APOE from astrocytes restores Alzheimer’s Aβ-pathology and DAM-like responses in APOE deficient microglia

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

Several groups have suggested lowering of APOE expression with APOE-antibodies or using antisense therapy as a therapeutic option for AD.

“Several groups have suggested lowering of APOE expression with APOE-antibodies (Gratuze et al, 2022; Liao et al, 2018; Xiong et al, 2021) or using antisense therapy (Huynh et al, 2017) as a therapeutic option for AD.”

• cites Gratuze et al, 2022 sourceDisputed

  The Discussion supports investigating the anti-ApoE antibody HAE-4 as a potential AD therapy, but explicitly says it does not lower overall ApoE levels. It identifies antisense oligonucleotides—not HAE-4—as an approach to lowering brain ApoE levels. The antisense-therapy clause in the citing sentence is attributed to a different citation. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites Liao et al, 2018 sourceDisputed

  The cited paper supports anti-apoE antibodies as a potential therapeutic approach, but not as a way to lower APOE expression. Its Discussion (p. 7) says HAE-4 did not significantly alter total apoE levels, and the Discussion (p. 8) explicitly distinguishes antibody targeting from simply lowering apoE levels. The antisense-therapy clause is attributed to a different citation. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites Xiong et al, 2021 sourcePartly supported

  The Introduction describes HAE-4 as an antibody targeting APOE in amyloid plaques, and the Discussion proposes its therapeutic potential for AD. The paper reports reduced amyloid pathology in mice, not lowered APOE expression. The antisense-therapy clause is attributed to a different citation. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

The major role of APOE in Aβ pathology is direct promotion of Aβ fibrillization.

“the major role is direct promotion of Aβ fibrillization (Castano et al, 1995; Ma et al, 1994; Wisniewski et al, 1994)”

• cites Castano et al, 1995 sourcePartly supported

  The Introduction reports that apoE accelerated Aβ fibril formation in vitro, and the Discussion says apoE may promote Aβ assembly. But the Discussion also describes apoE as one of several possible contributors whose importance varies among cases of Alzheimer’s disease; it does not establish direct fibrillization as APOE’s major role in Aβ pathology. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Ma et al, 1994Not matched

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

• cites Wisniewski et al, 1994Not matched

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

Classified as probably not a substantial claim.

APOE's role in promoting β-amyloid plaque pathology in Alzheimer's disease is well established.

“In AD, its specific role in promoting β-amyloid (Aβ) plaque pathology is, however, well established (Fleisher et al, 2013; Kok et al, 2009; Murphy et al, 2013; Schmechel et al, 1993).”

• cites Fleisher et al, 2013 sourceNot checked — no full text of the source
• cites Kok et al, 2009 sourceNot checked — no full text of the source
• cites Murphy et al, 2013 sourcePartly supported

  The Findings and Discussion report that ApoE ε4 is strongly associated with higher β-amyloid plaque density, including among people diagnosed with AD. The study describes this as an association and predictor of pathology; it does not establish that APOE causally promotes plaque formation. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

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

The APOE4 variant is the major genetic risk factor for Alzheimer's disease at the population level.

“The APOE4 variant of apolipoprotein E was identified 30 years ago as the major genetic risk factor for Alzheimer’s Disease (AD) at the population level (Bennett et al, 2009; Corder et al, 1993; Strittmatter et al, 1993)”

• cites Bennett et al, 2009 sourcePartly supported

  The ‘Relation of APOE to clinical AD’ section reports that APOE ε4 was associated with more than threefold higher odds of clinical AD, and the Discussion calls APOE a well-established risk factor in multiple populations. Our copy does not establish that ε4 is the major genetic risk factor at the population level or document its identification 30 years ago. Page numbers were not provided for these passages. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites Corder et al, 1993 sourceNot checked — no full text of the source
• cites Strittmatter et al, 1993 sourcePartly supported

  The Abstract (p. 1977) reports a significant association between APOE-e4 and late-onset familial Alzheimer disease. It does not establish that APOE4 is the major genetic risk factor for Alzheimer’s disease at the population level; the reported comparison is between affected members of families and age-matched controls. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

Experiments suggest the order APOE4 > APOE3 > APOE2 for effects on amyloid deposition.

“In general, experiments suggest the order of APOE4 > APOE3 > APOE2 for effects on amyloid deposition (Fagan et al, 2002; Castellano et al, 2011; Hudry et al, 2013; Zhao et al, 2016)”

• cites Fagan et al, 2002 sourceNot checked — no full text of the source
• cites Castellano et al, 2011 sourceSupported
• cites Hudry et al, 2013 sourceSupported

May combine two claims.

Mertk and Inpp5d are downregulated in APOE4.

“Also, Mertk which is necessary for detection and engulfment of amyloid plaques by microglia (Huang et al, 2021), and Inpp5d, a known AD risk factor which has a role in limiting plaque formation (Castranio et al, 2023), are downregulated in APOE4.”

• cites Huang et al, 2021 sourceSupported
• cites Castranio et al, 2023 sourceSupported

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

Having an APOE2 allele or the rare APOE3Ch (Christchurch variant) shields patients against dementia even in the presence of high amyloid plaque load.

“Interestingly, having an APOE2 allele (Berlau et al, 2007, 2009) or the rare isoform APOE3Ch (Christchurch variant) (Arboleda-Velasquez et al, 2019), shields patients against dementia even in the presence of high amyloid plaque load.”

• cites Berlau et al, 2007, 2009Not matched

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

• cites Arboleda-Velasquez et al, 2019 sourcePartly supported

  The Abstract reports one APOE3 Christchurch homozygote who remained resistant to dementia despite high amyloid plaque burden. It does not establish that having the variant generally shields patients: the authors say other factors may have contributed and suggest that homozygosity is required to postpone clinical onset. The separately cited APOE2 claim is not assessed here. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

Other studies have reported that APOE2 expression is associated with similar or even higher plaque load than APOE3.

“Nevertheless other studies have also reported that APOE2 expression is associated with similar or even higher plaque load than APOE3 (Rodriguez et al, 2014; Youmans et al, 2012; Bales et al, 2009; Dodart et al, 2005).”

• cites Rodriguez et al, 2014 sourceCould not verify

  The Results section on plaque morphology (page unavailable) reports a higher percentage of diffuse plaques in E2FAD than E3FAD mice, but percentages of plaque types do not establish total plaque load. Searches for E2FAD–E3FAD plaque burden and total Aβ load found no comparison showing that APOE2 has similar or higher plaque load than APOE3. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites Youmans et al, 2012 sourceSupported
• cites Bales et al, 2009 sourceNot checked — no full text of the source

May combine two claims.

Reconstitution of APOE expression in microglia only in an Apoe knockout background or microglia-specific knockout of APOE had no effect on amyloid plaque formation or microglial association with plaques.

