TREM2 Research Articles

Single-cell spatial transcriptomics reveals distinct patterns of dysregulation in non-neuronal and neuronal cells induced by the Trem2R47H Alzheimer's risk gene mutation.

The R47H missense mutation of the TREM2 gene is a known risk factor for development of Alzheimer's Disease. In this study, we analyze the impact of the Trem2R47H mutation on specific cell types in multiple cortical and subcortical brain regions in the context of wild-type and 5xFAD mouse background. We profile 19 mouse brain sections consisting of wild-type, Trem2R47H, 5xFAD and Trem2R47H; 5xFAD genotypes using MERFISH spatial transcriptomics, a technique that enables subcellular profiling of spatial gene expression. Spatial transcriptomics and neuropathology data are analyzed using our custom pipeline to identify plaque and Trem2R47H-induced transcriptomic dysregulation. We initially analyzecell type-specific transcriptomic alterations induced by plaque proximity. Next, we analyze spatial distributions of disease associated microglia and astrocytes, and how they vary between 5xFAD and Trem2R47H; 5xFAD mouse models. Finally, we analyze the impact of the Trem2R47H mutationon neuronal transcriptomes. The Trem2R47H mutation induces consistent upregulation of Bdnf and Ntrk2 across many cortical excitatory neuron types, independent of amyloid pathology. Spatial investigation of genotype enriched subclusters identified spatially localized neuronal subpopulations reduced in 5xFAD and Trem2R47H; 5xFAD mice. Overall, our MERFISH spatial transcriptomics analysis identifies glial and neuronal transcriptomic alterations induced independently by 5xFAD and Trem2R47H mutations, impacting inflammatory responses in microglia and astrocytes, and activity and BDNF signaling in neurons.

Abstract Tu021: Genome-wide CRISPR Screen In Vivo Decodes Monocyte Infiltration Associated With Abdominal Aortic Aneurysm

Introduction: Aortic aneurysm is the second most common aortic disease following atherosclerosis and accounts for the ninth-leading cause of death overall. The inflammatory response has a crucial role in the Abdominal Aortic Aneurysm (AAA) development. The infiltration of monocyte into aortic wall is a hallmark of aortic inflammation and it still requires further understanding of this complicated process. CRISPR screening is a powerful tool to investigate biological process, but it requires large pool of mutant cells and reliable selection procedure, which can usually only be performed in vitro or in xenograft models. To overcome this problem, we developed an unbiased genome-wide CRISPR-Cas Screen for Monocyte Infiltration in vivo (CRISPR-MI) and employed this new method in mouse AAA model to systematically study key genes regulating monocyte infiltration into aortic wall. Hypothesis: Monocyte infiltration is an important process in AAA development and CRISPR-MI could be utilized to study this process in vivo. Methods: To acquire enough monocytes for screen, we used the adoptive transfer method to transplant in vivo differentiated monocytes (bone marrow derived macrophage, BMDM) back into mouse circulation via tail vein injection. We optimized the lentivirus infection protocol, so that sgRNA library delivered by lentivirus can effectively knock out target genes in BMDMs. The BMDMs were infected with sgRNA library before adoptive transfer into AAA-model mice. After transfer, by comparing the sgRNA enrichment in different organs of receipt mice, we could identify key genes inhibiting monocyte infiltration. Results: CRISPR-MI identified novels genes regulating monocyte infiltration into aorta in AAA model, including Trem2 gene. In vitro experiments confirmed that Trem2 knockout increased adhesion molecules and chemokine receptors expression. Conclusions: CRISPR-MI can be used to identify new therapeutical targets for cardiovascular diseases.

Alzheimer's Disease in Lebanon: Exploring Genetic and Environmental Risk Factors-A Comprehensive Review.

Alzheimer's disease (AD) is a neurodegenerative condition that displays a high prevalence in Lebanon causing a local burden in healthcare and socio-economic sectors. Unfortunately, the lack of prevalence studies and clinical trials in Lebanon minimizes the improvement of AD patient health status. In this review, we include over 155 articles to cover the different aspects of AD ranging from mechanisms to possible treatment and management tools. We highlight some important modifiable and non-modifiable risk factors of the disease including genetics, age, cardiovascular diseases, smoking, etc. Finally, we propose a hypothetical genetic synergy model between APOE4 and TREM2 genes which constitutes a potential early diagnostic tool that helps in reducing the risk of AD based on preventative measures decades before cognitive decline. The studies on AD in Lebanon and the Middle East are scarce. This review points out the importance of genetic mapping in the understanding of disease pathology which is crucial for the emergence of novel diagnostic tools. Hence, we establish a rigid basis for further research to identify the most influential genetic and environmental risk factors for the purpose of using more specific diagnostic tools and possibly adopting a local management protocol.

