Senescence-related impairment of autophagy induces toxic intraneuronal amyloid-β accumulation in a mouse model of amyloid pathology

May combine two claims.

Progressive telomere attrition occurs in successive generations of Terc−/− mice compared with wild-type mice, with significant differences from generation 1 and a plateau at generations 3-4.

“As expected, progressive telomere attrition was observed in successive generations of Terc−/− mice, compared to WT mice, with already significant differences appearing from the first generation (G1) and reaching a plateau at G3-G4 (Fig. 1a).”

May combine two claims.

Non-neuronal cells from telomerase-deficient mice exhibit features of senescence, with a striking increase in the percentage of SA-β-gal-positive astrocytes.

“Non-neuronal cells from telomerase-deficient mice also exhibited features of senescence, as evidenced by a striking increase in the percentage of SA-β-gal-positive astrocytes (Additional file 1: Fig. S1).”

The quote only partly states this claim.

Primary neurons from G3Terc−/− mice have shorter telomeres than wild-type primary neurons, as evaluated by qPCR.

“Neurons bearing shorter telomeres, as evaluated by qPCR (Fig. 1c), exhibit a transcriptional upregulation of Il1b, Cxcl1, p16 and p21 (Fig. 1d).”

• AssumptionRests on an assumption: Primary neuronal cultures derived from postnatal day 0 pups are a valid model for studying senescence-related autophagy and intraneuronal amyloid-β accumulation in the aging brain.

May combine two claims.

In primary neurons obtained from 5xFAD mice, human APP expression was weak, and intraneuronal Aβ levels were below the detection limit even though human Aβ species were present at detectable levels in the media.

“Human APP expression in primary neurons obtained from 5xFAD mice was weak, and even though human Aβ species were present at detectable levels in the media, intraneuronal Aβ levels were below the detection limit (data not shown).”

May combine two claims.

In 5xFAD mice, GFAP protein levels and the area occupied by GFAP-positive astrocytes are increased in hippocampal and cortical regions.

“WB analysis indicated increased GFAP protein levels in hippocampal and cortical regions of 5xFAD mice (Additional file 1: Fig. S5a), which was further confirmed by immunohistological analyses of the area occupied by GFAP-positive astrocytes (Additional file 1: Fig. S5b).”

May combine two claims.

At 5 months, Aβ levels are decreased in hippocampal and cortical extracts of G3Terc−/− 5xFAD mice, while levels of full-length human APP are not altered in hippocampal extracts.

“Again, Aβ levels were found decreased both in hippocampal (Fig. 5c) and cortical (Fig. 5d) extracts of G3Terc−/− 5xFAD mice, while levels of full-length human APP were not altered (hippocampal extracts, Fig. 5c).”

May combine two claims.

In Western blot analyses of hippocampal protein extracts from 5-month-old 5xFAD mice, the W0-2 antibody recognizes APP, CTFβ and human Aβ peptides, whereas the C-terminal Aβ42 antibody clone H31L21 only recognizes the Aβ42 peptide.

“While the W0-2 antibody (raised against N-terminal anti-human Aβ) recognizes APP, CTFβ and Aβ peptides of human origin in 5xFAD mice, the C-terminal Aβ42 antibody (clone H31L21) only recognizes the Aβ42 peptide in the same samples (Additional file 1: Fig. S4a).”

May combine two claims.

In 5-month-old 5xFAD mice, the W0-2 antibody strongly labels the soma of cells in the CA1 region, probably due to APP recognition, while specific staining against Aβ42 yields no detectable signal.

“Our data demonstrated that the W0-2 antibody strongly labels the soma of cells located within the CA1 region, probably due to APP recognition, while specific staining against Aβ42 does not result in any detectable signal (Additional file 1: Fig. S4b).”

May combine two claims.

At 5 months, amyloid pathology is mainly represented by extracellular deposition of insoluble Aβ and not by intracellular accumulation as observed earlier at 1.5-2 months.

“At this stage (5 months of age), amyloid pathology was mainly represented by extracellular deposition of insoluble Aβ and not by intracellular accumulation as observed earlier (1.5–2 months of age).”

May combine two claims.

At 5 months, WT and G3Terc−/− mice show no positive amyloid staining, while 5XFAD mice exhibit substantial plaque burden.

“As expected, WT and G3Terc−/− mice showed no positive staining, but 5XFAD mice exhibited substantial plaque burden.”

May combine two claims.

Significant activation of microglia is observed in 5xFAD mice and appears to the same extent in G3Terc−/− 5xFAD mice, with only a trend toward increased Iba-1 protein levels in G3Terc−/− mice.

