APOE2, E3, and E4 differentially modulate cellular homeostasis, cholesterol metabolism, and inflammatory response in isogenic iPSC-derived astrocytes

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

APOE is known to affect many functional aspects of astrocytes, including uptake of glutamate and uptake and degradation of Aβ, as shown in mouse models.

“APOE is known to affect many functional aspects of astrocytes, including, but not limited to, homeostatic support such as uptake of glutamate as well as uptake and degradation of Aβ, as shown in mouse models (Verghese et al., 2013).”

• cites Verghese et al., 2013 sourcePartly supported

  The Significance section (page not provided) states that apoE isoforms affect Aβ uptake and subsequent degradation in astrocytes, and reports Aβ-clearance findings in a mouse model. The cited paper does not address astrocytic glutamate uptake, so it supports only the Aβ portion of the claim. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

The Cys112 residue in APOE2 reduces its receptor binding capacity, congruent with increased plasma lipoproteins, leading to hyperlipidemia III.

“The Cys112 residue in APOE2 reduces its receptor binding capacity, congruent with the increased levels of lipoproteins in plasma, leading to hyperlipidemia III (Fernandez et al., 2019).”

• cites Fernandez et al., 2019 sourcePartly supported

  In “Overview of APOE Isoforms,” the paper links impaired APOE2 receptor binding to delayed lipoprotein clearance and hyperlipoproteinemia III, characterized by accumulated plasma lipoproteins. But its Figure 1 description attributes deficient receptor binding to Cys158, not Cys112; it says Cys112 is present in both APOE2 and APOE3. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

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

APOE mainly exists in three allelic variants, APOE2, E3, and E4, which differ in a single amino acid change at position 112 or 158: APOE2 (Cys112, Cys158), APOE3 (Cys112, Arg158), and APOE4 (Arg112, Arg158).

“APOE mainly exists in three allelic variants, APOE2, E3, and E4, which differ in a single amino acid change at position 112 or 158: APOE2 (Cys112, Cys158), APOE3 (Cys112, Arg158), and APOE4 (Arg112, Arg158) (Liu et al., 2013).”

• cites Liu et al., 2013 sourceSupported

May combine two claims.

Isogenic APOE iPSCs obtained from EBiSC show mono-allelic expression of APOE, or APOE deletion.

“Isogenic APOE iPSCs were obtained from the European Bank for Induced Pluripotent Stem Cells (EBiSC), and show mono-allelic expression of APOE, or APOE deletion (Schmid et al., 2019, 2020).”

• cites Schmid et al., 2019 sourcePartly supported

  The Resource table identifies the isogenic APOE iPSC lines as EBiSC resources, and Resource details describes homozygous APOE-ε2/ε2, ε3/ε3 and ε4/ε4 lines plus an APOE knock-out line. The characterization passage confirms the knock-out lacks ApoE protein. None of these passages establishes mono-allelic APOE expression; a homozygous genotype does not by itself demonstrate expression from only one allele. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites Schmid et al., 2020 sourceNot checked — no full text of the source

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

APOE aids in cholesterol and lipid efflux from the cell by associating with lipoprotein particles upon formation.

“In turn, APOE aids in cholesterol and lipid efflux from the cell by associating with lipoprotein particles upon formation (Mahley, 2016).”

• cites Mahley, 2016 sourceSupported

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

ABCA1 is vital in facilitating cholesterol efflux and is upregulated in a positive feedback loop via LXR, or LXR-independent cholesterol efflux, as well as APOE lipidation and production of cholesterol-rich HDL particles.

“This protein is vital in facilitating cholesterol efflux and is upregulated in a positive feedback loop via liver X receptor (LXR), or LXR-independent cholesterol efflux, as well as APOE lipidation and production of cholesterol-rich high-density lipoprotein (HDL) particles (Fan et al., 2018).”

• cites Fan et al., 2018 sourcePartly supported

  The Introduction (p. 830) describes ABCA1-mediated cholesterol efflux and apoE lipidation, and p. 831 says LXR regulates ABCA1. But the paper does not describe a positive-feedback loop or establish production of cholesterol-rich HDL particles; its Results (p. 834) examine apoE-containing lipoproteins. The Discussion (p. 840) says the tested compounds require LXR activity to induce ABCA1 and apoE, despite not binding LXR directly, so their effects do not establish LXR-independent efflux. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

May combine two claims.

APOE4 and ABCA1 co-aggregate, reducing ABCA1 recycling to the membrane.

“Another study showed that APOE4 and ABCA1 co-aggregate, reducing ABCA1 recycling to the membrane (Rawat et al., 2019).”

• cites Rawat et al., 2019 sourceSupported

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

Previous research on humanized APOE mice showed reduced glutamine levels and reduced capacity to incorporate glutamate into the brain of APOE 4TR mice, while glutamate transporters were not significantly changed.

“Previous research on humanized APOE mice showed reduced glutamine levels, and reduced capacity to incorporate glutamate into the brain of APOE 4TR mice, while glutamate transporters were not significantly changed (Dumanis et al., 2013).”

