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

In the paper

“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.”

What the sources say

• Disputed

  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.

  cites Gratuze et al, 2022 sourceExperimental — accuracy not yet measured
• Disputed

  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.

  cites Liao et al, 2018 sourceExperimental — accuracy not yet measured
• Partly 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)

  cites Xiong et al, 2021 sourceExperimental — accuracy not yet measured

In the paper

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

What the sources say

• Partly 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)

  cites Castano et al, 1995 sourceExperimental — accuracy not yet measured
• ?Not matched

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

  cites Ma et al, 1994
• ?Not matched

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

  cites Wisniewski et al, 1994

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites Fleisher et al, 2013 source
• ?Not checked — no full text of the sourcecites Kok et al, 2009 source
• Partly 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)

  cites Murphy et al, 2013 sourceExperimental — accuracy not yet measured

In the paper

“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)”

What the sources say

• Partly 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)

  cites Bennett et al, 2009 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites Corder et al, 1993 source
• Partly 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)

  cites Strittmatter et al, 1993 sourceExperimental — accuracy not yet measured

In the paper

“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)”

What the sources say

• ?Not checked — no full text of the sourcecites Fagan et al, 2002 source
• Supported

  The Discussion reports that aged PDAPP/TRE mice developed Aβ/amyloid deposition in the order E4 > E3 > E2. It also reports that biomarkers of brain amyloid accumulation in cognitively normal people followed ε4 > ε3 > ε2. These passages support the cited sentence’s general, qualified claim. (LLM, conf=0.94)

  cites Castellano et al, 2011 source
• Supported

  In the Results (Expression of APOE isoforms modulates the kinetics of amyloidosis), plaque-growth measurements follow the order APOE4 > APOE3 > APOE2. The same section reports reduced amyloid-deposit density with APOE2 relative to both APOE3 and APOE4. These mouse experiments support the citing sentence’s hedged ordering. (LLM, conf=0.90)

  cites Hudry et al, 2013 source

In the paper

“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.”

What the sources say

• Supported

  The Huang et al. citation is attached to Mertk’s role in microglial detection and engulfment of plaques, not to the citing authors’ statement that the genes are downregulated in APOE4. The Results section, “Dense-core plaque burden in the absence of TAM signaling” (page unavailable), identifies Mer as performing most phagocytosis-dependent dense-core plaque construction; the paper also reports impaired plaque detection and engulfment when Axl and Mertk are both absent. (LLM, conf=0.88)

  cites Huang et al, 2021 source
• Supported

  The citation is attached to the description of INPP5D as an AD risk factor with a role in limiting plaque formation, not to the citing authors’ statement that it is downregulated in APOE4. The Discussion identifies an INPP5D variant that confers AD risk and reports that reducing microglial Inpp5d increased plaque burden in PSAPP mice. (LLM, conf=0.91)

  cites Castranio et al, 2023 source

In the paper

“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.”

What the sources say

• ?Not matched

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

  cites Berlau et al, 2007, 2009
• Partly 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)

  cites Arboleda-Velasquez et al, 2019 sourceExperimental — accuracy not yet measured

In the paper

“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).”

What the sources say

• ?Could 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)

  cites Rodriguez et al, 2014 sourceExperimental — accuracy not yet measured
• Supported

  The Results section (“Human APOE Genotype-specific Delay of Aβ Accumulation in 5xFAD,” Fig. 2C; page not specified in our copy) reports significantly higher plaque levels in E2FAD than E3FAD mice in the subiculum at 6 months. This supports the cited claim, though the reported comparison is specific to that brain region and age. (LLM, conf=0.92)

  cites Youmans et al, 2012 source
• ?Not checked — no full text of the sourcecites Bales et al, 2009 source

In the paper

“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).”

What the sources say

• ?Not matched

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

  cites Mancuso et al, 2024
• Supported

  The Abstract reports unchanged plaque load and microglial clustering around plaques after microglia-specific ApoE knockout in 5xFAD mice. The Results section on microglial number and plaque association also reports no difference in plaque-associated microglia. The study did find an approximately 35% increase in average plaque size, a distinct outcome from plaque formation and association. (LLM, conf=0.91)

  cites Henningfield et al, 2022 source

In the paper

“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”

What the sources say

• ?Not checked — no full text of the sourcecites Brown et al, 2002 source
• ?Not checked — no full text of the sourcecites Colton et al, 2005 source
• Supported

  The citation to Lanfranco et al. (2021) is attached specifically to the claim about a pro-inflammatory state in microglia, not to the sentence’s other listed effects. The Discussion reports higher basal TNFα in APOE4 microglia and says this supports greater inflammation in primary cells. (LLM, conf=0.93)

  cites Lanfranco et al, 2021 source

In the paper

“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).”

