Lipid-droplet-accumulating microglia represent a dysfunctional and proinflammatory state in the aging brain

May combine two claims.

In the aging brain, microglia lose their homeostatic molecular signature and show functional impairments such as increased pro-inflammatory cytokines, elevated ROS, and lysosomal deposits.

“In the aging brain and in neurodegeneration, microglia lose their homeostatic molecular signature and show profound functional impairments, such as increased production of pro-inflammatory cytokines, elevated generation of reactive oxygen species (ROS) and build-up of dysfunctional lysosomal deposits indicative of impaired phagocytosis2.”

• cites [2] Mosher, K. I. & Wyss-Coray, T. Microglial dysfunction in brain aging and Alzheimer’s disease. Biochem. Pharmacol. 88, 594–604 (2014). sourcePartly supported

  Section 4.4 reports generally increased pro-inflammatory cytokine release from aged microglia, altered marker expression, and increased oxidative stress with aging. Section 4.5 reports lysosomal inclusions in aged microglia, but says impaired degradation is a possible interpretation and that age-related effects on phagocytosis remain poorly defined. The source therefore supports the broad pattern, but not a definitive loss of a homeostatic molecular signature or the claim that the deposits establish impaired phagocytosis. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

Aged microglia show increased production of inflammatory cytokines such as TNF-α, IL-1β and IL-6 under baseline conditions and excessive cytokine release upon immune challenge.

“Another characteristic of aged microglia is the increased production of inflammatory cytokines such as TNF-α, IL-1β and IL-6 under baseline conditions and excessive cytokine release upon immune challenge2.”

• cites [2] Mosher, K. I. & Wyss-Coray, T. Microglial dysfunction in brain aging and Alzheimer’s disease. Biochem. Pharmacol. 88, 594–604 (2014). sourcePartly supported

  Section 4.4 (page unavailable) says proinflammatory cytokine production and release by aged microglia are generally increased, and identifies TNF-α, IL-1β and IL-6 as markers often elevated in aged mice. But it says secretion depends on the stimulant and notes studies finding no age-related activation differences without pathological insults; it does not establish increased production of all three under baseline conditions or consistently excessive release upon immune challenge. The Conclusion (page unavailable) presents over-reactivity to additional stimuli as a possibility. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Impaired phagocytosis is considered a hallmark of microglia aging.

“Impaired phagocytosis is considered a hallmark of microglia aging2.”

• cites [2] Mosher, K. I. & Wyss-Coray, T. Microglial dysfunction in brain aging and Alzheimer’s disease. Biochem. Pharmacol. 88, 594–604 (2014). sourceSupported

May combine two claims.

The optimized microglia isolation strategy uses mechanical tissue homogenization instead of enzymatic digestion, which keeps microglia largely in a non-activated state and prevents unwanted bias towards an activated pro-inflammatory signature.

“Of note, we used an optimized microglia isolation strategy that uses mechanical tissue homogenization instead of enzymatic digestion, which keeps microglia largely in a non-activated state and therefore prevents unwanted bias towards an activated pro-inflammatory signature19.”

• cites [19] Yu, Y., Ramachandran, P. V. & Wang, M. C. Shedding new light on lipid functions with CARS and SRS microscopy. Biochim. Biophys. Acta 1841, 1120–1129 (2014). sourceCould not verify

  Split into 2 atoms — 0 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 2 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Lipid droplets were induced in LPS-treated mouse hippocampal slice cultures and in the N9 microglia cell line.

“Lipid droplets were also induced in LPS-treated mouse hippocampal slice cultures and in the N9 microglia cell line15,16.”

• cites [15] Shimabukuro, M. K. et al. Lipid-laden cells differentially distributed in the aging brain are functionally active and correspond to distinct phenotypes. Sci. Rep. 6, 23795 (2016). sourceCould not verify

  The Results section describes lipid-laden cells in brain tissue from aging mice, and the Methods describe staining brain sections. Searches for LPS-treated hippocampal slice cultures and the N9 microglia cell line found no passage addressing either experiment in our copy. (LLM, conf=0.74)

  Experimental — accuracy not yet measured
• cites [16] Chang, P. K., Khatchadourian, A., McKinney, R. A. & Maysinger, D. Docosahexaenoic acid (DHA): a modulator of microglia activity and dendritic spine morphology. J. Neuroinflammation 12, 34 (2015). sourceSupported

Classified as probably not a substantial claim. May combine two claims.

GRN mutations are linked to the development of frontotemporal dementia, and GRN−/− mice are used as a model for FTD and are characterized by microglial changes, neuroinflammation, and cognitive deficits.

“GRN mutations are linked to the development of frontotemporal dementia (FTD)34, and GRN−/− mice are used as a model for FTD and are characterized by microglial changes, neuroinflammation, and cognitive deficits36.”