“However, reconstitution of APOE expression in microglia only in an Apoe knockout background (Mancuso et al, 2024) or microglia-specific knockout of APOE had no effect on amyloid plaque formation or microglial association with plaques (Henningfield et al, 2022).”

• cites Mancuso et al, 2024Not matched

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

• cites Henningfield et al, 2022 sourceSupported

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

APOE4 has been implicated in multiple pathophysiological functions including pro-inflammatory state in microglia and astrocytes, endolysosomal dysfunction, impaired autophagy, inducing and promoting spreading of tau pathology, neurodegeneration, maintaining integrity of pericytes and blood brain barrier, and impaired myelination in oligodendrocytes.

“Indeed, APOE4 has been implicated in the pro-inflammatory state in microglia (Brown et al, 2002; Colton et al, 2005; Lanfranco et al, 2021; Lin et al, 2018; Serrano-Pozo et al, 2021; Vitek et al, 2009) and astrocytes (Arnaud et al, 2022); endolysosomal dysfunction (Ji et al, 2002; Nuriel et al, 2017; Pohlkamp et al, 2021; Prasad and Rao, 2018; Xian et al, 2018); impaired autophagy (Parcon et al, 2”

• cites Brown et al, 2002 sourceNot checked — no full text of the source
• cites Colton et al, 2005 sourceNot checked — no full text of the source
• cites Lanfranco et al, 2021 sourceSupported

May combine two claims.

Restoration of APOE expression in astrocytes alone is sufficient to induce formation of fibrillar plaques and microglia reactivity.

“Restoration of APOE expression in astrocytes alone is sufficient to induce formation of fibrillar plaques and microglia reactivity, extending findings from previous studies (Holtzman et al, 2000a; Liu et al, 2017b; Mahan et al, 2022).”

• cites Holtzman et al, 2000a sourcePartly supported

  The Results section (“Expression of Human apoE Results in Fibrillar Aβ Deposition and Neuritic Plaques”; page unavailable) reports thioflavine-S-positive plaques in APPV717F mice expressing human apoE under an astrocyte-specific promoter. It does not report that restoring astrocytic apoE induced microglial reactivity; the Discussion mentions activated glial cells only as a general feature of neuritic plaques. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Liu et al, 2017b sourcePartly supported

  The Results sections on fibrillar plaques and Aβ-associated gliosis report that astrocytic apoE4 expression increased fibrillar plaque formation and Iba1-positive microglia in APP mice. The claim is broader than those findings: apoE3 did not increase either measure, and the study shows enhanced plaque formation in an amyloid-prone model, not that astrocytic APOE expression alone initiates plaques. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Mahan et al, 2022 sourcePartly supported

  The Discussion (page unavailable) reports that removing astrocytic apoE strongly reduced fibrillar plaques, and the Results section on microglial activation (page unavailable) reports reduced activation-associated gene expression after that removal. These findings support a role for astrocytic apoE in both outcomes, but the paper tested reduction, not restoration of APOE expression in astrocytes alone; it therefore does not establish the claimed sufficiency. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

APOE is not transferred over the blood brain barrier, suggesting astrocytes are the main contributor of APOE in the brain.

“However, APOE is not transferred over the blood brain barrier (Huynh et al, 2019; Lane-Donovan et al, 2016) suggesting that astrocytes are the main contributor of APOE in the brain (Boyles et al, 1985; Zhang et al, 2014).”

• cites Huynh et al, 2019 sourceSupported
• cites Lane-Donovan et al, 2016 sourceSupported
• cites Boyles et al, 1985 sourceSupported

May combine two claims.

Compared to APOE2, APOE4 had significant upregulation of several DAM marker genes (Cd74, Cst7, Cd63, Cd9, Ccl6), complement cascade genes (C1qa, C1qb, C1qc), and the pro-inflammatory gene Nfkbia.

“We observed that compared to APOE2, APOE4 had a significant upregulation of several DAM marker genes such as Cd74, Cst7, Cd63, Cd9, Ccl6, complement cascade genes (C1qa, C1qb, C1qc) and the pro-inflammatory gene Nfkbia (Sousa et al, 2018).”

• cites Sousa et al, 2018 sourcePartly supported

  In Results and Discussion (page unavailable), Sousa et al. compare inflammatory microglia with DAM, identify Cd9 as a disease-associated gene, and identify C1qa, C1qb, and C1qc as complement-system genes. The paper does not report the citing authors’ APOE4-versus-APOE2 result or establish the full listed set as DAM markers; it instead reports that those complement genes were downregulated in DAM relative to controls. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

• AssumptionRests on an assumption: APOE3 is neutral.

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

APOE2 expression leads to increased APOE levels in mouse models and human brain, which can influence amyloid pathology.

“It has also been shown that APOE2 expression leads to increased APOE levels in mouse models and human brain (Riddell et al, 2008; Bales et al, 2009; Conejero-Goldberg et al, 2014), which can influence amyloid pathology.”

• cites Riddell et al, 2008 sourcePartly supported

  The Figure 2 caption (indexed under Discussion; page unavailable) reports genotype-dependent apoE levels in the brains of targeted-replacement mice, including ε2/2 mice. The Discussion proposes that brain apoE levels may affect amyloid-β clearance and deposition. The paper does not demonstrate increased apoE levels in human brain with APOE2 expression, nor directly test the effect of APOE2 on amyloid pathology. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites Bales et al, 2009 sourceNot checked — no full text of the source
• cites Conejero-Goldberg et al, 2014 sourceNot checked — no full text of the source

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

Viral-vector mediated overexpression of APOE4 isoforms in AD mouse brains accelerates plaque formation.

“This has also been observed with viral-vector mediated overexpression of APOE4 isoforms in AD mouse brains (Dodart et al, 2005; Hudry et al, 2013; Zhao et al, 2016).”

• cites Dodart et al, 2005 sourcePartly supported

  The Discussion reports that lentiviral apoE4 increased Aβ deposition and congophilic amyloid within five weeks in PDAPP mice lacking mouse apoE. The claim omits that condition: the Results state that apoE4 did not increase hippocampal Aβ burden in PDAPP mice expressing mouse apoE. Page numbers were not available in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites Hudry et al, 2013 sourceSupported
• cites Zhao et al, 2016 sourceNot checked — no full text of the source

May combine two claims.

APOE4 affects the endosomal-lysosomal pathway and the APOE4 induced recycling block can be rescued by decreasing pH.

“APOE4 affects, however, the endosomal-lysosomal pathway (Nuriel et al, 2017) and the APOE4 induced recycling block can be rescued by decreasing pH (Xian et al, 2018).”

• cites Nuriel et al, 2017 sourceSupported
• cites Xian et al, 2018 sourceSupported

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

We did not explore the lipidation status of different APOE isoforms, which influences Aβ pathology.

