ApoE4 Impairs Neuron-Astrocyte Coupling of Fatty Acid Metabolism

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

ApoE is a lipid-binding protein that transports lipids across organs in the periphery and between different cell types in the brain.

“Apolipoprotein E (ApoE) is a lipid-binding protein that transports lipids across organs in the periphery and between different cell types in the brain (Hauser et al., 2011).”

• cites Hauser et al., 2011 sourceSupported

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

ApoE4 is the strongest genetic risk factor for late-onset AD.

“the ε4 isoform (ApoE4) is the strongest genetic risk factor for the predominant, late-onset form of Alzheimer’s disease (AD).”

• cites Liu et al., 2013 sourceSupported

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

ApoE4-positive cells show reduced cholesterol transport function.

“with ApoE4-positive cells showing reduced function (Rawat et al., 2019; Yamazaki et al., 2019).”

• cites Rawat et al., 2019 sourceSupported
• cites Yamazaki et al., 2019 sourceSupported

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

Neuron and astrocyte are coupled in glucose oxidation.

“As the two most abundant, and energetically most essential cell types in the brain, neuron and astrocyte are coupled in not only glucose oxidation (Magistretti and Allaman, 2018; Yellen, 2018), but also lipid metabolism (Barber and Raben, 2019).”

• cites Magistretti and Allaman, 2018 sourceNot checked — no full text of the source
• cites Yellen, 2018 sourcePartly supported

  In “The source of neuronal energy during activation” (page unavailable), Yellen describes possible exchange of glucose-derived metabolic products between astrocytes and neurons, but says there are no data showing significant exchange at rest. “Some key puzzles to solve” adds that the best direct evidence during neuronal stimulation favors neurons using glucose themselves, rather than the classical astrocyte-to-neuron lactate shuttle. The unqualified claim that the cells are coupled in glucose oxidation therefore overstates the paper’s position. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

ApoE4 disrupts brain mitochondrial function via multiple mechanisms.

“given that ApoE4 disrupts brain mitochondrial function via multiple mechanisms (Chen et al., 2011; Nuriel et al., 2017; Tambini et al., 2016; Zhao et al., 2017b)”

• cites Chen et al., 2011 sourcePartly supported

  The Discussion reports that ApoE4 causes mitochondrial respiratory dysfunction in neuronal cells through domain interaction and describes reduced expression of mitochondrial respiratory complexes. It also discusses other possible mechanisms, but says the mechanism is not known in detail. The paper therefore supports mitochondrial disruption in neuronal cells, not an established finding that ApoE4 disrupts brain mitochondrial function through multiple mechanisms. (LLM, conf=0.85)

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

  The Results section (“Increased energy-related metabolites in APOE4 EC”; page unavailable) reports APOE4-associated changes in mitochondrial and energy-related metabolites in aged mice. It does not establish that ApoE4 disrupts mitochondrial function via multiple mechanisms; it interprets those changes as correlates of hypermetabolism and neuronal hyperactivity. (LLM, conf=0.84)

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

  The Introduction and Results and Discussion report that ApoE4-containing astrocyte-conditioned media increased mitochondria-associated ER membrane activity and altered ER–mitochondrial communication in treated cells. They do not establish disruption of mitochondrial function in the brain through multiple mechanisms; the Results and Discussion also report similar respiration in ApoE4- and ApoE3-treated cells. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

A mechanism adapted by cells in peripheral organs to control lipotoxicity is to sequester free FAs as TAG in intracellular LDs.

“A mechanism adapted by cells in peripheral organs to control lipotoxicity is to sequester free FAs as triacylglycerol (TAG) in intracellular lipid droplets (LDs) (Nguyen et al., 2017).”

• cites Nguyen et al., 2017 sourceSupported

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

ApoE mediates inter-cellular cholesterol transport from non-neuronal to neuronal cells.

“Extensive studies have established the role of ApoE in mediating inter-cellular cholesterol transport from non-neuronal to neuronal cells (Björkhem et al., 1998; Mauch et al., 2001)”

• cites Björkhem et al., 1998 sourcePartly supported

  The “Flux of 24S-hydroxycholesterol from the brain” section discusses an ApoE-dependent mechanism for transporting cholesterol out of the human brain. It does not describe transport from non-neuronal cells to neurons, so the cited paper supports ApoE involvement in cholesterol transport but not the claimed direction between cell types. No page number is available in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites Mauch et al., 2001 sourceNot checked — no full text of the source

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

In the brain, LDs accumulate in glial cells upon neuronal oxidative stress and during aging.

“In the brain, LDs accumulate in glial cells upon neuronal oxidative stress and during aging (Liu et al., 2015; Marschallinger et al., 2020)”

• cites Liu et al., 2015 sourcePartly supported

  The Results sections on neuronal mitochondrial dysfunction and Ndufs4−/− mice support glial lipid-droplet accumulation associated with neuronal oxidative stress. They do not establish that aging itself causes this accumulation: the section on transient glial lipid droplets reports that droplet numbers decreased as the mutant flies aged. No page numbers are available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Marschallinger et al., 2020 sourcePartly supported

  The Abstract and Introduction report lipid-droplet accumulation in microglia in aging mouse and human brains, supporting the aging component. The Discussion section ‘What causes lipid droplet formation in LDAM?’ discusses ROS and stress as possible contributors, but does not establish that neuronal oxidative stress causes glial lipid droplets. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Astrocytes provide critical support to neurons for synapse formation/pruning, neurotransmitter modulation, and metabolic homeostasis.

