TYROBP/DAP12 knockout in Huntington's disease Q175 mice cell-autonomously decreases microglial expression of disease-associated genes and non-cell-autonomously mitigates astrogliosis and motor deterioration.

INTRODUCTION: Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat in the Huntingtin gene (HTT). Immune activation is abundant in the striatum of HD patients. Detection of active microglia at presymptomatic stages suggests that microgliosis is a key early driver of neuronal dysfunction and degeneration. Recent studies showed that deletion of Tyrobp, a microglial protein, ameliorates neuronal dysfunction in Alzheimer's disease amyloidopathy and tauopathy mouse models while decreasing components of the complement subnetwork. OBJECTIVE: While TYROBP/DAP12-mediated microglial activation is detrimental for some diseases such as peripheral nerve injury, it is beneficial for other diseases. We sought to determine whether the TYROBP network is implicated in HD and whether Tyrobp deletion impacts HD striatal function and transcriptomics. METHODS: To test the hypothesis that Tyrobp deficiency would be beneficial in an HD model, we placed the Q175 HD mouse model on a Tyrobp-null background. We characterized these mice with a combination of behavioral testing, immunohistochemistry, transcriptomic and proteomic profiling. Further, we evaluated the gene signature in isolated Q175 striatal microglia, with and without Tyrobp. RESULTS: Comprehensive analysis of publicly available human HD transcriptomic data revealed that the TYROBP network is overactivated in the HD putamen. The Q175 mice showed morphologic microglial activation, reduced levels of post-synaptic density-95 protein and motor deficits at 6 and 9 months of age, all of which were ameliorated on the Tyrobp-null background. Gene expression analysis revealed that lack of Tyrobp in the Q175 model does not prevent the decrease in the expression of striatal neuronal genes but reduces pro-inflammatory pathways that are specifically active in HD human brain, including genes identified as detrimental in neurodegenerative diseases, e.g. C1q and members of the Ccr5 signaling pathway. Integration of transcriptomic and proteomic data revealed that astrogliosis and complement system pathway were reduced after Tyrobp deletion, which was further validated by immunofluorescence analysis. CONCLUSIONS: Our data provide molecular and functional support demonstrating that Tyrobp deletion prevents many of the abnormalities in the HD Q175 mouse model, suggesting that the Tyrobp pathway is a potential therapeutic candidate for Huntington's disease.

BCI-838, an orally active mGluR2/3 receptor antagonist pro-drug, rescues learning behavior deficits in the PS19 MAPTP301S mouse model of tauopathy.

Tauopathies are a heterogeneous group of neurodegenerative disorders that are clinically and pathologically distinct from Alzheimer's disease (AD) having tau inclusions in neurons and/or glia as their most prominent neuropathological feature. BCI-838 (MGS00210) is a group II metabotropic glutamate receptor (mGluR2/3) antagonist pro-drug. Previously, we reported that orally administered BCI-838 improved learning behavior and reduced anxiety in Dutch (APPE693Q) transgenic mice, a model of the pathological accumulation of Aß oligomers found in AD. Herein, we investigated effects of BCI-838 on PS19 male mice that express the tauopathy mutation MAPTP301S associated with human frontotemporal lobar degeneration (FTLD). These mice develop an aging-related tauopathy without amyloid accumulation. Mice were divided into three experimental groups: (1) non-transgenic wild type mice treated with vehicle, (2) PS19 mice treated with vehicle and (3) PS19 mice treated with 5 mg/kg BCI-838. Groups of 10-13 mice were utilized. Vehicle or BCI-838 was administered by oral gavage for 4 weeks. Behavioral testing consisting of a novel object recognition task was conducted after drug administration. Two studies were performed beginning treatment of mice at 3 or 7 months of age. One month of BCI-838 treatment rescued deficits in recognition memory in PS19 mice whether treatment was begun at 3 or 7 months of age. These studies extend the potential utility of BCI-838 to neurodegenerative conditions that have tauopathy as their underlying basis. They also suggest an mGluR2/3 dependent mechanism as a basis for the behavioral deficits in PS19 mice.

Microglial INPP5D limits plaque formation and glial reactivity in the PSAPP mouse model of Alzheimer's disease.