• cites [34] Liu, P. et al. S113R mutation in SLC33A1 leads to neurodegeneration and augmented BMP signaling in a mouse model. Dis. Model. Mech. 10, 53–62 (2017). sourceCould not verify

  Citation 36 is attached to the claim about GRN−/− mice. The Results and Discussion passages concern Slc33a1-mutant mice and hereditary spastic paraplegia; searches for GRN, progranulin, FTD, microglial changes, neuroinflammation, and cognitive deficits found no passage addressing the GRN−/− model. The separate claim that GRN mutations are linked to FTD is attached to citation 34, not this source. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [36] Tsika, E. et al. Parkinson’s disease-linked mutations in VPS35 induce dopaminergic neurodegeneration. Hum. Mol. Genet. 23, 4621–4638 (2014). sourceCould not verify

  Citation 36 is attached to the claim about GRN−/− mice. The Discussion describes a rat model of VPS35-associated Parkinson’s disease, not a GRN−/− mouse model of FTD. Searches for GRN, FTD, microglial changes, neuroinflammation, and cognitive deficits found no passage addressing that claim. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Oil-red-O positive lipid-laden cells, including neurons, astrocytes, ependymal cells and Iba1+ cells, have been reported in mice and shown to increase with age.

“Oil-red-O positive “lipid-laden” cells, including neurons, astrocytes, ependymal cells and Iba1+ cells have recently been reported in mice and shown to increase with age 14.”

• cites [14] Liu, L. et al. Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration. Cell 160, 177–190 (2015). sourcePartly supported

  The Results section on Ndufs4−/− mice reports lipid droplets in mouse brain cells bearing astrocyte and microglia markers. It does not report Oil-red-O staining or lipid-laden ependymal cells or neurons. Nor does it show a general increase with age: droplets progressively disappeared in the vestibular nucleus as the mutant mice aged, although they persisted in the olfactory bulb (page unavailable in our copy). (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

Lipid droplets have not been studied functionally in brain myeloid cells in humans or vertebrates, and less than a handful of papers report the histological presence of lipid droplets in human brains.

“Surprisingly, lipid droplets have not been studied functionally in brain myeloid cells in humans or vertebrates, and less than a handful of papers report the histological presence of lipid droplets in human brains11,12.”

• cites [11] Childs, B. G. et al. Senescent intimal foam cells are deleterious at all stages of atherosclerosis. Science 354, 472–477 (2016). sourceCould not verify

  The passages on atherosclerotic plaques (section labeled Abstract; page unavailable) describe lipid-loaded macrophage-like cells in mouse arteries, not brain myeloid cells. Searches for brain microglia, lipid droplets, and human-brain histology found no passage addressing whether functional studies exist or how many papers report their histological presence. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [12] Castejon, O. J., Castellano, A., Arismendi, G. J. & Medina, Z. The inflammatory reaction in human traumatic oedematous cerebral cortex. J. Submicrosc. Cytol. Pathol. 37, 43–52 (2005).Not checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

In GRN−/− mice, neuroinflammation is most pronounced in the thalamus.

“This is of particular interest because it has been shown that in GRN−/− mice, neuroinflammation is most pronounced in the thalamus40.”

• cites [40] Rambold, A. S., Cohen, S. & Lippincott-Schwartz, J. Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics. Dev. Cell 32, 678–692 (2015). sourceCould not verify

  The Results passages concern fatty-acid trafficking in starved cells and mitochondrial-fusion-deficient cells, not neuroinflammation in GRN−/− mice. Searches for GRN−/− mice, thalamus, and progranulin-related neuroinflammation found no passage addressing the claim; page numbers were unavailable. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

GRN causes frontotemporal dementia in people with loss of function mutations.

“Excitingly, knockout of GRN, which causes frontotemporal dementia in people with loss of function mutations in this gene34, resulted in severe accumulation of lipid droplets in microglia in vivo.”

• cites [34] Liu, P. et al. S113R mutation in SLC33A1 leads to neurodegeneration and augmented BMP signaling in a mouse model. Dis. Model. Mech. 10, 53–62 (2017). sourceCould not verify

  The Results and Discussion passages concern SLC33A1 mutations, hereditary spastic paraplegia, and BMP signaling, not GRN. Searches for GRN, progranulin, frontotemporal dementia, and loss-of-function mutations found no passage in our copy addressing the attributed claim. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Lysosomes become dysfunctional in aged microglia.

“Lastly, there is emerging evidence that lysosomes become dysfunctional in aged microglia2.”

• cites [2] Mosher, K. I. & Wyss-Coray, T. Microglial dysfunction in brain aging and Alzheimer’s disease. Biochem. Pharmacol. 88, 594–604 (2014). sourcePartly supported

  Section 4.5 (Phagocytosis; page unavailable) reports lysosomal inclusions in aged rodent microglia and says these may indicate impaired degradation of ingested material. That is emerging evidence consistent with the claim, but the paper does not establish that the lysosomes themselves become dysfunctional. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Metabolic changes towards increased fatty acid production cause lipid droplet formation in immune cells and cancer cells.

“Besides inflammation, metabolic changes towards increased fatty acid production have been reported to cause lipid droplet formation in immune cells9 and also in cancer cells41.”

