A novel systems biology approach to evaluate mouse models of late-onset Alzheimer’s disease
ABSTRACT: Background: Late-onset Alzheimer’s disease (LOAD) is the most common form of dementia worldwide. To date, animal models of Alzheimer’s have focused on rare familial mutations, due to a lack of frank neuropathology from models based on common disease genes. Recent multi-cohort studies of postmortem human brain transcriptomes have identified a set of 30 gene co-expression modules associated with LOAD, providing a molecular catalog of relevant endophenotypes. Results: This resource enables precise gene-based alignment between new animal models and human molecular signatures of disease. Here, we describe a new resource to efficiently screen mouse models for LOAD relevance. A new NanoString nCounter® Mouse AD panel was designed to correlate key human disease processes and pathways with mRNA from mouse brains. Analysis of three mouse models based on LOAD genetics, carrying APOE4 and TREM2*R47H alleles and the 5xFAD mouse, a widely used amyloid pathology model, demonstrated overlaps with distinct human AD modules that, in turn, are functionally enriched in key disease-associated pathways. Comprehensive comparison with full transcriptome data from same-sample RNA-Seq shows strong correlation between gene expression changes independent of experimental platform. Conclusions: Taken together, we show that the nCounter Mouse AD panel offers a rapid, cost-effective and highly reproducible approach to assess disease relevance of potential LOAD mouse models Overall design: In this study, a cohort of 155 mice from four four different strains carrying Alzheimer associated genetic risk variants was used to to correlate key human disease processes and pathways with mRNA from mouse brains. Three novel mouse models, harboring two LOAD risk alleles, were used to translate co-expression profiles between human and mouse brain transcriptome data using our novel Mouse AD panel. Transcriptome analysis was performed for the APOE4 KI mouse, carrying a humanized version of the strongest LOAD associated risk allele (APOE-ε4) and the Trem2*R47H mouse, which harbors a rare deleterious variant in TREM2. The rare TREM2 R47H missense variant (rs75932628) has been previously associated with LOAD in multiple independent studies. In addition, we added the 5xFAD model, a commonly used mouse model carrying transgenes with a total of five AD-linked familial mutations in APP and PSEN1 to our analysis in order to compare our novel LOAD models to a model that resembles the early onset form of the disease with a high amyloid burden.Mouse transcriptome data for half brains was analyzed at different ages (2-14 months) to estimate the overlap with human post-mortem co-expression modules during aging.
INSTRUMENT(S): NanoString nCounter® Mouse AD panel
Project description:BACKGROUND:Late-onset Alzheimer's disease (LOAD) is the most common form of dementia worldwide. To date, animal models of Alzheimer's have focused on rare familial mutations, due to a lack of frank neuropathology from models based on common disease genes. Recent multi-cohort studies of postmortem human brain transcriptomes have identified a set of 30 gene co-expression modules associated with LOAD, providing a molecular catalog of relevant endophenotypes. RESULTS:This resource enables precise gene-based alignment between new animal models and human molecular signatures of disease. Here, we describe a new resource to efficiently screen mouse models for LOAD relevance. A new NanoString nCounter® Mouse AD panel was designed to correlate key human disease processes and pathways with mRNA from mouse brains. Analysis of the 5xFAD mouse, a widely used amyloid pathology model, and three mouse models based on LOAD genetics carrying APOE4 and TREM2*R47H alleles demonstrated overlaps with distinct human AD modules that, in turn, were functionally enriched in key disease-associated pathways. Comprehensive comparison with full transcriptome data from same-sample RNA-Seq showed strong correlation between gene expression changes independent of experimental platform. CONCLUSIONS:Taken together, we show that the nCounter Mouse AD panel offers a rapid, cost-effective and highly reproducible approach to assess disease relevance of potential LOAD mouse models.