• cites Dumanis et al., 2013 sourcePartly supported

  The Results section reports lower glutamate levels and lower incorporation of a glucose-derived 13C label into glutamate in APOE-ε4 TR mice, but it reports significantly higher—not reduced—glutamine levels. The Abstract says glutamate uptake transporters were unchanged; it also reports an increase in the vesicular glutamate transporter VGLUT1, so the claim is too broad about transporters. Page numbers were not provided in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

V-ATPase-regulated proton transport into the lysosomal lumen is crucial to maintaining the acidic pH, and defective acidification has been linked to AD or Parkinson disease.

“V-ATPase-regulated proton transport into the lysosomal lumen is crucial to maintaining the acidic pH, and defective acidification has been linked to AD or Parkinson disease (Colacurcio et al., 2018).”

• cites Colacurcio et al., 2018 sourceSupported

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

APOE4 interacts with insulin receptors and other transporters, relocating them to the endosomal compartment; a similar mechanism may affect EAAT and GLAST surface expression, ultimately decreasing glutamate uptake capacity in APOE4 iAstrocytes.

“APOE4 also interacts with insulin receptors and other transporters, relocating them to the endosomal compartment: a similar mechanism may also affect excitatory amino acid transporter (EAAT) and glutamate/aspartate transporter (GLAST) surface expression, ultimately decreasing glutamate uptake capacity in the APOE4 iAstrocytes (Yassine and Finch, 2020).”

• cites Yassine and Finch, 2020 sourcePartly supported

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

  Experimental — accuracy not yet measured

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

Astrocytes play a pivotal homeostatic role in the brain, providing neural cells with lipids and cholesterol, taking up excess neurotransmitters, and phagocytosing cellular debris and waste.

“Astrocytes play a pivotal homeostatic role in the brain, providing neural cells with cell membrane components such as lipids and cholesterol, taking up excess of neurotransmitters at the tripartite synapse to prevent excitotoxicity, and phagocytosing cellular debris and waste, alongside microglia (Zorec et al., 2017).”

• cites Zorec et al., 2017 sourceCould not verify

  No passage from our copy matched the claim. Searches for astrocyte homeostasis, cholesterol and lipid supply, glutamate uptake, and phagocytosis returned no passages, so the attributed functions cannot be verified from this copy. (LLM, conf=0.70)

  Experimental — accuracy not yet measured

May combine two claims.

The iPSC lines used harbor a single APOE2, APOE3, or APOE4 allele generated using CRISPR, or an APOE-KO generated by an insertion-deletion mutation in exon 2.

“iPSC lines were purchased from EBiSC (BIONi010-C-2, -C-3, C-4, and -C-6), harboring a single APOE2, APOE3, or APOE4 allele generated using CRISPR, or an APOE-KO generated by an insertion-deletion mutation in exon 2 (Schmid et al., 2019, 2020).”

• cites Schmid et al., 2019 sourcePartly supported

  The Resource table identifies the four EBiSC lines, and Resource details reports CRISPR-generated APOE-ε2/ε2, ε3/ε3 and ε4/ε4 lines plus an APOE knockout with a frameshift in exon 2. The variant lines are homozygous, not single-copy APOE allele lines. The paper describes deleting part of exon 2 but does not specify an insertion-deletion mutation. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites Schmid et al., 2020 sourceNot checked — no full text of the source

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

The TGF-β signaling downregulation in APOE4 is likely connected to its neuroprotective role.

“in line with a study showing dysfunction in TGF-β signaling in models of early AD and in human AD brain (Tesseur et al., 2006), likely connected to its neuroprotective role.”

• cites Tesseur et al., 2006 sourcePartly supported

  The Discussion reports reduced TGF-β receptor levels in human AD brain, including mild AD, and links impaired neuronal signaling to neurodegeneration in mouse models. It also proposes that increasing neuronal TGF-β signaling could be protective. The paper does not establish that TGF-β signaling is downregulated specifically in APOE4, so that part of the claim is unsupported. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

TCW and colleagues have shown activation of astrocytes by treatment with Aβ42 peptide.

“TCW and colleagues have shown activation of astrocytes by treatment with Aβ42 peptide.”

• cites TCW et al., 2017 sourceSupported

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

A decrease in LAMP2 levels causes a lack of lysosomal trafficking and decrease in autophagic flux.

“It is known that a decrease in LAMP2 levels causes a lack of lysosomal trafficking and decrease in autophagic flux (Cui et al., 2020).”

• cites Cui et al., 2020 sourcePartly supported

  The Discussion reports that Lamp2 knockdown blocked autophagic flux, and the Results show that Lamp2 restoration improved trafficking of the lysosomal cathepsins Cat B and Cat D. The paper does not establish that decreased Lamp2 causes a complete lack of lysosomal trafficking generally; its trafficking finding concerns those cathepsins in ischemic cardiomyocytes. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

May combine two claims.

APOE4-bound cholesterol, taken up by the cell, is released from the receptor in the late endosome or lysosome, and aggregation of APOE4 has been shown to cause endosomal congestion and dysfunction.

“APOE4-bound cholesterol, taken up by the cell, is released from the receptor in the late endosome or lysosome, and aggregation of APOE4 has been shown to cause endosomal congestion and dysfunction (Narayan et al., 2020; Nuriel et al., 2017; Rawat et al., 2019).”