“Although a trend towards an increase in Iba-1 protein levels was found in G3Terc−/− mice, significant activation of microglia was observed in 5xFAD mice and appeared to the same extent in G3Terc−/− 5xFAD mice.”

The quote only partly states this claim.

In primary neurons overexpressing hAPP3xmut, G3Terc−/− neurons show intracellular accumulation of both Aβ40 and Aβ42 compared to WT.

“Similar to what happens in vivo, our results indicate an intracellular accumulation of both Aβ40 and Aβ42 in G3Terc−/− primary neurons (Fig. 3c).”

The quote only partly states this claim.

At 2 months, a distinct intracellular Aβ staining pattern is observed in cortical layer V of 5xFAD and G3Terc−/− 5xFAD mice.

“while a distinct intracellular Aβ staining pattern was observed in the cortical layer V (Fig. 2c).”

May combine two claims.

At 1.5 months, 5XFAD and G3Terc−/− 5xFAD mice exhibit intracellular Aβ staining in cells within the subiculum, whereas WT and G3Terc−/− mice show no positive Aβ signal.

“While WT and G3Terc−/− mice showed no positive signal for Aβ, 5XFAD mice and G3Terc−/− 5xFAD mice exhibited intracellular Aβ staining in cells located within the subiculum.”

May combine two claims.

In G3Terc−/− 5xFAD mice, LC3-II and p62 accumulation becomes significant, suggesting an additive effect of both amyloid pathology and telomere attrition on autophagy impairment.

“This accumulation became significant in G3Terc−/− 5xFAD mice, which could suggest an additive effect of both amyloid pathology and telomere attrition on autophagy impairment.”

May combine two claims.

In primary neurons, chloroquine-induced accumulation of LC3-II/LC3-I and p62 is decreased in G3Terc−/− neurons compared to WT neurons (non-significant trend for LC3-II/LC3-I and significant difference for p62), suggesting a reduction in autophagic flux.

“WB analysis revealed that the accumulation of LC3-II/LC3-I and p62 levels after chloroquine treatment is decreased in G3Terc−/− neurons compared to WT neurons (a non-significant trend for LC3-II/LC3 and a significant difference for p62 is observed), suggesting a reduction in autophagic flux (Fig. 8c).”

May combine two claims.

p62 and LC3 partially colocalize with Aβ, suggesting that autophagosomes might be a reservoir of intracellular Aβ.

“In addition, immunostaining analyses showed that p62 and LC3 partially colocalized with Aβ (Fig. 7c), suggesting that autophagosomes might be a reservoir of intracellular Aβ.”

May combine two claims.

In primary neurons overexpressing hAPP3xmut, the G3Terc−/− genotype increases intraneuronal Aβ40 and Aβ42 levels, and rapamycin treatment significantly reduces their accumulation (ANOVA effects).

“Similarly, we found that the G3Terc−/− genotype increased intraneuronal levels of both Aβ isoforms, while rapamycin treatment significantly reduced their accumulation (Fig. 8e), as Two-way ANOVA analyses detected significant effects based on genotype (for Aβ40, F1, 10 = 13.44, P = 0.0043; for Aβ42, F1, 10 = 9.891, P = 0.0104) and treatment (for Aβ40, F1, 10 = 5.615, P = 0.0393; for Aβ42, F1, 10 =”

May combine two claims.

At 5 months, both telomere attrition and amyloid pathology significantly reduce neuronal density in the subiculum.

“Loss of neuronal density was even more apparent when measured in the subiculum of mice aged 5 months, and our results indicate that both telomere attrition and amyloid pathology significantly reduced neuronal density in this area (Fig. 6b).”

May combine two claims.

Rapamycin treatment activates autophagy in these neurons, evidenced by decreased p62 levels.

“Autophagy activation by rapamycin was evidenced by decreased levels of p62 (Additional file 1: Fig. S7).”

May combine two claims.

Brains and primary neurons from G3Terc−/− mice display telomere attrition and features of cellular senescence.

“We observed that the brains and primary neurons from G3Terc−/− mice display telomere attrition and features of cellular senescence.”

May combine two claims.

In pathologically defined preclinical AD (p-preAD) cases, there is a clear trend toward increased LC3-II and LC3-II/LC3-I levels, suggesting that autophagy alterations might appear early in disease progression.

“Additionally, there was a clear trend towards increased LC3-II and LC3-II/LC3-I levels in pathologically defined preclinical AD (p-preAD) cases, suggesting that autophagy alterations might appear early in the disease progression.”