What the sources say

• Partly 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)

  cites Holtzman et al, 2000a sourceExperimental — accuracy not yet measured
• Partly 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)

  cites Liu et al, 2017b sourceExperimental — accuracy not yet measured
• Partly 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)

  cites Mahan et al, 2022 sourceExperimental — accuracy not yet measured

In the paper

“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).”

What the sources say

• Supported

  Huynh et al. is cited for the blood–brain barrier clause, not the subsequent claim about astrocytes. The Background states that apoE cannot cross the BBB, and the Discussion says the study’s findings are consistent with the inability of peripheral apoE to cross it. (LLM, conf=0.94)

  cites Huynh et al, 2019 source
• Supported

  The paper states that ApoE typically cannot pass through the blood–brain barrier and that peripheral and brain ApoE pools are therefore largely separate (Peripheral and central sources of ApoE, reproduced in the Discussion passage). This supports the blood–brain barrier clause attributed to Lane-Donovan et al.; the astrocyte clause is cited to other sources. (LLM, conf=0.92)

  cites Lane-Donovan et al, 2016 source
• Supported

  The Discussion reports apo E in astrocytes, identifies it as an astrocytic secretory product, and considers microglia an unlikely source of the high apo E levels in normal brain. This supports the claim attributed to Boyles et al. that astrocytes are the main contributor; the blood–brain barrier statement is cited to other papers. (LLM, conf=0.90)

  cites Boyles et al, 1985 source

In the paper

“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).”

What the sources say

• Partly 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)

  cites Sousa et al, 2018 sourceExperimental — accuracy not yet measured

In the paper

“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.”

What the sources say

• Partly 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)

  cites Riddell et al, 2008 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites Bales et al, 2009 source
• ?Not checked — no full text of the sourcecites Conejero-Goldberg et al, 2014 source

In the paper

“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).”

What the sources say

• Partly 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)

  cites Dodart et al, 2005 sourceExperimental — accuracy not yet measured
• Supported

  The Results section, “Expression of APOE isoforms modulates the kinetics of amyloidosis” (page not provided), reports faster amyloid progression in APP/PS1 mice exposed to APOE4 after AAV4-mediated gene transfer. The plaque-density results also show more cortical plaques in AAV4-APOE4–injected mice. (LLM, conf=0.93)

  cites Hudry et al, 2013 source
• ?Not checked — no full text of the sourcecites Zhao et al, 2016 source

In the paper

“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).”

What the sources say

• Supported

  The Nuriel et al. citation is attached only to the claim that APOE4 affects the endosomal–lysosomal pathway. The Discussion reports age-dependent alterations in that system in APOE4 mice. The separate claim about rescuing a recycling block by decreasing pH is attributed to Xian et al. and is not part of this verdict. (LLM, conf=0.94)

  cites Nuriel et al, 2017 source
• Supported

  The Introduction states that lowering endosomal pH through NHE6 inhibition releases the ApoE4-induced recycling block. The Discussion likewise reports that NHE inhibition resolves the block and restores normal receptor recycling. The separate endosomal-lysosomal pathway clause is cited to Nuriel et al., not to this paper. (LLM, conf=0.94)

  cites Xian et al, 2018 source

In the paper

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

What the sources say

• ?Not checked — no full text of the sourcecites Koldamova et al, 2005 source
• Partly 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)

  cites Wahrle et al, 2005 sourceExperimental — accuracy not yet measured

In the paper

“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).”

What the sources say

• Supported

  The STAR★Methods section on marker gene set discovery says the paper generated marker sets for its cell-type clusters. That supports the citation as a source of previously identified cell-type signatures. The sentence describes AddModuleScore as the citing authors’ annotation method, not as a method used by Zeisel et al. (LLM, conf=0.88)

  cites Zeisel et al, 2018 source

In the paper

“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).”

What the sources say

• Partly 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)

  cites Stephen et al, 2019 sourceExperimental — accuracy not yet measured
• Partly 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)

  cites Thomas et al, 2016 sourceExperimental — accuracy not yet measured
• Partly 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)

  cites Sala Frigerio et al, 2019 sourceExperimental — accuracy not yet measured

In the paper

“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).”