• cites Narayan et al., 2020 sourcePartly supported

  The Results section on iPSC-derived astrocytes reports that APOE4 disrupts early endocytosis, supporting the general claim of endocytic dysfunction. It does not describe APOE4-bound cholesterol being released from a receptor in late endosomes or lysosomes. The yeast-model Results section instead reports that APOE4 did not accumulate in protein aggregates and that the observed defects were not a response to aggregation. Page numbers were not available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Nuriel et al., 2017 sourcePartly supported

  The Discussion reports that APOE4 expression causes endosomal–lysosomal dysregulation and enlarged early endosomes in aged mice. It does not show that APOE4 aggregation causes this dysfunction, or describe receptor release of APOE4-bound cholesterol in late endosomes or lysosomes. The Introduction discusses aggregation of Aβ, not aggregation of APOE4. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites Rawat et al., 2019 sourcePartly supported

  The Discussion (page unavailable) describes aggregated ApoE4 in acidic endosomes as a possible mechanism for endosomal congestion and reports impaired ApoE4 recycling. It does not establish that aggregation causes endosomal congestion and dysfunction, or describe APOE4-bound cholesterol being released from a receptor in a late endosome or lysosome. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

iAstrocytes drastically changed morphology from day 30 (with FCS) to day 44 (without FCS), and show increased membrane incorporation of gap junction protein GJA1, which is an indication of mature, adult astrocytes.

“Our iAstrocytes drastically changed morphology from day 30 (with FCS) to day 44 (without FCS), and show an increased membrane incorporation of gap junction protein GJA1, which is an indication of mature, adult astrocytes (Liang et al., 2020).”

• cites Liang et al., 2020 sourcePartly supported

  The ‘Structure and Distribution of Cx43’ section identifies Cx43 as a major gap-junction protein in astrocytes and describes its presence on astrocytic membranes in the adult mammalian brain. It does not say that increased membrane incorporation indicates astrocyte maturity. The day-30-to-day-44 observations are the citing authors’ own findings, not findings attributed to Liang et al. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

May combine two claims.

A flow-cytometry-based Aβ42 uptake assay showed an allele-dependent trend in uptake capacity (KO > E2 > E3 ≥ E4), although differences between APOE3 and APOE4 did not reach significance (p = 0.054).

“A flow-cytometry-based Aβ42 uptake assay, using pre-aggregated Hilyte488-tagged Aβ42, showed an allele-dependent trend in uptake capacity (KO > E2 > E3 ≥ E4), although differences between APOE3 and APOE4 did not reach significance (p = 0.054) (Figure 2F).”

May combine two claims.

Proteomic analysis showed most differently regulated proteins between APOE2 and APOE4 (173), less between APOE2 and APOE3 (130), and least between APOE4 and APOE3 (72).

“To investigate APOE-related phenotypes in isogenic iAstrocytes, proteomic analysis of APOE2, APOE3, and APOE4 iAstrocytes was performed showing most differently regulated proteins (fold change above 1 or below −1) between APOE2 and APOE4 (173), less between APOE2 and APOE3 (130), and least between APOE4 and APOE3 (72) (Figure 2A).”

May combine two claims.

APOE levels in iAstrocytes are regulated in an isoform-dependent manner (E2 > E3 > E4) and are absent in APOE-KO iAstrocytes.

“We analyzed APOE levels in iAstrocytes that were regulated in isoform-dependent manner (E2 > E3 > E4), and absent in APOE-KO iAstrocytes (Figures 2B and 2C).”

May combine two claims.

Glutamate uptake in iAstrocytes demonstrated an allele-dependent effect, with APOE4 iAstrocytes taking up the lowest amount while APOE2 and APOE-KO took up the highest amounts.

“Uptake of glutamate in iAstrocytes demonstrated an allele-dependent effect, with APOE4 iAstrocytes taking up the lowest amount of glutamate while APOE2 and APOE-KO took up the highest amounts (Figure 2D).”

• Also stated in: § Homeostatic functions: clearance and metabolism · plain

  “We show that APOE has an allele-dependent effect on glutamate uptake. APOE-KO and APOE2 showed similar uptake capacity, whereas APOE3 and APOE4 iAstrocytes displayed lowest uptake.”

May combine two claims.

GSEA showed significant downregulation of pathways involved in lipid biosynthesis/metabolism and sterol biosynthesis/metabolism in APOE4 compared with APOE3 or APOE2 iAstrocytes, while these were upregulated in APOE2 compared with APOE3.

“Gene set enrichment analysis (GSEA) of proteomic data showed significant downregulation of pathways involved in lipid biosynthesis/metabolism and sterol biosynthesis/metabolism in APOE4 compared with APOE3 or APOE2 iAstrocytes, while these were upregulated in APOE2 compared with APOE3 (Figure 3A).”

• Also stated in: § Abstract · plain

  “Mass-spectrometry-based proteomic analysis showed genotype-dependent reductions of cholesterol and lipid metabolic and biosynthetic pathways (reduction: APOE4 > E3 > E2).”

May combine two claims.

Astrocyte markers S100A6 and IQGAP1 were significantly upregulated in iAstrocytes and confirmed by western blot showing robust expression in iAstrocytes and absence in NPCs.

“Among the astrocyte markers significantly upregulated in iAstrocytes were calcium binding protein S100A6 and Rho-ATPase IQGAP1, which were confirmed with western blot showing robust expression in iAstrocytes, and absence in NPCs (Figure 1D).”