“Astrocytes provide critical support to neurons for synapse formation/pruning, neurotransmitter modulation, and metabolic homeostasis (Bélanger et al., 2011; Jha and Morrison, 2018).”

• cites Bélanger et al., 2011 sourceNot checked — no full text of the source
• cites Jha and Morrison, 2018 sourcePartly supported

  The Introduction and Conclusions (pages unavailable) describe glial support of tissue and neuronal metabolic homeostasis, specifically astrocyte-to-neuron lactate delivery. The Lactate section mentions synapse recruitment during stimulation, but does not establish astrocyte support for synapse formation or pruning. The passages found also do not establish that astrocytes modulate neurotransmitters. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

ApoE4 is associated with multiple neurological disorders including dementia with Lewy bodies, multiple sclerosis, and cerebrovascular disease.

“ApoE4 is also associated with multiple neurological disorders including dementia with Lewy bodies, multiple sclerosis, and cerebrovascular disease (Belloy et al., 2019)”

• cites Belloy et al., 2019 sourcePartly supported

  In “Other Neurodegenerative Disorders” (page unavailable), the review reports a strong link between APOE4 and dementia with Lewy bodies, but says that, on balance, APOE4 does not increase multiple sclerosis risk. In “Cerebrovascular Disease” (page unavailable), it reports increased ischemic stroke risk in APOE4 carriers. The multiple sclerosis association is therefore not supported as stated. (LLM, conf=0.93)

  Experimental — accuracy not yet measured

May combine two claims.

ApoE4-induced perturbation to fatty acid homeostasis underlies its negative effect on neuronal bioenergetic and synaptic function, in addition to its direct effect on mitochondria via domain interactions.

“Hence, our data suggest that ApoE4-induced perturbation to FA homeostasis underlies its negative effect on neuronal bioenergetic and synaptic function, besides its direct effect on mitochondria via domain interactions (Chen et al., 2011).”

• cites Chen et al., 2011 sourceSupported

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

Each ApoE4 allele increases AD risk by 3- to 4-fold while lowering the age at onset by ~8 years.

“Each ApoE4 allele increases AD risk by 3- to 4-fold while lowering the age at onset by ~8 years (Liu et al., 2013).”

• cites Liu et al., 2013 sourcePartly supported

  The “APOE ε4 as a strong risk factor for AD” section reports mean onset ages of 84, 76, and 68 years for noncarriers, heterozygotes, and homozygotes—an eight-year difference at each step. It also reports approximately threefold higher odds for one ε4 copy and 14.9-fold higher odds for two copies versus ε3/ε3, but those figures are for Caucasian subjects; the section notes that the association varies across populations. Thus, a universal 3- to 4-fold increase in risk per allele overstates the reported figures. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

Mitochondria are involved in both the formation and degradation of LDs in non-CNS cells.

“mitochondria are involved in both the formation and degradation of LDs in non-CNS cells (Benador et al., 2019)”

• cites Benador et al., 2019 sourceSupported

May combine two claims.

Both de novo FA synthesis and lipolysis contribute to neuronal LD formation.

“ACC inhibition by ND-630 (Harriman et al., 2016) or cPLA2 by MAFP (Longo et al., 1999) for 24 h reduced LD volume in both E3 and E4 neurons (Figure 1H), indicating both de novo FA synthesis and lipolysis contributed to LD formation.”

• cites Harriman et al., 2016 sourceSupported
• cites Longo et al., 1999 sourceNot checked — no full text of the source

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

ApoE is expressed in neurons under stress conditions, in neurons cultured in vitro, and in neurons of selected brain regions in vivo.

“because ApoE is also expressed in neurons under stress conditions (Xu et al., 2006), neurons cultured in vitro (Lin et al., 2018; Xu et al., 1999b), and neurons of selected brain regions in vivo (Han et al., 1994; Xu et al., 1999a)”

• cites Xu et al., 2006Not matched

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

• cites Lin et al., 2018 sourceSupported
• cites Han et al., 1994 sourceNot checked — no full text of the source

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

LDs store neutral lipids including TAG and sterol esters within a phospholipid monolayer and regenerate FAs for energetic needs upon starvation.

“These organelles store neutral lipids including TAG and sterol esters within a phospholipid monolayer and regenerate FAs for energetic needs upon starvation (Olzmann and Carvalho, 2019).”

• cites Olzmann and Carvalho, 2019 sourceSupported

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

Astrocytes promote neuronal function via various mechanisms, including fuel and cholesterol supply, release of neurotrophic factors, and donation of mitochondria.

“Astrocytes are known to promote neuronal function via various mechanisms, including fuel and cholesterol supply, release of neurotrophic factors, and donation of mitochondria (Hayakawa et al., 2016; Qi et al., 2020).”

• cites Hayakawa et al., 2016 sourcePartly supported

  The paper’s Abstract reports that astrocyte-derived mitochondria enter neurons and support neuronal survival after stroke. It also describes astrocyte-produced factors that protect or restore neurons, including vesicles containing VEGF and FGF-2. Our copy does not establish fuel or cholesterol supply, so it supports only part of the cited sentence. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Qi et al., 2020Not 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).