What the sources say

• Partly 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)

  cites Chung et al, 1999 sourceExperimental — accuracy not yet measured
• ?Not matched

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

  cites Hu et al, 2009
• Partly 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)

  cites Knauer et al, 1992 sourceExperimental — accuracy not yet measured

In the paper

“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).”

What the sources say

• Supported

  The ‘Enrichment analysis using g:GOSt’ section describes g:Profiler as a gene-list enrichment tool, lists GO Biological Process as a data source, and identifies g:SCS as its default multiple-testing correction. The cluster markers and 0.05 threshold describe the citing authors’ own analysis settings; the citation identifies the tool rather than serving as evidence that those settings were used. (LLM, conf=0.87)

  cites Kolberg et al, 2023 source

In the paper

“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”

What the sources say

• Partly 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)

  cites Bras et al, 2014 sourceExperimental — accuracy not yet measured
• Partly 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)

  cites Guerreiro et al, 2018 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites Huang et al, 2006 source

In the paper

“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).”

What the sources say

• Supported

  The citation to Zamanian et al. supports the astrocyte-cell-state gene sets, not the citing authors’ use of AddModuleScore or the separately cited plaque-induced and AD-risk gene sets. The Results section, “The reactive astrocyte transcriptomes show extensive induction of gene expression” (page unavailable), identifies reactive-astrocyte genes and uses them to distinguish astrocyte populations after different injuries. (LLM, conf=0.91)

  cites Zamanian et al, 2012 source
• ?Not checked — no full text of the sourcecites Hasel et al, 2021 source
• Supported

  The citation to Chen et al. is attached specifically to plaque-induced genes (PIGs), not to the scoring method or the other gene sets. In “Identification of the Plaque-Induced Genes (PIGs) module” (page unavailable), the paper identifies a 57-gene PIGs module associated with amyloid-β accumulation. (LLM, conf=0.94)

  cites Chen et al, 2020 source

In the paper

“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).”

What the sources say

• Partly 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)

  cites Marsh et al, 2022 sourceExperimental — accuracy not yet measured
• Partly 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)

  cites Perocchi et al, 2007 sourceExperimental — accuracy not yet measured

In the paper

“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).”

What the sources say

• Supported

  The Introduction reports that deleting microglial miR-155 in APP/PS1 mice induced an early response via IFNγ signaling, enhanced phagocytosis, and increased Aβ plaque compaction. The results passages also report enhanced IFN signaling and Aβ phagocytosis in the conditional-knockout mice. (LLM, conf=0.94)

  cites Yin et al, 2023a source

In the paper

“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).”

What the sources say

• Partly supported

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

  cites Sousa et al, 2018 sourceExperimental — accuracy not yet measured
• Supported

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

  cites Serrano-Pozo et al, 2021 source

In the paper

“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.”

What the sources say

• Supported

  The Results identify disease-associated microglia (DAM) near Aβ plaques and report that an early transition to the DAM state includes upregulation of Apoe. The paper thus supports the attributed plaque-associated DAM response and APOE induction. (LLM, conf=0.90)

  cites Keren-Shaul et al, 2017 source

In the paper

“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).”

What the sources say

• Supported

  The Results section (“Autophagy mRNAs in Alzheimer brain”; page not provided) reports that brain samples from AD patients with APOE ε4/ε4 had lower levels of three autophagy-gene transcripts than samples from AD patients with APOE ε3/ε3. The Discussion also reports lower levels of the corresponding autophagy proteins. The separate claim about astrocyte-mediated Aβ degradation is attributed to Simonovitch et al., not Parcon et al. (LLM, conf=0.94)

  cites Parcon et al, 2018 source
• ?Not matched

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

  cites Simonovitch et al, 2016, 2019

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites Doyle et al, 2008 source
• Partly 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)

  cites Gnep et al, 2017 sourceExperimental — accuracy not yet measured
• Partly 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)

  cites Hapsari et al, 2022 sourceExperimental — accuracy not yet measured

In the paper

“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.”