Unveiling macrophage diversity in myocardial ischemia-reperfusion injury: identification of a distinct lipid-associated macrophage subset.

Macrophages play a crucial and dichotomous role cardiac repair following myocardial ischemia-reperfusion, as they can both facilitate tissue healing and contribute to injury. This duality is intricately linked to environmental factors, and the identification of macrophage subtypes within the context of myocardial ischemia-reperfusion injury (MIRI) may offer insights for the development of more precise intervention strategies. Specific marker genes were used to identify macrophage subtypes in GSE227088 (mouse single-cell RNA sequencing dataset). Genome Set Enrichment Analysis (GSEA) was further employed to validate the identified LAM subtypes. Trajectory analysis and single-cell regulatory network inference were executed using the R packages Monocle2 and SCENIC, respectively. The conservation of LAM was verified using human ischemic cardiomyopathy heart failure samples from the GSE145154 (human single-cell RNA sequencing dataset). Fluorescent homologous double-labeling experiments were performed to determine the spatial localization of LAM-tagged gene expression in the MIRI mouse model. In this study, single-cell RNA sequencing (scRNA-seq) was employed to investigate the cellular landscape in ischemia-reperfusion injury (IRI). Macrophage subtypes, including a novel Lipid-Associated Macrophage (LAM) subtype characterized by high expression of Spp1, Trem2, and other genes, were identified. Enrichment and Progeny pathway analyses highlighted the distinctive functional role of the SPP1+ LAM subtype, particularly in lipid metabolism and the regulation of the MAPK pathway. Pseudotime analysis revealed the dynamic differentiation of macrophage subtypes during IRI, with the activation of pro-inflammatory pathways in specific clusters. Transcription factor analysis using SCENIC identified key regulators associated with macrophage differentiation. Furthermore, validation in human samples confirmed the presence of SPP1+ LAM. Co-staining experiments provided definitive evidence of LAM marker expression in the infarct zone. These findings shed light on the role of LAM in IRI and its potential as a therapeutic target. In conclusion, the study identifies SPP1+ LAM macrophages in ischemia-reperfusion injury and highlights their potential in cardiac remodeling.

TREM2 gene induces differentiation of induced pluripotent stem cells into dopaminergic neurons and promotes neuronal repair via TGF-β activation in 6-OHDA-lesioned mouse model of Parkinson's disease.

Induced pluripotent stem cells (iPSCs) hold a promising potential for rescuing dopaminergic neurons in therapy for Parkinson's disease (PD). This study clarifies a TREM2-dependent mechanism explaining the function of iPSC differentiation in neuronal repair of PD. PD-related differentially expressed genes were screened by bioinformatics analyses and their expression was verified using RT-qPCR in nigral tissues of 6-OHDA-lesioned mice. Following ectopic expression and depletion experiments in iPSCs, cell differentiation into dopaminergic neurons as well as the expression of dopaminergic neuronal markers TH and DAT was measured. Stereotaxic injection of 6-OHDA was used to develop a mouse model of PD, which was injected with iPSC suspension overexpressing TREM2 to verify the effect of TREM2 on neuronal repair. TREM2 was poorly expressed in the nigral tissues of 6-OHDA-lesioned mice. In the presence of TREM2 overexpression, the iPSCs showed increased expression of dopaminergic neuronal markers TH and DAT, which facilitated the differentiation of iPSCs into dopaminergic neurons. Mechanistic investigations indicated that TREM2 activated the TGF-β pathway and induced iPSC differentiation into dopaminergic neurons. Invivo data showed that iPSCs overexpressing TREM2 enhanced neuronal repair in 6-OHDA-lesioned mice. This work identifies a mechanistic insight for TREM2-mediated TGF-β activation in the regulation of neuronal repair in PD and suggests novel strategies for neurodegenerative disorders.

