative disorders, and identify associated molecular and cellular features.

Radiotherapy induces persistent innate immune reprogramming of microglia into a primed state.

Over half of patients with brain tumors experience debilitating and often progressive cognitive decline after radiotherapy treatment. Microglia, the resident macrophages in the brain, have been implicated in this decline. In response to various insults, microglia can develop innate immune memory (IIM), which can either enhance (priming or training) or repress (tolerance) the response to subsequent inflammatory challenges. Here, we investigate whether radiation affects the IIM of microglia by irradiating the brains of rats and later exposing them to a secondary inflammatory stimulus. Comparative transcriptomic profiling and protein validation of microglia isolated from irradiated rats show a stronger immune response to a secondary inflammatory insult, demonstrating that radiation can lead to long-lasting molecular reprogramming of microglia. Transcriptomic analysis of postmortem normal-appearing non-tumor brain tissue of patients with glioblastoma indicates that radiation-induced microglial priming is likely conserved in humans. Targeting microglial priming or avoiding further inflammatory insults could decrease radiotherapy-induced neurotoxicity.

SALL1 enforces microglia-specific DNA binding and function of SMADs to establish microglia identity.

Spalt-like transcription factor 1 (SALL1) is a critical regulator of organogenesis and microglia identity. Here we demonstrate that disruption of a conserved microglia-specific super-enhancer interacting with the Sall1 promoter results in complete and specific loss of Sall1 expression in microglia. By determining the genomic binding sites of SALL1 and leveraging Sall1 enhancer knockout mice, we provide evidence for functional interactions between SALL1 and SMAD4 required for microglia-specific gene expression. SMAD4 binds directly to the Sall1 super-enhancer and is required for Sall1 expression, consistent with an evolutionarily conserved requirement of the TGFß and SMAD homologs Dpp and Mad for cell-specific expression of Spalt in the Drosophila wing. Unexpectedly, SALL1 in turn promotes binding and function of SMAD4 at microglia-specific enhancers while simultaneously suppressing binding of SMAD4 to enhancers of genes that become inappropriately activated in enhancer knockout microglia, thereby enforcing microglia-specific functions of the TGFß-SMAD signaling axis.

Genetic insights into immune mechanisms of Alzheimer's and Parkinson's disease.

Microglia, the macrophages of the brain, are vital for brain homeostasis and have been implicated in a broad range of brain disorders. Neuroinflammation has gained traction as a possible therapeutic target for neurodegeneration, however, the precise function of microglia in specific neurodegenerative disorders is an ongoing area of research. Genetic studies offer valuable insights into understanding causality, rather than merely observing a correlation. Genome-wide association studies (GWAS) have identified many genetic loci that are linked to susceptibility to neurodegenerative disorders. (Post)-GWAS studies have determined that microglia likely play an important role in the development of Alzheimer's disease (AD) and Parkinson's disease (PD). The process of understanding how individual GWAS risk loci affect microglia function and mediate susceptibility is complex. A rapidly growing number of publications with genomic datasets and computational tools have formulated new hypotheses that guide the biological interpretation of AD and PD genetic risk. In this review, we discuss the key concepts and challenges in the post-GWAS interpretation of AD and PD GWAS risk alleles. Post-GWAS challenges include the identification of target cell (sub)type(s), causal variants, and target genes. Crucially, the prediction of GWAS-identified disease-risk cell types, variants and genes require validation and functional testing to understand the biological consequences within the pathology of the disorders. Many AD and PD risk genes are highly pleiotropic and perform multiple important functions that might not be equally relevant for the mechanisms by which GWAS risk alleles exert their effect(s). Ultimately, many GWAS risk alleles exert their effect by changing microglia function, thereby altering the pathophysiology of these disorders, and hence, we believe that modelling this context is crucial for a deepened understanding of these disorders.

Dissecting the limited genetic overlap of Parkinson's and Alzheimer's disease.

Parkinson's disease and Alzheimer's disease show overlapping features both clinically and neuropathologically and elucidating shared mechanisms could have important implications for therapeutic strategies. Evidence for genetic overlap is limited, although enrichment of heritability in genomic regions relevant to microglia has been demonstrated in both disorders. Using summary statistics from genome-wide association studies, we assessed genetic covariance stratified by cell types and local genetic correlation between Parkinson's and Alzheimer's disease. Significant covariance was observed for neurons only (p = 0.00046), and local genetic correlation was significant only in the human leukocyte antigen region (p = 1.0e-05). Our findings support a minor genetic overlap between these two disorders.