A recent proteomic network analysis of post-mortem brains revealed activation of a module enriched for glucose metabolism and glial proteins in Alzheimer's disease cases, with the ApoE4 homozygous subgroup most affected.

“Notably, a recent proteomic network analysis of post-mortal brains revealed the activation of a module enriched for glucose metabolism and glial proteins in AD cases, with the ApoE4 homozygous sub-group most affected (Johnson et al., 2020).”

• cites Johnson et al., 2020 sourcePartly supported

  The Introduction and Results describe an AD-associated M4 module enriched in sugar-metabolism and glial proteins, with increased expression in AD. But the Results report no strong APOE-genotype effects on module levels except an ε2 effect on a different module; they do not support the claim that APOE ε4 homozygotes were most affected. Page numbers were not provided in our copy. (LLM, conf=0.91)

  Experimental — accuracy not yet measured

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

Fatty acid oxidation, predominantly in astrocytes, contributes approximately 20% of total brain energy supply.

“FA oxidation—predominantly in astrocytes—contributes ~20% of total brain energy supply (Ebert et al., 2003).”

• cites Ebert et al., 2003 sourcePartly supported

  The Discussion reports that octanoate contributed approximately 20% of brain oxidative energy in intact rats and says the data support its oxidation in astrocytes. The claim generalizes that result to fatty acid oxidation and total brain energy supply without specifying the octanoate-infusion condition or rat population. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

The capacity in performing glycolysis and OXPHOS varies between neurons and astrocytes.

“The capacity in performing glycolysis and OXPHOS varies between neuron and astrocyte (Magistretti and Allaman, 2018; Yellen, 2018).”

• cites Magistretti and Allaman, 2018 sourceNot checked — no full text of the source
• cites Yellen, 2018 sourcePartly supported

  In “The source of neuronal energy during activation” (page unavailable), Yellen reports that astrocytes appear more glycolytic than neurons at rest and describes differences in their glycolytic-enzyme expression. Those passages support a difference in glycolytic behavior, but they do not establish a comparative capacity for oxidative phosphorylation between the two cell types. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

May combine two claims.

ApoE4 mice and Alzheimer's disease brains show dysfunctional neurons and activated astrocytes.

“This is consistent with dysfunctional neurons and activated astrocytes seen in both ApoE4 mice (Zhu et al., 2012) and AD brains (Long and Holtzman, 2019).”

• cites Zhu et al., 2012 sourceSupported
• cites Long and Holtzman, 2019 sourceSupported

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

Among the top risk genes for sporadic Alzheimer's disease, APOE, TREM2, APOJ, PICALM, ABCA7, and SORL1 are all directly involved in lipid trafficking or metabolism.

“among the top risk genes for sporadic AD, APOE, TREM2, APOJ, PICALM, ABCA7, and SORL1 are all directly involved in lipid trafficking or metabolism (Karch and Goate, 2015).”

• cites Karch and Goate, 2015 sourcePartly supported

  The Cholesterol Metabolism section identifies APOE, CLU (APOJ), ABCA7, and SORL1 as involved in cholesterol metabolism or lipid transport; the CLU subsection describes lipid transport, and the SORL1 subsection describes lipoprotein uptake. But the Immune Response > TREM2 subsection describes phagocytosis and inflammation, while Endocytosis > PICALM describes clathrin-mediated trafficking, not direct lipid trafficking or metabolism. The paper therefore does not support saying that all six genes have that direct role. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

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

Astrocyte-derived lactate is not required for the bioenergetic response to stimulation in neurons.

“This is consistent with a recent report that astrocyte-derived lactate is not required for the bioenergetic response to stimulation in neurons (Díaz-García et al., 2017).”

• cites Díaz-García et al., 2017 sourceSupported

May combine two claims.

In neuron-glia co-culture, mitochondrial OXPHOS inhibitor rotenone or knockout of Ndufs4 in neurons leads to glial lipid droplet accumulation.

“In neuron-glia co-culture, mitochondrial OXPHOS inhibitor rotenone or the knockout of Ndufs4 in neurons leads to glial LD accumulation (Liu et al., 2015).”

• cites Liu et al., 2015 sourcePartly supported

  The Results section on Ndufs4−/− mice reports glial lipid droplets in knockout mice and after neuronal Ndufs4 RNAi knockdown in flies (page unavailable). These findings support the link between Ndufs4 disruption and glial lipid droplets, but they are not neuron–glia co-culture experiments or a neuron-specific Ndufs4 knockout. Our copy does not report the claimed rotenone treatment. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

Loss of a mitochondrial fatty acid importer leads to lipid droplet accumulation in the Drosophila brain.

“These findings are in line with a previous report in Drosophila that loss of a mitochondrial FA importer leads to LD accumulation in the brain (Schulz et al., 2015).”

• cites Schulz et al., 2015 sourceSupported

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

ApoE4 shows both loss-of-function and gain-of-toxic-function across various Alzheimer's disease pathogenesis pathways.

“ApoE4 shows both loss-of-function and gain-of-toxic-function across various AD pathogenesis pathways (Yamazaki et al., 2019).”