“Additionally, we did not explore the lipidation status of different APOE isoforms, which influences Aβ pathology (Koldamova et al, 2005; Wahrle et al, 2005).”

• cites Koldamova et al, 2005 sourceNot checked — no full text of the source
• cites Wahrle et al, 2005 sourcePartly supported

  The Discussion (page not provided) links poorly lipidated apoE to increased Aβ deposition in PDAPP mice and describes isoform-dependent interactions with Aβ in vitro. It does not directly show that the lipidation status of different human APOE isoforms influences Aβ pathology; it presents the mechanism for its mouse findings as likely rather than established. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

Cell type annotation was done using AddModuleScore to assign cell scores based on previously identified signatures for different brain cell types.

“Cell type annotation was done using seurat::AddModuleScore() to assign cell scores based on previously identified signatures for different brain cell types (Zeisel et al, 2018).”

• cites Zeisel et al, 2018 sourceSupported

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

Previous reports have demonstrated that gender can affect AD pathology, microglia response and APOE expression, while the protective and disease causing effects of APOE2 and APOE4 are variable in different ethnic populations.

“Previous reports have demonstrated that gender can affect AD pathology, microglia response and APOE expression (Stephen et al, 2019; Thomas et al, 2016; Sala Frigerio et al, 2019), while the protective and disease causing effects of APOE2 and APOE4 are variable in different ethnic populations (Farrer et al, 1997; Naslavsky et al, 2022).”

• cites Stephen et al, 2019 sourcePartly supported

  The Results report that sex was associated with amyloid plaque burden and microglial responses in EFAD mice. The Discussion also describes sex differences in microglial plaque interactions, but the paper does not establish that sex affects APOE expression. The statement about ethnic populations is attributed to other citations, not Stephen et al. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites Thomas et al, 2016 sourcePartly supported

  The Introduction and the Discussion section on sex and APOE4-induced dysfunction report greater APOE4-associated AD risk in females and sex-related differences in AD-like pathology. The Discussion mentions microglial inflammatory responses and suggests females may have a stronger brain inflammatory response, but does not establish a sex-specific microglial response or a sex effect on APOE expression. The ethnicity clause is attributed to other citations, not Thomas et al.; the indexed passages have no page numbers. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites Sala Frigerio et al, 2019 sourcePartly supported

  The Results section (“Microglia Differentiate into Mutually Exclusive Response States”; page not provided) reports that female mice showed earlier, faster microglial responses to Aβ than males. The paper also reports Apoe upregulation in activated microglia, but the cited passages do not establish that sex changes Apoe expression or AD pathology. The ethnic-population claim is attached to other citations, not this paper. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

Aβ, when endocytosed, becomes concentrated in acidic endosomal compartments, which promotes amyloid fibril formation and then secretion.

“Aβ, when endocytosed, becomes concentrated in the acidic endosomal compartments, which promotes amyloid fibril formation and then secretion (Chung et al, 1999; Hu et al, 2009; Knauer et al, 1992).”

• cites Chung et al, 1999 sourcePartly supported

  The Results report that internalized soluble Aβ concentrates in organelles consistent with late endosomes and lysosomes before release. The Discussion says low pH should promote fibril growth, but treats incorporation of soluble Aβ into fibrils as a possibility. The paper reports release of internalized Aβ, not that newly formed fibrils are subsequently secreted; the claim presents that sequence as established. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites Hu et al, 2009Not matched

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

• cites Knauer et al, 1992 sourcePartly supported

  The abstract reports that endocytosed Aβ1–42 accumulated in cultured fibroblasts in an aggregated state, in a compartment overlapping late endosomes or secondary lysosomes. It does not show that acidity promotes fibril formation or that the accumulated peptide is subsequently secreted. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

Functional enrichment analysis was done using the g:Profiler web interface, querying cluster markers against the GO biological process database with g:SCS multiple testing correction and a significance threshold of 0.05.

“This was done using g:Profiler web interface, where the cluster markers were queried against GO biological process database, with g:SCS multiple testing correction method applying significance threshold of 0.05 (Kolberg et al, 2023).”

• cites Kolberg et al, 2023 sourceSupported

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

The other mechanisms of APOE4 dysfunction probably explain the association with neurodegenerative diseases in general, such as Parkinson's disease, Lewy body dementia, TDP-43 proteinopathy, and vascular dementia.

“The other mechanisms of APOE4 dysfunction probably explain the association with neurodegenerative diseases in general, such as Parkinson’s disease (odds ratio ~1.6–4.19) and Lewy body dementia (odds ratio ~2.4–6.1) (Bras et al, 2014; Guerreiro et al, 2018; Huang et al, 2006; Irwin et al, 2012; Tropea et al, 2018; Tsuang et al, 2013); TDP-43 proteinopathy (odds ratio ~2) (Josephs et al, 2014; Wenn”

• cites Bras et al, 2014 sourcePartly supported

  The Results and Discussion report an association between APOE ε4 and dementia with Lewy bodies. But the Discussion says this association is likely driven by the variant’s amyloid-β-promoting effect and only notes that other mechanisms cannot be excluded. The cited passages do not establish that other APOE4 mechanisms explain an association with Parkinson’s disease or substantiate the quoted odds-ratio ranges. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Guerreiro et al, 2018 sourcePartly supported

  The Results report an APOE association with dementia with Lewy bodies (OR 2.40), supporting the lower end of the cited range. The Discussion says APOE ε4 has been implicated in cognitive impairment within Parkinson’s disease, but not in Parkinson’s disease risk itself. The paper does not establish that other mechanisms of APOE4 dysfunction explain these associations. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Huang et al, 2006 sourceNot checked — no full text of the source

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

Astrocyte clusters were scored using AddModuleScore for markers from previously published genesets of astrocyte cell states, plaque-induced genes, and AD risk genes.

“Astrocyte clusters were scored using seurat::AddModuleScore(), for markers from previously published genesets of astrocyte cell states (Zamanian et al, 2012; Hasel et al, 2021), plaque-induced genes (PIGs) (Chen et al, 2020) and AD risk genes (Bellenguez et al, 2022).”

• cites Zamanian et al, 2012 sourceSupported
• cites Hasel et al, 2021 sourceNot checked — no full text of the source
• cites Chen et al, 2020 sourceSupported

Classified as probably not a substantial claim.

5 μM Actinomycin D was kept during collection and enzymatic dissociation of the tissue to preserve transcriptional state and prevent artificial induction of stress-response genes during tissue dissociation.

“Note that 5 μM ActD was kept during collection and enzymatic dissociation of the tissue to preserve transcriptional state and prevent artificial induction stress-response genes during tissue dissociation (Marsh et al, 2022; Perocchi et al, 2007).”