Project description:The R47H variant in the triggering receptor expressed on myeloid cells 2 gene (TREM2), a modulator of the immune response of microglia, is a strong genetic risk factor for Alzheimer disease (AD) and possibly other neurodegenerative disorders.To investigate a large family with late-onset AD (LOAD), in which R47H cosegregated with 75% of cases.This study includes genetic and pathologic studies of families with LOAD from 1985 to 2014. A total of 131 families with LOAD (751 individuals) were included from the University of Washington Alzheimer Disease Research Center. To identify LOAD genes/risk factors in the LOAD123 family with 21 affected members and 12 autopsies, we sequenced 4 exomes. Candidate variants were tested for cosegregation with the disease. TREM2 R47H was genotyped in an additional 130 families with LOAD. We performed clinical and neuropathological assessments of patients with and without R47H and evaluated the variant's effect on brain pathology, cellular morphology, and expression of microglial markers.We assessed the effect of TREM2 genotype on age at onset and disease duration. We compared Braak and Consortium to Establish a Registry for Alzheimer's Disease scores, presence of ?-synuclein and TAR DNA-binding protein 43 aggregates, and additional vascular or Parkinson pathology in TREM2 R47H carriers vs noncarriers. Microglial activation was assessed by quantitative immunohistochemistry and morphometry.Twelve of 16 patients with AD in the LOAD123 family carried R47H. Eleven patients with dementia had apolipoprotein E 4 (ApoE4) and R47H genotypes. We also found a rare missense variant, D353N, in a nominated AD risk gene, unc-5 homolog C (UNC5C), in 5 affected individuals in the LOAD123 family. R47H carriers demonstrated a shortened disease duration (mean [SD], 6.7 [2.8] vs 11.1 [6.6] years; 2-tailed t test; P?=?.04) and more frequent ?-synucleinopathy. The panmicroglial marker ionized calcium-binding adapter molecule 1 was decreased in all AD cases and the decrease was most pronounced in R47H carriers (mean [SD], in the hilus: 0.114 [0.13] for R47H_AD vs 0.574 [0.26] for control individuals; 2-tailed t test; P?=?.005 and vs 0.465 [0.32] for AD; P?=?.02; in frontal cortex gray matter: 0.006 [0.004] for R47H_AD vs 0.016 [0.01] for AD; P?=?.04 and vs 0.033 [0.013] for control individuals; P?<?.001). Major histocompatibility complex class II, a marker of microglial activation, was increased in all patients with AD (AD: 2.5, R47H_AD: 2.7, and control: 1.0; P?<?.01).Our results demonstrate a complex genetic landscape of LOAD, even in a single pedigree with an apparent autosomal dominant pattern of inheritance. ApoE4, TREM2 R47H, and rare variants in other genes, such as UNC5C D353N, are likely responsible for the notable occurrence of AD in this family. Our findings support the role of the TREM2 receptor in microglial clearance of aggregation-prone proteins that is compromised in R47H carriers and may accelerate the course of disease.
Project description:BACKGROUND:The R47H variant of the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) significantly increases the risk for late onset Alzheimer's disease. Mouse models accurately reproducing phenotypes observed in Alzheimer' disease patients carrying the R47H coding variant are required to understand the TREM2 related dysfunctions responsible for the enhanced risk for late onset Alzheimer's disease. METHODS:A CRISPR/Cas9-assisted gene targeting strategy was used to generate Trem2 R47H knock-in mice. Trem2 mRNA and protein levels as well as Trem2 splicing patterns were assessed in these mice, in iPSC-derived human microglia-like cells, and in human brains from Alzheimer's patients carrying the TREM2 R47H risk factor. RESULTS:Two independent Trem2 R47H knock-in mouse models show reduced Trem2 mRNA and protein production. In both mouse models Trem2 haploinsufficiency was due to atypical splicing of mouse Trem2 R47H, which introduced a premature stop codon. Cellular splicing assays using minigene constructs demonstrate that the R47H variant induced abnormal splicing only occurs in mice but not in humans. TREM2 mRNA levels and splicing patterns were both normal in iPSC-derived human microglia-like cells and patient brains with the TREM2 R47H variant. CONCLUSIONS:The Trem2 R47H variant activates a cryptic splice site that generates miss-spliced transcripts leading to Trem2 haploinsufficiency only in mice but not in humans. Since Trem2 R47H related phenotypes are mouse specific and do not occur in humans, humanized TREM2 R47H knock-in mice should be generated to study the cellular consequences caused by the human TREM2 R47H coding variant. Currently described phenotypes of Trem2 R47H knock-in mice can therefore not be translated to humans.
Project description:Alzheimer's disease (AD) is a neurodegenerative disease that causes late-onset dementia. The R47H variant of the microglial receptor TREM2 triples AD risk in genome-wide association studies. In mouse AD models, TREM2-deficient microglia fail to proliferate and cluster around the amyloid-? plaques characteristic of AD. In vitro, the common variant (CV) of TREM2 binds anionic lipids, whereas R47H mutation impairs binding. However, in vivo, the identity of TREM2 ligands and effect of the R47H variant remain unknown. We generated transgenic mice expressing human CV or R47H TREM2 and lacking endogenous TREM2 in the 5XFAD AD model. Only the CV transgene restored amyloid-?-induced microgliosis and microglial activation, indicating that R47H impairs TREM2 function in vivo. Remarkably, soluble TREM2 was found on neurons and plaques in CV- but not R47H-expressing 5XFAD brains, although in vitro CV and R47H were shed similarly via Adam17 proteolytic activity. These results demonstrate that TREM2 interacts with neurons and plaques duing amyloid-? accumulation and R47H impairs this interaction.