• cites Yamazaki et al., 2019 sourceSupported

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

Neurons synthesize and secrete as much as 20% of brain ApoE under physiological conditions.

“neurons were found to synthesize and secrete as much as 20% of brain ApoE under physiological conditions (Knoferle et al., 2014)”

• cites Knoferle et al., 2014 sourcePartly supported

  The Results section (page unavailable) reports that deleting APOE in neurons reduced total ApoE protein by about 20% in mouse cortex, supporting a neuronal contribution to ApoE levels. The paper does not establish that neurons secrete that amount, and its hippocampal estimates differ by ApoE variant; the claim generalizes the cortical finding to brain ApoE. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

ApoE4 lowers FA oxidation and leads to lipid accumulation in both astrocyte and hippocampus.

“Further, ApoE4 lowers FA oxidation and leads to lipid accumulation in both astrocyte and the hippocampus.”

May combine two claims.

Disruption to Golgi secretory pathway by brefeldin A or to endocytosis by Pitstop 2 substantially reduces FA transfer.

“Our analyses suggested that disruption to Golgi secretory pathway by brefeldin A (BFA) (Figure 4E) or to endocytosis by Pitstop 2 (Figure 4F) substantially reduced FA transfer.”

May combine two claims.

E3 astrocytes trigger increased length, area, and branch counts of neurite, but effects are significantly lessened with E4 astrocytes.

“Although E3 astrocytes triggered increased length (Figures 5G and 5H), area (Figure S4G), and branch counts (Figure S4H) of neurite, effects were significantly lessened with E4 astrocytes.”

May combine two claims.

The shift in preference of E4 astrocytes toward glucose metabolism is demonstrated by higher basal respiration on glucose, higher glycolytic rate, and increased expression of multiple subunits of OXPHOS complexes and HK1.

“The shift in the preference of E4 astrocytes toward glucose metabolism was further demonstrated by their higher basal respiration on glucose (Figures 6I and 6J), higher glycolytic rate (Figure 6K), and increased expression of multiple subunits of OXPHOS complexes (Figures 6L and S5E) and HK1 (Figures 6M and S5F).”

May combine two claims.

The ApoE isoform effect on extracellular acidification rate under glycemic conditions represented variance in glycolytic rate, as confirmed by minimum ECAR values under aglycemic conditions.

“The minimum ECAR values under aglycemic conditions confirmed that the ApoE isoform effect under glycemic condition represented the variance in glycolytic rate.”

May combine two claims.

Key regulators of glucose metabolism (phospho-Akt) and mitochondrial biogenesis (PGC1α/β) are upregulated by astrocytes, and an ApoE isoform effect in astrocytes is seen for Akt activation in E3 neurons.

“In parallel, key regulators of glucose metabolism (phospho-Akt) and mitochondrial biogenesis (PGC1α/β) were upregulated by astrocytes, and an ApoE isoform effect in astrocytes was seen for Akt activation in E3 neurons (Figures 5F and S4F).”

May combine two claims.

E4 astrocytes have lower ability in uptaking LDL, consistent with a reduced capacity in internalizing FAs.

“Because LDL receptors are involved in neuron-to-astrocyte FA transfer (Ioannou et al., 2019), the lower ability of E4 astrocytes in uptaking LDL (Figure S3E) is consistent with a reduced capacity of E4 astrocytes in internalizing FAs.”

• cites Ioannou et al., 2019 sourceNot checked — no full text of the source

May combine two claims.

Both mitochondrial function and aerobic glycolysis in E3 neurons are elevated by E3 astrocytes, as evidenced by elevated respiration, ECAR, and lactate levels.

“Metabolically, both mitochondrial function and aerobic glycolysis in E3 neurons was elevated by E3 astrocytes, as evidenced by elevated respiration (Figures 5A and 5B), ECAR (Figure 5C), and lactate levels (Figure 5D).”

May combine two claims.

Key components of FA synthesis are downregulated in E4 neurons: higher inhibitory phosphorylation of ACC at Ser79 (+27.8%), reduced expression of FAS (−16.3%) and PPAR-γ (20.2%).

“Key components of FA synthesis were downregulated in E4 neurons, as suggested by the higher inhibitory phosphorylation of ACC at Ser79 (+27.8%), and reduced expression of FA synthase (FAS, −16.3%) and PPAR-γ (20.2%) (Figures 1I and S1J).”

May combine two claims.

Either E3 or E4 ACM is sufficient to reduce LD in neurons, with E3 ACM inducing a greater decline than E4 ACM.

“Indeed, either E3 or E4 ACM was sufficient to reduce LD in neurons, with E3 ACM inducing a greater decline than E4 ACM (Figures 5K, 5L, and S4M).”

May combine two claims.

Glucose metabolism is impaired in ApoE4 neurons, as shown by reduced glucose uptake and a decline in OXPHOS when glucose is the only fuel substrate.

“Glucose metabolism was also impaired in ApoE4 neurons as suggested by reduced glucose uptake (Figure 2K) and a decline in OXPHOS when glucose was the only fuel substrate (no pyruvate that bypasses glycolysis) (Figure 2L).”

May combine two claims.

Length, branch counts, and area of neurites are significantly lower in E4 versus E3 neurons.

“the length, branch counts, and area of neurites were significantly lower in E4 versus E3 neurons (Figures 2M and 2N)”

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

Glutamate in culture medium did not alter neuronal LD accumulation.