May combine two claims.

Quantification of LRP1 and VLDLR showed no significant difference, albeit a minor decrease of average LRP1 levels in APOE4 cells.

“Quantification of LRP1 and VLDLR showed no significant difference, albeit a minor decrease of average LRP1 levels in APOE4 cells (Figures 2G and 2H).”

May combine two claims.

MS proteomic analysis of iAstrocytes and NPCs from four genotypes detected 3,765 proteins, of which 2,432 were differentially expressed in iAstrocytes compared with NPCs (FDR <0.1).

“Unlabeled mass spectrometry (MS)-based proteomic analysis of iAstrocytes and NPCs from four different genotypes (APOE2, APOE3, APOE4, and APOE-KO; Figure S1A) resulted in a total of 3,765 detected proteins, of which the majority, 2,432 proteins, were differentially expressed in iAstrocytes compared with NPCs (false discovery rate [FDR] <0.1)”

May combine two claims.

Immunocytochemical analysis showed all cells positive for mature astrocyte markers S100β and GJA1 at day 44, as well as for GFAP.

“Immunocytochemical analysis of mature astrocyte markers, calcium binding protein S100β, and gap junction protein GJA1 showed all cells being positive at day 44 (Figure 1E), as well as for intermediate filament protein GFAP (Figure S1B).”

May combine two claims.

APOE-KO iAstrocytes showed higher variation in PCA and a substantially lower number of peptides and proteins in MS analysis.

“It should be noted that the three biological replicates of APOE-KO iAstrocytes are more dispersed in the PCA plot than the other genotypes, indicating higher variation in APOE-KO cells, and that we observed a substantially lower number of peptides and proteins in MS analysis.”

May combine two claims.

Total as well as cellular and secreted cholesterol was decreased in APOE4 compared with APOE3 and APOE2 iAstrocytes, while showing similar levels to APOE-KO (E2 = E3 > E4 = KO).

“In line with the proteomic data, total as well as cellular and secreted cholesterol was decreased in APOE4 compared with APOE3 and APOE2 iAstrocytes, while showing similar levels to APOE-KO (E2 = E3 > E4 = KO) (Figures 3D, S2A, and S2B)”

May combine two claims.

There is an allele-dependent effect on iAstrocyte homeostatic functions (KO = E2 > E3 > E4), where APOE2 closely resembled APOE-KO iAstrocytes.

“We further show an allele-dependent effect on iAstrocyte homeostatic functions (KO = E2 > E3 > E4), where APOE2 closely resembled APOE-KO iAstrocytes.”

May combine two claims.

Compared with day 44, day 30 iAstrocytes were smaller and expressed most GJA1 in the cytosol, while day 44 cells had GJA1 prominently on the cell surface and a significant increase in cell volume.

“Compared with iAstrocytes at day 44, cells on day 30 displayed a different morphology (Figures S1C and S1D); the latter were smaller and expressed most GJA1 in the cytosol, while GJA1 was prominently expressed on the cell surface on day 44, in addition to the significant increase in cell volume demonstrated in the S100β staining.”

May combine two claims.

A larger amount of APOE protein was observed in the secreted fraction, where the difference between genotypes was higher than in the lysate.

“In line with the extracellular lipoprotein-binding function of APOE, we observed a larger amount of APOE protein in the secreted fraction, where the difference between genotypes was higher than in the lysate.”

May combine two claims.

Cholesterol and CEs showed an allele-dependent decrease in cellular content (E2 = E3 > E4 = KO) and efflux (E2 = E3 = KO > E4).

“Cholesterol and CEs showed an allele-dependent decrease in cellular content (E2 = E3 > E4 = KO) and efflux (E2 = E3 = KO > E4), in line with our proteomic findings.”

May combine two claims.

No major abnormalities in endosomal and lysosomal compartment morphology were observed, while LAMP2 protein levels and lysosomal activity were affected by APOE genotype.

“Thus, no major abnormalities in endosomal and lysosomal compartment morphology were observed, while LAMP2 protein levels and lysosomal activity were affected by APOE genotype.”

May combine two claims.

Endosomal compartments labeled with RAB5 did not show a significant difference in percentage of cell area between genotypes, and neither did RAB5 protein levels.

“Endosomal compartments labeled with RAB5 did not show a significant difference in percentage of cell area between genotypes, and neither did RAB5 protein levels (Figures 4B, 4D, and 4E).”

May combine two claims.

No significant changes in RAB5 protein levels or cell area covered were detected.

“Although we did not detect significant changes in RAB5 protein levels or cell area covered”

May combine two claims.

Total cholesterol and cholesteryl esters were lowest in APOE4 and highest in APOE3 iAstrocytes after IL-1β treatment.

“Total cholesterol and CE were lowest in APOE4 and highest in APOE3 iAstrocytes (Figure 6B).”

May combine two claims.

IL-6 was increased upon stimulation, but also showed detectable levels at baseline in APOE4 iAstrocytes.

“Interestingly, IL-6 was increased upon stimulation, but also showed detectable levels at baseline in APOE4 iAstrocytes.”

May combine two claims.

In APOE4 and APOE3 iAstrocytes, inflammatory pathways were predominantly upregulated after IL-1β treatment, while the opposite was found in APOE2 cells.