• cites [9] Bozza, P. T. & Viola, J. P. Lipid droplets in inflammation and cancer. Prostaglandins Leukot. Essent Fatty Acids 82, 243–250 (2010). sourceNot checked — no full text of the source
• cites [41] Ghoshal, N., Dearborn, J. T., Wozniak, D. F. & Cairns, N. J. Core features of frontotemporal dementia recapitulated in progranulin knockout mice. Neurobiol. Dis. 45, 395–408 (2012). sourceCould not verify

  The Results section on microglial activation reports increased microglia/macrophages in progranulin-knockout mice, and the ubiquitin-immunoreactivity section mentions a few foamy macrophages. Neither passage addresses increased fatty acid production or lipid droplet formation in immune cells. Searches for those terms found no supporting passage; the cancer-cell portion is attached to a different citation. (LLM, conf=0.75)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Lipid droplets in microglia have been detected in LPS-treated cells of the microglia-like cell line N9.

“For example, lipid droplets in microglia have been detected in adult mice after induction of ROS by rotenone13, in hippocampal slice culture from P6–8 mice upon treatment with LPS, and in LPS-treated cells of the microglia-like cell line N916.”

• cites [16] Chang, P. K., Khatchadourian, A., McKinney, R. A. & Maysinger, D. Docosahexaenoic acid (DHA): a modulator of microglia activity and dendritic spine morphology. J. Neuroinflammation 12, 34 (2015). sourceSupported

May combine two claims.

ACLY is a key enzyme in the shift from the TCA cycle to lipid formation and has been shown to cause lipid droplet accumulation.

“For example, expression of ACLY, a key enzyme in the shift from the TCA cycle to lipid formation that has been shown to cause lipid droplet accumulation42, is significantly higher in LDAM than in microglia without lipid droplets.”

• cites [42] Currie, E., Schulze, A., Zechner, R., Walther, T. C. & Farese, R. V. Jr. Cellular fatty acid metabolism and cancer. Cell Metab. 18, 153–161 (2013). sourcePartly supported

  The ACLY subsection of “Blocking Fatty Acid Synthesis” says ACLY links glucose metabolism to fatty-acid synthesis and that knocking it down reduces conversion of glucose to lipid. The “Diverting Fatty Acids to Storage” subsection discusses lipid droplets, but does not say ACLY causes their accumulation. Page numbers are unavailable in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Several subsets of microglia with unique molecular and functional phenotypes exist in the healthy, aged, and degenerating brain.

“Recently, it has been shown that several subsets of microglia with unique molecular and functional phenotypes exist in the healthy, aged, and degenerating brain3–5.”

• cites [3] Keren-Shaul, H. et al. A unique microglia type associated with restricting development of Alzheimer’s disease. Cell 169, 1276–1290.e17 (2017). sourcePartly supported

  The Results identify distinct microglial states in an Alzheimer’s mouse model, with altered gene expression and phagocytic characteristics; they also compare these cells with wild-type controls. The paper does not establish several distinct subsets across healthy, normally aged, and degenerating brains: its disease-associated states were observed in the AD model, not the wild-type controls. No page numbers are available in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [4] Krasemann, S. et al. The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity 47, 566–581.e9 (2017). sourceSupported
• cites [5] Li, Q. et al. Developmental heterogeneity of microglia and brain myeloid cells revealed by deep single-cell RNA sequencing. Neuron 101, 207–223.e10 (2019). sourcePartly supported

  The Results describe distinct microglial populations in the healthy developing mouse brain, and the Discussion compares one of them with disease-associated microglia. But the paper does not establish several distinct subsets in aged brains; its Discussion instead reports little transcriptomic variation among classical adult microglia. The claim extends the paper’s findings beyond the populations it studied. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Lipid droplets can form due to various environmental and cellular conditions, including elevated concentrations of extracellular lipids, inflammatory events, increased ROS levels and intracellular metabolic changes.

“Lipid droplets can form due to various environmental and cellular conditions, including elevated concentrations of extracellular lipids, inflammatory events, increased ROS levels and intracellular metabolic changes38,39.”

• cites [38] Vieira-de-Abreu, A. et al. Cross-talk between macrophage migration inhibitory factor and eotaxin in allergic eosinophil activation forms leukotriene C(4)-synthesizing lipid bodies. Am. J. Respir. Cell Mol. Biol. 44, 509–516 (2011). sourcePartly supported

  The copy’s Abstract reports lipid-droplet formation during allergic pulmonary inflammation and after inflammatory MIF stimulation of eosinophils. It does not address elevated extracellular lipids, increased ROS, or intracellular metabolic changes as causes; searches for those terms returned no supporting passage. No page number is available in the indexed copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [39] Hu, X., Xu, B. & Ge, W. The role of lipid bodies in the microglial aging process and related diseases. Neurochem. Res. 42, 3140–3148 (2017). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Lipid droplet containing immune cells in the periphery are detrimental, including foamy macrophages in atherosclerosis or lipid droplet-rich eosinophils in experimental models of allergy.

“This is in line with findings which have shown that lipid droplet containing immune cells in the periphery are detrimental, including foamy macrophages in atherosclerosis10 or lipid droplet-rich eosinophils in experimental models of allergy37.”