What the sources say

• Supported

  The Results section reports that deleting Apoe in microglia represses disease-associated genes and restores homeostatic gene expression in phagocytic microglia (Figure 4F). It also explicitly concludes from global and microglia-specific deletion experiments that microglial Apoe has an essential role in the MGnD phenotype (Figures 4H–I). (LLM, conf=0.90)

  cites Krasemann et al, 2017 source
• Supported

  The Discussion (page not provided) says the ARMs response overlaps with DAM and that deleting Apoe severely reduces the number of microglia with an ARMs signature. The Results section on Apoe deletion likewise reports that it impairs deployment of the ARMs response. The citation is attached to the dependence claim, not to the subsequent statement about microglial APOE being essential. (LLM, conf=0.90)

  cites Sala Frigerio et al, 2019 source

In the paper

“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).”

What the sources say

• Supported

  The Discussion (p. 12) explicitly describes a cell-autonomous negative effect of microglial APOE4 on acquiring the neurodegenerative microglial (MGnD) phenotype. The Results (p. 3) show that APOE4-expressing microglia failed to induce this response compared with APOE3-expressing microglia, and that deleting microglial APOE4 restored it. This supports the citing sentence’s characterization of an impaired disease-associated microglial response. (LLM, conf=0.91)

  cites Yin et al, 2023b source
• Supported

  The Introduction identifies activated response microglia (ARM) as also termed disease-associated microglia (DAM). The Discussion reports that microglial apoE3 shifts cells toward ARM states under amyloid pathology, whereas apoE4 fails to do so; the Results likewise find that apoE3, but not apoE4, increases microglial clustering around plaques. This supports the hedged claim of a microglia-specific, isoform-dependent restriction of the DAM response. (LLM, conf=0.92)

  cites Liu et al, 2023 source

In the paper

“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).”

What the sources say

• Supported

  The citation is attached to the term “core” gene, not to the citing authors’ proposal about APOE4. In Results, “The Omnigenic Model” (page unavailable), Liu et al. define core genes as genes that can affect disease risk directly. The APOE4 classification, amyloid explanation, and odds ratio are the citing authors’ own claims, not claims attributed to Liu et al. by this citation. (LLM, conf=0.93)

  cites Liu et al, 2019 source

In the paper

“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.”

What the sources say

• Supported

  The Discussion identifies SuperExactTest as an R package for statistical testing and visualization of set intersections. The Methods (Software Availability) shows commands for both testing overlaps and plotting them. The APOE3 comparison describes the citing authors’ use of the package, not an analysis attributed to Wang et al. (LLM, conf=0.92)

  cites Wang et al, 2015 source

In the paper

“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.”

What the sources say

• Supported

  The citation identifies the plaque-induced genes (PIGs), not the citing authors’ enrichment test. The source’s “Identification of the Plaque-Induced Genes (PIGs) module” section defines PIGs as a 57-gene module. The hypergeometric test and Bonferroni correction are described as the citing authors’ method and are not attributed to Chen et al. (LLM, conf=0.94)

  cites Chen et al, 2020 source

In the paper

“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)”

In the paper

“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).”

In the paper

“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.”

In the paper

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

In the paper

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

In the paper

“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.”

In the paper

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

In the paper

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

In the paper

“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).”

In the paper

“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).”

In the paper

“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)”

In the paper

“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)”

In the paper

“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.”

In the paper

“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).”

In the paper

“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.”

In the paper

“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).”

In the paper

“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.”

In the paper

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

In the paper

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

In the paper

“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.”

In the paper

“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.”

In the paper

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

In the paper

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

What the sources say

• ?Not checked — no full text of the sourcecites Corder et al, 1994 source
• ?Not checked — no full text of the sourcecites Nagy et al, 1995 source

In the paper

“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.”

In the paper

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

In the paper

“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.”

In the paper

“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.”

In the paper

“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).”

In the paper

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

In the paper

“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).”

In the paper

“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).”

What the sources say

• ?Not matched

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

  cites Chen et al, 2023

In the paper

“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.”

In the paper

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

In the paper

“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).”

In the paper

“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.”

In the paper

“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).”

In the paper

“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.”

In the paper

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

In the paper

“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.”

In the paper

“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.”

In the paper

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

In the paper

“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.”

In the paper

“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.”

In the paper

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

In the paper

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

In the paper

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

In the paper

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

In the paper

“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.”

In the paper

“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).”

In the paper

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

In the paper

“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.”

In the paper

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

In the paper

“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.”

In the paper

“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.”

In the paper

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

In the paper

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

In the paper

“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.”

In the paper

“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.”

In the paper

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

In the paper

“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).”

In the paper

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

In the paper

“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).”

In the paper

“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).”