“In APOE4 and APOE3 iAstrocytes, inflammatory pathways were predominantly upregulated after IL-1β treatment, while the opposite was found in APOE2 cells.”

May combine two claims.

iAstrocytes showed a reduced proliferation rate, providing evidence for further progress into a mature, resting state.

“Further, iAstrocytes showed a reduced proliferation rate, providing evidence for a further progress into a mature, resting state.”

May combine two claims.

Triacylglycerols show a genotype-dependent increase both at baseline and after activation, with lowest cellular TAG levels in APOE-KO and highest in APOE4 iAstrocytes.

“TAGs show a genotype-dependent increase both at baseline and after activation, with lowest cellular TAG levels in APOE-KO and highest in APOE4 iAstrocytes (Figures S2A and S5A).”

May combine two claims.

Immune regulatory pathways in iAstrocytes at baseline were negatively enriched in APOE2 versus E3, highly positively enriched in APOE4 versus E3, and moderately enriched in APOE4 versus E2.

“Immune regulatory pathways in iAstrocytes at baseline were negatively enriched in APOE2 versus E3, highly positively enriched in APOE4 versus E3, and moderately enriched in APOE4 versus E2.”

May combine two claims.

APOE3 cells showed a decrease in sterol metabolic pathways upon activation, while lipid and fatty acid metabolism were not regulated uniformly.

“APOE3 cells showed a decrease in sterol metabolic pathways upon activation, while lipid and fatty acid metabolism were not regulated uniformly.”

May combine two claims.

After IL-1β stimulation, cholesterol, fatty acid, and lipid metabolism/biosynthesis pathways were enriched in APOE4, downregulated in APOE2, whereas APOE3 showed differential regulation compared with baseline.

“These pathways were mostly downregulated in APOE4 versus APOE2 and APOE3 at baseline (Figure 3A), but after IL-1β stimulation they were enriched in APOE4, and downregulated in APOE2, whereas APOE3 showed differential regulation of these pathways compared with baseline (Figure 6A).”

May combine two claims.

IL-8 already showed detectable release at baseline, following a similar trend (E4 > E3 > E2 = KO).

“This cytokine already showed detectable release at baseline, following a similar trend (E4 > E3 > E2 = KO).”

May combine two claims.

Activation of iAstrocytes resulted in an increase in pathways classified as lipid, fatty acid, and cholesterol metabolic processes in APOE4 iAstrocytes, while the opposite effect was seen in APOE2 iAstrocytes.

“Activation of iAstrocytes also resulted in an increase in pathways classified as lipid, fatty acid, and cholesterol metabolic processes in APOE4 iAstrocytes, while the opposite effect was seen in APOE2 iAstrocytes.”

May combine two claims.

GSEA of the proteomic dataset revealed downregulation of lipid/sterol metabolism and biosynthesis in APOE4 compared with APOE3 and APOE2 iAstrocytes, whereas lipid catabolic processes show opposite effects.

“GSEA of the proteomic dataset revealed downregulation of lipid/sterol metabolism and biosynthesis in APOE4 compared with APOE3 and APOE2 iAstrocytes, whereas lipid catabolic processes show opposite effects.”

May combine two claims.

The significant difference between intracellular and secreted cholesterol in APOE4 iAstrocytes was abolished after IL-1β treatment but had no effect on CE or PE.

“The significant difference between intracellular and secreted cholesterol in APOE4 iAstrocytes was abolished after IL-1β treatment but had no effect on CE or PE”

May combine two claims.

Using a calorimetric NF-κB assay, equal levels of NF-κB were shown at baseline, while NF-κB activity was lower in E4 and E3 iAstrocytes after IL-1β treatment compared with KO iAstrocytes.

“Using a calorimetric NF-κB assay, we showed equal levels of NF-κB at baseline, while NF-κB activity was lower in E4 and E3 iAstrocytes after IL-1β treatment, compared with KO iAstrocytes (Figure 5D).”

May combine two claims.

Positive regulation of ion transport and ion homeostasis pathways were enriched in APOE4 compared with APOE3, and other ion regulatory pathways were not significantly regulated.

“In addition, positive regulation of ion transport and ion homeostasis pathways were enriched in APOE4 compared with APOE3, and other ion regulatory pathways were not significantly regulated.”

May combine two claims.

IL-1β-treated E3 iAstrocytes also had lower NF-κB activity, while no changes in NF-κB proteomic pathways were observed.

“However, IL-1β-treated E3 iAstrocytes also had lower NF-κB activity, while no changes in NF-κB proteomic pathways were observed.”

May combine two claims.

There is a strong allele-dependent increase in inflammatory cytokine release upon iAstrocyte activation (E4 > E3 > E2 > KO), and even IL-6 and IL-8 release from APOE4 at baseline.

“Using MSD analysis, we observed a strong allele-dependent increase in inflammatory cytokine release upon iAstrocyte activation (E4 > E3 > E2 > KO), and even IL-6 and IL-8 release from APOE4 at baseline.”

May combine two claims.

Colocalization of Filipin III with Dextran-Alexa 555 was highest in APOE4 iAstrocytes, indicating higher cholesterol levels in lysosomes of APOE4 iAstrocytes (E4 > E2).