Molecular basis of dementia

Dementia is a set of symptoms characterized by deterioration of memory and cognitive functions. Dementia diseases include Alzheimer's disease, dementia with Lewy bodies, frontotemporal dementia, vascular dementia, and mixed dementia. This disease represents an escalating social issue, particularly in a society with an increasing elderly population. In 2019, 271,998 people succumbed to dementia, making Alzheimer's disease the sixth most prevalent cause of death. The pathophysiology of Alzheimer's disease is complex and not fully understood. It is a multifaceted disease, with its pathogenesis influenced by a combination of genetic, environmental, and lifestyle factors. One of the genes involved in the pathogenesis of the disease is the apolipoprotein E (APOE) gene, which is one of the most common risk factors for Alzheimer's disease. The significance of other genes, including presenilin genes (PSEN1 and PSEN2), the TREM2 gene, the MAPT gene, and the APP gene, linked to various forms of dementia, is also emphasized. Another issue is the growing number of identified genetic variants within genes implicated in the onset of dementia. Dementia diseases are also characterized by chemical alterations in the brain, including the accumulation of abnormal excitotoxic proteins, varying degrees of inflammation, and metabolic disorders. This review summarizes current research in the field of dementia and highlights the significance of molecular factors in its pathogenesis. Gaining insight into the pathogenic mechanisms of dementia may allow for faster diagnosis of the disease and facilitate the creation of more efficient patient care plans.

Analysis of macrophage polarization and regulation characteristics in ovarian tissues of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) can lead to infertility and increase the risk of endometrial cancer. Analyzing the macrophage polarization characteristics in ovarian tissues of PCOS is crucial for clinical treatment. We obtained 13 PCOS and nine control ovarian samples from the CEO database and analyzed differentially expressed genes (DEGs). Macrophage polarization-related genes (MPRGs) were sourced from the GeneCards and MSigDB databases. Intersection of DEGs with MPRGs identified DEGs associated with macrophage polarization (MPRDEGs). Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein-protein interaction (PPI) Network analysis were conducted on MPRDEGs. Moreover, the top 10 genes from three algorithms were identified as the hub genes of MPRGs. In addition, miRNAs, transcription factors (TFs), and drugs were retrieved from relevant databases for regulatory network analysis of mRNA-miRNA, mRNA-TF, and mRNA-Drug interactions. Immune cell composition analysis between the PCOS and control groups was performed using the CIBERSORT algorithm to calculate correlations across 22 immune cell types. A total of 13 PCOS samples and nine control ovarian samples were obtained in this study. We identified 714 DEGs between the two groups, with 394 up-regulated and 320 down-regulated. Additionally, we identified 774 MPRGs, from which we derived 30 MPRDEGs by intersecting with DEGs, among which 21 exhibited interaction relationships. GO and KEGG analyses revealed the enrichment of MPRDEGs in five biological processes, five cell components, five molecular functions, and three biological pathways. Immune infiltration analysis indicated a strong positive correlation between activated nature killer (NK) cells and memory B cells, while neutrophils and monocytes showed the strongest negative correlation. Further investigation of MPRDEGs identified nine hub genes associated with 41 TFs, 82 miRNAs, and 44 drugs or molecular compounds. Additionally, qRT-PCR results demonstrated overexpression of the CD163, TREM1, and TREM2 genes in ovarian tissues from the PCOS group. This study elucidated the polarization status and regulatory characteristics of macrophages in ovarian tissues of the PCOS subjects, confirming significant overexpression of CD163, TREM1, and TREM2. These findings contribute to a deeper understanding of the pathogenesis of PCOS.

Elovanoids and Precursor Free Fatty Acid 34:6 Confer Robust Neuroprotection after Experimental Traumatic Brain Injury

One-third of the close to 70 million people suffering from traumatic brain injury (TBI) have an increased risk for developing chronic cognitive impairments, including Alzheimer’s-like Dementia (AD). We studied neuroprotection by Elovanoid-34:6 (ELV-N34) and its precursor free-fatty- acid-34:6 (FFA34:6) following TBI in male Sprague-Dawley rats using fluid percussion injury (FPI) or controlled cortical impact (CCI) models. At 1 and 24 hours post-surgery, rats were intranasally administered saline, ELV-N34, or FFA34:6 (only in the CCI model). Rats underwent composite neurological testing to assess sensorimotor integration. On day 14, FPI animals were sacrificed, and MRI and diffuse tensor imaging (DTI) were performed to assess lesion size and white matter connectivity. On day 3, CCI animals were used for multi-omic analysis using 10x Chromium, Illumina Sequencing, and Seurat v4.0.1. Total neurological scores for animals administered ELV-N34 and FFA34:6 were lower than controls, and scores for FFA34:6 treated rats were lower than those of ELV34:6 treated rats (p<0.05). MRI and DTI showed smaller lesions and preserved white matter connectivity in the ELV34:6 group. Single cell analysis with ELV-N34 treatment revealed downregulation of Spp1, Fos, and Gfap genes and upregulation of Trem2 and Pld5 genes. FFA34:6 treatment revealed downregulation of inflammatory genes Spp1, Ccl2, and Vim. These changing gene cluster patterns are implicated in neurodegenerative diseases. We demonstrated that ELV-N34 and FFA34:6 convey behavioral, structural, and genetic neuroprotective bioactivity in experimental TBI.