May combine two claims.

p62 expression is very low in control samples, with some accumulation in p-preAD cases, and AD cases show neurons with strong somatic p62 staining and intracellular inclusions resembling neurofibrillary tangles.

“For p62, we observed very low expression in control samples, but a certain degree of accumulation in some p-preAD cases. In AD cases, we detected the presence of neurons with strong p62 staining in the soma, with some intracellular inclusions highly resembling neurofibrillary tangles.”

May combine two claims.

At 2 months, compared to WT mice, there is a trend toward decreased neuronal density in the subiculum of G3Terc−/− and 5xFAD mice, with significant neuronal loss in G3Terc−/− 5xFAD mice, suggesting an additive effect for both genotypes.

“Compared to WT mice, a trend towards decreased neuronal density was found in G3Terc−/− and 5xFAD mice, with significant neuronal loss observed in G3Terc−/− 5xFAD mice, suggesting an additive effect for both genotypes (Fig. 6a).”

May combine two claims.

At 5 months, hippocampal regions lacking initial intraneuronal Aβ accumulation, such as CA1, CA3 and dentate gyrus, show no significant differences in neuron number between groups, despite amyloid plaques being observable throughout the hippocampus of 5xFAD mice.

“In contrast, hippocampal regions lacking initial intraneuronal Aβ accumulation, such as the CA1, CA3 and dentate gyrus, showed no significant differences in neuron number between groups when evaluated at 5 months of age (Fig. 6d–f), an age at which amyloid plaques can be observed throughout the hippocampus of 5xFAD mice.”

May combine two claims.

Intraneuronal Aβ is observed in the subiculum, while the reduction of amyloid burden was observed in different but anatomically interconnected brain regions, such as the cortex and hippocampus.

“However, while intraneuronal Aβ is observed in the subiculum, the reduction of amyloid burden was observed in different but anatomically interconnected brain regions, such as the cortex and hippocampus.”

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

Neurons accumulate high amounts of DNA damage over time that can trigger a senescence-like phenotype, with increased SA-β-gal activity and elevated expression of proinflammatory interleukins.

“Still, neurons accumulate high amounts of DNA damage over time that can trigger a senescence-like phenotype, with increased SA-β-gal activity and elevated expression of proinflammatory interleukins [59]”

• cites [59] Jurk D, Wang C, Miwa S, Maddick M, Korolchuk V, Tsolou A et al (2012) Postmitotic neurons develop a p21-dependent senescence-like phenotype driven by a DNA damage response. Aging Cell 11:996–1004. https://doi.org/10.1111/j.1474-9726.2012.00870.x sourceNot checked yet

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

Cellular senescence is a key biological process linked to aging that can contribute to or cause age-related pathologies like Alzheimer's disease.

“In this regard, cellular senescence emerges as a key biological process linked to aging that can contribute to or cause the appearance of age-related pathologies like AD [5].”

• cites [5] Baker DJ, Petersen RC (2018) Cellular senescence in brain aging and neurodegenerative diseases: evidence and perspectives. J Clin Invest 128:1208–1216. https://doi.org/10.1172/jci95145 sourceNot checked yet

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Classified as probably not a substantial claim.

Aging is the primary driver of many neurodegenerative diseases, including Alzheimer's disease.

“Aging is known to be the primary driver of many neurodegenerative diseases, including Alzheimer’s disease (AD) [53].”

• cites [53] Hou Y, Dan X, Babbar M, Wei Y, Hasselbalch SG, Croteau DL et al (2019) Ageing as a risk factor for neurodegenerative disease. Nat Rev Neurol 15:565–581 sourceNot checked yet

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

A variety of stressors, including oncogenic activation, reactive oxygen species and mitochondrial dysfunction, can stimulate conversion into a state of senescence, mostly through generation of DNA damage.

“It has been demonstrated that a variety of stressors, including oncogenic activation, reactive oxygen species (ROS) and mitochondrial dysfunction, can stimulate the conversion into a state of senescence, mostly through the generation of DNA damage [5].”

• cites [5] Baker DJ, Petersen RC (2018) Cellular senescence in brain aging and neurodegenerative diseases: evidence and perspectives. J Clin Invest 128:1208–1216. https://doi.org/10.1172/jci95145 sourceNot checked yet

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

Senescent cells accumulate with age throughout the body and eventually promote tissue deterioration, producing a low-grade chronic inflammatory context and acquiring a phenotype that prevents their clearance.