• cites [10] den Brok, M. H., Raaijmakers, T. K., Collado-Camps, E. & Adema, G. J. Lipid droplets as immune modulators in myeloid cells. Trends Immunol. 39, 380–392 (2018). sourceNot checked — no full text of the source
• cites [37] Yin, F. et al. Behavioral deficits and progressive neuropathology in progranulin-deficient mice: a mouse model of frontotemporal dementia. FASEB J. 24, 4639–4647 (2010). sourceCould not verify

  The citation is attached to the example of lipid droplet-rich eosinophils in experimental allergy models. Our copy’s Discussion addresses inflammation and immune responses in progranulin-deficient mice, but searches for eosinophils, lipid droplets, and allergy found no passage addressing that example. Page numbers were not available in the indexed copy. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Lipid-associated macrophages control metabolic homeostasis in a TREM2 dependent manner and are transcriptionally similar to DAM.

“Likewise, LDAM showed only low overlap with the transcriptome of recently identified “lipid-associated macrophages” from mouse and human adipose tissue, a cell population that controls metabolic homeostasis in a TREM2 dependent manner and is transcriptionally similar to DAM48.”

• cites [48] Chandak, P. G. et al. Efficient phagocytosis requires triacylglycerol hydrolysis by adipose triglyceride lipase. J. Biol. Chem. 285, 20192–20201 (2010). sourceCould not verify

  The Results discuss ATGL expression in macrophages and foam cells, while the Introduction and Discussion focus on lipid storage and phagocytosis (page numbers unavailable). Searches for TREM2-dependent metabolic homeostasis and transcriptional similarity to DAM found no passage addressing either claim. (LLM, conf=0.75)

  Experimental — accuracy not yet measured

May combine two claims.

Almost 2,000 genes were differentially expressed between LD-hi and LD-lo GRN−/− microglia and linked to metabolic processes and cellular response to stress, pathways also enriched in LDAM.

“Almost 2,000 genes were differentially expressed between LD-hi and LD-lo microglia (Fig. 7h) and linked to metabolic processes and cellular response to stress (Fig. 7i), pathways that are also enriched in LDAM (Fig, 2e, Extended Data Fig. 2b).”

May combine two claims.

Phagosome maturation and Production of NO and ROS are the most significant pathways associated with LD-hi microglia.

“Pathway Analysis of differentially expressed genes revealed Phagosome maturation and Production of NO and ROS, two key functions of microglia that become dysregulated with age, as the most significant pathways associated with LD-hi microglia (Fig. 2e).”

May combine two claims.

Lipid droplets from whole hippocampus and from aged microglia show nearly identical lipid distribution and are mainly composed of glycerolipids (hippocampus 41.3%; microglia 44.4%), with cholesteryl esters almost absent (hippocampus 1.1%; microglia 0.7%).

“Lipidomics analysis revealed that lipid droplets from the whole hippocampus and from aged microglia show a nearly identical lipid distribution and are mainly composed of glycerolipids (hippocampus: 41.3%; microglia: 44.4%), while cholesteryl esters were almost absent (hippocampus: 1.1%; microglia: 0.7%) (Fig. 1k).”

May combine two claims.

BV2 cells with sgRNAs targeting GRN and SLC33A1 had significantly increased ROS generation under baseline conditions, and SLC33A1 sgRNA expressing cells had increased ROS upon LPS treatment.

“We used flow cytometry to measure ROS levels and found significantly increased ROS generation in BV2 cells with sgRNAs targeting GRN and SLC33A1 cells under baseline conditions and in SLC33A1 sgRNA expressing cells upon LPS treatment (Fig. 6h).”

May combine two claims.

Production of NO and ROS was the second most significantly regulated pathway in LDAM with over 90% of the differentially expressed genes increased.

“Production of NO and ROS was the second most significantly regulated pathway in LDAM (Fig. 2e) with over 90% of the differentially expressed genes increased (Fig. 5a).”

May combine two claims.

Aged mouse microglia contain characteristic lipid droplets, frequently adjacent to dense lysosomal material, whereas young microglia rarely do.

“Interestingly, we observed characteristic lipid droplets in aged but rarely in young microglia and they were frequently adjacent to dense lysosomal material (Fig. 1a).”

May combine two claims.

Unsupervised cluster analysis segregated LD-lo from LD-hi microglia and revealed 692 significantly differentially expressed genes.

“Unsupervised cluster analysis segregated LD-lo from LD-hi microglia and revealed prominent differences between their transcriptome with 692 significantly differentially expressed genes (Fig. 2c,d).”

May combine two claims.

Microglia in the hippocampus of GRN−/− mice contain high numbers of lipid droplets, resulting in a twofold higher percentage of BODIPY+ microglia and twice as many lipid droplets per cell compared with wild type littermates.

“We found that microglia in the hippocampus of GRN−/− mice contained high numbers of lipid droplets, resulting in a twofold higher percentage of BODIPY+ microglia and twice as many lipid droplets per cell in GRN−/− mice compared with wild type littermates (Fig. 7a–c).”