Project description:Genes associated with immune response and inflammation have been identified as genetic risk factors for late-onset Alzheimer´s disease (LOAD). The rare R47H variant within triggering receptor expressed on myeloid cells 2 (TREM2) has been shown to increase the risk for developing Alzheimer's disease (AD) 2-3-fold. Here, we report the generation and characterization of a model of late-onset Alzheimer's disease (LOAD) using lymphoblast-derived induced pluripotent stem cells (iPSCs) from patients carrying the TREM2 R47H mutation, as well as from control individuals without dementia. All iPSCs efficiently differentiated into mature neuronal cultures, however AD neuronal cultures showed a distinct gene expression profile. Furthermore, manipulation of the iPSC-derived neuronal cultures with an A?-S8C dimer highlighted metabolic pathways, phagosome and immune response as the most perturbed pathways in AD neuronal cultures. Through the construction of an A?-induced gene regulatory network, we were able to identify an A? signature linked to protein processing in the endoplasmic reticulum (ER), which emphasized ER-stress, as a potential causal role in LOAD. Overall, this study has shown that our AD-iPSC based model can be used for in-depth studies to better understand the molecular mechanisms underlying the etiology of LOAD and provides new opportunities for screening of potential therapeutic targets.
Project description:The R47H variant of the Triggering-Receptor-Expressed on Myeloid cells 2 (TREM2) increases the risk of Alzheimer's disease (AD). Mutagenesis of exon 2?in Knock-in (KI) mouse models of the R47H variant introduced a cryptic splice site, leading to nonsense mediated decay. Since haploinsufficiency does not model Trem2-R47H function, a new rat KI model, the Trem2R47H KI rat was created. Human A? has higher propensity to form toxic A? species, which are considered the main pathogenic entity in AD, as compared to rodent A?, the rat Amyloid Precursor Protein (App) gene was mutated to produce human A?. Trem2 splicing and expression was measured in Trem2R47H KI rat brains and microglia by qualitative and quantitative RT-PCR. Trem2 levels and Trem2 processing was assessed by Western analysis. APP metabolite levels were determined by enzyme-linked immunosorbent assay (ELISA), for Human A? and soluble APP, and Western analysis, for full length APP, ?CTF and ?CTF. Trem2 expression and Trem2 levels are unchanged in Trem2R47H KI rats. The artifactual splicing seen in KI mouse models is not present; additionally, two novel isoforms of rat Trem2 are described. Trem2R47H rat brains have lower human A?38, sAPP? and sAPP? levels. Thus, Trem2R47H KI rats may prove valuable to define pathogenic mechanisms triggered by the Trem2 R47H variant, including those mediated by toxic species of human A? peptides.
Project description:BACKGROUND:The R47H variant of Triggering Receptor Expressed on Myeloid cells 2 (TREM2) confers greatly increased risk for Alzheimer's disease (AD), reflective of a central role for myeloid cells in neurodegeneration. Understanding how this variant confers AD risk promises to provide important insights into how myeloid cells contribute to AD pathogenesis and progression. METHODS:In order to investigate this mechanism, CRISPR/Cas9 was used to generate a mouse model of AD harboring one copy of the single nucleotide polymorphism (SNP) encoding the R47H variant in murine Trem2. TREM2 expression, myeloid cell responses to amyloid deposition, plaque burden, and neuritic dystrophy were assessed at 4 months of age. RESULTS:AD mice heterozygous for the Trem2 R47H allele exhibited reduced total Trem2 mRNA expression, reduced TREM2 expression around plaques, and reduced association of myeloid cells with plaques. These results were comparable to AD mice lacking one copy of Trem2. AD mice heterozygous for the Trem2 R47H allele also showed reduced myeloid cell responses to amyloid deposition, including a reduction in proliferation and a reduction in CD45 expression around plaques. Expression of the Trem2 R47H variant also reduced dense core plaque number but increased plaque-associated neuritic dystrophy. CONCLUSIONS:These data suggest that the AD-associated TREM2 R47H variant increases risk for AD by conferring a loss of TREM2 function and enhancing neuritic dystrophy around plaques.
