TREM2 Regulates Microglial Cholesterol Metabolism upon Chronic Phagocytic Challenge

In the paper

“Microglial dysfunction is a hallmark of the aging brain, as cells accumulate excessive myelin debris and acquire a DAM transcriptional state”

What the sources say

• Partly supported

  Section 2 describes age-related microglial deterioration, and Section 3.3 describes a distinct gene-expression signature and a shift toward a neurodegenerative phenotype in aging. But Section 3.2 reports myelin-debris accumulation in a cuprizone demyelination model with defective TREM2 function, not as a general feature of the aging brain. The paper does not identify the aged-cell signature as a DAM state. (LLM, conf=0.88)

  cites Mecca et al., 2018 sourceExperimental — accuracy not yet measured
• Partly supported

  The paper’s Abstract reports that age-related myelin fragmentation produces lysosomal inclusions in microglia and contributes to microglial senescence and immune dysfunction. It also reports gene-expression changes in aged microglia, but does not identify those changes as a DAM transcriptional state. No page number is available in our copy. (LLM, conf=0.88)

  cites Safaiyan et al., 2016 sourceExperimental — accuracy not yet measured
• Partly supported

  The Results report a DAM population in aged mice that was not detected in young mice, supporting an association between aging and a DAM transcriptional state. The paper does not report accumulation of myelin debris, and the Discussion describes DAM as potentially protective rather than establishing microglial dysfunction as a hallmark of aging. Page numbers were not provided in our copy. (LLM, conf=0.88)

  cites Keren-Shaul et al., 2017 sourceExperimental — accuracy not yet measured

In the paper

“DAM 1 genes were upregulated in Trem2+/+ CPZ, Trem2+/– CPZ, and Trem2–/– CPZ microglia, although Trem2–/– showed reduced upregulation.”

What the sources say

• Partly supported

  The Results report that stage-1 DAM genes, including Apoe and B2m, are upregulated through a Trem2-independent process in 5XFAD mice, including Trem2 knockouts. They do not report this result in CPZ-treated microglia or establish the claimed pattern across Trem2+/+, Trem2+/−, and Trem2−/− CPZ groups, including reduced upregulation in the knockout. (LLM, conf=0.85)

  cites Keren-Shaul et al., 2017 sourceExperimental — accuracy not yet measured

In the paper

“Myelin contains the majority (>80%) of the brain’s free cholesterol”

What the sources say

• Partly supported

  The Introduction says a large fraction of brain cholesterol is present in the myelin sheath. It does not specify that the fraction exceeds 80% or that the statement concerns free cholesterol; searches for those details found no supporting passage. Our copy provides no page number for the passage. (LLM, conf=0.86)

  cites Martín et al., 2014 sourceExperimental — accuracy not yet measured

In the paper

“Upon lipid ligand binding, TREM2 recruits SYK via DAP12’s ITAM domain, leading to SYK phosphorylation”

What the sources say

• Partly supported

  The DAP12 section says phosphorylated tyrosines in its ITAM recruit SYK and/or Zap70, and the TREM-2 section describes signaling through DAP12. It does not identify the ligand as a lipid or report that ligand binding leads to SYK phosphorylation; the phosphorylation it describes is of DAP12’s ITAM tyrosines. (LLM, conf=0.87)

  cites Hamerman et al., 2006 sourceExperimental — accuracy not yet measured

In the paper

“Although a link has been established between TREM2 and lipid metabolism, previous studies have focused primarily on lipids as candidate TREM2 ligands”

What the sources say

• Supported

  In the ‘ApoA1 and ApoB Also Bind to TREM2’ section (page not provided), the paper reports that TREM2 binds apoE, apoA1, and apoB, and identifies apoE and apoA1 as components of HDL. These findings support the broad claim of a link between TREM2 and lipid metabolism; the claim does not assert that TREM2 directly regulates metabolism. (LLM, conf=0.87)

  cites Atagi et al., 2015 source
• ?Not checked — no full text of the sourcecites Bailey et al., 2015 source
• Partly supported

  The section on R47H lipid binding reports that TREM2 binds phospholipids and that phosphatidylserine stimulation affects TREM2 signaling. This establishes a link to lipids, but the paper does not establish a role for TREM2 in lipid metabolism specifically. (LLM, conf=0.83)

  cites Sudom et al., 2018 sourceExperimental — accuracy not yet measured

In the paper

“previous studies have focused primarily on lipids as candidate TREM2 ligands, in the form of either lipoprotein particles or cell surface-exposed signals”