Diet-regulated production of PDGFcc by macrophages controls energy storage.

The mechanisms by which macrophages regulate energy storage remain poorly understood. We identify in a genetic screen a platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF)-family ortholog, Pvf3, that is produced by macrophages and is required for lipid storage in fat-body cells of Drosophila larvae. Genetic and pharmacological experiments indicate that the mouse Pvf3 ortholog PDGFcc, produced by adipose tissue-resident macrophages, controls lipid storage in adipocytes in a leptin receptor- and C-C chemokine receptor type 2-independent manner. PDGFcc production is regulated by diet and acts in a paracrine manner to control lipid storage in adipose tissues of newborn and adult mice. At the organismal level upon PDGFcc blockade, excess lipids are redirected toward thermogenesis in brown fat. These data identify a macrophage-dependent mechanism, conducive to the design of pharmacological interventions, that controls energy storage in metazoans.

Mechanisms underlying divergent responses of genetically distinct macrophages to IL-4.

Mechanisms by which noncoding genetic variation influences gene expression remain only partially understood but are considered to be major determinants of phenotypic diversity and disease risk. Here, we evaluated effects of >50 million single-nucleotide polymorphisms and short insertions/deletions provided by five inbred strains of mice on the responses of macrophages to interleukin-4 (IL-4), a cytokine that plays pleiotropic roles in immunity and tissue homeostasis. Of >600 genes induced >2-fold by IL-4 across the five strains, only 26 genes reached this threshold in all strains. By applying deep learning and motif mutation analyses to epigenetic data for macrophages from each strain, we identified the dominant combinations of lineage-determining and signal-dependent transcription factors driving IL-4 enhancer activation. These studies further revealed mechanisms by which noncoding genetic variation influences absolute levels of enhancer activity and their dynamic responses to IL-4, thereby contributing to strain-differential patterns of gene expression and phenotypic diversity.

Heritability Enrichment Implicates Microglia in Parkinson's Disease Pathogenesis.

OBJECTIVE: Understanding how different parts of the immune system contribute to pathogenesis in Parkinson's disease is a burning challenge with important therapeutic implications. We studied enrichment of common variant heritability for Parkinson's disease stratified by immune and brain cell types. METHODS: We used summary statistics from the most recent meta-analysis of genomewide association studies in Parkinson's disease and partitioned heritability using linkage disequilibrium score regression, stratified for specific cell types, as defined by open chromatin regions. We also validated enrichment results using a polygenic risk score approach and intersected disease-associated variants with epigenetic data and expression quantitative loci to nominate and explore a putative microglial locus. RESULTS: We found significant enrichment of Parkinson's disease risk heritability in open chromatin regions of microglia and monocytes. Genomic annotations overlapped substantially between these 2 cell types, and only the enrichment signal for microglia remained significant in a joint model. We present evidence suggesting P2RY12, a key microglial gene and target for the antithrombotic agent clopidogrel, as the likely driver of a significant Parkinson's disease association signal on chromosome 3. INTERPRETATION: Our results provide further support for the importance of immune mechanisms in Parkinson's disease pathogenesis, highlight microglial dysregulation as a contributing etiological factor, and nominate a targetable microglial gene candidate as a pathogenic player. Immune processes can be modulated by therapy, with potentially important clinical implications for future treatment in Parkinson's disease. ANN NEUROL 2021;89:942-951.

Type I interferon-activated microglia are critical for neuromyelitis optica pathology.

Neuromyelitis optica (NMO) is an inflammatory disease of the central nervous system (CNS) most frequently mediated by serum autoantibodies against the water channel aquaporin 4, expressed on CNS astrocytes, resulting in primary astrocytopathy. There is no cure for NMO, and treatment with Type I interferon (IFNI)-IFNß is ineffective or even detrimental. We have previously shown that both NMO lesions and associated microglial activation were reduced in mice lacking the receptor for IFNß. However, the role of microglia in NMO is not well understood. In this study, we clarify the pathomechanism for IFNI dependence of and the role of microglia in experimental NMO. Transcriptome analysis showed a strong IFNI footprint in affected CNS tissue as well as in microglial subpopulations. Treatment with IFNß led to exacerbated pathology and further microglial activation as evidenced by expansion of a CD11c+ subset of microglia. Importantly, depletion of microglia led to suppression of pathology and decrease of IFNI signature genes. Our data show a pro-pathologic role for IFNI-activated microglia in NMO and open new perspectives for microglia-targeted therapies.