“We also confirmed that glutamate in culture medium did not alter LD accumulation (Figure S1A)”

May combine two claims.

Lipid peroxidation was not affected by ApoE genotype or glutamate levels.

“and lipid peroxidation was not affected by ApoE genotype or glutamate levels (Figure S1B).”

May combine two claims.

Astrocyte exposure upregulates HK1 and HK2 expression while reducing inhibitory phosphorylation of PDH (pSer293) in neurons, with E4 astrocytes less effective than E3 astrocytes.

“Consistent with the metabolic data, astrocyte exposure upregulated HK1 and HK2 expression while it reduced the inhibitory phosphorylation of PDH (pSer293) in neurons, with E4 astrocytes less effective than E3 astrocytes (Figures 5E and S4E).”

May combine two claims.

Blocking neuronal secretory pathway prevents ACM-induced LD removal and abolishes the increase in neuronal mitochondrial function.

“Intriguingly, blocking neuronal secretary pathway not only prevented ACM-induced LD removal (Figure 5O) but also abolished the increase in neuronal mitochondrial function (Figure 5P).”

May combine two claims.

Saturated FAs including palmitic acid, myristic acid, and stearic acid are higher, and ApoE proteins are lower (−41.0%), in E4 versus E3 astrocytes.

“Nevertheless, as seen in neurons, saturated FAs including palmitic acid, myristic acid, and stearic acid were higher (Figure 4G), and ApoE proteins were lower (−41.0%) (Figure S5B), in E4 versus E3 astrocytes.”

The quote only partly states this claim.

E4 neurons have lower ApoE protein (35.9%) than E3 neurons.

“After confirming lower ApoE protein in E4 neurons (35.9%) (Figures 2A and S2A)”

May combine two claims.

Neuronal LD-like structures are located predominantly in the soma and surrounded by perilipin-2.

“These structures were located predominantly in the soma and surrounded by LD surface marker perilipin-2 (Plin2) (Figure 1A, lower panel).”

May combine two claims.

E4 astrocytes have a lower dependence and capacity of using FAs, whereas their ability to use glucose is higher than E3 astrocytes.

“Further, metabolic flexibility assay using inhibitors of different fuel pathways including glucose, glutamine, and FA revealed that E4 astrocytes had a lower dependence and capacity of using FAs, whereas their ability to use glucose was higher than E3 astrocytes (Figure 6H).”

May combine two claims.

When mitochondrial function is inhibited by FCCP, LDs in astrocytes increase substantially, a response not observed in neurons.

“Consistently, when mitochondrial function was inhibited by FCCP, LDs in astrocytes increased substantially (Figures 6G and S5D), a response not observed in neurons (Figure 5Q).”

May combine two claims.

rApoE3 further reduces LDs in neurons cultured with E3-ACM, indicating a positive effect of ApoE3 in clearing neuron LDs; rApoE4 is unable to reduce neuronal LD.

“Interestingly, rApoE3 further reduced LDs in neurons cultured with E3-ACM (Figure 5R), indicating a positive effect of ApoE3 in clearing neuron LDs; in contrast, rApoE4 was unable to reduce neuronal LD.”

May combine two claims.

PSD-95 and SNAP-25 levels are increased by astrocyte co-culture, but E4 astrocytes are less capable in upregulating PSD-95 in E3 neurons.

“In parallel, PSD-95 and SNAP-25 levels were increased by astrocyte co-culture, but E4 astrocytes were less capable in upregulating PSD-95 in E3 neurons (Figures 5I and S4I).”

May combine two claims.

E4 neurons have reduced levels of postsynaptic marker PSD-95 (−38.8%) and presynaptic marker SNAP-25 (−20.3%).

“which were in parallel with reduced levels of postsynaptic marker PSD-95 (−38.8%) and presynaptic marker SNAP-25 (−20.3%) (Figures 2O and S2E).”

May combine two claims.

Neuronal LDs are not due to the introduction of humanized ApoE, because LDs in wild-type neurons are comparable to E3 neurons.

“We confirmed that neuronal LDs are not due to the introduction of humanized ApoE, because LDs in wild-type neurons were comparable to E3 neurons (Figure S1I).”

May combine two claims.

ApoE levels in shared medium of E3-neuron-E3-astrocyte co-culture are lower than that of E3 neurons alone and higher than E3 astrocytes alone; E4 astrocytes are not able to alter ApoE in E3 neuron medium.

“Consistently, ApoE levels in the shared medium of E3-neuron-E3-astrocyte co-culture were lower than that of E3 neurons alone and higher than E3 astrocytes alone; however, E4 astrocytes were not able to alter ApoE in E3 neuron medium (Figure 4L).”

May combine two claims.

E4 neurons have lower mitochondrial membrane potential and cellular ATPs.

“which were accompanied by lower mitochondrial membrane potential (Δψm) (Figure 2D) and cellular ATPs (Figure 2E).”

Classified as probably not a substantial claim.

AMPK suppresses de novo synthesis of fatty acids by phosphorylating and inhibiting ACC.

“which suppresses de novo synthesis of FAs by phosphorylating and inhibiting ACC”

May combine two claims.

E4 neurons exhibit lower maximal respiration and spare respiratory capacity ratio but indistinguishable basal respiration compared to E3 neurons.