What the sources say

• Partly supported

  The Discussion (page unavailable) describes earlier work on anionic, zwitterionic, and myelin-associated lipids as TREM2 ligands. It also reports binding to lipidated apoE, which can form a lipoprotein particle, but presents that as this study's finding. The cited passages do not establish that previous studies focused primarily on lipids in lipoprotein particles or as cell surface-exposed signals. (LLM, conf=0.82)

  cites Atagi et al., 2015 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites Bailey et al., 2015 source
• Partly supported

  The Abstract reports anionic lipids among putative TREM2 ligands, and the section on R47H lipid binding describes TREM2 binding to several phospholipids. Our copy does not establish that previous studies focused primarily on lipids or describe those candidate ligands specifically as lipoprotein particles or cell surface-exposed signals. Page numbers were not supplied for these passages. (LLM, conf=0.82)

  cites Sudom et al., 2018 sourceExperimental — accuracy not yet measured

In the paper

“Importantly, genetic ablation and pharmacological inhibition of ACAT1, which lowers CE levels, is protective in amyloid and tauopathy models through various mechanisms, including autophagy activation”

What the sources say

• Partly supported

  Section 2.1 reports that ACAT ablation improved AD-like pathology and reduced Aβ42 in mice, while ACAT inhibitor treatment reduced amyloid plaques, brain cholesteryl esters (CE), and Aβ. The copy does not report protection in tauopathy models or identify autophagy activation as a mechanism; section 2.2 instead discusses possible effects on APP processing and says the mechanism remains uncertain. Page numbers were not available in the indexed passages. (LLM, conf=0.88)

  cites Bhattacharyya and Kovacs, 2010 sourceExperimental — accuracy not yet measured
• Partly supported

  The Results report that ACAT1 ablation reduced amyloid pathology in 3XTg-AD mice, and the Discussion reports reduced amyloid plaques after treatment with ACAT inhibitors. But the Results found no significant change in neurofibrillary tangles after ACAT1 ablation, suggesting no attenuation of tau pathology; the paper does not establish autophagy activation as a mechanism. (LLM, conf=0.90)

  cites Bryleva et al., 2010 sourceExperimental — accuracy not yet measured
• Partly supported

  The section on cholesterol metabolism and amyloidogenesis reports that ACAT inhibition reduces cholesteryl esters and Aβ, while ACAT1 ablation reduces Aβ pathology and cognitive impairment in an AD mouse model. The therapeutic-approaches section also reports efficacy of ACAT1 inhibitors in preclinical AD models. The copy does not report protection by ACAT1 intervention in tauopathy models or link ACAT1 to autophagy activation; its tau-pathology section discusses autophagy in connection with Vps34 instead. Page numbers were not provided. (LLM, conf=0.90)

  cites Di Paolo and Kim, 2011 sourceExperimental — accuracy not yet measured

In the paper

“Gene ontology analysis of the top upregulated marker genes within this cluster are enriched for mitochondrial cellular compartments, such as “inner mitochondrial membrane protein complex” (GO:0098800, FDR: 0.01), “mitochondrial membrane part” (GO:0044455, FDR: 0.02), among others”

What the sources say

• Partly supported

  The Results section, “Biological features of low quality cells” (page unavailable), reports marginal upregulation of mitochondrial and membrane-related genes in broken cells. It does not report an analysis of cluster 3’s marker genes, the two specified GO terms, or their stated FDR values. (LLM, conf=0.83)

  cites Ilicic et al., 2016 sourceExperimental — accuracy not yet measured

In the paper

“The fact that human iPSC-derived APOE4/E4 astrocytes were recently reported to accumulate endolysosomal cholesterol”

What the sources say

• Partly supported

  The Results section reports higher cellular cholesterol staining in human iPSC-derived APOE4 astrocytes than in APOE3 astrocytes, and the Discussion describes intracellular cholesterol accumulation. Neither passage establishes that the cholesterol accumulated specifically in endolysosomes; the lysosomal experiment in the Results concerns Aβ, not cholesterol. Page numbers were not available in our copy. (LLM, conf=0.88)

  cites Lin et al., 2018 sourceExperimental — accuracy not yet measured

In the paper

“Aged wild-type microglia downregulated a number of homeostatic genes and upregulated DAM 1 and DAM 2 genes compared to young microglia, albeit to a lesser degree than modulation in the CPZ model (Figures S1G and S1H).”

In the paper

“CPZ triggered the TREM2-dependent transition from a homeostatic to damage-associated microglia state with upregulation of genes controlling lipid transport and metabolism.”

In the paper

“These changes were restricted primarily to Trem2+/+ and Trem2+/– microglia, while Trem2–/– microglia largely failed to respond to CPZ (Figures 1B and 1C).”