Brain cell type-specific enhancer-promoter interactome maps and disease-risk association.

Noncoding genetic variation is a major driver of phenotypic diversity, but functional interpretation is challenging. To better understand common genetic variation associated with brain diseases, we defined noncoding regulatory regions for major cell types of the human brain. Whereas psychiatric disorders were primarily associated with variants in transcriptional enhancers and promoters in neurons, sporadic Alzheimer's disease (AD) variants were largely confined to microglia enhancers. Interactome maps connecting disease-risk variants in cell-type-specific enhancers to promoters revealed an extended microglia gene network in AD. Deletion of a microglia-specific enhancer harboring AD-risk variants ablated BIN1 expression in microglia, but not in neurons or astrocytes. These findings revise and expand the list of genes likely to be influenced by noncoding variants in AD and suggest the probable cell types in which they function.

Analysis of Genetically Diverse Macrophages Reveals Local and Domain-wide Mechanisms that Control Transcription Factor Binding and Function.

Non-coding genetic variation is a major driver of phenotypic diversity and allows the investigation of mechanisms that control gene expression. Here, we systematically investigated the effects of >50 million variations from five strains of mice on mRNA, nascent transcription, transcription start sites, and transcription factor binding in resting and activated macrophages. We observed substantial differences associated with distinct molecular pathways. Evaluating genetic variation provided evidence for roles of ∼100 TFs in shaping lineage-determining factor binding. Unexpectedly, a substantial fraction of strain-specific factor binding could not be explained by local mutations. Integration of genomic features with chromatin interaction data provided evidence for hundreds of connected cis-regulatory domains associated with differences in transcription factor binding and gene expression. This system and the >250 datasets establish a substantial new resource for investigation of how genetic variation affects cellular phenotypes.

A novel microglial subset plays a key role in myelinogenesis in developing brain.

Microglia are resident macrophages of the central nervous system that contribute to homeostasis and neuroinflammation. Although known to play an important role in brain development, their exact function has not been fully described. Here, we show that in contrast to healthy adult and inflammation-activated cells, neonatal microglia show a unique myelinogenic and neurogenic phenotype. A CD11c+ microglial subset that predominates in primary myelinating areas of the developing brain expresses genes for neuronal and glial survival, migration, and differentiation. These cells are the major source of insulin-like growth factor 1, and its selective depletion from CD11c+ microglia leads to impairment of primary myelination. CD11c-targeted toxin regimens induced a selective transcriptional response in neonates, distinct from adult microglia. CD11c+ microglia are also found in clusters of repopulating microglia after experimental ablation and in neuroinflammation in adult mice, but despite some similarities, they do not recapitulate neonatal microglial characteristics. We therefore identify a unique phenotype of neonatal microglia that deliver signals necessary for myelination and neurogenesis.

Transcriptional control of microglia phenotypes in health and disease.

Microglia are the main resident macrophage population of the CNS and perform numerous functions required for CNS development, homeostasis, immunity, and repair. Many lines of evidence also indicate that dysregulation of microglia contributes to the pathogenesis of neurodegenerative and behavioral diseases. These observations provide a compelling argument to more clearly define the mechanisms that control microglia identity and function in health and disease. In this Review, we present a conceptual framework for how different classes of transcription factors interact to select and activate regulatory elements that control microglia development and their responses to internal and external signals. We then describe functions of specific transcription factors in normal and pathological contexts and conclude with a consideration of open questions to be addressed in the future.

Increased White Matter Inflammation in Aging- and Alzheimer's Disease Brain.