The influence of rs75932628 and rs2234253 polymorphisms of the TREM2 gene and the mTOR signaling pathway in the development of Alzheimer's disease

Annotation. According to the data of the World Health Organization, as of March 15, 2023, more than 55 million cases of dementia disorders were recorded in the world. Among them, Alzheimer’s disease remains a global medical challenge, which is particularly relevant for low- and middle-income countries. This pathology already ranks 7th among the causes of death among the elderly population and has a significant social and economic impact. The purpose of the study – to determine the role of the TREM2 gene and its polymorphisms, in particular rs75932628 and rs2234253, in susceptibility to Alzheimer’s disease. To analyze the role of the mTOR signaling pathway in the neurodegeneration of microglia. Literature sources were searched using the key words “Alzheimer’s disease”, “microglia”, “dementia”, etc. The study was conducted based on an interactive group approach using a comprehensive literature review in the Web of Science, Scopus, Pubmed, Elsevier, and Springer databases. As a result of the analysis, it was found that one of the key participants in the process of neuroinflammation is microglia, which plays an important role in phagocytosis, transmission of neuronal signals and local immune response. The TREM2 gene, which normally promotes the phagocytosis of β-amyloid protein, was found to be a significant factor associated with dementia. Due to its effect on galectin-3, it causes the activation of microglia, which is of key importance in the development of neurodegenerative diseases. Another important factor turned out to be the mTOR signaling pathway, which is considered a key regulator of the main cellular and metabolic processes in microglia, however, the data of practical works on this pathway remain controversial. Thus, the study confirms the importance of the TREM2 gene and the mTOR signaling pathway in the development of Alzheimer's disease, which is related to their effects on microglia. The obtained results can be used for further research and development of strategies for the treatment of Alzheimer's disease.

TREM2 mediated A clearance dysfunction in microglia cells by circadian disruption in Alzheimer's disease

Genome-wide association studies (GWAS) have shown that the Trem2 gene is a risk factor associated with the onset of Alzheimer's Disease (AD). The Trem2 gene is highly expressed on the surface membrane of microglia cells in the central neuron system (CNS), which mediates neuron inflammation and the clearance of axons and synapses. As a growing amount of evidence is connecting Circadian disruptions with increased levels of CNS inflammation, this review aims to find a connection between circadian disruption and amyloid-leading Alzheimers disease through Trem2 in Microglia cells. This review summarizes past research independently about the circadian disruption with microglia activation, microglia activation with Trem2, and Trem2-affected MG cells impacting the development of Alzheimers Disease. Further research may focus on specifying the importance of Trem2 microglia cells caused neuron inflammation in the development of Alzheimer's pathology, for whether the early A-beta plaquing is triggered by Trem2 microglia cells, or Trem2 is just contributing but not the causation of AD in its Mild Cognitive impairment (MCI) stage.

Aging × Genetics × Environment: Characterization of Precision Disease Models for Preclinical Testing for Late‐onset Alzheimer’s disease

AbstractBackgroundThe ability to effectively translate therapeutic efficacy from the bench to clinical success for Alzheimer’s disease (AD) has been hampered in part due to limited recapitulation of the complexity of the disease in animal models. While analogous AD risk mutations have been engineered into animal models and have dominated the research field, these have primarily been familial, early onset risk alleles which do not capture the risk for AD for the majority of patients that present with sporadic late onset AD (LOAD).MethodThe IU/JAX/PITT MODEL‐AD consortium is focused on developing mouse models with genetic risk variants associated with LOAD, in combination with environmental risk factors and aging to enable improved translation. The present studies aimed to characterize mice expressing humanized Aβ in combination with multiple genetic risk factors (APOE4 and the R47H risk variant in the Trem2 gene; LOAD2) and aged in the presence of a high‐fat, high‐sugar diet (HFD).ResultLOAD2 mice exposed to HFD from adolescence (LOAD2+HFD) demonstrated aging changes relative to LOAD2 mice in the absence of HFD, including presentation of insoluble Aβ42 in brain and plasma, and increased inflammatory cytokines. 12‐month aged LOAD2+HFD mice also demonstrated increased NfL in CSF, as well as vascular and perfusion changes as measured by PET/MRI. By 18 months, LOAD2+HFD mice demonstrated reductions in hippocampal neurons as well as cognitive impairment relative to LOAD2 mice in the absence of HFD on a translational touchscreen task. Intriguingly, gene expression profiles and proteomic signatures of aged LOAD2+HFD mice aligned with ‘omics signatures of AD patients in the absence of core neuritic plaques, which were not detected up to 24 months of age.ConclusionMice with genetic risk for LOAD coupled with environmental risk factors demonstrate aging‐dependent changes in line with a spectrum and trajectory of features of clinical LOAD. From a precision medicine approach, our MODEL‐AD Preclinical Testing Core and our TREAT‐AD colleagues are prioritizing LOAD2+HFD mice as an important model system for evaluating the therapeutic potential of non‐amyloid targeting therapeutics as well as for prophylactic interventions initiated prior to significant amyloid accumulation.