• cites Marsh et al, 2022 sourcePartly supported

  The Methods section, “Inhibitor cocktail to prevent activation” (page not provided), specifies actinomycin D in both dissection and digestion buffers, and the Main section says the inhibitor protocol prevents artifactual transcription during dissociation. The specified concentration is 5 μg ml−1, not 5 μM, and the protocol uses actinomycin D alongside other inhibitors rather than establishing that actinomycin D alone has this effect. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites Perocchi et al, 2007 sourcePartly supported

  The Introduction (page unavailable) states that ActD inhibits transcription, which supports the general rationale for limiting new transcription. But the paper uses ActD during reverse transcription of yeast RNA, not at 5 μM during tissue collection or enzymatic dissociation; it does not show prevention of dissociation-induced stress-response genes. Materials and Methods (page unavailable) instead specifies 6 μg/ml ActD during cDNA synthesis. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

Conditional knockout of microRNA miR155 in microglia in APP/PS1 mice leads to activation of interferon-mediated signalling, which was associated with increased phagocytosis and plaque compaction.

“Recently it was reported that conditional knockout of microRNA miR155 in microglia in APP/PS1 mice leads to activation of interferon-mediated signalling, which was associated with increased phagocytosis and plaque compaction (Yin et al, 2023a).”

• cites Yin et al, 2023a sourceSupported

May combine two claims.

At transcriptional level, microglia from the APOE4 group upregulated several DAM markers (Cd74, Cst7, Cd63, Cd9, Ccl6), complement cascade genes (C1qa, C1qb, C1qc) and the pro-inflammatory gene Nfkbia, similar to transcriptomic changes in microglia from APOE4 AD individuals.

“At transcriptional level, microglia from the APOE4 group upregulated several DAM markers (Cd74, Cst7, Cd63, Cd9, Ccl6), complement cascade genes (C1qa, C1qb, C1qc) and the pro-inflammatory gene Nfkbia (Sousa et al, 2018), similar to transcriptomic changes in microglia from APOE4 AD individuals (Serrano-Pozo et al, 2021).”

• cites Sousa et al, 2018 sourcePartly supported

  Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites Serrano-Pozo et al, 2021 sourceSupported

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

Microglia are the main responders to amyloid plaques, mounting a DAM response, which also causes induction of APOE expression in these cells.

“Microglia are the main responders to amyloid plaques, mounting a DAM response (disease associated microglial cell-state response) (Keren-Shaul et al, 2017), which also causes the induction of APOE expression in these cells.”

• cites Keren-Shaul et al, 2017 sourceSupported

May combine two claims.

This is in line with evidence from brain samples of APOE4 patients showing downregulation of autophagy components and supports the role of astrocyte-mediated autophagy in degradation of Aβ.

“This is in line with evidence from brain samples of APOE4 patients showing downregulation of autophagy components (Parcon et al, 2018) and supports the role of astrocyte-mediated autophagy in degradation of Aβ (Simonovitch et al, 2016, 2019).”

• cites Parcon et al, 2018 sourceSupported
• cites Simonovitch et al, 2016, 2019Not matched

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

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

Haralick's features are a well-established set of rotation invariant image statistics used in medical classification across many fields and imaging modalities.

“Haralick’s features are a well-established set of rotation invariant image statistics that have been used in medical classification issues across many fields and imaging modalities (Doyle et al, 2008; Gnep et al, 2017; Hapsari et al, 2022).”

• cites Doyle et al, 2008 sourceNot checked — no full text of the source
• cites Gnep et al, 2017 sourcePartly supported

  The MRI Feature Extraction section describes Haralick features as image statistics derived from gray-level co-occurrence matrices and says the study averaged values across four directions. The Discussion describes their use in several cancers, but only in a few MRI studies. The paper does not establish that the features are inherently rotation invariant or used for medical classification across many fields and imaging modalities. Page numbers were not available in our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites Hapsari et al, 2022 sourcePartly supported

  The Introduction describes Haralick features as GLCM-based texture statistics used for disease classification, and the Discussion reports tests on iris, brain MRI, and chest X-ray images. But the paper’s invariant features are invariant to gray-level quantization, not stated to be rotation invariant (Discussion and Conclusions; page numbers unavailable). (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

The DAM response is dependent on APOE expression but it was thought that APOE expression in microglia was essential.

“The DAM response is dependent on APOE expression (Krasemann et al, 2017; Sala Frigerio et al, 2019) but it was thought that APOE expression in microglia was essential.”

• cites Krasemann et al, 2017 sourceSupported
• cites Sala Frigerio et al, 2019 sourceSupported

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

Recent data suggests a cell autonomous role of microglia-specific APOE4 expression in blocking the DAM response, compared to APOE3.

“Recent data also suggests a cell autonomous role of microglia-specific APOE4 expression in blocking the DAM response, compared to APOE3 (Yin et al, 2023b; Liu et al, 2023).”

• cites Yin et al, 2023b sourceSupported
• cites Liu et al, 2023 sourceSupported

May combine two claims.

APOE4 is a 'core' gene of AD pathogenesis in the sense that the strong effect on amyloid pathology, a core pathway of AD, explains the strong specific association with AD (odds ratio ~3.7-14.5).

“Therefore, we propose that APOE4 is a “core” gene (Liu et al, 2019) of AD pathogenesis in the sense that the strong effect on amyloid pathology, a core pathway of AD, explains the strong specific association with AD (odds ratio ~3.7–14.5).”

• cites Liu et al, 2019 sourceSupported

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

SuperExactTest was used to calculate statistical significance and visualize overlap of differentially expressed genes while comparing each group with APOE3.

“SuperExactTest (Wang et al, 2015) was used to calculate statistical significance (Datasets EV3, EV6) and visualize overlap of differentially expressed genes while comparing each group with APOE3.”

• cites Wang et al, 2015 sourceSupported

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

Enrichment of plaque-induced genes in the intersection of differentially expressed genes in the microglia dataset was tested using a hypergeometric test with Bonferroni correction of p-values.

“Enrichment of plaque-induced genes (PIGs) (Chen et al, 2020) in intersection of differentially expressed genes in microglia dataset was tested using hypergeometric test with Bonferroni correction of p-values.”

• cites Chen et al, 2020 sourceSupported

May combine two claims.

The increase in microglia population with plaque load is driven by increased number of plaque-associated microglia, while the non-plaque-associated population remains relatively stable.

“this increase in microglia population with plaque load was driven by increased number of plaque-associated microglia (R2 = 0.97, slope = 973.13, p = 2e−16) while the non-plaque-associated population remained relatively stable despite increase in plaque load (R2 = 0.5, slope = −242.04, p = 3.5e−05)”

• AssumptionRests on an assumption: The AppNL-G-F x Apoe−/− mouse model is a valid model for studying APOE’s role in amyloid plaque pathology in Alzheimer’s disease.