Project description:Background TREM2 is a microglial cell surface receptor, with risk mutations linked to Alzheimer’s disease (AD), including R47H. TREM2 signalling via SYK aids phagocytosis, chemotaxis, survival, and changes to microglial activation state. In AD mouse models, knockout (KO) of TREM2 impairs microglial clustering around amyloid and prevents microglial activation. The R47H mutation is proposed to reduce TREM2 ligand binding. We investigated cell phenotypes of the R47H mutant and TREM2 KO in a model of human microglia, and compared their transcriptional signatures, to determine the mechanism by which R47H TREM2 disrupts function. Methods We generated human microglia-like iPSC-macrophages (pMac) from isogenic induced pluripotent stem cell (iPSC) lines, with homozygous R47H mutation or TREM2 knockout (KO). We firstly validated the effect of the R47H mutant on TREM2 surface and subcellular localization in pMac. To assess microglial phenotypic function, we measured phagocytosis of dead neurons, cell morphology, directed migration, survival, and LPS-induced inflammation. We performed bulk RNA-seq, comparing significant differentially expressed genes (DEGs; p?<?0.05) between the R47H and KO versus WT, and bioinformatically predicted potential upstream regulators of TREM2-mediated gene expression. Results R47H modified surface expression and shedding of TREM2, but did not impair TREM2-mediated signalling, or gross phenotypes that were dysregulated in the TREM2 KO (phagocytosis, motility, survival). However, altered gene expression in the R47H TREM2 pMac overlapped by 90% with the TREM2 KO and was characterised by dysregulation of genes involved with immunity, proliferation, activation, chemotaxis, and adhesion. Downregulated mediators of ECM adhesion included the vitronectin receptor ?V?3, and consequently, R47H TREM2 pMac adhered weakly to vitronectin compared with WT pMac. To counteract these transcriptional defects, we investigated TGF?1, as a candidate upstream regulator. TGF?1 failed to rescue vitronectin adhesion of pMac, although it improved ?V?3 expression. Conclusions The R47H mutation is not sufficient to cause gross phenotypic defects of human pMac under standard culture conditions. However, overlapping transcriptional defects with TREM2 KO supports the hypothesised partial loss-of-function effects of the R47H mutation. Furthermore, transcriptomics can guide us to more subtle phenotypic defects in the R47H cells, such as reduced cell adhesion, and can be used to predict targets for therapeutic intervention.
Project description:Alzheimer's disease (AD) is a late-onset dementia characterized by the deposition of amyloid plaques and formation of neurofibrillary tangles (NFTs) which lead to neuronal loss and cognitive deficits. Abnormal protein aggregates in the AD brain are also associated with reactive microglia and astrocytes. Whether this glial response is beneficial or detrimental in AD pathology is under debate. Microglia are the resident innate immune cells in the central nervous system (CNS) that survey the surrounding environment. Genome-wide association studies (GWAS) have identified the R47H variant of triggering receptor expressed on myeloid cell 2 (TREM2) as a risk factor for late-onset AD (LOAD) with an odds ratio of 4.5. TREM2 is an immunoreceptor primarily present on microglia in the CNS that binds to polyanionic molecules. The transmembrane domain of TREM2 signals through DAP12, an adaptor protein that contains an immunoreceptor tyrosine-based activation motif (ITAM), which mediates TREM2 signaling and promotes microglial activation and survival. In mouse models of AD, Trem2 haplodeficiency and deficiency lead to reduced microglial clustering around amyloid ? (A?) plaques, suggesting TREM2 is required for plaque-associated microglial responses. Recently, TREM2 has been shown to enhance microglial metabolism through the mammalian target of rapamycin (mTOR) pathway. Although aberrant metabolism has long been associated with AD, not much was known regarding how metabolism in microglia might affect disease progression. In this review, we discuss the role of TREM2 and metabolism in AD pathology, highlighting how TREM2-mediated microglial metabolism modulates AD pathogenesis.
Project description:Glia have been implicated in Alzheimer's disease (AD) pathogenesis. Variants of the microglia receptor triggering receptor expressed on myeloid cells 2 (TREM2) increase AD risk, and activation of disease-associated microglia (DAM) is dependent on TREM2 in mouse models of AD. We surveyed gene-expression changes associated with AD pathology and TREM2 in 5XFAD mice and in human AD by single-nucleus RNA sequencing. We confirmed the presence of Trem2-dependent DAM and identified a previously undiscovered Serpina3n+C4b+ reactive oligodendrocyte population in mice. Interestingly, remarkably different glial phenotypes were evident in human AD. Microglia signature was reminiscent of IRF8-driven reactive microglia in peripheral-nerve injury. Oligodendrocyte signatures suggested impaired axonal myelination and metabolic adaptation to neuronal degeneration. Astrocyte profiles indicated weakened metabolic coordination with neurons. Notably, the reactive phenotype of microglia was less evident in TREM2-R47H and TREM2-R62H carriers than in non-carriers, demonstrating a TREM2 requirement in both mouse and human AD, despite the marked species-specific differences.