Effects of homo‐ and heterozygous TREM2 mutations on natural killer cell activity

AbstractBackgroundNasu‐Hakola disease (NHD) is a rare genetic neurodegenerative disorder characterised by progressive presenile dementia and bone cysts. Dysfunctions in myeloid‐derived innate immune cells such as microglia due to loss‐of‐function mutations in the TREM2 (trigger receptor 2 expressed on myeloid cells) gene cause the clinical manifestations. Heterozygous carriers have strongly increased risk of developing Alzheimer’s Disease but do not present with NHD symptoms. TREM2 is associated with DAP12 protein, which contains an immunoreceptor tyrosine‐based activation motif that is structurally similar to natural killer (NK) cell receptors. In this study, we aimed to analyse the effects of TREM2 mutation and its effect on the peripheral immune system represented by NK cell functions.MethodSix patients with NHD with a defined homozygous TREM2 mutation, 7 carriers with heterozygous TREM2 mutations and 10 age‐ and gender‐matched controls were included. NK cell subsets, KIR expressions (NKG2A, NKG2D, NKp46), cytotoxic activity (CD107a, Perforin, Granzyme A) and cytokine secretions (IL‐4, IFN‐γ, TNF‐α, IL‐10, IL‐17, TGF‐β) were analysed by flow cytometry.ResultCD3−CD56dimCD16− (p = 0.003) subsets were found to be increased in the NHD group compared to carriers. Activating C‐type lectin receptor NKG2D expression was increased in CD8+ (p = 0.003) NK cell subsets in the NHD group. Inhibiting C‐type lectin receptor NKG2A expression was increased in total (p = 0.011) as well as CD8+ (p = 0.007) NK cell subsets in carriers compared to controls. In total NK cells, intracellular TNF‐α (p = 0.005) and TGF‐β (p = 0.013) levels were decreased, IL‐17 (p = 0.05) and IL‐4 (p = 0.005) levels were increased in the NHD group. When NK cells were grouped due to their CD8 expression, TNF‐α levels were decreased, and IL‐17 levels were increased in both subsets in the NHD group. Cytotoxic activity showed no differentiation in both unstimulated and K562 stimulated conditions between groups.ConclusionUnlike previous case series, NK cell ratios did not differ between groups. Over‐expression of inhibiting receptors in the carriers group, unlike high levels of activating receptors in the NHD, might explain the lack of symptoms in the carriers. The increased ratio of CD3−CD56dimCD16− NK cell subsets that overexpress inhibitory checkpoints might explain the low levels of pro‐inflammatory cytokines in the NHD group.

Pathogenic characteristics of genetically modified cells with TREM2 T44A mutation

AbstractBackgroundTriggering Receptor Expressed on Myeloid Cells‐2 (TREM2) is known as a risk gene for Alzheimer’s disease (AD). and affect AD pathogenesis through beta‐amyloid (Aβ)‐related neuroinflammations and phagocytosis. TREM2 R47H mutation was the first reported from an AD patient in 2013. Although the TREM2 R47H variant was revealed to increase the risk of AD by three times, further mutations still need to study. Here, we found TREM2 T44A mutation from a patient of a 65‐year‐old AD patient. In order to confirm the pathogenicity of TREM2 T44A, the genetically modified cells were produced and investigated the association of the TREM2 gene with Aβ‐related pathology.MethodTREM2 Wild type (WT) and T44A were transfected onto the HEK 293, SHSY5Y, U87 cells by using a plasmid transfection system and selected using geneticin (G418). Gene expression and TREM2 protein was measured. For measuring Aβ uptakes, Aβ was measured after incubation with cells. Cell phagocytosis was conducted with treating pHrodo labeled with Aβ and obtained cell images and video.ResultTREM2 gene was overexpressed in transfected cells and TREM2 protein was elevated, but TREM2 T44A lowered compared to WT. TREM2 T44A showed higher Aβ42/40 ratio, with increasing extracellular Aβ42. The Aβ42 uptake were relatively lowered than TREM2 WT. Through images and video, TREM2 T44A showed lower red fluorescence than WT, but similar to MOCK.ConclusionIn this study, we transfected TREM2 WT and T44A in a variety of cell lines (HEK 293, SHSY5Y, U87) to investigate the pathogenicity of TREM2 T44A. TREM2 T44A was shown to increase extracellular Aβ42 and decrease Aβ42 uptake. This suggests that the Aβ uptake and elimination function of TREM2 was loosed by the mutation, and therefore Aβ was not completely removed, resulting in a slight increase in extracellular Aβ in the mutant cell. The loss of the Aβ uptake correlated with confocal and video results, the red fluorescence signals were decreased in TREM2 T44A, suggesting relatively lower Aβ phagocytosis. Thus, Aβ phagocytosis was inhibited by TREM2 mutation, and the lowered Aβ uptake could not clear it, suggesting that Aβ growth and accumulation may influence the pathogenicity of AD.