May combine two claims.

Microglia depletion increased APOE levels in APOE3 and APOE4 mice, with a non-significant trend in APOE2 mice.

“It is notable that especially in the APOE3 mice, the APOE levels are increased in the microglia-depleted brains (Appendix Fig. S5Bii). This increase in APOE levels was present in APOE4 mice as well (Appendix Fig. S5Biii), with APOE2 mice showing a similar non-significant trend (Appendix Fig. S5Bi).”

May combine two claims.

APOE2 expression only in astrocytes did not reduce amyloid plaque formation compared to APOE3, even when APOE4 had significantly higher levels than both.

“Interestingly, in our experimental paradigm, APOE2 expression only in astrocytes did not reduce the amyloid plaque formation compared to APOE3, even when APOE4 had significantly higher levels than both.”

• AssumptionRests on an assumption: The AppNL-G-F x Apoe−/− mouse model is a valid model for studying APOE’s role in amyloid plaque pathology in Alzheimer’s disease.

May combine two claims.

Astrocytes expressing APOE show altered transcriptional responses, with potential impairment of autophagy and proteostasis in those expressing APOE4.

“Astrocytes expressing APOE showed altered transcriptional responses, with potential impairment of autophagy and proteostasis in those expressing APOE4.”

• AssumptionRests on an assumption: The AppNL-G-F x Apoe−/− mouse model is a valid model for studying APOE’s role in amyloid plaque pathology in Alzheimer’s disease.

May combine two claims.

The APOE4 group shows low levels of soluble Aβ aggregates despite high fibrillar plaque deposition.

“Interestingly, the APOE4 group which showed a high level of fibrillar plaque deposition, showed low levels of soluble Aβ aggregates (Fig. 1G).”

• AssumptionRests on an assumption: The AppNL-G-F x Apoe−/− mouse model is a valid model for studying APOE’s role in amyloid plaque pathology in Alzheimer’s disease.

May combine two claims.

The APOE effect of astrocytes on amyloid plaques is largely cell autonomous, as in the absence of microglia, amyloid plaques are still generated.

“Moreover, we show that the APOE effect of astrocytes on amyloid plaques is largely cell autonomous, as in the absence of microglia, amyloid plaques are still generated.”

• AssumptionRests on an assumption: The AppNL-G-F x Apoe−/− mouse model is a valid model for studying APOE’s role in amyloid plaque pathology in Alzheimer’s disease.

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

Unbiased clustering analysis identified 7 sub-populations of microglia.

“Unbiased clustering analysis led to the identification of 7 sub-populations of microglia (Figs. EV2A,B, Dataset EV4–EV5).”

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

Differential gene expression analysis revealed shifts in gene signatures among experimental groups.

“However, we observed interesting shifts in gene signatures by performing differential gene expression analysis (Fig. EV3A, Dataset EV6).”

May combine two claims.

Both the APOE2 group (β = −148.2, SE = 32.22, p = 0.0013) and the APOE4 group (β = −357.1, SE = 60.64, p = 0.0002) showed a statistically significant decrease in mean Aβ42 levels with PLX-treatment compared to non-treated group.

“On the other hand, both the APOE2 group (β = −148.2, SE = 32.22, p = 0.0013) and the APOE4 group (β = −357.1, SE = 60.64, p = 0.0002) showed a statistically significant decrease in mean Aβ42 levels with PLX-treatment compared to the non-treated group (Fig. 5D).”

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

Droplet-based scRNAseq on CD11b+/CD45-low microglia isolated at 6 months of age from mouse brains expressing different APOE isoforms in astrocytes retained 18,569 Apoe-deficient microglia across four experimental groups (APOEKO, APOE2, APOE3, APOE4).

“After filtering out non-microglial cells and low-quality cells, we retained 18,569 Apoe-deficient microglia over the four experimental groups (APOEKO, APOE2, APOE3 and APOE4).”

May combine two claims.

Plaque-associated microglia have significantly lower distance to neighbouring microglia, lower territory size, and lower total process length compared to non-plaque-associated microglia.

“Plaque-associated microglia clustered together with a significantly lower distance to neighbouring microglia, compared to the well-tiled microglia present away from plaques (Appendix Fig. S4E). Plaque-associated microglia also had significantly lower territory size (convex area) (Appendix Fig. S4Fi) and total process length (Appendix Fig. S4Fii)”

• AssumptionRests on an assumption: The AppNL-G-F x Apoe−/− mouse model is a valid model for studying APOE’s role in amyloid plaque pathology in Alzheimer’s disease.

May combine two claims.

The highest intersection of DEGs was observed between APOEKO and APOE4 (p = 4.2e−78), with 52 genes commonly upregulated in the two groups.

“Interestingly, among the different sets of DEGs, the highest intersection was observed between APOEKO and APOE4 (p = 4.2e−78), where 52 genes were commonly upregulated in the two groups (Fig. EV3A, EV3C, Dataset EV6)”

May combine two claims.

For the APOE3 group, there was no significant association between Aβ42 levels and APOE levels and no significant shift in mean Aβ42 levels (β = −28.53, SE = 38.31, p = 0.4708) with PLX-treatment.

“Interestingly, for the APOE3 group, like above (Fig. 1E), we found no significant association between Aβ42 levels and APOE levels and there was no significant shift in mean Aβ42 levels (β = −28.53, SE = 38.31, p = 0.4708) with PLX-treatment.”

May combine two claims.

In the AppNL-G-F mouse, Apoe deletion almost annihilates the generation of fibrillar amyloid plaques, while increasing soluble Aβ aggregate species.

“We confirm here in the AppNL-G-F mouse that despite the presence of strong aggregation promoting mutations in this particular Aβ sequence, Apoe deletion almost annihilates the generation of fibrillar amyloid plaques, while increasing the soluble Aβ aggregate species (Fig. 1).”

May combine two claims.

At morphological level, we demonstrated increased microglial clustering and specific staining of DAM markers in Apoe−/− microglia surrounding the amyloid plaques.

“At morphological level, we demonstrated increased microglial clustering (Fig. 4A), and specific staining of DAM markers (Fig. 4B) in Apoe−/− microglia surrounding the amyloid plaques.”

May combine two claims.

The APOE2 allele causes more amyloid plaques than APOE3 in our model, while it also leaves more soluble Aβ aggregates in the extracellular milieu.

“At first glance, increased fibrilization might explain decreased levels of soluble Aβ aggregates, but the APOE2 allele causes more amyloid plaques than APOE3 in our model, while it also leaves more soluble Aβ aggregates in the extracellular milieu (Fig. 1C,G).”

• AssumptionRests on an assumption: APOE3 is neutral.

May combine two claims.

APOE3 expression in astrocytes in our experiments results in restoration of amyloid plaque formation at 6 months and decrease in soluble Aβ aggregates.