In the paper

“CPZ induced transcriptional changes in Trem2+/+ and Trem2+/– microglia, whereas CPZ-challenged Trem2–/– microglia clustered with those of untreated mice (principal-component analysis [PCA]; Figure 1A).”

In the paper

“Cluster 4 showed the highest enrichment for DAM 2 gene expression, followed by cluster 8 (Figure 2B, right)”

In the paper

“Apoe expression was 8-fold higher with CPZ treatment of Trem2+/+ and Trem2+/– mice after 12 weeks but was attenuated in Trem2–/– mice”

In the paper

“Relative expression of the top 15 up- and downregulated genes per cluster confirmed that these clusters are distinct (Figure 2C), although genes exclusive to a single cluster are rare.”

In the paper

“CE accumulation was rescued by inhibition of acetyl-CoA acetyltransferase 1 (ACAT1), which converts cholesterol to CE, and by a liver X receptor (LXR) agonist”

In the paper

“Cluster 8 similarly consisted of cells with upregulated lysosome- and lipid metabolism-related genes, such as Ctsb, Ctsd, and Apoe, but their expression was lower than that seen in cluster 4”

In the paper

“Microglia from cluster 8 were largely absent in Trem2+/+ controls (< 2.3%), mildly increased in the Trem2+/+ CPZ and Trem2+/– CPZ mice (∼10%), and most abundant in the Trem2–/– CPZ mice (∼20%) (Data S2).”

In the paper

“12 week CPZ increased levels of certain lipid species in microglia of all genotypes compared with untreated genotype controls, but Trem2–/– microglia exhibited more dramatic increases compared with Trem2+/+ and Trem2+/– microglia (Figure 4A; Data S3; two-way ANOVA, FDR < 0.05).”

In the paper

“Increased pSYK response to sulfatide liposomes was abolished by addition of recombinant hTREM2 but not hTREM1 ECD”

In the paper

“Upon 5 week CPZ, minimal changes were detected, with an enhancement in CE and oxCE levels in all three genotypes (Figure S3E).”

In the paper

“CPZ led to an increase in CE18:1, 20:4, and 22:6 species in Apoe–/– versus Apoe+/+ forebrain (Figure 5B; main effect from two-way ANOVA, p < 0.001; FCs were 6.6, 1.4, and 6.7, respectively), and APOE deficiency significantly exacerbated the treatment effects for CE18:1 and CE20:4 (genotype-treatment interaction p < 0.05).”

In the paper

“CE22:6 showed the most striking increase in Trem2–/– brain with chronic demyelination, upward of 38-fold compared with Trem2–/– control brain and 2.5-fold compared with Trem2+/+ 12 week CPZ brain (Figure 3C).”

In the paper

“The increase trended toward attenuation by pre-incubating oxLDL with high concentrations of recombinant hTREM2 ECD at 9 μM (p = 0.1, two-way ANOVA, Tukey test) but not at lower concentrations such as those used in liposome/hTREM2 competition experiments (3 μM)”

In the paper

“Lipids selectively increased in Trem2–/– microglia upon chronic demyelination included many species dysregulated in the forebrain (e.g., CE20:4, CE22:6) (Figures 4D and 4E; interaction p < 0.001 for 12 week CPZ, two-way ANOVA), which were elevated 8- to 20-fold in Trem2–/– microglia upon 12 week CPZ diet compared with controls.”

In the paper

“Lipid changes were unique to microglia, as astrocytes or CSF did not display any genotype- or CPZ treatment-specific alterations (Figures 4B and 4C; two-way ANOVA, FDR < 0.05).”

In the paper

“Myelin lipids HexCer and sulfatides were similarly increased in wild-type and Apoe–/– microglia with 12 week CPZ, but there were no treatment effects in astrocytes (Figures S5B and S5C; Data S4).”

In the paper

“hTREM2 exhibited similar binding affinity and response at the highest analyte concentration to sulfatide- and PS-containing liposomes: KD = 6.8 μM, response units (RU) = 704, and KD = 5.6 μM, RU = 631, respectively (Figures 6D and 6E).”

In the paper

“hTREM2 R47H showed reduced affinity and lower binding response (i.e., RU) for sulfatide and PS liposomes: KD = 20 μM, RU = 191, and KD = 14 μM, RU = 267, respectively”

In the paper

“cluster 3 showed a mild increase in abundance under CPZ treatment (∼1.2% of Trem2+/+ cells with control diet, to ∼2.7% Trem2+/+ cells with CPZ diet, ∼2.5% Trem2+/– cells with CPZ diet, ∼2.0% Trem2–/–”

In the paper

“The vast majority of CD11b+ cells were CD45low with the CD11b+ CD45high cells representing less than 1% of the total live cells.”