“Further colocalization of Filipin III with Dextran-Alexa 555 was highest in APOE4 iAstrocytes, indicating higher cholesterol levels in lysosomes of APOE4 iAstrocytes (E4 > E2) (Figure 3G).”

May combine two claims.

Proton and cation transmembrane transport were highest in APOE4 iAstrocytes and lowest in E2.

“Proton and cation transmembrane transport were highest in APOE4 iAstrocytes, and lowest in E2 (Figure 4A).”

May combine two claims.

APOE4 iAstrocytes showed the most differentially expressed pathways (both up- and downregulated), more than APOE3, and APOE2 iAstrocytes showed almost no significantly regulated pathways, indicating that APOE4 iAstrocytes are most susceptible to pro-inflammatory stimuli.

“Interestingly, APOE4 iAstrocytes showed the most differentially expressed pathways (both up- and downregulated), more than APOE3, and APOE2 iAstrocytes showed almost no significantly regulated pathways, indicating that APOE4 iAstrocytes are most susceptible to pro-inflammatory stimuli (Figure 5C).”

May combine two claims.

iAstrocytes can be stimulated with low levels of pro-inflammatory cytokine, while being mostly non-reactive at baseline.

“In addition, iAstrocytes can be stimulated with low levels of pro-inflammatory cytokine, while being mostly non-reactive at baseline.”

May combine two claims.

Day 44 iAstrocytes are not susceptible to activation by Aβ42, possibly pointing out that these cells need to be primed in order to be reactive to Aβ.

“Interestingly, our day 44 iAstrocytes are not susceptible to activation by Aβ42, possibly pointing out that these cells need to be primed in order to be reactive to Aβ.”

May combine two claims.

Cell-cell junction was significantly downregulated in E4 but only slightly in E2 and E3.

“Lastly, cell-cell junction was significantly downregulated in E4 but only slightly in E2 and E3”

May combine two claims.

Two of the pathways downregulated in APOE4 iAstrocytes were NIK/NF-κB- and IL-1-mediated signaling, which were not significantly regulated in APOE3 or E2.

“Interestingly, two of the pathways downregulated in APOE4 iAstrocytes were NIK/NF-κB- and IL-1-mediated signaling, which were not significantly regulated in APOE3 or E2.”

May combine two claims.

The cell aging pathway was significantly upregulated in E4; enriched, but not significantly, in E3; and even less in E2.

“Interestingly, the cell aging pathway was significantly upregulated in E4; enriched, but not significantly, in E3; and even less in E2.”

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

FDFT1 (squalene synthase) is the first committed enzyme in the de novo biosynthesis pathway of cholesterol, or mevalonate pathway.

“One of the proteins significantly regulated was FDFT1 (squalene synthase), the first committed enzyme in the de novo biosynthesis pathway of cholesterol, or mevalonate pathway (Do et al., 2009).”

• cites Do et al., 2009Not matched

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

May combine two claims.

Early AD pathology is signified by a complex cellular phase with synapse loss, elevated immune response, defective clearance by the endolysosomal system, ER stress, and an increase in cholesteryl esters, cholesterol, and decreased glycerophospholipids.

“Early AD pathology is signified by a complex cellular phase, presenting a myriad of dysfunctions such as synapse loss, elevated immune response, defective clearance by the endolysosomal system, ER stress, and an increase in cholesteryl esters, cholesterol, and decreased levels of glycerophospholipids (De Strooper and Karran, 2016; Tackenberg et al., 2009).”

• cites De Strooper and Karran, 2016 sourceNot checked — no full text of the source
• cites Tackenberg et al., 2009 sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

The accumulation and aggregation of Aβ, caused by reduced clearance mechanisms, plays a significant role in the progression of AD pathogenesis.

“The accumulation and aggregation of Aβ, caused by reduced clearance mechanisms, plays a significant role in the progression of AD pathogenesis (Winblad et al., 2016).”

• cites Winblad et al., 2016 sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Considering that APOE is primarily expressed in astrocytes, a key role is implied for astrocytic APOE in the initial phase of AD pathophysiology.

“Considering that APOE is primarily expressed in astrocytes, a key role is implied for astrocytic APOE in the initial phase of AD pathophysiology.”

• AssumptionRests on an assumption: In the brain, APOE is mainly expressed by astrocytes, and in low levels by microglia.

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

The biological role of APOE2 is heavily understudied and not yet understood.

“The APOE2 allele is protective for AD; however, its biological role is heavily understudied and not yet understood (Michaelson, 2014).”

• cites Michaelson, 2014 sourceNot checked — no full text of the source

May combine two claims.

Reduced glutamate uptake in APOE4 iAstrocytes was not caused by lower APOE levels, as APOE-KO iAstrocytes showed similar glutamate uptake capacity to APOE2.

“Reduced glutamate uptake was not caused by the lower APOE levels in APOE4 iAstrocytes, as the APOE-KO iAstrocytes showed similar glutamate uptake capacity to APOE2.”

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

APOE binds lipoprotein complexes and facilitates their uptake through LDLRs or LRP1, providing lipids and cholesterol to the cell.

“Apolipoprotein E (APOE) is part of a family of lipoprotein chaperones, binding lipoprotein complexes and facilitating their uptake through low-density lipoprotein receptors (LDLRs), or lipoprotein receptor 1 (LRP1), providing lipids and cholesterol to the cell (Kockx et al., 2018).”