“Compared to E3 controls, E4 neurons exhibited lower maximal respiration (respiration after mitochondrial uncoupling) (Figure S2B) and spare respiratory capacity ratio (SCR, the ratio of maximal to basal respiration, an indicator for utilization of OXPHOS capacity) but indistinguishable basal respiration (Figures 2B and 2C)”

May combine two claims.

In neurons cultured with E4-ACM, additional rApoE3 promotes LD clearance but rApoE4 completely abolishes the effect elicited by E4-ACM.

“Similar results were obtained in neurons cultured with E4-ACM, where additional rApoE3 promoted LD clearance but rApoE4 completely abolished the effect elicited by E4-ACM (Figure 5S).”

May combine two claims.

BODIPY-C12 localizes to neuronal LDs and is tracked to astrocytes and their LDs in 4 h after co-culture.

“After incubation, BODIPY-C12 were localized to neuronal LDs (Figure 4B); subsequent co-culture of these labeled neurons with astrocytes tracked BODIPY-C12 to astrocytes and localized to their LDs in 4 h (Figure 4C).”

May combine two claims.

ATP levels are higher in E4 astrocytes, consistent with a lower activation of AMPK (−31.8%).

“ATP levels were higher in E4 astrocytes (Figure 6N), which was consistent with a lower activation of AMPK (−31.8%) (Figures 6O and S5G).”

May combine two claims.

ApoE4 was not in E3 neurons co-cultured with E4 astrocytes but was detected in E3 astrocytes co-cultured with E4 neurons.

“As shown in Figure S3I, ApoE4 was not in E3 neurons co-cultured with E4 astrocytes but was detected in E3 astrocytes co-cultured with E4 neurons (Figure S3J).”

May combine two claims.

Neuronal LDs are subcellular structures delimited by a single membrane leaflet, and most are adjacent to mitochondria.

“Transmission electron microscopy (TEM) further visualized neuronal LDs as subcellular structures delimited by a single membrane leaflet, and most of them were adjacent to mitochondria (Figure 1E).”

May combine two claims.

Inhibition of de novo FA synthesis completely prevents LD accumulation in both E3 and E4 astrocytes, suggesting that FA biosynthesis predominantly accounts for astrocytic LD formation.

“The inhibition of de novo FA synthesis completely prevented LD accumulation in both E3 and E4 astrocytes (Figure 6D), suggesting that FA biosynthesis predominantly accounts for astrocytic LD formation.”

May combine two claims.

In acute hippocampal slices from E3 and E4 knockin mice, E4 slices exhibited higher basal respiration than E3 when glucose (10 mM) was present, but not when fatty acid was present.

“E4 slices exhibited a higher basal respiration than E3 when glucose (10 mM), but not FA, was present (Figure 7B).”

May combine two claims.

E4 neurons accumulate fewer and smaller LDs than E3 neurons.

“Intriguingly, compared to E3 neurons, E4 neurons accumulated fewer and smaller LDs (Figures 1B–1D)”

May combine two claims.

Either E3 or E4 astrocytes dramatically reduce the number and volume of LDs in E3 or E4 neurons at DIV8.

“Either E3 or E4 astrocytes dramatically reduced the number and volume of LDs in E3 or E4 neurons at DIV8”

May combine two claims.

Mitochondria in E4 astrocytes are more fragmented and form less tubule structures compared to E3 controls.

“Mitochondria in E4 astrocytes were more fragmented and formed less tubule structures compared to E3 controls (Figure 6P)”

May combine two claims.

E4 astrocytes show higher FA levels than E3 controls with or without neuronal co-culture, indicating a disruption to FA degradation in E4 astrocytes.

“Interestingly, despite a reduced uptake ability, E4 astrocytes showed higher FA levels than E3 controls with or without neuronal co-culture (Figures 4G and S3F), indicating a disruption to FA degradation in E4 astrocytes.”

May combine two claims.

Neuronal levels of saturated, medium- to long-chain FAs (C8-C19) are reduced by astrocytes, with pentadecanoic acid (C15) and heptadecanoic acid (C17) reaching statistical significance upon E3 astrocyte co-culture.

“Targeted metabolomic analysis confirmed that neuronal levels of saturated, medium- to long-chain FAs (C8–C19) were reduced by astrocyte (Figure 3I), with pentadecanoic acid (C15) and heptadecanoic acid (C17) reaching statistical significance upon E3 astrocyte co-culture (Figure 3J).”

May combine two claims.

E4 brains showed a higher rate of aerobic glycolysis both with and without fatty acid.

“E4 brains showed a higher rate of aerobic glycolysis either with (Figure 7F) or without FA (Figure 7G).”

May combine two claims.

AMPK activation regulates neuronal LD formation: AICAR diminishes and compound C promotes LD formation.

“The regulatory role of AMPK in neuronal LD formation was further confirmed that its activator 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR) diminished, while its inhibitor compound C promoted, LD formation (Figure 1J).”

May combine two claims.

Reduced FA oxidative capacity and elevated neutral lipid levels were manifested in ApoE4 mouse hippocampi.

“In accordance with that, reduced FA oxidative capacity and elevated neutral lipid levels were manifested in ApoE4 mouse hippocampi.”

May combine two claims.