Identification of RS2234253 T96K polymorphism of TREM2 gene in Tunisian Alzheimer's disease population

Identification of RS2234253 T96K polymorphism of TREM2 gene in Tunisian Alzheimer's disease population

Docosahexaenoic Acid (DHA) Reduces LPS-Induced Inflammatory Response Via ATF3 Transcription Factor and Stimulates Src/Syk Signaling-Dependent Phagocytosis in Microglia.

Microglial cells play a crucial role in the development of neuroinflammation in response to harmful stimuli, such as infection, ischemia or injury. Their chronic activation, however, is associated with a progression of neurodegenerative diseases. Therefore, looking for potential factors limiting microglial activation, the effect of docosahexaenoic acid (DHA) on the inflammatory response and TREM2-dependent phagocytic activity in microglia was investigated. In LPS-induced primary microglia preincubated with DHA, or without preincubation the expression of ATF3 and TREM2 genes and TREM2, Syk, Akt proteins were determined by RT-PCR and WB, respectively. Cell viability was assayed by MTT and cytokine and chemokine expression was determined by the Proteome Profiler assay. Moreover, the phagocytic activity of microglia was assayed using immunofluorescence. We found that DHA significantly increased the expression of ATF3 , and decreased the levels of CINC-1, CINC-2αβ, CINC-3 chemokines, IL-1α and IL-1β cytokines, and ICAM-1 adhesion protein. Additionally, preincubation of microglia with DHA resulted in increased Src/Syk kinases activation associated with increased phagocytic microglia activity. These findings indicate that DHA efficiently inhibits ATF3-dependent release of proinflammatory mediators and enhances phagocytic activity of microglia. The study provides a new mechanism of DHA action in reactive microglia, which may help limit neuronal damage caused by the pro-inflammatory milieu in the brain.

STREM2 is a plasma biomarker for human NASH and promotes hepatocyte lipid accumulation.

Pathogenetic mechanisms of the progression of NAFL to advanced NASH coupled with potential noninvasive biomarkers and novel therapeutic targets are active areas of investigation. The recent finding that increased plasma levels of a protein shed by myeloid cells -soluble Triggering Receptor Expressed on Myeloid cells 2 (sTREM2) -may be a biomarker for NASH has received much interest. We aimed to test sTREM2 as a biomarker for human NASH and investigate the role of sTREM2 in the pathogenesis of NASH. We conducted studies in both humans (comparing patients with NASH vs. NAFL) and in mice (comparing different mouse models of NASH) involving measurements of TREM2 gene and protein expression levels in the liver as well as circulating sTREM2 levels in plasma. We investigated the pathogenetic role of sTREM2 in hepatic steatosis using primary hepatocytes and bone marrow derived macrophages. RNA sequencing analysis of livers from patients with NASH or NAFL as well as livers from 2 mouse models of NASH revealed elevated TREM2 expression in patients/mice with NASH as compared with NAFL. Plasma levels of sTREM2 were significantly higher in a well-characterized cohort of patients with biopsy-proven NASH versus NAFL (area under receiver-operating curve 0.807). Mechanistic studies revealed that cocultures of primary hepatocytes and macrophages with an impaired ability to shed sTREM2 resulted in reduced hepatocyte lipid droplet formation on palmitate stimulation, an effect that was counteracted by the addition of exogenous sTREM2 chimeric protein. Conversely, exogenous sTREM2 chimeric protein increased lipid droplet formation, triglyceride content, and expression of the lipid transporter CD36 in hepatocytes. Furthermore, inhibition of CD36 markedly attenuated sTREM2-induced lipid droplet formation in mouse primary hepatocytes. Elevated levels of sTREM2 due to TREM2 shedding may directly contribute to the pathogenesis of NAFLD by promoting hepatocyte lipid accumulation, as well as serving as a biomarker for distinguishing patients with NASH versus NAFL. Further investigation of sTREM2 as a clinically useful diagnostic biomarker and of the therapeutic effects of targeting sTREM2 in NASH is warranted.