In the paper

“Our results establish a key role for TREM2 in the control of microglial gene expression and cholesterol transport upon chronic myelin phagocytosis.”

In the paper

“Proper conversion to reactive states during chronic demyelination may enable lysosomal degradation and/or efflux of myelin lipids.”

In the paper

“suggesting that Trem2+/+ CPZ and Trem2+/– CPZ microglia exhibit a DAM 2-like transition in response to CPZ that is attenuated in Trem2–/– CPZ microglia.”

In the paper

“Our data indicate a role of TREM2 in endolysosomal function and lipid metabolism, with a clear implication of cholesterol transport and metabolism.”

In the paper

“TREM2 senses lipids and mediates myelin phagocytosis, but its role in microglial lipid metabolism is unknown.”

In the paper

“Thus, upon chronic demyelination, Trem2–/– microglia appear to be arrested in their transition to expression states seen in Trem2+/+ and Trem2+/– microglia and are unable to upregulate transcription of DAM genes, including lysosome- and lipid metabolism-related genes”

In the paper

“consistent with the fact that oxLDL is known to bind and stimulate multiple immune receptors”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites Choi et al., 2017 source

In the paper

“Thus, both intracellular and extracellular cholesterol transport may be defective in Trem2–/– microglia with CPZ.”

In the paper

“suggesting that CPZ increases the levels of most CE species similarly in wild-type and Apoe–/– microglia, thus preserving genotype differences.”

In the paper

“Lower binding response was due to a faster off-rate of the interaction, leading to shorter residency of mutant TREM2 on the lipid surface.”

In the paper

“This maladaptive state leads to neuritic dystrophy and Nf-L release”

In the paper

“This transcriptional program encompasses a gene “toolbox” that may enable microglia to enhance not only their chemotactic and phagocytic properties but also their lysosomal degradative capacity and ability to facilitate intracellular and extracellular cholesterol transport, including efflux from microglia.”

In the paper

“These data show that sulfatide binds and signals via TREM2 and that the R47H LOAD variant is significantly impaired in its interaction with this lipid.”

In the paper

“Thus, Trem2–/– microglia are able to phagocytose myelin debris during demyelination but are unable to properly metabolize or mediate the efflux of myelin lipids.”

In the paper

“Thus, high levels of M-CSF may provide compensatory upregulation of phagocytic pathways in Trem2–/– BMDMs and reveal that the phagocytosis defects in TREM2-deficient cells can be context dependent.”

In the paper

“Familial cases of atherosclerosis linked to mutations in the LIPA gene (encoding the lysosomal acid lipase) lead to a CE storage disorder”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Du and Grabowski, 2004

In the paper

“some of which (e.g., sulfatide) are myelin enriched”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Du and Grabowski, 2004

In the paper

“Our data suggest that TREM2 deficiency causes cholesterol efflux defects, leading to accumulation of an ACAT1 inhibitor-sensitive pool of CE in human iMG.”

In the paper

“suggesting that lipid accumulation observed in bulk forebrain tissue does not reflect extracellular accumulation, although changes in interstitial fluid cannot be ruled out.”

In the paper

“As a negative control, 10μM Cytochalasin D was added to cells 1 hr before myelin and retained throughout uptake assays.”

In the paper

“Biochemical results did not differ between littermate controls and C57BL/6J mice.”

In the paper

“APOE isoform-specific differences in the handling of lipids by glial cells may also account for the lower and higher genetic risk of APOE2 and APOE4 (relative to APOE3) to LOAD, respectively.”

In the paper

“Lipids were quantified using a mixture of non-endogenous internal standards as reported in Table S1.”

In the paper

“Reduced metabolic flux of CE may expose it to oxidation, resulting in secondary accumulation of oxCE species”

In the paper

“For manual quantification, puncta of APP staining in HC neuropil were counted in blinded images from the selected HC regions.”

In the paper

“Brain-penetrant LXR agonists have also been considered as therapeutics on the basis of anti-inflammatory and amyloid-lowering properties, resulting from their ability to enhance cholesterol efflux.”

In the paper

“littermates of the same gender were randomly assigned to experimental groups.”

In the paper

“All mouse husbandry and experimental procedures were approved by Denali Institutional Animal Care and Use Committee.”

In the paper

“it was comprised of very few cells and closer analysis suggests this cluster of cells to be lower quality cells that slipped through our aforementioned quality control.”

In the paper

“alteration of cholesterol and CE metabolism in microglia may be a unifying mechanism accounting for disease risk, perhaps via impacts on Aβ, tau, inflammation, or other AD-relevant pathogenic processes.”

In the paper

“viability was verified to be > 70%.”