INTRODUCTION: The inositol polyphosphate-5-phosphatase D (INPP5D) gene encodes a dual-specificity phosphatase that can dephosphorylate both phospholipids and phosphoproteins. Single nucleotide polymorphisms in INPP5D impact risk for developing late onset sporadic Alzheimer's disease (LOAD). METHODS: To assess the consequences of inducible Inpp5d knockdown in microglia of APPKM670/671NL /PSEN1Δexon9 (PSAPP) mice, we injected 3-month-old Inpp5dfl/fl /Cx3cr1CreER/+ and PSAPP/Inpp5dfl/fl /Cx3cr1CreER/+ mice with either tamoxifen (TAM) or corn oil (CO) to induce recombination. RESULTS: At age 6 months, we found that the percent area of 6E10+ deposits and plaque-associated microglia in Inpp5d knockdown mice were increased compared to controls. Spatial transcriptomics identified a plaque-specific expression profile that was extensively altered by Inpp5d knockdown. DISCUSSION: These results demonstrate that conditional Inpp5d downregulation in the PSAPP mouse increases plaque burden and recruitment of microglia to plaques. Spatial transcriptomics highlighted an extended gene expression signature associated with plaques and identified CST7 (cystatin F) as a novel marker of plaques. HIGHLIGHTS: Inpp5d knockdown increases plaque burden and plaque-associated microglia number. Spatial transcriptomics identifies an expanded plaque-specific gene expression profile. Plaque-induced gene expression is altered by Inpp5d knockdown in microglia. Our plaque-associated gene signature overlaps with human Alzheimer's disease gene networks.

Microglial TYROBP/DAP12 in Alzheimer's disease: Transduction of physiological and pathological signals across TREM2.

TYROBP (also known as DAP12 or KARAP) is a transmembrane adaptor protein initially described as a receptor-activating subunit component of natural killer (NK) cells. TYROBP is expressed in numerous cell types, including peripheral blood monocytes, macrophages, dendritic cells, and osteoclasts, but a key point of recent interest is related to the critical role played by TYROBP in the function of many receptors expressed on the plasma membrane of microglia. TYROBP is the downstream adaptor and putative signaling partner for several receptors implicated in Alzheimer's disease (AD), including SIRP1ß, CD33, CR3, and TREM2. TYROBP has received much of its current notoriety because of its importance in brain homeostasis by signal transduction across those receptors. In this review, we provide an overview of evidence indicating that the biology of TYROBP extends beyond its interaction with these four ligand-binding ectodomain-intramembranous domain molecules. In addition to reviewing the structure and localization of TYROBP, we discuss our recent progress using mouse models of either cerebral amyloidosis or tauopathy that were engineered to be TYROBP-deficient or TYROBP-overexpressing. Remarkably, constitutively TYROBP-deficient mice provided a model of genetic resilience to either of the defining proteinopathies of AD. Learning behavior and synaptic electrophysiological function were preserved at normal physiological levels even in the face of robust cerebral amyloidosis (in APP/PSEN1;Tyrobp-/- mice) or tauopathy (in MAPTP301S;Tyrobp-/- mice). A fundamental underpinning of the functional synaptic dysfunction associated with each proteotype was an accumulation of complement C1q. TYROBP deficiency prevented C1q accumulation associated with either proteinopathy. Based on these data, we speculate that TYROBP plays a key role in the microglial sensome and the emergence of the disease-associated microglia (DAM) phenotype. TYROBP may also play a key role in the loss of markers of synaptic integrity (e.g., synaptophysin-like immunoreactivity) that has long been held to be the feature of human AD molecular neuropathology that most closely correlates with concurrent clinical cognitive function.

Transcriptomic Changes Highly Similar to Alzheimer's Disease Are Observed in a Subpopulation of Individuals During Normal Brain Aging.

Aging is a major risk factor for late-onset Alzheimer's disease (LOAD). How aging contributes to the development of LOAD remains elusive. In this study, we examined multiple large-scale transcriptomic datasets from both normal aging and LOAD brains to understand the molecular interconnection between aging and LOAD. We found that shared gene expression changes between aging and LOAD are mostly seen in the hippocampal and several cortical regions. In the hippocampus, the expression of phosphoprotein, alternative splicing and cytoskeleton genes are commonly changed in both aging and AD, while synapse, ion transport, and synaptic vesicle genes are commonly down-regulated. Aging-specific changes are associated with acetylation and methylation, while LOAD-specific changes are more related to glycoprotein (both up- and down-regulations), inflammatory response (up-regulation), myelin sheath and lipoprotein (down-regulation). We also found that normal aging brain transcriptomes from relatively young donors (45-70 years old) clustered into several subgroups and some subgroups showed gene expression changes highly similar to those seen in LOAD brains. Using brain transcriptomic datasets from another cohort of older individuals (>70 years), we found that samples from cognitively normal older individuals clustered with the "healthy aging" subgroup while AD samples mainly clustered with the "AD similar" subgroups. This may imply that individuals in the healthy aging subgroup will likely remain cognitively normal when they become older and vice versa. In summary, our results suggest that on the transcriptome level, aging and LOAD have strong interconnections in some brain regions in a subpopulation of cognitively normal aging individuals. This supports the theory that the initiation of LOAD occurs decades earlier than the manifestation of clinical phenotype and it may be essential to closely study the "normal brain aging" to identify the very early molecular events that may lead to LOAD development.