May combine two claims.

Lipid droplets from BV2 cells and aged microglia show similar chain length distribution of TAG-associated fatty acids with peaks at 38:2 and 44:5, while liver lipid droplets have a shift towards longer TAG chain fatty acids with a peak at 52:2 carbon atoms.

“In addition, lipid droplets from BV2 cells and from aged microglia showed a similar chain length distribution of TAG-associated fatty acids with peaks at 38:2 and 44:5, while liver lipid droplets had a shift towards longer TAG chain fatty acids with a peak at 52:2 carbon atoms, which is in agreement with previous reports26 (Fig. 3g,h).”

May combine two claims.

Phagosome maturation was the top regulated pathway associated with the transcriptome of LDAM, characterized by upregulation of endosomal and lysosomal genes and increased CD68 immunoreactivity in BODIPY+ microglia in the hippocampus of aged mice.

“Interestingly, phagosome maturation was the top regulated pathway associated with the transcriptome of LDAM (Fig. 2e), characterized by upregulation of endosomal and lysosomal genes (Fig. 4a) and increased immunoreactivity for the lysosome-associated protein CD68 in BODIPY+ microglia in the hippocampus of aged mice (Fig. 4b,c).”

May combine two claims.

Only nine common genes were differentially expressed in all three data sets (aging, GRN−/−, and LPS-treated young mice), one of them PLIN3.

“We found that although only nine common genes were differentially expressed in all three data sets (one of them was the lipid droplet specific gene PLIN3), the directionality of expression changes of LDAM genes was remarkably similar for all groups and almost identical for LDAM and LD-hi GRN−/− microglia (Fig. 7l,m; Extended Data Figure 6).”

May combine two claims.

Lipid droplets were primarily found in microglia but not in other cell types.

“Lipid droplets were primarily found in microglia but not in other cell types.”

May combine two claims.

Lipid droplets from BV2 cells and aged microglia have highly similar overall lipid content and similar composition of neutral lipids, with low cholesteryl esters and high glycerolipids.

“Interestingly, they had a highly similar overall lipid content and a similar composition of neutral lipids, with low amounts of cholesteryl esters but high levels of glycerolipids (MAG, TAG).”

May combine two claims.

sgRNAs targeting GRN and SLC33A1 induced significant defects in phagocytosis and increased ROS production in BV2 cells.

“In addition, sgRNAs targeting GRN and SLC33A1 induced significant defects in phagocytosis and increased ROS production in BV2 cells, hence recapitulating the functional impairments of LDAM.”

May combine two claims.

LPS treatment led to lipid droplet formation in hippocampal microglia in young mice in vivo and in the microglia cell line BV2 in vitro.

“Interestingly, we found that LPS treatment, which provokes an acute inflammatory response in the brain, led to lipid droplet formation in hippocampal microglia in young mice in vivo and in the microglia cell line BV2 in vitro.”

May combine two claims.

Lipidomics analysis revealed that lipid droplets in LDAM contained mainly glycerolipids and only low amounts of cholesteryl ester.

“We did not find ultrastructural evidence for cholesterol crystals, and lipidomics analysis revealed that these lipid droplets contained mainly glycerolipids and only low amounts of cholesteryl ester.”

May combine two claims.

sgRNAs targeting these genes were able to increase (SLC33A1, GRN, VPS35) and decrease (SNX17) lipid droplet load in microglial BV2 cells.

“Indeed, sgRNAs targeting these genes were able to increase (SLC33A1, GRN, VPS35) and decrease (SNX17) lipid droplet load in microglial BV2 cells.”

May combine two claims.

LDAM contain high numbers of lysosomes, and these lysosomes accumulate in close contact to lipid droplets.

“Intriguingly, we observed that LDAM contain high numbers of lysosomes, and moreover, these lysosomes accumulate in close contact to lipid droplets.”

May combine two claims.

Lipid droplet rich microglia in GRN−/− mice show a similar transcriptome signature and the same functional impairments as LDAM.

“These lipid droplet rich microglia in GRN−/− mice show a similar transcriptome signature and the same functional impairments as LDAM.”

May combine two claims.

Directionality of expression changes of LDAM genes was remarkably similar for all groups and almost identical for LDAM and LD-hi GRN−/− microglia.

“We found that although only nine common genes were differentially expressed in all three data sets (one of them was the lipid droplet specific gene PLIN3), the directionality of expression changes of LDAM genes was remarkably similar for all groups and almost identical for LDAM and LD-hi GRN−/− microglia (Fig. 7l,m; Extended Data Figure 6).”

May combine two claims.

BODIPY+/Iba1+ cells were frequently found in the thalamus and were detected to a lesser extent in cortex and corpus callosum of 9-month-old male GRN−/− mice.

“BODIPY+/Iba1+ cells were frequently found in the thalamus and were detected to a lesser extent in cortex and corpus callosum.”

May combine two claims.

Lipid droplet accumulating microglia are abundant in the hippocampus but rare in other brain regions of aged mice.