“Senescent cells accumulate with age throughout the body and, although playing a protective role by preventing the appearance of cancer cells, they eventually promote tissue deterioration, producing a low-grade but chronic inflammatory context and acquiring a phenotype that prevents their clearance from the tissue in which they reside [14, 114].”

• cites [14] Campisi J, d’Adda di Fagagna F (2007) Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol 8:729–740. https://doi.org/10.1038/nrm2233 sourceNot checked yet

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• cites [114] van Deursen JM (2014) The role of senescent cells in ageing. Nature 509:439–446. https://doi.org/10.1038/nature13193 sourceNot checked yet

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Markers of cellular senescence have been found in dividing cells such as glial cells or neural stem cells in the context of aging and neurodegenerative diseases.

“Indeed, markers of cellular senescence have been found in those cell types in the context of aging and neurodegenerative diseases [5].”

• cites [5] Baker DJ, Petersen RC (2018) Cellular senescence in brain aging and neurodegenerative diseases: evidence and perspectives. J Clin Invest 128:1208–1216. https://doi.org/10.1172/jci95145 sourceNot checked yet

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

Telomere attrition gradually appears with increasing number of cell divisions, triggering replicative senescence.

“As a consequence, telomere attrition gradually appears with increasing number of cell divisions, triggering replicative senescence [2, 48].”

• cites [2] Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB et al (1992) Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci U S A 89:10114–10118. https://doi.org/10.1073/pnas.89.21.10114 sourceNot checked yet

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• cites [48] Hayflick L, Moorhead PS (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25:585–621. https://doi.org/10.1016/0014-4827(61)90192-6 sourceNot checked yet

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Classified as probably not a substantial claim.

Reduced autophagic activity during aging is correlated to the onset of age-related diseases.

“Reduced autophagic activity during aging, which might contribute to the accumulation of damaged proteins and organelles, has been correlated to the onset of age-related diseases [71]”

• cites [71] Leidal AM, Levine B, Debnath J (2018) Autophagy and the cell biology of age-related disease. Nat Cell Biol 20:1338–1348. https://doi.org/10.1038/s41556-018-0235-8 sourceNot checked yet

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Senescent cells share detectable features including activation of senescence-associated β-galactosidase and transcriptional upregulation of cyclin-dependent kinase inhibitors p16Ink4a, p19Arf and p21waf1/cip1.

“Senescent cells share distinct detectable features, including the activation of the senescence-associated β-galactosidase (SA-β-gal) [28] and the transcriptional upregulation of cyclin-dependent kinase inhibitors (i.e., p16Ink4a, p19Arf and p21waf1/cip1) [65, 74].”

• cites [28] Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C et al (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92:9363–9367. https://doi.org/10.1073/pnas.92.20.9363 sourceNot checked yet

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• cites [65] Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L et al (2004) Ink4a/Arf expression is a biomarker of aging. J Clin Invest 114:1299–1307. https://doi.org/10.1172/jci22475 sourceNot checked yet

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• cites [74] Liu JY, Souroullas GP, Diekman BO, Krishnamurthy J, Hall BM, Sorrentino JA et al (2019) Cells exhibiting strong p16(INK4a) promoter activation in vivo display features of senescence. Proc Natl Acad Sci U S A 116:2603–2611. https://doi.org/10.1073/pnas.1818313116 sourceNot checked yet

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Recent reports suggested a causal relationship between shorter telomeres and Alzheimer's disease risk.

“Additionally, recent reports suggested a causal relationship between shorter telomeres and AD risk [102].”

• cites [102] Scarabino D, Broggio E, Gambina G, Pelliccia F, Corbo RM (2017) Common variants of human TERT and TERC genes and susceptibility to sporadic Alzheimers disease. Exp Gerontol 88:19–24. https://doi.org/10.1016/j.exger.2016.12.017 sourceNot checked yet

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

Alzheimer's disease is characterized by gradual appearance of cognitive deficits, neurodegeneration, and disease-defining pathological features consisting of extracellular senile plaques containing amyloid-β and intracellular neurofibrillary tangles mainly composed of hyperphosphorylated tau.

“It is characterized by the gradual appearance of cognitive deficits, neurodegeneration, and the disease-defining pathological features consisting in extracellular senile plaques, containing the amyloid-β (Aβ) peptide, and intracellular neurofibrillary tangles, mainly composed of hyperphosphorylated tau [105].”