• cites Kockx et al., 2018 sourceNot checked — no full text of the source

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

Many astrocyte homeostatic functions have been found to be altered or dysfunctional in AD, such as a decrease in glutamate uptake.

“Many of these functions have been found to be altered or dysfunctional in AD, such as a decrease in glutamate uptake (Scott et al., 2011).”

• cites Scott et al., 2011 sourceNot checked — no full text of the source

May combine two claims.

APOE-dependent Aβ42 uptake may be the primary mode of degradation, even though an APOE-independent route, as seen in APOE-KO iAstrocytes, might be more efficient.

“APOE-dependent Aβ42 uptake may be the primary mode of degradation, even though an APOE-independent route, as seen in APOE-KO iAstrocytes, might be more efficient.”

May combine two claims.

The increase in non-membrane-bound cholesterol is in line with the decrease in cholesterol efflux in APOE4 cells; APOE-mediated efflux may be ineffective in APOE4 due to the lack of ABCA1.

“The increase in non-membrane-bound cholesterol is in line with the decrease in cholesterol efflux in APOE4 cells; membrane-bound cholesterol can bind to HDL particles and be secreted, and APOE-mediated efflux may be ineffective in APOE4 due to the lack of ABCA1.”

May combine two claims.

The TAG increase may lead to an imbalance in lipid metabolism/biosynthesis and catabolism, which are counter-regulated in APOE2 and APOE4 iAstrocytes.

“This may lead to an imbalance in lipid metabolism/biosynthesis and catabolism, which are counter-regulated in APOE2 and APOE4 iAstrocytes.”

May combine two claims.

Aβ42 uptake is likely facilitated by APOE-bound Aβ42 clearance, with complex formation being genotype dependent (E2 > E3 > E4).

“which is likely facilitated by APOE-bound Aβ42 clearance; a complex formation that is genotype dependent (E2 > E3 > E4) (Aleshkov et al., 1997).”

• cites Aleshkov et al., 1997 sourceNot checked — no full text of the source

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

The LXR/RXR complex is linked to PPARγ, which can also induce ABCA1 and APOE expression.

“The LXR/RXR complex is in turn linked to transcription factor peroxisome proliferator-activated receptor gamma (PPARγ), which can also induce ABCA1 and APOE expression.”

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

Cytokine stimulation can regulate cholesterol synthesis by activating SREBP1.

“It has been shown that cytokine stimulation can regulate cholesterol synthesis by activating SREBP1 (Gierens et al., 2000).”

• cites Gierens et al., 2000 sourceNot checked — no full text of the source

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

SREBP1 induces de novo biosynthesis of cholesterol and increases expression of LRP1 or VLDLR to increase cholesterol uptake.

“This transcription factor in turn induces de novo biosynthesis of cholesterol and increases the expression of LRP1 or VLDLR to increase cholesterol uptake.”

• cites Gierens et al., 2000 sourceNot checked — no full text of the source

May combine two claims.

APOE2, E3, E4, and KO iAstrocytes show distinct phenotypes in homeostatic functions, cholesterol and lipid metabolism, lysosomal function, and inflammatory regulation.

“Together, we show that APOE2, E3, E4, and KO iAstrocytes show distinct phenotypes in homeostatic functions, cholesterol and lipid metabolism, lysosomal function, and inflammatory regulation.”

• AssumptionRests on an assumption: The four isogenic iPSC lines differ only in APOE genotype and not in other genetic or epigenetic factors that affect the measured phenotypes.
• AssumptionRests on an assumption: The in vitro iPSC-derived iAstrocyte model recapitulates relevant aspects of human astrocyte biology and APOE-related AD mechanisms.

May combine two claims.

The effect of IL-1β seen is due to an increase of total cholesterol in the APOE2 and E3 iAstrocytes, but not in APOE4.

“we conclude that the effect of IL-1β seen is due to an increase of total cholesterol in the APOE2 and E3 iAstrocytes, but not in APOE4.”

• AssumptionRests on an assumption: The four isogenic iPSC lines differ only in APOE genotype and not in other genetic or epigenetic factors that affect the measured phenotypes.

May combine two claims.

It is not a mere decrease of function in APOE4 cells: APOE2, APOE3, and APOE4 iAstrocytes may utilize different mechanisms altogether.

“the interplay between cholesterol metabolism and immune regulation indicates that it is not a mere decrease of function in APOE4 cells: APOE2, APOE3, and APOE4 iAstrocytes may utilize different mechanisms altogether.”

• AssumptionRests on an assumption: The four isogenic iPSC lines differ only in APOE genotype and not in other genetic or epigenetic factors that affect the measured phenotypes.
• AssumptionRests on an assumption: The in vitro iPSC-derived iAstrocyte model recapitulates relevant aspects of human astrocyte biology and APOE-related AD mechanisms.

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

Chronic inflammation is associated with an earlier onset of AD in APOE4 carriers.

“This is in line with research showing that chronic inflammation is associated with an earlier onset of AD in APOE4 carriers.”

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

The absence of significant RAB5 changes does not exclude functional changes in endosomes or may require prior activation of astrocytes to detect endosomal deficits.