“Lipid droplet accumulating microglia are abundant in the hippocampus but rare in other brain regions of aged mice.”

Probably not a substantial claim: may not pass S3 (load-bearing).

ApoE expression does not differ significantly between LD-lo and LD-hi microglia.

“Paired dot plot showing FPKM values of LD-lo and LD-hi microglia for ApoE (paired Student’s t-test; P= 0.423).”

Probably not a substantial claim: may not pass S4 (based).

Triacylglycerides (TAG) are a major component of lipid droplets in LDAM.

“Since triacylglycerides (TAG) are a major component of lipid droplets in LDAM, it is possible that lipid droplet accumulation in GRN deficient microglia contributes to elevated TAG levels in brains lacking GRN.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The lipid composition of lipid droplets from young and aged microglia is highly similar, but differs between young and aged liver tissue.

“Pie charts showing that the lipid composition of lipid droplets from young and aged microglia is highly similar, but differs between young and aged liver tissue.”

May combine two claims.

LPS treatment induces lipid droplet formation in microglia and in BV2 cells.

“LPS treatment induces lipid droplet formation in microglia and in BV2 cells.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Lipid droplets are production and storage sites for eicosanoids and inflammatory cytokines, and are involved in antigen presentation and pathogen clearance.

“Here, lipid droplets are production and storage sites for eicosanoids and inflammatory cytokines, and are further involved in antigen presentation and pathogen clearance9.”

• cites [9] Bozza, P. T. & Viola, J. P. Lipid droplets in inflammation and cancer. Prostaglandins Leukot. Essent Fatty Acids 82, 243–250 (2010). sourceNot checked — no full text of the source

Classified as probably not a substantial claim. May combine two claims.

Lipid droplets are lipid storing organelles that contain neutral lipids such as glycerolipids and cholesterol, and are increasingly accepted as structural markers of inflammation.

“In particular, lipid droplets, which are lipid storing organelles that contain neutral lipids such as glycerolipids and cholesterol, are increasingly accepted as structural markers of inflammation8.”

• cites [8] Fowler, S. D., Mayer, E. P. & Greenspan, P. Foam cells and atherogenesis. Ann. NY Acad. Sci. 454, 79–90 (1985). sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Myeloid cells form lipid droplets in response to inflammation and stress.

“Myeloid cells form lipid droplets in response to inflammation and stress, including the aforementioned macrophages in atherosclerotic lesions, leukocytes in inflammatory arthritis, and eosinophils in allergic inflammation.”

• cites [9] Bozza, P. T. & Viola, J. P. Lipid droplets in inflammation and cancer. Prostaglandins Leukot. Essent Fatty Acids 82, 243–250 (2010). sourceNot checked — no full text of the source

May combine two claims.

Lipid droplet-rich microglia have phagocytosis deficits and increased lipid storage is associated with impaired phagocytosis.

“Collectively, these data suggest that lipid droplet-rich microglia have phagocytosis deficits and that increased lipid storage is associated with impaired phagocytosis.”

May combine two claims.

LDAM are in a primed activation state that becomes hyperactivated upon stimulation with LPS.

“These findings suggest that LDAM are in a primed activation state that becomes hyperactivated upon stimulation with LPS.”

May combine two claims.

LDAM have elevated concentrations of ROS and lipid droplets mediate LPS-induced ROS generation.

“Together, these findings demonstrate that LDAM have elevated concentrations of ROS and suggest that lipid droplets mediate LPS-induced ROS generation.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Lipid-droplet rich foam cells in atherosclerosis show hallmarks of senescent cells and seem deleterious at all stages of disease.

“Importantly, lipid-droplet rich foam cells in atherosclerosis show hallmarks of senescent cells and they seem deleterious at all stages of disease10.”

• cites [10] den Brok, M. H., Raaijmakers, T. K., Collado-Camps, E. & Adema, G. J. Lipid droplets as immune modulators in myeloid cells. Trends Immunol. 39, 380–392 (2018). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Lipid droplet formation has been reported in glia at the onset of neurodegeneration in a drosophila model.

“That they may have important functions in disease has recently been suggested in a drosophila model where lipid droplet formation has been reported in glia at the onset of neurodegeneration13.”

• cites [13] Lee, S. C., Moore, G. R., Golenwsky, G. & Raine, C. S. Multiple sclerosis: a role for astroglia in active demyelination suggested by class II MHC expression and ultrastructural study. J. Neuropathol. Exp. Neurol. 49, 122–136 (1990). sourceNot checked — no full text of the source

May combine two claims.

LPS triggers lipid droplet formation in microglia in vitro and in vivo.

“Together, these findings demonstrate that LPS triggers lipid droplet formation in microglia in vitro and in vivo, and lipid droplet containing microglia in young LPS-treated mice show a transcriptional signature that partially overlaps with LDAM.”

May combine two claims.

LPS-induced lipid droplets in BV2 cells are highly similar to lipid droplets in aged microglia, making this in vitro assay a useful model to study lipid droplets in LDAM.

“Furthermore, LPS-induced lipid droplets in BV2 cells are highly similar to lipid droplets in aged microglia, thus making this in vitro assay a useful model to study lipid droplets in LDAM.”