Chronic neuroinflammation, which is primarily mediated by microglia, plays an essential role in aging and neurodegeneration. It is still unclear whether this microglia-induced neuroinflammation occurs globally or is confined to distinct brain regions. In this study, we investigated microglia activity in various brain regions upon healthy aging and Alzheimer's disease (AD)-related pathology in both human and mouse samples. In purified microglia isolated from aging mouse brains, we found a profound gene expression pattern related to pro-inflammatory processes, phagocytosis, and lipid homeostasis. Particularly in white matter microglia of 24-month-old mice, abundant expression of phagocytic markers including Mac-2, Axl, CD16/32, Dectin1, CD11c, and CD36 was detected. Interestingly, in white matter of human brain tissue the first signs of inflammatory activity were already detected during middle age. Thus quantification of microglial proteins, such as CD68 (commonly associated with phagocytosis) and HLA-DR (associated with antigen presentation), in postmortem human white matter brain tissue showed an age-dependent increase in immunoreactivity already in middle-aged people (53.2 ± 2.0 years). This early inflammation was also detectable by non-invasive positron emission tomography imaging using [11C]-(R)-PK11195, a ligand that binds to activated microglia. Increased microglia activity was also prominently present in the white matter of human postmortem early-onset AD (EOAD) brain tissue. Interestingly, microglia activity in the white matter of late-onset AD (LOAD) CNS was similar to that of the aged clinically silent AD cases. These data indicate that microglia-induced neuroinflammation is predominant in the white matter of aging mice and humans as well as in EOAD brains. This white matter inflammation may contribute to the progression of neurodegeneration, and have prognostic value for detecting the onset and progression of aging and neurodegeneration.

Transcriptomic analysis of purified human cortical microglia reveals age-associated changes.

Microglia are essential for CNS homeostasis and innate neuroimmune function, and play important roles in neurodegeneration and brain aging. Here we present gene expression profiles of purified microglia isolated at autopsy from the parietal cortex of 39 human subjects with intact cognition. Overall, genes expressed by human microglia were similar to those in mouse, including established microglial genes CX3CR1, P2RY12 and ITGAM (CD11B). However, a number of immune genes, not identified as part of the mouse microglial signature, were abundantly expressed in human microglia, including TLR, Fcγ and SIGLEC receptors, as well as TAL1 and IFI16, regulators of proliferation and cell cycle. Age-associated changes in human microglia were enriched for genes involved in cell adhesion, axonal guidance, cell surface receptor expression and actin (dis)assembly. Limited overlap was observed in microglial genes regulated during aging between mice and humans, indicating that human and mouse microglia age differently.

An environment-dependent transcriptional network specifies human microglia identity.

Microglia play essential roles in central nervous system (CNS) homeostasis and influence diverse aspects of neuronal function. However, the transcriptional mechanisms that specify human microglia phenotypes are largely unknown. We examined the transcriptomes and epigenetic landscapes of human microglia isolated from surgically resected brain tissue ex vivo and after transition to an in vitro environment. Transfer to a tissue culture environment resulted in rapid and extensive down-regulation of microglia-specific genes that were induced in primitive mouse macrophages after migration into the fetal brain. Substantial subsets of these genes exhibited altered expression in neurodegenerative and behavioral diseases and were associated with noncoding risk variants. These findings reveal an environment-dependent transcriptional network specifying microglia-specific programs of gene expression and facilitate efforts to understand the roles of microglia in human brain diseases.

Immune hyperreactivity of Aß plaque-associated microglia in Alzheimer's disease.

Alzheimer's disease (AD) is strongly associated with microglia-induced neuroinflammation. Particularly, Aß plaque-associated microglia take on an "activated" morphology. However, the function and phenotype of these Aß plaque-associated microglia are not well understood. We show hyperreactivity of Aß plaque-associated microglia upon systemic inflammation in transgenic AD mouse models (i.e., 5XFAD and APP23). Gene expression profiling of Aß plaque-associated microglia (major histocompatibility complex II+ microglia) isolated from 5XFAD mice revealed a proinflammatory phenotype. The upregulated genes involved in the biological processes (gene ontology terms) included: "immune response to external stimulus" such as Axl, Cd63, Egr2, and Lgals3, "cell motility", such as Ccl3, Ccl4, Cxcr4, and Sdc3, "cell differentiation", and "system development", such as St14, Trpm1, and Spp1. In human AD tissue with similar Braak stages, expression of phagocytic markers and AD-associated genes, including HLA-DRA, APOE, AXL, TREM2, and TYROBP, was higher in laser-captured early-onset AD (EOAD) plaques than in late-onset AD plaques. Interestingly, the nonplaque parenchyma of both EOAD and late-onset AD brains, the expression of above-mentioned markers were similarly low. Here, we provide evidence that Aß plaque-associated microglia are hyperreactive in their immune response and phagocytosis in the transgenic AD mice as well as in EOAD brain tissue. We suggest that Aß plaque-associated microglia are the primary source of neuroinflammation related to AD pathology.

Identification of highly connected hub genes in the protective response program of human macrophages and microglia activated by alpha B-crystallin.

The glial stress protein alpha B-crystallin (HSPB5) is an endogenous agonist for Toll-like receptor 2 in CD14+ cells. Following systemic administration, HSPB5 acts as a potent inhibitor of neuroinflammation in animal models and reduces lesion development in multiple sclerosis patients. Here, we show that systemically administered HSPB5 rapidly crosses the blood-brain barrier, implicating microglia as additional targets for HSPB5 along with peripheral monocytes and macrophages. To compare key players in the HSPB5-induced protective response of human macrophages and microglia, we applied weighted gene co-expression network analysis on transcript expression data obtained 1 and 4 h after activation. This approach identified networks of genes that are co-expressed in all datasets, thus reducing the complexity of the nonsynchronous waves of transcripts that appear after activation by HSPB5. In both cell types, HSPB5 activates a network of highly connected genes that appear to be functionally equivalent and consistent with the therapeutic effects of HSPB5 in vivo, since both networks include factors that suppress apoptosis, the production of proinflammatory factors, and the development of adaptive immunity. Yet, hub genes at the core of the network in either cell type were strikingly different. They prominently feature the well-known tolerance-promoting programmed-death ligand 1 as a key player in the macrophage response to HSPB5, and the immune-regulatory enzyme cyclooxygenase-2 (COX-2) in that of microglia. This latter finding indicates that despite its reputation as a potential target for nonsteroidal anti-inflammatory drugs, microglial COX-2 plays a central role in the therapeutic effects of HSPB5 during neuroinflammation. GLIA 2017;65:460-473.

Aging, microglia and cytoskeletal regulation are key factors in the pathological evolution of the APP23 mouse model for Alzheimer's disease.

Aging is the key risk factor for Alzheimer's disease (AD). In addition, the amyloid-beta (Aß) peptide is considered a critical neurotoxic agent in AD pathology. However, the connection between these factors is unclear. We aimed to provide an extensive characterization of the gene expression profiles of the amyloidosis APP23 model for AD and control mice and to evaluate the effect of aging on these profiles. We also correlated our findings to changes in soluble Aß-levels and other pathological and symptomatic features of the model. We observed a clear biphasic expression profile. The first phase displayed a maturation profile, which resembled features found in young carriers of familial AD mutations. The second phase reflected aging processes and showed similarities to the progression of human AD pathology. During this phase, the model displayed a clear upregulation of microglial activation and lysosomal pathways and downregulation of neuron differentiation and axon guidance pathways. Interestingly, the changes in expression were all correlated to aging in general, but appeared more extensive/accelerated in APP23 mice.

Identification of a conserved and acute neurodegeneration-specific microglial transcriptome in the zebrafish.

Microglia are brain resident macrophages important for brain development, connectivity, homeostasis and disease. However, it is still largely unclear how microglia functions and their identity are regulated at the molecular level. Although recent transcriptomic studies have identified genes specifically expressed in microglia, the function of most of these genes in microglia is still unknown. Here, we performed RNA sequencing on microglia acutely isolated from healthy and neurodegenerative zebrafish brains. We found that a large fraction of the mouse microglial signature is conserved in the zebrafish, corroborating the use of zebrafish to help understand microglial genetics in mammals in addition to studying basic microglia biology. Second, our transcriptome analysis of microglia following neuronal ablation suggested primarily a proliferative response of microglia, which we confirmed by immunohistochemistry and in vivo imaging. Together with the recent improvements in genome editing technology in zebrafish, these data offer opportunities to facilitate functional genetic research on microglia in vivo in the healthy as well as in the diseased brain. GLIA 2016;65:138-149.