Inflammation and Brain Structure in Alzheimer's Disease and Other Neurodegenerative Disorders: a Mendelian Randomization Study.

Previous in vitro and post-mortem studies have reported the role of inflammation in neurodegenerative disorders. However, the association between inflammation and brain structure in vivo and the transcriptome-driven functional basis with relevance to neurodegenerative disorders remains elusive. The aim of the present study is to identify the association among inflammation, brain structure, and neurodegenerative disorders at genetic and transcriptomic levels. Genetic variants associated with inflammatory cytokines were selected from the latest and largest genome-wide association studies of European ancestry. Neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and dementia with Lewy bodies (DLB) and brain structure imaging measures were selected as the outcomes. Two-sample Mendelian randomization analyses were conducted to identify the causal associations. Single-nucleus transcriptome data of the occipitotemporal cortex was further analyzed to identify the differential expressed genes in AD, which were tested for biological processes and protein interaction network. MR analysis indicated that genetically predicted TREM2 and sTREM2 were significantly associated with AD (TREM2: z-score = -9.088, p-value = 1.02 × 10-19; sTREM2: z-score = -7.495, p-value = 6.61 × 10-14). The present study found no evidence to support the causal associations between other inflammatory cytokines and the risks of AD, PD, ALS, or DLB. Genetically predicted TREM2 was significantly associated with the cortical thickness of inferior temporal (z-score = -4.238, p-value = 2.26 × 10-5) and pole temporal (z-score = -4.549, p-value = 5.40 × 10-6). In the occipitotemporal cortex samples, microglia were the main source of TREM2 gene and showed increasing expression of genes associated with inflammation and immunity. The present study has leveraged genetic and transcriptomic data to identify the association among TREM2, temporal lobe, and AD and the underlying cellular and molecular basis, thus providing a new perspective on the role of TREM2 in AD and insights into the complex associations among inflammation, brain structure, and neurodegenerative disorders, particularly AD.

Interrogation of human microglial phagocytosis by CRISPR genome editing.

Microglia are an integral part of central nervous system, but our understanding of microglial biology is limited due to the challenges in obtaining and culturing primary human microglia. HMC3 is an important cell line for studying human microglia because it is readily accessible and straightforward to maintain in standard laboratories. Although HMC3 is widely used for microglial research, a robust genetic method has not been described. Here, we report a CRISPR genome editing platform, by the electroporation of Cas9 ribonucleoproteins (Cas9 RNP) and synthetic DNA repair templates, to enable rapid and precise genetic modifications of HMC3. For proof-of-concept demonstrations, we targeted the genes implicated in the regulation of amyloid beta (Aβ) and glioblastoma phagocytosis in microglia. We showed that CRISPR genome editing could enhance the phagocytic activities of HMC3. We performed CRISPR gene knockout (KO) in HMC3 by the electroporation of pre-assembled Cas9 RNP. Co-introduction of DNA repair templates allowed site-specific knock-in (KI) of an epitope tag, a synthetic promoter and a fluorescent reporter gene. The editing efficiencies were determined genotypically by DNA sequencing and phenotypically by immunofluorescent staining and flow cytometry. The gene-edited HMC3 cells were examined in vitro by fluorescent Aβ and glioblastoma phagocytosis assays. Our platform enabled robust single (>90%) and double (>70%) KO without detectable off-target editing by high throughput DNA sequencing. We also inserted a synthetic SFFV promoter to efficiently upregulate the expression of endogenous CD14 and TREM2 genes associated with microglial phagocytosis. The CRISPR-edited HMC3 showed stable phenotypes and enhanced phagocytosis of fluorescence-labeled Aβ1-42 peptides. Confocal microscopy further confirmed the localization of Aβ1-42 aggregates in the acidified lysosomes. HMC3 mutants also changed the phagocytic characteristic toward apoptotic glioblastoma cells. CRISPR genome editing by Cas9 RNP electroporation is a robust approach to genetically modify HMC3 for functional studies such as the interrogation of Aβ and tumor phagocytosis, and is readily adoptable to investigate other aspects of microglial biology.