May combine two claims.

Lipid droplet-containing aged microglia show transcriptional changes in genes related to phagocytosis, ROS production and immune signaling, yet have a unique transcriptome signature distinct from previously described microglia states in aging and neurodegeneration.

“Overall, these data suggest that lipid droplet-containing aged microglia show transcriptional changes of genes related to key microglia functions such as phagocytosis, ROS production and immune signaling, yet they have a unique transcriptome signature that is distinct from previously described microglia states observed in aging and neurodegeneration.”

Classified as probably not a substantial claim.

In a drosophila model for neurodegeneration, lipid droplets in glia form due to an APOE dependent transfer of lipids from neurons to glia.

“In a drosophila model for neurodegeneration13, lipid droplets in glia form due to an APOE dependent transfer of lipids from neurons to glia.”

• cites [13] Lee, S. C., Moore, G. R., Golenwsky, G. & Raine, C. S. Multiple sclerosis: a role for astroglia in active demyelination suggested by class II MHC expression and ultrastructural study. J. Neuropathol. Exp. Neurol. 49, 122–136 (1990). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

At sites of atherosclerotic lesions, lipid droplet rich foamy macrophages show decreased phagocytosis activity compared to macrophages without lipid droplets.

“This finding is in line with a previous report which observed that at the sites of atherosclerotic lesions, lipid droplet rich foamy macrophages show decreased phagocytosis activity compared to macrophages without lipid droplets46.”

• cites [46] Nadjar, A. Role of metabolic programming in the modulation of microglia phagocytosis by lipids. Prostaglandins Leukot. Essent. Fatty Acids 135, 63–73 (2018). sourceNot checked — no full text of the source

May combine two claims.

These findings validate the in vivo relevance of the in vitro CRISPR screen and confirm that microglia from GRN−/− mice contain lipid droplets.

“Together, these findings validate the in vivo relevance of our in vitro CRISPR screen and confirm that microglia from GRN−/− mice contain lipid droplets.”

May combine two claims.

These findings indicate that LDAM represent a dysfunctional and pro-inflammatory microglia state in the aging brain.

“Together, these findings indicate that LDAM represent a dysfunctional and pro-inflammatory microglia state in the aging brain, and further suggest that LDAM might contribute to neurodegenerative diseases.”

May combine two claims.

In this study, we identify a novel LDAM state in the aging brain characterized by a unique transcriptional signature, severe functional deficits and a pro-inflammatory phenotype.

“In this study, we identify a novel, lipid droplet accumulating microglia (LDAM) state in the aging brain and show that LDAM are characterized by a unique transcriptional signature, severe functional deficits and a pro-inflammatory phenotype.”

May combine two claims.

LDAM and DAM show different functional phenotypes; DAM are actively phagocytic populations, while LDAM are severely impaired in phagocytosis.

“LDAM and DAM also show different functional phenotypes; while DAM are actively phagocytic populations, LDAM are severely impaired in this function.”

• AssumptionRests on an assumption: DAM are actively phagocytic populations.

May combine two claims.

LD-hi microglia from GRN−/− mice and LDAM in aging have similar functional and transcriptional phenotypes, suggesting that lipid droplet containing GRN−/− microglia share the LDAM state.

“Remarkably, LD-hi microglia from GRN−/− mice and LDAM in aging have similar functional and transcriptional phenotypes, suggesting that lipid droplet containing GRN−/− microglia share the LDAM state.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In the brain, a protective role has been reported for lipid droplets in glia in a drosophila model for neurodegeneration.

“In the brain, a protective role has been reported for lipid droplets in glia in a drosophila model for neurodegeneration13.”

• cites [13] Lee, S. C., Moore, G. R., Golenwsky, G. & Raine, C. S. Multiple sclerosis: a role for astroglia in active demyelination suggested by class II MHC expression and ultrastructural study. J. Neuropathol. Exp. Neurol. 49, 122–136 (1990). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Lipid droplets have also been correlated with beneficial functions, such as improved host defense and antigen cross presentation in myeloid cells.

“However, lipid droplets have also been correlated with beneficial functions, such as improved host defense and antigen cross presentation in myeloid cells9.”

• cites [9] Bozza, P. T. & Viola, J. P. Lipid droplets in inflammation and cancer. Prostaglandins Leukot. Essent Fatty Acids 82, 243–250 (2010). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

In peripheral immune cells, lipid droplets form often as a response to inflammation and stress.

“In peripheral immune cells, lipid droplets form often as a response to inflammation and stress9.”

• cites [9] Bozza, P. T. & Viola, J. P. Lipid droplets in inflammation and cancer. Prostaglandins Leukot. Essent Fatty Acids 82, 243–250 (2010). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Lipid droplets in microglia have been detected in adult mice after induction of ROS by rotenone.

“For example, lipid droplets in microglia have been detected in adult mice after induction of ROS by rotenone13, in hippocampal slice culture from P6–8 mice upon treatment with LPS, and in LPS-treated cells of the microglia-like cell line N916.”