Reactive or transgenic increase in microglial TYROBP reveals a TREM2-independent TYROBP-APOE link in wild-type and Alzheimer's-related mice.

INTRODUCTION: Microglial TYROBP (DAP12) is a network hub and driver in sporadic late-onset Alzheimer's disease (AD). TYROBP is a cytoplasmic adaptor for TREM2 and other receptors, but little is known about its roles and actions in AD. Herein, we demonstrate that endogenous Tyrobp transcription is specifically increased in recruited microglia. METHODS: Using a novel transgenic mouse overexpressing TYROBP in microglia, we observed a decrease of the amyloid burden and an increase of TAU phosphorylation stoichiometry when crossed with APP/PSEN1 or MAPTP301S mice, respectively. Characterization of these mice revealed Tyrobp-related modulation of apolipoprotein E (Apoe) transcription. We also showed that Tyrobp and Apoe mRNAs were increased in Trem2-null microglia recruited around either amyloid beta deposits or a cortical stab injury. Conversely, microglial Apoe transcription was dramatically diminished when Tyrobp was absent. CONCLUSIONS: Our results provide evidence that TYROBP-APOE signaling does not require TREM2 and could be an initiating step in establishment of the disease-associated microglia (DAM) phenotype.

Multiscale causal networks identify VGF as a key regulator of Alzheimer's disease.

Though discovered over 100 years ago, the molecular foundation of sporadic Alzheimer's disease (AD) remains elusive. To better characterize the complex nature of AD, we constructed multiscale causal networks on a large human AD multi-omics dataset, integrating clinical features of AD, DNA variation, and gene- and protein-expression. These probabilistic causal models enabled detection, prioritization and replication of high-confidence master regulators of AD-associated networks, including the top predicted regulator, VGF. Overexpression of neuropeptide precursor VGF in 5xFAD mice partially rescued beta-amyloid-mediated memory impairment and neuropathology. Molecular validation of network predictions downstream of VGF was also achieved in this AD model, with significant enrichment for homologous genes identified as differentially expressed in 5xFAD brains overexpressing VGF. Our findings support a causal role for VGF in protecting against AD pathogenesis and progression.

miR155 regulation of behavior, neuropathology, and cortical transcriptomics in Alzheimer's disease.

MicroRNAs are recognized as important regulators of many facets of physiological brain function while also being implicated in the pathogenesis of several neurological disorders. Dysregulation of miR155 is widely reported across a variety of neurodegenerative conditions, including Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis, and traumatic brain injury. In previous work, we observed that experimentally validated miR155 gene targets were consistently enriched among genes identified as differentially expressed across multiple brain tissue and disease contexts. In particular, we found that human herpesvirus-6A (HHV-6A) suppressed miR155, recapitulating reports of miR155 inhibition by HHV-6A in infected T-cells, thyrocytes, and natural killer cells. In earlier studies, we also reported the effects of constitutive deletion of miR155 on accelerating the accumulation of Aß deposits in 4-month-old APP/PSEN1 mice. Herein, we complete the cumulative characterization of transcriptomic, electrophysiological, neuropathological, and learning behavior profiles from 4-, 8- and 10-month-old WT and APP/PSEN1 mice in the absence or presence of miR155. We also integrated human post-mortem brain RNA-sequences from four independent AD consortium studies, together comprising 928 samples collected from six brain regions. We report that gene expression perturbations associated with miR155 deletion in mouse cortex are in aggregate observed to be concordant with AD-associated changes across these independent human late-onset AD (LOAD) data sets, supporting the relevance of our findings to human disease. LOAD has recently been formulated as the clinicopathological manifestation of a multiplex of genetic underpinnings and pathophysiological mechanisms. Our accumulated data are consistent with such a formulation, indicating that miR155 may be uniquely positioned at the intersection of at least four components of this LOAD "multiplex": (1) innate immune response pathways; (2) viral response gene networks; (3) synaptic pathology; and (4) proamyloidogenic pathways involving the amyloid ß peptide (Aß).

Meta-Analysis of the Alzheimer's Disease Human Brain Transcriptome and Functional Dissection in Mouse Models.