• cites [105] Selkoe DJ (2001) Alzheimer’s disease: genes, proteins, and therapy. Physiol Rev 81:741–766. https://doi.org/10.1152/physrev.2001.81.2.741 sourceNot checked yet

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

Extracellular amyloid-β deposits like senile plaques might represent a relatively innocuous endpoint of the amyloid-β aggregation process, while soluble and smaller amyloid-β aggregates show higher toxicity and a greater correlation with cognitive decline and neurodegeneration.

“Extracellular Aβ deposits like senile plaques might represent a relatively innocuous endpoint of the Aβ aggregation process, while soluble and smaller Aβ aggregates show higher toxicity and a greater correlation with cognitive decline and neurodegeneration [58, 76].”

• cites [58] Jongbloed W, Bruggink KA, Kester MI, Visser PJ, Scheltens P, Blankenstein MA et al (2015) Amyloid-β oligomers relate to cognitive decline in Alzheimer’s disease. J Alzheimers Dis 45:35–43. https://doi.org/10.3233/jad-142136 sourceNot checked yet

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• cites [76] Lue LF, Kuo YM, Roher AE, Brachova L, Shen Y, Sue L et al (1999) Soluble amyloid beta peptide concentration as a predictor of synaptic change in Alzheimer’s disease. Am J Pathol 155:853–862. https://doi.org/10.1016/s0002-9440(10)65184-x sourceNot checked yet

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

Neurodegeneration in Alzheimer's disease animal models and human patients seems to be more restricted to specific brain regions in the hippocampus and cortex, and the molecular basis for this selective vulnerability remains unknown.

“Additionally, despite the near-ubiquitous expression of APP in the brain, neurodegeneration in AD animal models and human patients seems to be more restricted to specific brain regions in the hippocampus and the cortex, and the molecular basis for this selective vulnerability remains unknown [98].”

• cites [98] Roussarie JP, Yao V, Rodriguez-Rodriguez P, Oughtred R, Rust J, Plautz Z et al (2020) Selective neuronal vulnerability in Alzheimer’s disease: a network-based analysis. Neuron 107:821-835.e812. https://doi.org/10.1016/j.neuron.2020.06.010 sourceNot checked yet

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

Amyloid-β is produced by sequential cleavage of amyloid precursor protein by β-secretases and γ-secretases and is a central and early actor in Alzheimer's disease pathogenesis, triggering tau pathology and associated neurodegeneration.

“Aβ, which is produced by sequential cleavage of the amyloid precursor protein (APP) by β-secretases and γ-secretases, is a central and early actor in AD pathogenesis, triggering tau pathology and associated neurodegeneration [45, 46].”

• cites [45] Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297:353–356. https://doi.org/10.1126/science.1072994 sourceNot checked yet

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• cites [46] Hardy JA, Higgins GA (1992) Alzheimer’s disease: the amyloid cascade hypothesis. Science 256:184–185. https://doi.org/10.1126/science.1566067 sourceNot checked yet

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Genetic variations in genes involved in telomere maintenance correlate with Alzheimer's disease susceptibility.

“Supporting this idea, genetic variations in genes involved in telomere maintenance have been shown to correlate with AD susceptibility [121].”

• cites [121] Zhan Y, Song C, Karlsson R, Tillander A, Reynolds CA, Pedersen NL et al (2015) Telomere length shortening and Alzheimer disease–a Mendelian randomization study. JAMA Neurol 72:1202–1203. https://doi.org/10.1001/jamaneurol.2015.1513 sourceNot checked yet

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Recent data show increased expression of senescence markers in brain regions of Terc−/− mice.

“with recent data showing the increased expression of senescence markers in brain regions of these mice [59].”

• cites [59] Jurk D, Wang C, Miwa S, Maddick M, Korolchuk V, Tsolou A et al (2012) Postmitotic neurons develop a p21-dependent senescence-like phenotype driven by a DNA damage response. Aging Cell 11:996–1004. https://doi.org/10.1111/j.1474-9726.2012.00870.x sourceNot checked yet

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Classified as probably not a substantial claim.

Early increases in intracellular amyloid-β levels have been observed in vulnerable neurons of Alzheimer's disease human patients and animal models, preceding the appearance of amyloid-β plaques.

“Apart from extracellular deposition, early increases in intracellular Aβ levels have been observed in vulnerable neurons of AD human patients [41, 78] and animal models [22, 87, 110], preceding the appearance of Aβ plaques.”