“APOE3 expression in astrocytes in our experiments results in two phenomena: (1) the restoration of amyloid plaque formation at 6 months and (2) the decrease in soluble Aβ aggregates in these animals.”

• AssumptionRests on an assumption: APOE3 is neutral.

May combine two claims.

APOE2, but not APOE3, shows a dose-dependent relationship between level of expression and amyloid aggregation, as APOE4 does.

“In addition, APOE2, but not APOE3, shows a dose-dependent relationship between level of expression and amyloid aggregation (Fig. 1E), as APOE4 does.”

• AssumptionRests on an assumption: APOE3 is neutral.

Classified as probably not a substantial claim.

In our model, APOE expression with the AAV was induced in different subtypes of astrocytes in the mouse brain.

“In our model, APOE expression with the AAV was induced in different subtypes of astrocytes in the mouse brain (Fig. 2A).”

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

Enrichment of APOEKO group in Cluster a0, which is marked by processes related to lipid metabolism, is expected in the absence of a lipoprotein like APOE.

“Enrichment of APOEKO group in Cluster a0, which is marked by processes related to lipid metabolism is expected in the absence of a lipoprotein like APOE.”

May combine two claims.

Cluster a6 is exclusively present in APOE-expressing groups and is enriched for genes modulating inflammatory pathways which might play a role in astrocyte reactivity towards amyloid pathology.

“Cluster a6 which is exclusively present in the APOE-expressing groups, and thus also the plaque-bearing brains, is enriched for genes modulating inflammatory pathways which might play a role in astrocyte reactivity towards amyloid pathology.”

May combine two claims.

This was accompanied with limited plaque formation, compact plaques and low levels of oligomeric Aβ in the mice brain.

“This was accompanied with limited plaque formation, compact plaques and low levels of oligomeric Aβ in the mice brain.”

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

The APOE2 variant is a protective allele against Alzheimer's disease.

“while the APOE2 variant was the first identified protective allele against AD (Corder et al, 1994; Nagy et al, 1995).”

• cites Corder et al, 1994 sourceNot checked — no full text of the source
• cites Nagy et al, 1995 sourceNot checked — no full text of the source

May combine two claims.

At morphological level, microglial responses to fibrillar plaques do not depend on the isoform of APOE produced by astrocytes but are likely mediated directly by the plaque load.

“Therefore, at morphological level, microglial responses to fibrillar plaques do not seem to depend on the isoform of APOE produced by astrocytes but are likely mediated directly by the plaque load.”

• AssumptionRests on an assumption: The AppNL-G-F x Apoe−/− mouse model is a valid model for studying APOE’s role in amyloid plaque pathology in Alzheimer’s disease.

May combine two claims.

The AAV2/8 vector with ALDH1L1 promoter did not transduce neurons, oligodendrocytes, or microglia, as confirmed by lack of mCherry expression in NeuN+, Apc+, and Iba1+ cells.

“However, we confirmed by immunofluorescence the lack of mCherry expression in NeuN+ neurons, Apc+ oligodendrocytes and Iba1+ microglia (Appendix Fig. S1D).”

May combine two claims.

While APOE from astrocytes is sufficient for plaque deposition at 6 months of age in this model, microglia play a role in modulating Aβ pathology in APOE2 and APOE4 expressing mice.

“Therefore, this suggests that while APOE from astrocytes is sufficient for plaque deposition at 6 months of age in this model, microglia play a role in modulating Aβ pathology in APOE2 and APOE4 expressing mice.”

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

We cannot rule out that microglia played a role in APOE-induced initial seeding of plaques or that remaining microglia are sufficient to induce plaque seeds.

“As depletion of microglia was performed at 2 months and initial amyloid plaque seeding might have occurred already before that time point, we cannot rule out the possibility that microglia played a role in the APOE induced initial seeding of the plaques or that the remaining microglia are sufficient to induce plaque seeds.”

May combine two claims.

Around 14% of ACSA2+ astrocytes expressed mCherry and around 90% of mCherry-positive cells were positive for astrocyte marker ACSA2.

“Using flow cytometry, we found that around 14% of ACSA2+ astrocytes expressed mCherry (Appendix Fig. S1Cii) and around 90% of the mCherry-positive cells were positive for astrocyte marker ACSA2 (Appendix Fig. S1Ciii).”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

No consensus exists whether and how additional pathophysiological functions of APOE2 and APOE4 contribute to the specific risk of AD.

“No consensus exists whether and how additional pathophysiological functions of APOE2 and APOE4 contribute to the specific risk of AD.”

Classified as probably not a substantial claim.

Multiple regression analysis was used to account for the confounding effect of APOE levels when comparing guanidine-soluble Aβ42 levels between PLX3397-treated and untreated animals in each experimental group.

“Since we have earlier seen that APOE levels also influence Aβ plaque deposition (Fig. 1E), we used multiple regression analysis to account for the confounding effect of APOE levels while comparing guanidine-soluble Aβ42 levels between PLX3397-treated and untreated animals in each experimental group (Fig. 5D).”

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

The protective effects of APOE3Ch downstream of amyloid plaque formation were also seen in an AD amyloid mouse model where overexpression had stronger effect in reducing tau seeding and propagation along with neuronal dystrophy around plaques upon injection of AD-tau brain extract.

“The protective effects of APOE3Ch downstream of the amyloid plaque formation (on which a relative modest effect was observed) were also seen in an AD amyloid mouse model where overexpression had stronger effect in reducing tau seeding and propagation along with neuronal dystrophy around plaques upon injection of AD-tau brain extract (Chen et al, 2023).”

• cites Chen et al, 2023Not matched

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

May combine two claims.

Different APOE isoforms induce different transcriptomic responses in astrocytes in the AppNL-G-F mouse model, with potential impairment of proteostasis and autophagy in APOE4-expressing astrocytes.

“Thus, different APOE isoforms induce different transcriptomic responses in astrocytes in the AppNL-G-F mouse model, with potential impairment of proteostasis and autophagy in APOE4-expressing astrocytes.”

• AssumptionRests on an assumption: The AppNL-G-F x Apoe−/− mouse model is a valid model for studying APOE’s role in amyloid plaque pathology in Alzheimer’s disease.

May combine two claims.

APOE expression was confirmed in mCherry+ cells, demonstrating reconstitution of human APOE expression in astrocytes.

“We also confirmed APOE expression in mCherry+ cells (Appendix Fig. S1E), demonstrating that we reconstituted human APOE expression in astrocytes.”

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

All experiments were conducted in accordance with institutional and national guidelines and regulations, following approval of Ethical Committee for Animal Experimentation at KU Leuven.

“All experiments were conducted in accordance with institutional and national guidelines and regulations, following approval of Ethical Committee for Animal Experimentation at the University of Leuven (KU Leuven) (permit number P007/2017 and P072/2019).”