We present a consensus atlas of the human brain transcriptome in Alzheimer's disease (AD), based on meta-analysis of differential gene expression in 2,114 postmortem samples. We discover 30 brain coexpression modules from seven regions as the major source of AD transcriptional perturbations. We next examine overlap with 251 brain differentially expressed gene sets from mouse models of AD and other neurodegenerative disorders. Human-mouse overlaps highlight responses to amyloid versus tau pathology and reveal age- and sex-dependent expression signatures for disease progression. Human coexpression modules enriched for neuronal and/or microglial genes broadly overlap with mouse models of AD, Huntington's disease, amyotrophic lateral sclerosis, and aging. Other human coexpression modules, including those implicated in proteostasis, are not activated in AD models but rather following other, unexpected genetic manipulations. Our results comprise a cross-species resource, highlighting transcriptional networks altered by human brain pathophysiology and identifying correspondences with mouse models for AD preclinical studies.

Correction: Integrative approach to sporadic Alzheimer's disease: deficiency of TYROBP in cerebral Aß amyloidosis mouse normalizes clinical phenotype and complement subnetwork molecular pathology without reducing Aß burden.

This article was originally published under standard licence, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly.

Integrative approach to sporadic Alzheimer's disease: deficiency of TYROBP in a tauopathy mouse model reduces C1q and normalizes clinical phenotype while increasing spread and state of phosphorylation of tau.

TYROBP/DAP12 forms complexes with ectodomains of immune receptors (TREM2, SIRPß1, CR3) associated with Alzheimer's disease (AD) and is a network hub and driver in the complement subnetwork identified by multi-scale gene network studies of postmortem human AD brain. Using transgenic or viral approaches, we characterized in mice the effects of TYROBP deficiency on the phenotypic and pathological evolution of tauopathy. Biomarkers usually associated with worsening clinical phenotype (i.e., hyperphosphorylation and increased tauopathy spreading) were unexpectedly increased in MAPTP301S;Tyrobp-/- mice despite the improved learning behavior and synaptic function relative to controls with normal levels of TYROBP. Notably, levels of complement cascade initiator C1q were reduced in MAPTP301S;Tyrobp-/- mice, consistent with the prediction that C1q reduction exerts a neuroprotective effect. These observations suggest a model wherein TYROBP-KO-(knock-out)-associated reduction in C1q is associated with normalized learning behavior and electrophysiological properties in tauopathy model mice despite a paradoxical evolution of biomarker signatures usually associated with neurological decline.

Integrative approach to sporadic Alzheimer's disease: deficiency of TYROBP in cerebral Aß amyloidosis mouse normalizes clinical phenotype and complement subnetwork molecular pathology without reducing Aß burden.

Integrative gene network approaches enable new avenues of exploration that implicate causal genes in sporadic late-onset Alzheimer's disease (LOAD) pathogenesis, thereby offering novel insights for drug-discovery programs. We previously constructed a probabilistic causal network model of sporadic LOAD and identified TYROBP/DAP12, encoding a microglial transmembrane signaling polypeptide and direct adapter of TREM2, as the most robust key driver gene in the network. Here, we show that absence of TYROBP/DAP12 in a mouse model of AD-type cerebral Aß amyloidosis (APPKM670/671NL/PSEN1Δexon9) recapitulates the expected network characteristics by normalizing the transcriptome of APP/PSEN1 mice and repressing the induction of genes involved in the switch from homeostatic microglia to disease-associated microglia (DAM), including Trem2, complement (C1qa, C1qb, C1qc, and Itgax), Clec7a and Cst7. Importantly, we show that constitutive absence of TYROBP/DAP12 in the amyloidosis mouse model prevented appearance of the electrophysiological and learning behavior alterations associated with the phenotype of APPKM670/671NL/PSEN1Δexon9 mice. Our results suggest that TYROBP/DAP12 could represent a novel therapeutic target to slow, arrest, or prevent the development of sporadic LOAD. These data establish that the network pathology observed in postmortem human LOAD brain can be faithfully recapitulated in the brain of a genetically manipulated mouse. These data also validate our multiscale gene networks by demonstrating how the networks intersect with the standard neuropathological features of LOAD.

Intestinal epithelial Notch-1 protects from colorectal mucinous adenocarcinoma.