Soluble TREM2 Concentrations in the Cerebrospinal Fluid Correlate with the Severity of Neurofibrillary Degeneration, Cognitive Impairment, and Inflammasome Activation in Alzheimer's Disease.

Individuals with specific TREM2 gene variants that encode for a Triggering Receptor Expressed on Myeloid cells 2 have a higher prevalence of Alzheimer's disease (AD). By interacting with amyloid and apolipoproteins, the TREM2 receptor regulates the number of myeloid cells, phagocytosis, and the inflammatory response. Higher TREM2 expression has been suggested to protect against AD. However, it is extremely difficult to comprehend TREM2 signaling in the context of AD. Previous results are variable and show distinct effects on diverse pathological changes in AD, differences between soluble and membrane isoform signaling, and inconsistency between animal models and humans. In addition, the relationship between TREM2 and inflammasome activation pathways is not yet entirely understood. This study aimed to determine the relationship between soluble TREM2 (sTREM2) levels in cerebrospinal fluid (CSF) and plasma samples and other indicators of AD pathology. Using the Enzyme-Linked Immunosorbent Assay (ELISA), we analyzed 98 samples of AD plasma, 35 samples of plasma from individuals with mild cognitive impairment (MCI), and 11 samples of plasma from healthy controls (HC), as well as 155 samples of AD CSF, 90 samples of MCI CSF, and 50 samples of HC CSF. CSF sTREM2 levels were significantly correlated with neurofibrillary degeneration, cognitive decline, and inflammasome activity in AD patients. In contrast to plasma sTREM2, CSF sTREM2 levels in the AD group were higher than those in the MCI and HC groups. Moreover, concentrations of sTREM2 in CSF were substantially higher in the MCI group than in the HC group, indicating that CSF sTREM2 levels could be used not only to distinguish between HC and AD patients but also as a biomarker to detect earlier changes in the MCI stage. The results indicate CSF sTREM2 levels reliably predict neurofibrillary degeneration, cognitive decline, and inflammasome activation, and also have a high diagnostic potential for distinguishing diseased from healthy individuals. To add sTREM2 to the list of required AD biomarkers, future studies will need to include a larger number of patients and utilize a standardized methodology.

Danggui-Shaoyao-San Promotes Amyloid-β Clearance through Regulating Microglia Polarization via Trem2 in BV2 Cells.

Alzheimer's disease (AD) is a chronic neurodegenerative brain disorder currently without satisfactory therapeutic treatments. Triggering receptors expressed on a myeloid cells-2 (Trem2) gene mutation has been reported as a powerful AD risk factor that induces Trem2 gene deletion aggravated microglia disfunction and Amyloid-β (Aβ) aggregation in the brain. The traditional Chinese medicine (TCM) formula Danggui-Shaoyao-San (DSS) has shown therapeutic effect on alleviating the symptoms of AD. However, the neuroprotective effect and underlying mechanism of DSS against AD is still far from fully understood. Double-label immunofluorescence and Western blotting were employed to evaluate the different polarization states of mouse BV2 microglial (BV2) cells after lipopolysaccharide (LPS) or interleukin (IL)-4 treatment. Trem2 over-expression lentiviral vector and Trem2 siRNA were used respectively to evaluate the effect of Trem2 on microglia polarization via detecting the proteins expression of iNOS and arginase1 (Arg1) by Western blotting while the Aβ-scavenging capacity of BV2 cells was assessed by flow cytometry. Cell counting kit-8 (CCK8) assay was performed to assess the effect of DSS on the viability of BV2 cells. Flow cytometry was used to investigate the effect of DSS on the Aβ-scavenging capacity of BV2 cells treated with corresponding concentration of DSS-containing serum. Protein of Trem2 and the gene expression of the M1 or M2 phenotype in BV2 cells treated with DSS after Trem2 over-expression or silence were detected by Western blot and RT-qPCR, respectively. In vitro experiments. DSS exhibited anti-inflammatory and neuroprotective functions. It was found that Trem2 had an effect on inducing a shift of M1 microglia towards the M2 phenotype and enhanced the Aβ-scavenging capacity of BV2 cells, further that DSS administration relieved inflammation by engulfing Aβ through the activities of Trem2. Importantly, DSS treatment effectively increased the Aβ-scavenging capacity of BV2 cells through accelerating the shift of M1 microglia towards an M2 phenotype via increasing Trem2 expression. Results demonstrated that DSS promoted the clearance of Aβ through the regulation of microglia polarization via increased expression of Trem2 in BV2 cells.