Both lipolysis and lipophagy contribute to ACM-induced mobilization of neuronal LDs.

“In contrast to neurons cultured alone (Figures 1K and S1K), both lipolysis and lipophagy contributed to ACM-induced mobilization of neuronal LDs as either atglistatin (Figure 5M) or 3-MA (Figure 5N) partially abolished LD clearance.”

May combine two claims.

ApoE protein is increased in astrocytes after co-culture with mRNA levels unchanged, suggesting that elevated astrocytic ApoE could be transported from neurons together with FAs.

“Simultaneously, ApoE protein was increased in astrocytes after co-culture (Figures 4K and S3H) with mRNA levels remained unchanged (Figure 4I), suggesting that elevated astrocytic ApoE could be transported from neurons together with FAs.”

May combine two claims.

SCR, but not maximal respiration, is lower in E4 astrocytes, largely due to higher basal respiration.

“SCR, but not maximal respiration, was lower in E4 astrocytes (Figures S5H and S5I), largely due to the higher basal respiration (Figure 6J).”

May combine two claims.

Mitochondria in E4 neurons are smaller and more dispersed in the cytoplasm.

“In addition, TEM imaging revealed that mitochondria in E4 neurons are smaller and more dispersed in the cytoplasm (Figure 2H).”

May combine two claims.

Neuron-to-astrocyte lipid transport is altered by ApoE4 in both cell types: E4 astrocytes exhibit lower BODIPY-C12 positive rate than E3 astrocytes when co-cultured with E3 neurons, and E3 astrocytes receive less lipids from E4 neurons than from E3 neurons.

“Importantly, neuron-to-astrocyte lipid transport was altered by ApoE4 in both cell types (Figure 4D and S3D): E4 astrocytes exhibited lower BODIPY-C12 positive rate than E3 astrocytes when co-cultured with E3 neurons, whereas E3 astrocytes received less lipids from E4 neurons than from E3 neurons (Figure 4D).”

The quote only partly states this claim.

E4 astrocytes are less efficient in internalizing fatty acids than E3 astrocytes.

“with E4 neurons and E4 astrocytes less efficient in exporting and internalizing FAs, respectively.”

May combine two claims.

In co-culture, ApoE protein levels decrease in neurons and increase in astrocytes.

“ApoE protein levels decrease in neurons and increase in co-cultured astrocytes”

May combine two claims.

E4 astrocytes with decreased fatty acid catabolizing capacity exhibit increased lipid droplet levels, similar to astrocytes with OXPHOS inhibition.

“E4 astrocytes, with decreased FA catabolizing capacity, exhibit increased LD levels, as seen in astrocytes with OXPHOS inhibition.”

The quote only partly states this claim.

E4 neurons are less efficient in exporting fatty acids than E3 neurons.

“with E4 neurons and E4 astrocytes less efficient in exporting and internalizing FAs, respectively.”

May combine two claims.

ApoE4 perturbs brain lipid homeostasis and energy transduction.

“Alzheimer’s disease (AD) risk gene ApoE4 perturbs brain lipid homeostasis and energy transduction.”

May combine two claims.

Astrocytes eliminate neuronal LDs and E4 astrocytes have reduced capacity in clearing neuronal lipids.

“Together, these results support the role of astrocyte in eliminating neuronal LDs and reveal the reduced capacity of E4 astrocytes in clearing neuronal lipids.”

May combine two claims.

Neuronal E4 may impair LD mobilization and/or export.

“suggesting that neuronal E4 may also impair LD mobilization and/or export.”

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

The reduction in neuronal ApoE mRNA suggests that neurotrophic support by astrocytes may reduce neuronal ApoE expression by alleviating cellular stress.

“The reduction in neuronal ApoE mRNA also suggests that the neurotrophic support by astrocytes may reduce neuronal ApoE expression by alleviating cellular stress (Xu et al., 2006).”

• cites Xu et al., 2006Not matched

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

May combine two claims.

Excessive FAs induce mitochondrial uncoupling and dysfunction.

“Because neurons have minimum capacity to catabolize FAs, and excessive FAs induce mitochondrial uncoupling and dysfunction, fine control and timely elimination of neuronal free FAs is critical to protect against neurodegeneration (Schönfeld and Reiser, 2017).”

• cites Schönfeld and Reiser, 2017 sourceNot checked — no full text of the source

May combine two claims.

ApoE4 in neurons dampens mitochondrial OXPHOS, glucose metabolism, neurite growth, and synaptic density, coupled with lower cellular ApoE and impaired FA sequestering in LD.

“Collectively, these findings suggest that ApoE4 in neurons dampened mitochondrial OXPHOS, glucose metabolism, neurite growth, and synaptic density, which were coupled with lower cellular levels of ApoE and impaired FA sequestering in LD.”

Classified as probably not a substantial claim.

ApoE transports peroxidized FAs from neuron to astrocyte upon neuronal stimulation.

“recent reports revealed a role of ApoE in transporting peroxidized FAs from neuron to astrocyte upon neuronal stimulation (Ioannou et al., 2019)”

• cites Ioannou et al., 2019 sourceNot checked — no full text of the source

May combine two claims.

E4 brains have higher glucose metabolism but a reduced ability to use non-glucose fuels.