• cites [13] Lee, S. C., Moore, G. R., Golenwsky, G. & Raine, C. S. Multiple sclerosis: a role for astroglia in active demyelination suggested by class II MHC expression and ultrastructural study. J. Neuropathol. Exp. Neurol. 49, 122–136 (1990). sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Cholesterol accumulation has been observed in phagocytes in an EAE demyelination model in aged mice as a result of excessive uptake of myelin debris.

“Cholesterol accumulation has recently been observed in phagocytes in an EAE demyelination model in aged mice as a result of excessive uptake of myelin debris43.”

• cites [43] Burke, A. C. & Huff, M. W. ATP-citrate lyase: genetics, molecular biology and therapeutic target for dyslipidemia. Curr. Opin. Lipidol. 28, 193–200 (2017). sourceNot checked — no full text of the source

May combine two claims.

Engulfment of cellular debris relies on the availability of free fatty acids, which are released upon degradation of lipid droplets, linking effective lysis of lipid droplets with successful phagocytosis.

“In this context, a study in macrophages has shown that engulfment of cellular debris relies on the availability of free fatty acids, which are released upon degradation of lipid droplets, thus linking effective lysis of lipid droplets with successful phagocytosis47.”

• cites [47] Chinetti-Gbaguidi, G. et al. Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPARγ and LXRα pathways. Circ. Res. 108, 985–995 (2011). sourceNot checked — no full text of the source

May combine two claims.

Microglia in the LDAM state, but not microglia without lipid droplets, show typical age-related functional impairments and a primed phenotype, suggesting that LDAM may be the primary detrimental microglia state in the aging brain.

“Microglia in the LDAM state, which account for more than 50% of all microglia in the aged hippocampus, but not microglia without lipid droplets, show typical age-related functional impairments and a primed phenotype, suggesting that LDAM may be the primary detrimental microglia state in the aging brain.”

May combine two claims.

LDAM demonstrate a novel state of microglia with a unique transcriptional signature and functional impairments in the aging brain.

“Together, we show that LDAM demonstrate a novel state of microglia with a unique transcriptional signature and functional impairments in the aging brain, and we identified lipid droplet containing microglia in a GRN−/− model for chronic neuroinflammation, and in an LPS-induced acutely inflamed brain milieu.”

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

Data in Extended Data Fig. 5 were replicated in at least three independent experiments.

“Data were replicated in at least three independent experiments.”

Classified as probably not a substantial claim.

All samples used for human post-mortem brain tissue were obtained from individuals without any neurological or psychiatric diagnoses.

“All samples used were obtained from individuals without any neurological or psychiatric diagnoses.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Data in Extended Data Fig. 3a-b were replicated in at least two independent experiments.

“Data in a-b were replicated in at least two independent experiments.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Data collection was randomized for all experiments.

“Data collection was randomized for all experiments.”

Classified as probably not a substantial claim.

Analyses for quantification of BODIPY+ microglia, CD68hi microglia, lipid droplet numbers and lipid droplet size were performed blinded on coded slides.

“For quantification of BODIPY+ microglia, of CD68hi microglia, of BODIPY+ lipid droplet numbers and of lipid droplet size, analyses were performed blinded on coded slides.”

Probably not a substantial claim: may not pass S3 (load-bearing).

No sample-size calculations were performed.

“No sample-size calculations were performed.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Sample sizes were chosen based on prior literature using similar experimental paradigms.

“Further, sample sizes were chosen based on prior literature using similar experimental paradigms.”

Classified as probably not a substantial claim.

Six randomly selected visual fields per animal were photographed and used for quantitative analysis.

“Six randomly selected visual fields per animal were photographed using a confocal scanning laser microscope (LSM 700, Zeiss) with LSM software (ZEN 2011) and used for quantitative analysis.”

Classified as probably not a substantial claim.

For quantification of CARS signal in microglia, 20 randomly selected microglia in the dentate gyrus per animal were imaged.

“For quantification of CARS signal in microglia, 20 randomly selected microglia in the dentate gyrus per animal were imaged.”

Probably not a substantial claim: may not pass S3 (load-bearing).

False discovery rate was estimated using Benjamini and Hochberg approach.

“False discovery rate was estimated using Benjamini and Hochberg approach (Benjamini and Hochberg, 1995).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Experimenters were blinded for imaging and data analysis.

“Experimenters were blinded for imaging and data analysis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All RNA samples passed a quality control threshold (RIN ≥ 9.0) to proceed to library preparations and RNAseq.

“All samples passed a quality control threshold (RIN ≥ 9.0) to proceed to library preparations and RNAseq.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Sample size was determined to be adequate based on the magnitude and consistency of measurable differences between groups.

“Sample size was determined to be adequate based on the magnitude and consistency of measurable differences between groups.”

Probably not a substantial claim: may not pass S3 (load-bearing).

After selection, more than 90% of BV2 cells expressed the mCherry reporter, confirming sgRNA infection.

“sgRNA infection was confirmed by flow cytometry, which indicated >90% of cells expressed the mCherry reporter.”