“it does not exclude the presence of functional changes in endosomes or it may require prior activation of the astrocytes to detect endosomal deficits, which would thus not be present in our cells at baseline.”

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

Cholesterol biosynthesis and lipid efflux machinery are regulated via LXR/RXR complex activation; when intracellular cholesterol is increased, LXR/RXR increases ABCA1 expression to induce lipid efflux, APOE expression, and inhibition of SREBP activation.

“Cholesterol biosynthesis and lipid efflux machinery are regulated via LXR and retinoid X receptor (RXR) complex activation; when intracellular cholesterol is increased, LXR/RXR increases ABCA1 expression to induce lipid efflux, APOE expression, and inhibition of SREBP activation (Shimano and Sato, 2017).”

• cites Shimano and Sato, 2017 sourceNot checked — no full text of the source

May combine two claims.

The APOE allele-dependent effect on glutamate uptake and LRP1-dependent Aβ42 uptake can provide a mechanism explaining the biological role of APOE in homeostatic functions such as regulation of glutamate levels, but also pathological events such as extracellular Aβ42 buildup.

“The APOE allele-dependent effect on glutamate uptake and LRP1-dependent Aβ42 uptake can provide a mechanism explaining the biological role of APOE in homeostatic functions such as regulation of glutamate levels, but also pathological events such as extracellular Aβ42 buildup.”

• AssumptionRests on an assumption: The in vitro iPSC-derived iAstrocyte model recapitulates relevant aspects of human astrocyte biology and APOE-related AD mechanisms.

May combine two claims.

APOE4 iAstrocytes already have an increased inflammatory phenotype, which is exacerbated in reactive iAstrocytes.

“This indicates APOE4 iAstrocytes already have an increased inflammatory phenotype, which is exacerbated in reactive iAstrocytes.”

• AssumptionRests on an assumption: The four isogenic iPSC lines differ only in APOE genotype and not in other genetic or epigenetic factors that affect the measured phenotypes.

May combine two claims.

These data indicate a role of the inflammatory microenvironment on cholesterol regulation, with APOE2 cells being unaffected, APOE3 cells increasing their cholesterol content, and APOE4 cells showing altered cholesterol regulation upon IL-1β treatment.

“Altogether, these data indicate a role of the inflammatory microenvironment on cholesterol regulation, with APOE2 cells being unaffected, APOE3 cells increasing their cholesterol content, and APOE4 cells showing altered cholesterol regulation upon IL-1β treatment.”

May combine two claims.

PPARγ signaling was decreased in APOE4 transgenic mouse brain, and increased in APOE2.

“In line with our results, PPARγ signaling was decreased in APOE4 transgenic mouse brain, and increased in APOE2 (Wu et al., 2018).”

• cites Wu et al., 2018Not 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). May combine two claims.

Genetic variability of LXR may contribute to the risk for AD, in addition to the ability of LXR agonists to attenuate Aβ pathology.

“Furthermore, genetic variability of LXR may contribute to the risk for AD, in addition to the ability of LXR agonists to attenuate Aβ pathology (Kang and Rivest, 2012).”

• cites Kang and Rivest, 2012 sourceNot checked — no full text of the source

May combine two claims.

LXR/RXR are likely not entirely dysfunctional as cellular/secreted cholesterol is not significantly different after IL-1β treatment in contrast to baseline, suggesting residual LXR activity allowing for the relative increase in efflux of cholesterol.

“However, LXR/RXR are likely not entirely dysfunctional as cellular/secreted cholesterol is not significantly different after IL-1β treatment in contrast to baseline, suggesting residual LXR activity allowing for the relative increase in efflux of cholesterol.”

• AssumptionRests on an assumption: The four isogenic iPSC lines differ only in APOE genotype and not in other genetic or epigenetic factors that affect the measured phenotypes.

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

An increase in proton and cation transmembrane transport might indicate a compensatory mechanism for underlying dysfunction.

“An increase in proton and cation transmembrane transport might indicate a compensatory mechanism for underlying dysfunction.”

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

Many studies show an increase in NF-κB in the AD brain.

“Many studies show an increase in NF-κB in the AD brain (Liddelow and Barres, 2017).”

• cites Liddelow and Barres, 2017 sourceNot checked — no full text of the source

May combine two claims.

Taken together, APOE4 iAstrocytes reveal a disease-relevant phenotype, causing dysregulated cholesterol/lipid homeostasis, increased inflammatory signaling, and reduced β-amyloid uptake, while APOE2 iAstrocytes show opposing effects.

“Taken together, APOE4 iAstrocytes reveal a disease-relevant phenotype, causing dysregulated cholesterol/lipid homeostasis, increased inflammatory signaling, and reduced β-amyloid uptake, while APOE2 iAstrocytes show opposing effects.”

May combine two claims.

These data suggest that APOE4 iAstrocytes have an inflammatory phenotype already at baseline, which is exacerbated upon activation, while APOE2 cells display a rather anti-inflammatory or neutral phenotype, and APOE3 represents an intermediate state.

“These data suggest that APOE4 iAstrocytes have an inflammatory phenotype already at baseline, which is exacerbated upon activation, while APOE2 cells display a rather anti-inflammatory or neutral phenotype, and APOE3 represents an intermediate state.”

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

Experiments were performed at least in triplicate.

“Experiments were performed at least in triplicate”