• cites [41] Gouras GK, Tsai J, Naslund J, Vincent B, Edgar M, Checler F et al (2000) Intraneuronal Abeta42 accumulation in human brain. Am J Pathol 156:15–20. https://doi.org/10.1016/s0002-9440(10)64700-1 sourceNot checked yet

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• cites [78] Mochizuki A, Tamaoka A, Shimohata A, Komatsuzaki Y, Shoji S (2000) Abeta42-positive non-pyramidal neurons around amyloid plaques in Alzheimer’s disease. Lancet 355:42–43. https://doi.org/10.1016/s0140-6736(99)04937-5 sourceNot checked yet

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• cites [22] Chui DH, Tanahashi H, Ozawa K, Ikeda S, Checler F, Ueda O et al (1999) Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation. Nat Med 5:560–564. https://doi.org/10.1038/8438 sourceNot checked yet

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• cites [87] Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J et al (2006) Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation. J Neurosci 26:10129–10140. https://doi.org/10.1523/jneurosci.1202-06.2006 sourceNot checked yet

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• cites [110] Takahashi RH, Milner TA, Li F, Nam EE, Edgar MA, Yamaguchi H et al (2002) Intraneuronal Alzheimer abeta42 accumulates in multivesicular bodies and is associated with synaptic pathology. Am J Pathol 161:1869–1879. https://doi.org/10.1016/s0002-9440(10)64463-x sourceNot checked yet

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May combine two claims.

Mice carrying a homozygous germline deletion of the telomerase RNA component gene (Terc−/−) exhibit critically short telomeres and premature development of age-related pathologies from the second or third generation.

“In particular, mice carrying a homozygous germline deletion of the telomerase RNA component gene (Terc−/−) exhibit critically short telomeres and a premature development of age-related pathologies from the second or third generation [8, 18–20, 51, 70, 72, 101, 103]”

• cites [8] Blasco MA, Lee HW, Hande MP, Samper E, Lansdorp PM, DePinho RA et al (1997) Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91:25–34. https://doi.org/10.1016/s0092-8674(01)80006-4 sourceNot checked yet

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• cites [18] Chang S, Multani AS, Cabrera NG, Naylor ML, Laud P, Lombard D et al (2004) Essential role of limiting telomeres in the pathogenesis of Werner syndrome. Nat Genet 36:877–882. https://doi.org/10.1038/ng1389 sourceNot checked yet

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• cites [19] Chen R, Zhang K, Chen H, Zhao X, Wang J, Li L et al (2015) Telomerase deficiency causes alveolar stem cell senescence-associated low-grade inflammation in lungs. J Biol Chem 290:30813–30829. https://doi.org/10.1074/jbc.M115.681619 sourceNot checked yet

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• cites [20] Cheng H, Fan X, Lawson WE, Paueksakon P, Harris RC (2015) Telomerase deficiency delays renal recovery in mice after ischemia-reperfusion injury by impairing autophagy. Kidney Int 88:85–94. https://doi.org/10.1038/ki.2015.69 sourceNot checked yet

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• cites [51] Herrera E, Samper E, Martín-Caballero J, Flores JM, Lee HW, Blasco MA (1999) Disease states associated with telomerase deficiency appear earlier in mice with short telomeres. Embo J 18:2950–2960. https://doi.org/10.1093/emboj/18.11.2950 sourceNot checked yet

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• cites [70] Lee HW, Blasco MA, Gottlieb GJ, Horner JW 2nd, Greider CW, DePinho RA (1998) Essential role of mouse telomerase in highly proliferative organs. Nature 392:569–574. https://doi.org/10.1038/33345 sourceNot checked yet

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• cites [72] Leri A, Franco S, Zacheo A, Barlucchi L, Chimenti S, Limana F et al (2003) Ablation of telomerase and telomere loss leads to cardiac dilatation and heart failure associated with p53 upregulation. Embo j 22:131–139. https://doi.org/10.1093/emboj/cdg013 sourceNot checked yet

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• cites [101] Samper E, Flores JM, Blasco MA (2001) Restoration of telomerase activity rescues chromosomal instability and premature aging in Terc-/- mice with short telomeres. EMBO Rep 2:800–807. https://doi.org/10.1093/embo-reports/kve174 sourceNot checked yet

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• cites [103] Scheffold A, Holtman IR, Dieni S, Brouwer N, Katz SF, Jebaraj BM et al (2016) Telomere shortening leads to an acceleration of synucleinopathy and impaired microglia response in a genetic mouse model. Acta Neuropathol Commun 4:87. https://doi.org/10.1186/s40478-016-0364-x sourceNot checked yet

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May combine two claims.

APP and CTFβ are much more abundant than Aβ inside cells, especially in FAD mutant mice such as 5xFAD mice expressing the Swedish mutation, which triggers β-cleavage of APP.