Increasing evidence links Notch-1 signaling with the maintenance of intestinal architecture and homeostasis. Dysfunction in the common Notch-1 pathway transcription factor recombinant binding protein suppressor of hairless (RBP-J) is associated with loss of epithelial barrier integrity and aberrant conversion of proliferative crypt cells into goblet cells. Furthermore, we have recently discovered that epithelial Notch-1 is indispensable in bridging innate and adaptive immunity in the gut and is required for supporting protective epithelial pro-inflammatory responses. Yet, the epithelial specific function of Notch-1 in intestinal tumorigenesis remains unknown. We generated Villin-Cre/Notch-1 fl/fl (VN -/- ) mice that are selectively deficient in Notch-1 in intestinal epithelial cells. Intestinal epithelial Notch-1 preserved barrier function and integrity, whereas lack of epithelial Notch-1 induced goblet cell hyperplasia, spontaneous serrated lesions, multifocal low- and high-grade dysplasia and colonic mucinous neoplasms in mice. Over time, VN -/- mice displayed high occurrence of colorectal mucinous adenocarcinomas, which correlated with increased levels of mitogenic, angiogenic and pro-tumorigenic gene expression. Finally, we found that the expression of Notch-1 is significantly reduced in human colorectal mucinous adenocarcinoma when compared to colorectal adenocarcinoma. Taken together, our findings reveal a novel and critical protective role for Notch-1 in controlling intestinal tumorigenesis.

Multiscale Analysis of Independent Alzheimer's Cohorts Finds Disruption of Molecular, Genetic, and Clinical Networks by Human Herpesvirus.

Investigators have long suspected that pathogenic microbes might contribute to the onset and progression of Alzheimer's disease (AD) although definitive evidence has not been presented. Whether such findings represent a causal contribution, or reflect opportunistic passengers of neurodegeneration, is also difficult to resolve. We constructed multiscale networks of the late-onset AD-associated virome, integrating genomic, transcriptomic, proteomic, and histopathological data across four brain regions from human post-mortem tissue. We observed increased human herpesvirus 6A (HHV-6A) and human herpesvirus 7 (HHV-7) from subjects with AD compared with controls. These results were replicated in two additional, independent and geographically dispersed cohorts. We observed regulatory relationships linking viral abundance and modulators of APP metabolism, including induction of APBB2, APPBP2, BIN1, BACE1, CLU, PICALM, and PSEN1 by HHV-6A. This study elucidates networks linking molecular, clinical, and neuropathological features with viral activity and is consistent with viral activity constituting a general feature of AD.

Deficiency of TYROBP, an adapter protein for TREM2 and CR3 receptors, is neuroprotective in a mouse model of early Alzheimer's pathology.

Conventional genetic approaches and computational strategies have converged on immune-inflammatory pathways as key events in the pathogenesis of late onset sporadic Alzheimer's disease (LOAD). Mutations and/or differential expression of microglial specific receptors such as TREM2, CD33, and CR3 have been associated with strong increased risk for developing Alzheimer's disease (AD). DAP12 (DNAX-activating protein 12)/TYROBP, a molecule localized to microglia, is a direct partner/adapter for TREM2, CD33, and CR3. We and others have previously shown that TYROBP expression is increased in AD patients and in mouse models. Moreover, missense mutations in the coding region of TYROBP have recently been identified in some AD patients. These lines of evidence, along with computational analysis of LOAD brain gene expression, point to DAP12/TYROBP as a potential hub or driver protein in the pathogenesis of AD. Using a comprehensive panel of biochemical, physiological, behavioral, and transcriptomic assays, we evaluated in a mouse model the role of TYROBP in early stage AD. We crossed an Alzheimer's model mutant APP KM670/671NL /PSEN1 Δexon9 (APP/PSEN1) mouse model with Tyrobp -/- mice to generate AD model mice deficient or null for TYROBP (APP/PSEN1; Tyrobp +/- or APP/PSEN1; Tyrobp -/-). While we observed relatively minor effects of TYROBP deficiency on steady-state levels of amyloid-ß peptides, there was an effect of Tyrobp deficiency on the morphology of amyloid deposits resembling that reported by others for Trem2 -/- mice. We identified modulatory effects of TYROBP deficiency on the level of phosphorylation of TAU that was accompanied by a reduction in the severity of neuritic dystrophy. TYROBP deficiency also altered the expression of several AD related genes, including Cd33. Electrophysiological abnormalities and learning behavior deficits associated with APP/PSEN1 transgenes were greatly attenuated on a Tyrobp-null background. Some modulatory effects of TYROBP on Alzheimer's-related genes were only apparent on a background of mice with cerebral amyloidosis due to overexpression of mutant APP/PSEN1. These results suggest that reduction of TYROBP gene expression and/or protein levels could represent an immune-inflammatory therapeutic opportunity for modulating early stage LOAD, potentially leading to slowing or arresting the progression to full-blown clinical and pathological LOAD.