May combine two claims.

This process is receptor-mediated and therefore likely saturable, hence there was no concentration-dependent effect of APOE3 on uptake of Aβ in the overexpression condition of our experiment.

“This process is receptor-mediated and therefore likely saturable, hence there was no concentration-dependent effect of APOE3 on uptake of Aβ in the overexpression condition of our experiment.”

• AssumptionRests on an assumption: APOE is secreted by astrocytes and taken up via LDL receptors into endosomal compartments of all cell types in the brain, including microglia and astrocytes.

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

Contaminating cell types in the astrocyte dataset were identified as ependymal cells, mural cells, microglia, endothelial cells, and a population of undetermined identity.

“Contaminating cell types were identified (Appendix Fig. S6D, E) as ependymal cells (astrocyte QC Cluster 5), mural cells (astrocyte QC Cluster 7), microglia (astrocyte QC Cluster 9), endothelial cells (astrocyte QC Cluster 10) and a population of undetermined identity (astrocyte QC Cluster 8).”

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

Cell hashing did not work efficiently for astrocytes because a good proportion of cells identified as negatives had read counts in the same range as singlet cells.

“Cell hashing did not work efficiently for astrocytes, as a good proportion of cells identified as negatives had read counts in the same range as Singlet cells.”

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

Integration anchors were identified and batch correction was performed.

“Integration anchors were identified (Seurat::FindIntegrationAnchors()) and batch correction was performed (Seurat::IntegrateData()).”

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

PLX3397/Pexidartinib was formulated in standard chow and administered at 600 mg/kg for 4 months starting at 2 months of age before onset of plaque deposition.

“Modified chow containing PLX3397 (600 mg/kg of chow) was administered to the mice for 4 months, starting at 2 months of age before onset of plaque deposition.”

May combine two claims.

Our work shows that even in a relatively circumscribed and limited part of Alzheimer's disease pathogenesis, i.e., the formation of amyloid plaques and the microglia response to it, the different isoforms of APOE act in diverse and unanticipated ways, affecting amyloid plaques, Aβ soluble aggregates, but also the cellular reactions of microglia and astrocytes.

“Our work shows that even in a relative circumscribed and limited part of Alzheimer’s disease pathogenesis, i.e., the formation of amyloid plaques and the microglia response to it, the different isoforms of APOE act in diverse and unanticipated ways, affecting amyloid plaques, Aβ soluble aggregates, but also the cellular reactions of microglia and astrocytes.”

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

After cell type annotation, about 7% of cells in the astrocyte dataset had non-astrocyte identity.

“After cell type annotation as described above, ~7% of cells had non-astrocyte identity.”

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

APOE expression and Aβ pathology are closely associated in our model which makes it difficult to determine the factors contributing directly or indirectly to the DEG changes seen in microglia and astrocytes.

“APOE expression and Aβ pathology are closely associated in our model which makes it difficult to determine the factors contributing directly or indirectly to the DEG changes seen in microglia and astrocytes.”

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

Reverse transcription of mRNA was done using Superscript II reverse transcriptase and real-time semi-quantitative PCR was performed using SensiFast Sybr No-Rox kit.

“Reverse transcription of mRNA was done using the Superscript II reverse transcriptase. Real-time semi-quantitative PCR was performed using the SensiFast Sybr No-Rox kit for cDNA.”

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

About 9% of cells in the microglia dataset had non-microglial identity, with half of those having macrophage identity.

“~9% of cells had non-microglial identity with half of those having macrophage identity.”

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

To remove batch effects between libraries during cell clustering, the Seurat object was split into individual libraries.

“To remove batch effects between libraries while cell clustering, the Seurat Object was split into individual libraries using Seurat::SplitObject().”

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

After quality control, 18,667 astrocytes were retained for analysis as good-quality cells.

“Finally, 18,667 astrocytes were retained for analysis as good-quality cells.”

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

Only singlets were retained because doublets and singlets had abnormally high and low numbers of reads, respectively, indicative of poor-quality cells.

“Only singlets were retained, as doublets and singlets had abnormally high and low number of reads, respectively, indicative of poor-quality cells.”

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

It is possible that APOE-Aβ co-aggregates are taken up by microglia for clearance, and when microglia is depleted, this leads to increased levels of APOE in the mouse brain.

“Thus, it is possible that APOE-Aβ co-aggregates are taken up by microglia for clearance, and when microglia is depleted, this leads to increased levels of APOE in the mouse brain.”

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

Contaminating cell types in the microglia dataset were identified as macrophages, endothelial cells, a mix of astrocyte and microglial cells, monocytes, and oligodendrocytes.

“Contaminating cell types were identified (Appendix Fig. S6A, B) as macrophages (microglia QC Cluster 4), endothelial cells (microglia QC Cluster 5), mix of astrocyte and microglial cells (microglia QC Cluster 6), monocytes (microglia QC Clusters 7 and 9), and oligodendrocytes (microglia QC Cluster 10).”

May combine two claims.

A downregulation of APOE expression is not mimicking the protective effects of APOE2, and APOE2 protective action is complex and remains poorly understood.

“A downregulation of APOE expression is not mimicking the protective effects of APOE2, and APOE2 protective action is complex and remains poorly understood.”

May combine two claims.

This approach allowed to establish the dose-dependent effects of different APOE isoforms on amyloid deposition, helping to understand the effect of dose versus isoform.

“However, this approach allowed to establish the dose-dependent effects of different APOE isoforms on amyloid deposition, helping to understand the effect of dose versus isoform.”

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

Mice were randomly assigned to experimental groups (unblinded).

“Mice were randomly assigned to experimental groups (unblinded).”

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

Labkit was particularly good at identifying which pixels belonged to microglia but failed to distinguish individual microglia from each other, especially around plaques where microglia cluster densely.

“the established method Labkit was found to be particularly good at identifying which pixels belonged to microglia but failed to distinguish individual microglia from each-other, especially around plaques, where microglia tend to cluster densely.”

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

Among Haralick's features, ASM was the statistic most congruent with the visual differences between more diffuse and more dense plaques.

“Among the different Haralick’s features, ASM was found to be the statistic most congruent with the visual differences between more diffuse and more dense plaques.”

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

For representative images, brightness was adjusted equally across all experimental groups when necessary for visualization.

“For representative images provided in Figures, brightness was adjusted, if necessary (by equal measure across all experimental groups), for better visualization.”

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

For sample collection, mice were euthanized using an overdose of carbon dioxide and perfused with ice-cold 1x DPBS.

“For sample collection, mice were euthanized using an overdose of carbon dioxide and perfused with ice-cold 1x DPBS.”

May combine two claims.

In the absence of APOE, clearance of Aβ is lowered, resulting in increased soluble Aβ aggregates in the medium as demonstrated with the APOEKO.

“Thus, in the absence of APOE, clearance of Aβ is lowered, resulting in increased soluble Aβ aggregates in the medium as demonstrated with the APOEKO.”

• AssumptionRests on an assumption: APOE is secreted by astrocytes and taken up via LDL receptors into endosomal compartments of all cell types in the brain, including microglia and astrocytes.

May combine two claims.

When a critical concentration of Aβ is reached in the endosomes, Aβ fibrilization is induced, leading to release of Aβ fibrils and generation of Aβ plaques extracellularly.

“When a critical concentration of Aβ is reached in the endosomes, Aβ fibrilization is induced, leading to the release of Aβ fibrils and the generation of Aβ plaques extracellularly as seen in Fig. 1B,C and measured as an increase in guanidine extractable Aβ42 in Fig. 1E.”

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

We did not investigate possible gender- and ethnicity-specific effects of the different APOE isoforms.

“We also did not investigate possible gender- and ethnicity-specific effects of the different APOE isoforms.”

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

About 31% of the remaining astrocyte cells separated out as clusters with low number of reads and gene count.

“Out of the rest, ~31% separated out as clusters (astrocyte QC Clusters 1 and 6) with low number of reads and gene count (Appendix Fig. S6F).”

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

A pipeline combining the strengths of Labkit and Cellpose was built to achieve the highest accuracy in microglia segmentation.

“To achieve the highest accuracy, a pipeline was built to combine the strengths of both methods.”

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

About 10% of the remaining microglia cells separated out as a cluster with low number of reads and gene count.

“Out of the rest ~10% separated out as a cluster (microglia QC Cluster 2) with low number of reads and gene count (Appendix Fig. S6C).”

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

To avoid over-clustering or under-clustering, multiple resolutions were used and clustree was used to select an optimum resolution.

“To avoid over-clustering or under-clustering, multiple resolutions were used for the FindCluster() function and clustree::clustree() was used for selecting an optimum resolution (astrocytes = 0.7; microglia = 0.4).”

May combine two claims.

APOE plays an important role in the interaction between astrocytes and microglia, with astrocytic APOE being a major driver of amyloid plaque pathology which in its turn modifies the microglia response.

“It is clear that APOE plays an important role in the interaction between these cell types, with astrocytic APOE being a major driver of the amyloid plaque pathology which in its turn modifies the microglia response.”

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

For calculating p-values, MAST was used, a GLM framework that treats cellular detection rate as a covariate.

“For calculating p-values, MAST was used which is a GLM-framework that treats cellular detection rate as a covariate.”

May combine two claims.

It was not possible to isolate microglia from the AAV-transduced regions only, which has likely diluted the effect size of changes seen in microglia at transcriptomic level.

“Finally, it was not possible to isolate microglia from the AAV-transduced regions only. This has likely diluted the effect size of changes we see in the microglia at transcriptomic level. This problem was not present for astrocytes where we could use the reporter to specifically enrich for the transduced cells.”

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

Following forward and reverse primers were used for human APOE: 5′-GTTGCTGGTCACATTCCTGG-3′ and 3′-GCAGGTAATCCCAAAAGCGAC-5′.

“Following forward and reverse primers were used for human APOE: 5′-GTTGCTGGTCACATTCCTGG-3′ and 3′-GCAGGTAATCCCAAAAGCGAC-5′.”

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

Actinomycin D was removed from the myelin removal step to prevent toxicity from long-term exposure.

“ActD was removed from the myelin removal step to prevent toxicity derived from long-term exposure.”

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

Imaris was unable to accurately segment physically touching and overlapping cell bodies with intertwining branches in the absence of nuclei staining.

“This more tailored solution was chosen over more prevalent methods like Imaris as it was unable to accurately segment physically touching and overlapping cell bodies with intertwining branches, in the absence of nuclei staining.”

May combine two claims.

Depletion of microglia demonstrated that even though astrocyte-derived APOE is sufficient to induce plaque formation by 6 months of age, there was still a significant decrease in amyloid level in the absence of microglia, suggesting that they modulate amyloid plaque formation.

“Depletion of microglia in our model, demonstrated that even though astrocyte-derived APOE is sufficient to induce plaque formation by 6 months of age, there was still a significant decrease in amyloid level in the absence of microglia, suggesting that they modulate amyloid plaque formation.”

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

After quality control, 18,569 microglia were retained for analysis as good-quality cells.

“Finally, 18,569 microglia were retained for analysis as good-quality cells.”

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

Because there were differences in the gender ratio between groups, gender identity was regressed out during differential expression.

“Since there were differences in the gender ratio between the groups, gender identity was regressed out during differential expression using latent.vars parameter.”

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

Sample size estimation was based on pilot experiments.

“Sample size estimation was based on pilot experiments.”

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

Following forward and reverse primers were used for housekeeping genes mouse Gapdh and Actin.

“Following forward and reverse primers were used for housekeeping genes mouse Gapdh: 5′-TTGATGGCAACAATCTCCAC-3′ and 3′-CGTCCCGTAGACAAAATGGT-5′, and Actin: 5′- GTGACGTTGACATCCGTAAAGA-3′ and 3′-GCCGGACTCATCGTACTCC-5′.”

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

For comparison of ASM values in Figs. 2 and 5, a linear mixed-effects model was used with mouse ID and plaque diameter as confounding factors.

“For comparison of ASM values in Figs. 2 and 5, the linear mixed-effects model was used with mouse ID and diameter of plaques as confounding factors.”

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

Differential abundance of plaque-associated microglia around individual plaques in Fig. 4 was tested using a binomial generalized linear mixed-effects model with mouse ID as a confounding factor.

“Differential abundance of plaque-associated microglia around individual plaques in Fig. 4 was tested using binomial generalized linear mixed-effects model with mouse ID as confounding factor.”

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

Differences in total number of differentially expressed genes between groups compared to APOE3 were tested using Fisher's exact test with Bonferroni correction of p-values.

“Differences in total number of differentially expressed genes between groups (compared to APOE3) was tested using Fisher’s exact test with Bonferroni correction of p-values.”

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

The ASM response variable was log-transformed to improve the normality of residuals.

“ASM response variable was also log-transformed to improve the normality of the residuals.”

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

For analyses where individual plaques or microglia were plotted as data points, mixed effects models were used to account for variability between individual mice.

“mixed effects models were used to account for the variability between individual mice”

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

Morphological differences between plaque-associated and non-plaque-associated microglia in Fig. EV1 were tested using a linear mixed-effects model with mouse ID as a confounding factor.

“Morphological differences between plaque-associated microglia and non-plaque-associated microglia in Fig. EV1 was tested using linear mixed-effects model with mouse ID as confounding factor.”

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

Q-Q plots were used to assess the normality of residuals.

“Q-Q plots were used to assess the normality of the residuals”