“Both APP and CTFβ are much more abundant than Aβ inside the cells [36], especially in FAD mutant mice (such as 5xFAD mice) expressing the Swedish mutation, which is known to trigger the β-cleavage of APP [31].”

• cites [36] G S (2011) Intraneuronal Aβ: Was It APP All Along? | ALZFORUM Accessed 18 Dec 2022Not checked yet

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• cites [31] Felsenstein KM, Hunihan LW, Roberts SB (1994) Altered cleavage and secretion of a recombinant beta-APP bearing the Swedish familial Alzheimer’s disease mutation. Nat Genet 6:251–255. https://doi.org/10.1038/ng0394-251 sourceNot checked yet

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Classified as probably not a substantial claim.

The accumulation and oligomerization of amyloid-β peptide is an early event in Alzheimer's disease pathology.

“The accumulation and oligomerization of the Aβ peptide is an early event in AD pathology [45, 46].”

• cites [45] Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297:353–356. https://doi.org/10.1126/science.1072994 sourceNot checked yet

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• cites [46] Hardy JA, Higgins GA (1992) Alzheimer’s disease: the amyloid cascade hypothesis. Science 256:184–185. https://doi.org/10.1126/science.1566067 sourceNot checked yet

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

Negative control samples processed without primary antibody showed no signal.

“As negative controls, samples were processed as described in the absence of primary antibody and no signal was detected.”

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

In 5XFAD mice, early intraneuronal Aβ accumulation has also been reported in large pyramidal neurons of cortical layer V.

“Apart from the subiculum, early intraneuronal Aβ accumulation in 5xFAD mice was also reported in large pyramidal neurons of cortical layer V [30, 87].”

• cites [30] Eimer WA, Vassar R (2013) Neuron loss in the 5XFAD mouse model of Alzheimer’s disease correlates with intraneuronal Aβ42 accumulation and Caspase-3 activation. Mol Neurodegener 8:2. https://doi.org/10.1186/1750-1326-8-2 sourceNot checked yet

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• cites [87] Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J et al (2006) Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation. J Neurosci 26:10129–10140. https://doi.org/10.1523/jneurosci.1202-06.2006 sourceNot checked yet

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

Intraneuronal Aβ accumulation is neurotoxic and induces neuronal apoptosis.

“Intraneuronal Aβ accumulation was reported to be neurotoxic and induce neuronal apoptosis [30, 61].”

• cites [30] Eimer WA, Vassar R (2013) Neuron loss in the 5XFAD mouse model of Alzheimer’s disease correlates with intraneuronal Aβ42 accumulation and Caspase-3 activation. Mol Neurodegener 8:2. https://doi.org/10.1186/1750-1326-8-2 sourceNot checked yet

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• cites [61] Kienlen-Campard P, Miolet S, Tasiaux B, Octave JN (2002) Intracellular amyloid-beta 1–42, but not extracellular soluble amyloid-beta peptides, induces neuronal apoptosis. J Biol Chem 277:15666–15670. https://doi.org/10.1074/jbc.M200887200 sourceNot checked yet

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

5XFAD mice present high levels of the Aβ42 peptide, which starts to accumulate intracellularly at 1.5 months of age, prior to amyloid deposition and gliosis, which begins at two months of age.

“It has been reported that 5XFAD mice present high levels of the Aβ42 peptide, which starts to accumulate intracellularly at 1.5 months of age, prior to amyloid deposition and gliosis, which begins at two months of age [87].”

• cites [87] Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J et al (2006) Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation. J Neurosci 26:10129–10140. https://doi.org/10.1523/jneurosci.1202-06.2006 sourceNot checked yet

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

Microglia and astrocytes play a key role in Aβ clearance and degradation.

“Indeed, microglia and astrocytes have been shown to play a key role in Aβ clearance and degradation [92].”

• cites [92] Ries M, Sastre M (2016) Mechanisms of Aβ clearance and degradation by glial cells. Front Aging Neurosci 8:160. https://doi.org/10.3389/fnagi.2016.00160 sourceNot checked yet

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May combine two claims.

In 5xFAD mice, Aβ deposition starts to appear in the subiculum from 2 months of age, but can only be observed in the hippocampus and cortical regions from 4 months of age.

“Aβ deposition starts to appear in the subiculum of 5xFAD mice from 2 months of age (Fig. 2b), but it can only be observed in the hippocampus and cortical regions of 5xFAD mice from 4 months of age [87].”

• cites [87] Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J et al (2006) Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation. J Neurosci 26:10129–10140. https://doi.org/10.1523/jneurosci.1202-06.2006 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Age- and sex-matched littermates were used for analysis.

“Age- and sex-matched littermates were used for analysis.”

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

The CA1 hippocampal pyramidal cell layer has been reported to lack intraneuronal Aβ immunoreactivity while presenting elevated levels of intracellular human APP.

“In particular, the CA1 hippocampal pyramidal cell layer was investigated as it has been reported to lack intraneuronal Aβ immunoreactivity while presenting elevated levels of intracellular human APP [52, 57].”

• cites [52] Herzer S, Hagan C, von Gerichten J, Dieterle V, Munteanu B, Sandhoff R et al (2018) Deletion of specific sphingolipids in distinct neurons improves spatial memory in a mouse model of Alzheimer’s disease. Front Mol Neurosci 11:206. https://doi.org/10.3389/fnmol.2018.00206 sourceNot checked yet

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• cites [57] Jawhar S, Trawicka A, Jenneckens C, Bayer TA, Wirths O (2012) Motor deficits, neuron loss, and reduced anxiety coinciding with axonal degeneration and intraneuronal Aβ aggregation in the 5XFAD mouse model of Alzheimer’s disease. Neurobiol Aging 33:196.e129–140. https://doi.org/10.1016/j.neurobiolaging.2010.05.027 sourceNot checked yet

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May combine two claims.

In 5-month-old G3Terc−/− 5xFAD mice, the senescent context reduces Aβ levels and amyloid load without affecting APP expression or processing and therefore Aβ generation.

“Altogether, our results suggest that the senescent context reduces Aβ levels and amyloid load in 5-month-old G3Terc−/− 5xFAD mice without affecting APP expression or processing and therefore Aβ generation.”

May combine two claims.

The positive intracellular Aβ signals observed in Fig. 2 are not due to antibody cross-recognition but are related to Aβ42 accumulation.

“Thus, our results confirmed that positive signals observed in Fig. 2 are not due to antibody cross-recognition patterns but are indeed readily related to Aβ42 accumulation.”

May combine two claims.

When autophagy is impaired, Aβ peptides can accumulate intracellularly while their extracellular release decreases.

“Evidence indicates that Aβ peptides can accumulate intracellularly while their extracellular release decreases when autophagy is impaired [85, 93].”

• cites [85] Nilsson P, Loganathan K, Sekiguchi M, Matsuba Y, Hui K, Tsubuki S et al (2013) Aβ secretion and plaque formation depend on autophagy. Cell Rep 5:61–69. https://doi.org/10.1016/j.celrep.2013.08.042 sourceNot checked yet

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• cites [93] Rocchi A, Yamamoto S, Ting T, Fan Y, Sadleir K, Wang Y et al (2017) A Becn1 mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in Alzheimer’s disease. PLoS Genet 13:e1006962. https://doi.org/10.1371/journal.pgen.1006962 sourceNot checked yet

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Classified as probably not a substantial claim.

Accumulation of the Aβ peptide is an early event that initiates AD pathophysiology.

“The accumulation of the Aβ peptide is an early event that initiates AD pathophysiology [42, 45, 46].”

• cites [42] Haass C, Selkoe DJ (2007) Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid beta-peptide. Nat Rev Mol Cell Biol 8:101–112. https://doi.org/10.1038/nrm2101 sourceNot checked yet

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• cites [45] Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297:353–356. https://doi.org/10.1126/science.1072994 sourceNot checked yet

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• cites [46] Hardy JA, Higgins GA (1992) Alzheimer’s disease: the amyloid cascade hypothesis. Science 256:184–185. https://doi.org/10.1126/science.1566067 sourceNot checked yet

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Classified as probably not a substantial claim.

The difference in the levels of autophagy markers before and after chloroquine treatment correlates with the number of autophagosomes being degraded upon fusion with the lysosome during a specific time frame.

“The difference in the levels of these autophagy markers before and after treatment with chloroquine correlates with the number of autophagosomes that are being degraded upon fusion with the lysosome during a specific time frame [119].”

• cites [119] Yoshii SR, Mizushima N (2017) Monitoring and measuring autophagy. Int J Mol Sci. https://doi.org/10.3390/ijms18091865 sourceNot checked yet

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Classified as probably not a substantial claim.

LC3-positive punctate dots do not always represent autophagic structures, particularly in cells presenting protein aggregates.

“However, it is important to be cautious with the interpretation of the LC3 staining, as studies previously demonstrated that LC3-positive punctate dots do not always represent autophagic structures, particularly in cells presenting protein aggregates [67].”

• cites [67] Kuma A, