Parenchymal border macrophages regulate the flow dynamics of the cerebrospinal fluid.

Macrophages are important players in the maintenance of tissue homeostasis1. Perivascular and leptomeningeal macrophages reside near the central nervous system (CNS) parenchyma2, and their role in CNS physiology has not been sufficiently well studied. Given their continuous interaction with the cerebrospinal fluid (CSF) and strategic positioning, we refer to these cells collectively as parenchymal border macrophages (PBMs). Here we demonstrate that PBMs regulate CSF flow dynamics. We identify a subpopulation of PBMs that express high levels of CD163 and LYVE1 (scavenger receptor proteins), closely associated with the brain arterial tree, and show that LYVE1+ PBMs regulate arterial motion that drives CSF flow. Pharmacological or genetic depletion of PBMs led to accumulation of extracellular matrix proteins, obstructing CSF access to perivascular spaces and impairing CNS perfusion and clearance. Ageing-associated alterations in PBMs and impairment of CSF dynamics were restored after intracisternal injection of macrophage colony-stimulating factor. Single-nucleus RNA sequencing data obtained from patients with Alzheimer's disease (AD) and from non-AD individuals point to changes in phagocytosis, endocytosis and interferon-γ signalling on PBMs, pathways that are corroborated in a mouse model of AD. Collectively, our results identify PBMs as new cellular regulators of CSF flow dynamics, which could be targeted pharmacologically to alleviate brain clearance deficits associated with ageing and AD.

Brain high-throughput multi-omics data reveal molecular heterogeneity in Alzheimer's disease.

Unbiased data-driven omic approaches are revealing the molecular heterogeneity of Alzheimer disease. Here, we used machine learning approaches to integrate high-throughput transcriptomic, proteomic, metabolomic, and lipidomic profiles with clinical and neuropathological data from multiple human AD cohorts. We discovered 4 unique multimodal molecular profiles, one of them showing signs of poor cognitive function, a faster pace of disease progression, shorter survival with the disease, severe neurodegeneration and astrogliosis, and reduced levels of metabolomic profiles. We found this molecular profile to be present in multiple affected cortical regions associated with higher Braak tau scores and significant dysregulation of synapse-related genes, endocytosis, phagosome, and mTOR signaling pathways altered in AD early and late stages. AD cross-omics data integration with transcriptomic data from an SNCA mouse model revealed an overlapping signature. Furthermore, we leveraged single-nuclei RNA-seq data to identify distinct cell-types that most likely mediate molecular profiles. Lastly, we identified that the multimodal clusters uncovered cerebrospinal fluid biomarkers poised to monitor AD progression and possibly cognition. Our cross-omics analyses provide novel critical molecular insights into AD.

Meningeal lymphatics affect microglia responses and anti-Aß immunotherapy.

Alzheimer's disease (AD) is the most prevalent cause of dementia1. Although there is no effective treatment for AD, passive immunotherapy with monoclonal antibodies against amyloid beta (Aß) is a promising therapeutic strategy2,3. Meningeal lymphatic drainage has an important role in the accumulation of Aß in the brain4, but it is not known whether modulation of meningeal lymphatic function can influence the outcome of immunotherapy in AD. Here we show that ablation of meningeal lymphatic vessels in 5xFAD mice (a mouse model of amyloid deposition that expresses five mutations found in familial AD) worsened the outcome of mice treated with anti-Aß passive immunotherapy by exacerbating the deposition of Aß, microgliosis, neurovascular dysfunction, and behavioural deficits. By contrast, therapeutic delivery of vascular endothelial growth factor C improved clearance of Aß by monoclonal antibodies. Notably, there was a substantial overlap between the gene signature of microglia from 5xFAD mice with impaired meningeal lymphatic function and the transcriptional profile of activated microglia from the brains of individuals with AD. Overall, our data demonstrate that impaired meningeal lymphatic drainage exacerbates the microglial inflammatory response in AD and that enhancement of meningeal lymphatic function combined with immunotherapies could lead to better clinical outcomes.

Preclinical Alzheimer's disease biomarkers accurately predict cognitive and neuropathological outcomes.

Alzheimer's disease biomarkers are widely accepted as surrogate markers of underlying neuropathological changes. However, few studies have evaluated whether preclinical Alzheimer's disease biomarkers predict Alzheimer's neuropathology at autopsy. We sought to determine whether amyloid PET imaging or CSF biomarkers accurately predict cognitive outcomes and Alzheimer's disease neuropathological findings. This study included 720 participants, 42-91 years of age, who were enrolled in longitudinal studies of memory and aging in the Washington University Knight Alzheimer Disease Research Center and were cognitively normal at baseline, underwent amyloid PET imaging and/or CSF collection within 1 year of baseline clinical assessment, and had subsequent clinical follow-up. Cognitive status was assessed longitudinally by Clinical Dementia Rating®. Biomarker status was assessed using predefined cut-offs for amyloid PET imaging or CSF p-tau181/amyloid-ß42. Subsequently, 57 participants died and underwent neuropathologic examination. Alzheimer's disease neuropathological changes were assessed using standard criteria. We assessed the predictive value of Alzheimer's disease biomarker status on progression to cognitive impairment and for presence of Alzheimer's disease neuropathological changes. Among cognitively normal participants with positive biomarkers, 34.4% developed cognitive impairment (Clinical Dementia Rating > 0) as compared to 8.4% of those with negative biomarkers. Cox proportional hazards modelling indicated that preclinical Alzheimer's disease biomarker status, APOE ɛ4 carrier status, polygenic risk score and centred age influenced risk of developing cognitive impairment. Among autopsied participants, 90.9% of biomarker-positive participants and 8.6% of biomarker-negative participants had Alzheimer's disease neuropathological changes. Sensitivity was 87.0%, specificity 94.1%, positive predictive value 90.9% and negative predictive value 91.4% for detection of Alzheimer's disease neuropathological changes by preclinical biomarkers. Single CSF and amyloid PET baseline biomarkers were also predictive of Alzheimer's disease neuropathological changes, as well as Thal phase and Braak stage of pathology at autopsy. Biomarker-negative participants who developed cognitive impairment were more likely to exhibit non-Alzheimer's disease pathology at autopsy. The detection of preclinical Alzheimer's disease biomarkers is strongly predictive of future cognitive impairment and accurately predicts presence of Alzheimer's disease neuropathology at autopsy.

Multi-ancestry GWAS reveals excitotoxicity associated with outcome after ischaemic stroke.

During the first hours after stroke onset, neurological deficits can be highly unstable: some patients rapidly improve, while others deteriorate. This early neurological instability has a major impact on long-term outcome. Here, we aimed to determine the genetic architecture of early neurological instability measured by the difference between the National Institutes of Health Stroke Scale (NIHSS) within 6 h of stroke onset and NIHSS at 24 h. A total of 5876 individuals from seven countries (Spain, Finland, Poland, USA, Costa Rica, Mexico and Korea) were studied using a multi-ancestry meta-analyses. We found that 8.7% of NIHSS at 24 h of variance was explained by common genetic variations, and also that early neurological instability has a different genetic architecture from that of stroke risk. Eight loci (1p21.1, 1q42.2, 2p25.1, 2q31.2, 2q33.3, 5q33.2, 7p21.2 and 13q31.1) were genome-wide significant and explained 1.8% of the variability suggesting that additional variants influence early change in neurological deficits. We used functional genomics and bioinformatic annotation to identify the genes driving the association from each locus. Expression quantitative trait loci mapping and summary data-based Mendelian randomization indicate that ADAM23 (log Bayes factor = 5.41) was driving the association for 2q33.3. Gene-based analyses suggested that GRIA1 (log Bayes factor = 5.19), which is predominantly expressed in the brain, is the gene driving the association for the 5q33.2 locus. These analyses also nominated GNPAT (log Bayes factor = 7.64) ABCB5 (log Bayes factor = 5.97) for the 1p21.1 and 7p21.1 loci. Human brain single-nuclei RNA-sequencing indicates that the gene expression of ADAM23 and GRIA1 is enriched in neurons. ADAM23, a presynaptic protein and GRIA1, a protein subunit of the AMPA receptor, are part of a synaptic protein complex that modulates neuronal excitability. These data provide the first genetic evidence in humans that excitotoxicity may contribute to early neurological instability after acute ischaemic stroke.

Metabolomic and lipidomic signatures in autosomal dominant and late-onset Alzheimer's disease brains.

INTRODUCTION: The identification of multiple genetic risk factors for Alzheimer's disease (AD) suggests that many pathways contribute to AD onset and progression. However, the metabolomic and lipidomic profiles in carriers of distinct genetic risk factors are not fully understood. The metabolome can provide a direct image of dysregulated pathways in the brain. METHODS: We interrogated metabolomic signatures in the AD brain, including carriers of pathogenic variants in APP, PSEN1, and PSEN2 (autosomal dominant AD; ADAD), APOE ɛ4, and TREM2 risk variant carriers, and sporadic AD (sAD). RESULTS: We identified 133 unique and shared metabolites associated with ADAD, TREM2, and sAD. We identified a signature of 16 metabolites significantly altered between groups and associated with AD duration. DISCUSSION: AD genetic variants show distinct metabolic perturbations. Investigation of these metabolites may provide greater insight into the etiology of AD and its impact on clinical presentation. HIGHLIGHTS: APP/PSEN1/PSEN2 and TREM2 variant carriers show distinct metabolic changes. A total of 133 metabolites were differentially abundant in AD genetic groups. ß-citrylglutamate is differentially abundant in autosomal dominant, TREM2, and sporadic AD. A 16-metabolite profile shows differences between Alzheimer's disease (AD) genetic groups. The identified metabolic profile is associated with duration of disease.

Alzheimer's disease alters oligodendrocytic glycolytic and ketolytic gene expression.

INTRODUCTION: Sporadic Alzheimer's disease (AD) is strongly correlated with impaired brain glucose metabolism, which may affect AD onset and progression. Ketolysis has been suggested as an alternative pathway to fuel the brain. METHODS: RNA-seq profiles of post mortem AD brains were used to determine whether dysfunctional AD brain metabolism can be determined by impairments in glycolytic and ketolytic gene expression. Data were obtained from the Knight Alzheimer's Disease Research Center (62 cases; 13 controls), Mount Sinai Brain Bank (110 cases; 44 controls), and the Mayo Clinic Brain Bank (80 cases; 76 controls), and were normalized to cell type: astrocytes, microglia, neurons, oligodendrocytes. RESULTS: In oligodendrocytes, both glycolytic and ketolytic pathways were significantly impaired in AD brains. Ketolytic gene expression was not significantly altered in neurons, astrocytes, and microglia. DISCUSSION: Oligodendrocytes may contribute to brain hypometabolism observed in AD. These results are suggestive of a potential link between hypometabolism and dysmyelination in disease physiology. Additionally, ketones may be therapeutic in AD due to their ability to fuel neurons despite impaired glycolytic metabolism.

Overlapping genetic architecture between Parkinson disease and melanoma.

Epidemiologic studies have reported inconsistent results regarding an association between Parkinson disease (PD) and cutaneous melanoma (melanoma). Identifying shared genetic architecture between these diseases can support epidemiologic findings and identify common risk genes and biological pathways. Here, we apply polygenic, linkage disequilibrium-informed methods to the largest available case-control, genome-wide association study summary statistic data for melanoma and PD. We identify positive and significant genetic correlation (correlation: 0.17, 95% CI 0.10-0.24; P = 4.09 × 10-06) between melanoma and PD. We further demonstrate melanoma and PD-inferred gene expression to overlap across tissues (correlation: 0.14, 95% CI 0.06 to 0.22; P = 7.87 × 10-04) and highlight seven genes including PIEZO1, TRAPPC2L, and SOX6 as potential mediators of the genetic correlation between melanoma and PD. These findings demonstrate specific, shared genetic architecture between PD and melanoma that manifests at the level of gene expression.

Correction to: Overlapping genetic architecture between Parkinson disease and melanoma.

The original version of this article unfortunately contained a mistake. Supplementary Tables 3 and 4 are not available with the rest of the supplementary material available online.

TREM2 activation on microglia promotes myelin debris clearance and remyelination in a model of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) triggered by autoimmune mechanisms. Microglia are critical for the clearance of myelin debris in areas of demyelination, a key step to allow remyelination. TREM2 is expressed by microglia and promotes microglial survival, proliferation, and phagocytic activity. Herein we demonstrate that TREM2 was highly expressed on myelin-laden phagocytes in active demyelinating lesions in the CNS of subjects with MS. In gene expression studies, macrophages from subjects with TREM2 genetic deficiency displayed a defect in phagocytic pathways. Treatment with a new TREM2 agonistic antibody promoted the clearance of myelin debris in the cuprizone model of CNS demyelination. Effects included enhancement of myelin uptake and degradation, resulting in accelerated myelin debris removal by microglia. Most importantly, antibody-dependent TREM2 activation on microglia increased density of oligodendrocyte precursors in areas of demyelination, as well as the formation of mature oligodendrocytes thus enhancing remyelination and axonal integrity. These results are relevant as they propose TREM2 on microglia as a potential new target to promote remyelination.

The TMEM106B FTLD-protective variant, rs1990621, is also associated with increased neuronal proportion.

Apart from amyloid ß deposition and tau neurofibrillary tangles, Alzheimer's disease (AD) is a neurodegenerative disorder characterized by neuronal loss and astrocytosis in the cerebral cortex. The goal of this study is to investigate genetic factors associated with the neuronal proportion in health and disease. To identify cell-autonomous genetic variants associated with neuronal proportion in cortical tissues, we inferred cellular population structure from bulk RNA-Seq derived from 1536 individuals. We identified the variant rs1990621 located in the TMEM106B gene region as significantly associated with neuronal proportion (p value = 6.40 × 10-07) and replicated this finding in an independent dataset (p value = 7.41 × 10-04) surpassing the genome-wide threshold in the meta-analysis (p value = 9.42 × 10-09). This variant is in high LD with the TMEM106B non-synonymous variant p.T185S (rs3173615; r2 = 0.98) which was previously identified as a protective variant for frontotemporal lobar degeneration (FTLD). We stratified the samples by disease status, and discovered that this variant modulates neuronal proportion not only in AD cases, but also several neurodegenerative diseases and in elderly cognitively healthy controls. Furthermore, we did not find a significant association in younger controls or schizophrenia patients, suggesting that this variant might increase neuronal survival or confer resilience to the neurodegenerative process. The single variant and gene-based analyses also identified an overall genetic association between neuronal proportion, AD and FTLD risk. These results suggest that common pathways are implicated in these neurodegenerative diseases, that implicate neuronal survival. In summary, we identified a protective variant in the TMEM106B gene that may have a neuronal protection effect against general aging, independent of disease status, which could help elucidate the relationship between aging and neuronal survival in the presence or absence of neurodegenerative disorders. Our findings suggest that TMEM106B could be a potential target for neuronal protection therapies to ameliorate cognitive and functional deficits.

Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease.

Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimer's disease (LOAD). These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case-control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer's disease in seven independent case-control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3, reveal that several variants in this gene increase risk for Alzheimer's disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer's disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer's disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-ß precursor protein (APP) and extracellular Aß42 and Aß40 (the 42- and 40-residue isoforms of the amyloid-ß peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aß42 and Aß40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex traits.

Analysis of neurodegenerative Mendelian genes in clinically diagnosed Alzheimer Disease.

Alzheimer disease (AD), Frontotemporal lobar degeneration (FTD), Amyotrophic lateral sclerosis (ALS) and Parkinson disease (PD) have a certain degree of clinical, pathological and molecular overlap. Previous studies indicate that causative mutations in AD and FTD/ALS genes can be found in clinical familial AD. We examined the presence of causative and low frequency coding variants in the AD, FTD, ALS and PD Mendelian genes, in over 450 families with clinical history of AD and over 11,710 sporadic cases and cognitive normal participants from North America. Known pathogenic mutations were found in 1.05% of the sporadic cases, in 0.69% of the cognitively normal participants and in 4.22% of the families. A trend towards enrichment, albeit non-significant, was observed for most AD, FTD and PD genes. Only PSEN1 and PINK1 showed consistent association with AD cases when we used ExAC as the control population. These results suggest that current study designs may contain heterogeneity and contamination of the control population, and that current statistical methods for the discovery of novel genes with real pathogenic variants in complex late onset diseases may be inadequate or underpowered to identify genes carrying pathogenic mutations.

Polygenic risk score of sporadic late-onset Alzheimer's disease reveals a shared architecture with the familial and early-onset forms.

OBJECTIVE: To determine whether the extent of overlap of the genetic architecture among the sporadic late-onset Alzheimer's Disease (sLOAD), familial late-onset AD (fLOAD), sporadic early-onset AD (sEOAD), and autosomal dominant early-onset AD (eADAD). METHODS: Polygenic risk scores (PRSs) were constructed using previously identified 21 genome-wide significant loci for LOAD risk. RESULTS: We found that there is an overlap in the genetic architecture among sEOAD, fLOAD, and sLOAD. The highest association of the PRS and risk (odds ratio [OR] = 2.27; P = 1.29 × 10-7) was observed in sEOAD, followed by fLOAD (OR = 1.75; P = 1.12 × 10-7) and sLOAD (OR = 1.40; P = 1.21 × 10-3). The PRS was associated with cerebrospinal fluid ptau181-Aß42 on eADAD (P = 4.36 × 10-2). CONCLUSION: Our analysis confirms that the genetic factors identified for LOAD modulate risk in sLOAD and fLOAD and also sEOAD cohorts. Specifically, our results suggest that the burden of these risk variants is associated with familial clustering and earlier onset of AD. Although these variants are not associated with risk in the eADAD, they may be modulating age at onset.

Genome-wide association study identifies four novel loci associated with Alzheimer's endophenotypes and disease modifiers.

More than 20 genetic loci have been associated with risk for Alzheimer's disease (AD), but reported genome-wide significant loci do not account for all the estimated heritability and provide little information about underlying biological mechanisms. Genetic studies using intermediate quantitative traits such as biomarkers, or endophenotypes, benefit from increased statistical power to identify variants that may not pass the stringent multiple test correction in case-control studies. Endophenotypes also contain additional information helpful for identifying variants and genes associated with other aspects of disease, such as rate of progression or onset, and provide context to interpret the results from genome-wide association studies (GWAS). We conducted GWAS of amyloid beta (Aß42), tau, and phosphorylated tau (ptau181) levels in cerebrospinal fluid (CSF) from 3146 participants across nine studies to identify novel variants associated with AD. Five genome-wide significant loci (two novel) were associated with ptau181, including loci that have also been associated with AD risk or brain-related phenotypes. Two novel loci associated with Aß42 near GLIS1 on 1p32.3 (ß = -0.059, P = 2.08 × 10-8) and within SERPINB1 on 6p25 (ß = -0.025, P = 1.72 × 10-8) were also associated with AD risk (GLIS1: OR = 1.105, P = 3.43 × 10-2), disease progression (GLIS1: ß = 0.277, P = 1.92 × 10-2), and age at onset (SERPINB1: ß = 0.043, P = 4.62 × 10-3). Bioinformatics indicate that the intronic SERPINB1 variant (rs316341) affects expression of SERPINB1 in various tissues, including the hippocampus, suggesting that SERPINB1 influences AD through an Aß-associated mechanism. Analyses of known AD risk loci suggest CLU and FERMT2 may influence CSF Aß42 (P = 0.001 and P = 0.009, respectively) and the INPP5D locus may affect ptau181 levels (P = 0.009); larger studies are necessary to verify these results. Together the findings from this study can be used to inform future AD studies.

Parkinson disease polygenic risk score is associated with Parkinson disease status and age at onset but not with alpha-synuclein cerebrospinal fluid levels.

BACKGROUND: The genetic architecture of Parkinson's Disease (PD) is complex and not completely understood. Multiple genetic studies to date have identified multiple causal genes and risk loci. Nevertheless, most of the expected genetic heritability remains unexplained. Polygenic risk scores (PRS) may provide greater statistical power and inform about the genetic architecture of multiple phenotypes. The aim of this study was to test the association between PRS and PD risk, age at onset and cerebrospinal fluid (CSF) biomarkers (α-synuclein, Aß1-42, t-tau and p-tau). METHODS: The weighted PRS was created using the genome-wide loci from Nalls et al., 2014 PD GWAs meta-analysis. The PRS was tested for association with PD status, age at onset and CSF biomarker levels in 829 cases and 432 controls of European ancestry. RESULTS: The PRS was associated with PD status (p = 5.83×10-08) and age at onset (p = 5.70×10-07). The CSF t-tau levels showed a nominal association with the PRS (p = 0.02). However, CSF α-synuclein, amyloid beta and phosphorylated tau were not found to be associated with the PRS. CONCLUSION: Our study suggests that there is an overlap in the genetic architecture of PD risk and onset, although the different loci present different weights for those phenotypes. In our dataset we found a marginal association of the PRS with CSF t-tau but not with α-synuclein CSF levels, suggesting that the genetic architecture for the CSF biomarker levels is different from that of PD risk.

Genome-wide association study of CSF levels of 59 alzheimer's disease candidate proteins: significant associations with proteins involved in amyloid processing and inflammation.

Cerebrospinal fluid (CSF) 42 amino acid species of amyloid beta (Aß42) and tau levels are strongly correlated with the presence of Alzheimer's disease (AD) neuropathology including amyloid plaques and neurodegeneration and have been successfully used as endophenotypes for genetic studies of AD. Additional CSF analytes may also serve as useful endophenotypes that capture other aspects of AD pathophysiology. Here we have conducted a genome-wide association study of CSF levels of 59 AD-related analytes. All analytes were measured using the Rules Based Medicine Human DiscoveryMAP Panel, which includes analytes relevant to several disease-related processes. Data from two independently collected and measured datasets, the Knight Alzheimer's Disease Research Center (ADRC) and Alzheimer's Disease Neuroimaging Initiative (ADNI), were analyzed separately, and combined results were obtained using meta-analysis. We identified genetic associations with CSF levels of 5 proteins (Angiotensin-converting enzyme (ACE), Chemokine (C-C motif) ligand 2 (CCL2), Chemokine (C-C motif) ligand 4 (CCL4), Interleukin 6 receptor (IL6R) and Matrix metalloproteinase-3 (MMP3)) with study-wide significant p-values (p<1.46×10-10) and significant, consistent evidence for association in both the Knight ADRC and the ADNI samples. These proteins are involved in amyloid processing and pro-inflammatory signaling. SNPs associated with ACE, IL6R and MMP3 protein levels are located within the coding regions of the corresponding structural gene. The SNPs associated with CSF levels of CCL4 and CCL2 are located in known chemokine binding proteins. The genetic associations reported here are novel and suggest mechanisms for genetic control of CSF and plasma levels of these disease-related proteins. Significant SNPs in ACE and MMP3 also showed association with AD risk. Our findings suggest that these proteins/pathways may be valuable therapeutic targets for AD. Robust associations in cognitively normal individuals suggest that these SNPs also influence regulation of these proteins more generally and may therefore be relevant to other diseases.

Coding variants in TREM2 increase risk for Alzheimer's disease.

The triggering receptor expressed on myeloid 2 (TREM2) is an immune phagocytic receptor expressed on brain microglia known to trigger phagocytosis and regulate the inflammatory response. Homozygous mutations in TREM2 cause Nasu-Hakola disease, a rare recessive form of dementia. A heterozygous TREM2 variant, p.R47H, was recently shown to increase Alzheimer''s disease (AD) risk. We hypothesized that if TREM2 is truly an AD risk gene, there would be additional rare variants in TREM2 that substantially affect AD risk. To test this hypothesis, we performed pooled sequencing of TREM2 coding regions in 2082 AD cases and 1648 cognitively normal elderly controls of European American descent. We identified 16 non-synonymous variants, six of which were not identified in previous AD studies. Two variants, p.R47H [P = 9.17 × 10(-4), odds ratio (OR) = 2.63 (1.44-4.81)] and p.R62H [P = 2.36 × 10(-4), OR = 2.36 (1.47-3.80)] were significantly associated with disease risk in single-variant analyses. Gene-based tests demonstrate variants in TREM2 are genome-wide significantly associated with AD [PSKAT-O = 5.37 × 10(-7); OR = 2.55 (1.80-3.67)]. The association of TREM2 variants with AD is still highly significant after excluding p.R47H [PSKAT-O = 7.72 × 10(-5); OR = 2.47 (1.62-3.87)], indicating that additional TREM2 variants affect AD risk. Genotyping in available family members of probands suggested that p.R47H (P = 4.65 × 10(-2)) and p.R62H (P = 6.87 × 10(-3)) were more frequently seen in AD cases versus controls within these families. Gel electrophoresis analysis confirms that at least three TREM2 transcripts are expressed in human brains, including one encoding a soluble form of TREM2.

Use of a predictive model derived from in vivo endophenotype measurements to demonstrate associations with a complex locus, CYP2A6.

This study demonstrates a novel approach to test associations between highly heterogeneous genetic loci and complex phenotypes. Previous investigations of the relationship between Cytochrome P450 2A6 (CYP2A6) genotype and smoking phenotypes made comparisons by dividing subjects into broad categories based on assumptions that simplify the range of function of different CYP2A6 alleles, their numerous possible diplotype combinations and non-additive allele effects. A predictive model that translates CYP2A6 diplotype into a single continuous variable was previously derived from an in vivo metabolism experiment in 189 European Americans. Here, we apply this model to assess associations between genotype, inferred nicotine metabolism and smoking behaviors in larger samples without direct nicotine metabolism measurements. CYP2A6 genotype is not associated with nicotine dependence, as defined by the Fagerström Test of Nicotine Dependence, demonstrating that cigarettes smoked per day (CPD) and nicotine dependence have distinct genetic correlates. The predicted metric is significantly associated with CPD among African Americans and European American dependent smokers. Individual slow metabolizing genotypes are associated with lower CPD, but the predicted metric is the best predictor of CPD. Furthermore, optimizing the predictive model by including additional CYP2A6 alleles improves the fit of the model in an independent data set and provides a novel method of predicting the functional impact of alleles without direct metabolism measurements. Lastly, comprehensive genotyping and in vivo metabolism data are used to demonstrate that genome-wide significant associations between CPD and single nucleotide polymorphisms are the result of synthetic associations.

Comparative gene regulatory networks modulating APOE expression in microglia and astrocytes.

Background: Single-cell technologies have unveiled various transcriptional states in different brain cell types. Transcription factors (TFs) regulate the expression of related gene sets, thereby controlling these diverse expression states. Apolipoprotein E (APOE), a pivotal risk-modifying gene in Alzheimer's disease (AD), is expressed in specific glial transcriptional states associated with AD. However, it is still unknown whether the upstream regulatory programs that modulate its expression are shared across brain cell types or specific to microglia and astrocytes. Methods: We used pySCENIC to construct state-specific gene regulatory networks (GRNs) for resting and activated cell states within microglia and astrocytes based on single-nucleus RNA sequencing data from AD patients' cortices from the Knight ADRC-DIAN cohort. We then identified replicating TF using data from the ROSMAP cohort. We identified sets of genes co-regulated with APOE by clustering the GRN target genes and identifying genes differentially expressed after the virtual knockout of TFs regulating APOE. We performed enrichment analyses on these gene sets and evaluated their overlap with genes found in AD GWAS loci. Results: We identified an average of 96 replicating regulators for each microglial and astrocyte cell state. Our analysis identified the CEBP, JUN, FOS, and FOXO TF families as key regulators of microglial APOE expression. The steroid/thyroid hormone receptor families, including the THR TF family, consistently regulated APOE across astrocyte states, while CEBP and JUN TF families were also involved in resting astrocytes. AD GWAS-associated genes (PGRN, FCGR3A, CTSH, ABCA1, MARCKS, CTSB, SQSTM1, TSC22D4, FCER1G, and HLA genes) are co-regulated with APOE. We also uncovered that APOE-regulating TFs were linked to circadian rhythm (BHLHE40, DBP, XBP1, CREM, SREBF1, FOXO3, and NR2F1). Conclusions: Our findings reveal a novel perspective on the transcriptional regulation of APOE in the human brain. We found a comprehensive and cell-type-specific regulatory landscape for APOE, revealing distinct and shared regulatory mechanisms across microglia and astrocytes, underscoring the complexity of APOE regulation. APOE-co-regulated genes might also affect AD risk. Furthermore, our study uncovers a potential link between circadian rhythm disruption and APOE regulation, shedding new light on the pathogenesis of AD.