“These data suggest that E4 brains have higher glucose metabolism but a reduced ability to use non-glucose fuels.”

May combine two claims.

In astrocytes, E4-induced mitochondria fission is coupled with a decline in degradation of FAs that leads to FA and LD accumulation, and a shift toward increased basal glucose metabolism that could be connected with compromised metabolic support to neurons.

“Together, these findings suggest that in astrocyte, E4-induced mitochondria fission is coupled with (1) a decline in degradation of FAs—either synthesized by astrocytes or transferred from neurons—that leads to FA and LD accumulation, and (2) a shift toward increased basal glucose metabolism that could be connected with their compromised metabolic support to neurons.”

May combine two claims.

The ability of astrocyte to potentiate neuronal bioenergetic function is associated with, and dependent on, the removal of excessive neuronal lipids, whereas the mobilization of neuronal LDs by astrocyte does not rely on mitochondrial OXPHOS.

“These results suggest that the ability of astrocyte to potentiate neuronal bioenergetic function is associated with, and dependent on, the removal of excessive neuronal lipids, whereas the mobilization of neuronal LDs by astrocyte does not rely on mitochondrial OXPHOS.”

May combine two claims.

ApoE isoform is essential in mediating neuronal lipid homeostasis; ApoE3 is an enhancer of neuronal LD clearance and excessive ApoE4 is a negative regulator of the process.

“Together, these data confirm the essential role of ApoE isoform in mediating neuronal lipid homeostasis and suggest ApoE3 as an enhancer of neuronal LD clearance and excessive ApoE4 as a negative regulator of the process.”

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

Remaining astrocytes were verified for negative microglia staining (Iba1) before experimental use.

“the remaining astrocytes are verified for negative microglia staining (Iba1) before experimental use.”

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

Data were presented as the means ± SEM from at least three independent experiments.

“Data were presented as the means ± SEM from at least three independent experiments.”

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

Autophagy contributes to lipid droplet formation in NMDA-stimulated neurons.

“while autophagy contributes to LD formation in NMDA-stimulated neurons (Ioannou et al., 2019)”

• cites Ioannou et al., 2019 sourceNot checked — no full text of the source

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

Human studies have detected neuronal ApoE mRNA and protein.

“human studies also detected neuronal ApoE mRNA (Xu et al., 1999a) and protein (Han et al., 1994).”

• cites Xu et al., 1999aNot matched

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

• cites Han et al., 1994 sourceNot checked — no full text of the source

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

All animal experiments were approved by the University of Arizona Institutional Animal Care and Use Committee (IACUC).

“All animal experiments were approved by the University of Arizona Institutional Animal Care and Use Committee (IACUC).”

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

Saturated pixel highlighting feature of the instrument was used to prevent image overexposure for chemiluminescence.

“Saturated pixel highlighting feature of the instrument was used to prevent image overexposure for chemiluminescence.”

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

Equal amounts of proteins (10-20 μg) were loaded and separated on a 4%-15% Criterion Precast Midi Protein Gel and transferred to a PVDF membrane for immunoblotting.

“Equal amounts of proteins (10–20 μg) were loaded and separated on a 4%–15% Criterion Precast Midi Protein Gel (Bio-Rad) and then transferred to a 0.45 μm pore size polyvinylidene difluoride (PVDF) membrane and immunoblotted with the appropriate primary antibodies.”

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

Data shown are representative results from at least 3 independent batches/animals of cells.

“Data shown are representative result from at least 3 independent batches/animals of cells.”

May combine two claims.

Astrocytic mitochondria play a critical role in performing β-oxidation and thereby modulating brain lipid homeostasis.

“Overall, our data support the critical role of astrocytic mitochondria in performing β-oxidation and thereby modulating brain lipid homeostasis.”

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

Experiments from cells isolated from at least three animals were performed in these assays.

“Experiments from cells isolated from at least three animals were performed in these assays.”

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

A quality control (QC) sample was pooled from all the study samples.

“A quality control (QC) sample was pooled from all the study samples.”

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

The isoform-dependent dose-response relationship suggests that a direct comparison of ApoE3 and ApoE4 protein levels could be complicated by their structural and functional distinctions.

“This isoform-dependent dose-response relationship suggests that a direct comparison of ApoE3 and ApoE4 protein levels could be complicated by their structural and functional distinctions.”

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

Cultured neurons were >94% pure and GFAP-positive cells were <1%, as determined by NeuN, GFAP and DAPI immunostaining on DIV8.

“The purity of cultured neurons was > 94% while GFAP-positive cells are < 1% as determined by NeuN, GFAP and DAPI immunostaining on DIV8.”

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

High-powered (400 x) fields of micrographs from three independent experiments per group were analyzed.

“High-powered (400 x) fields of micrographs from three independent experiments per group were analyzed.”

May combine two claims.

The neuron-astrocyte coupling of fatty acid metabolism is a highly regulated pathway activated by astrocyte-originated signals.

“This suggests that the neuron-astrocyte coupling of FA metabolism is a highly regulated pathway activated by astrocyte-originated signals”

May combine two claims.

ApoE4 elicits a decline in glucose-dependent OXPHOS in neurons but an increase in astrocytes.

“Our findings suggest that ApoE4 elicits a decline in glucose-dependent OXPHOS in neurons but an increase in astrocytes.”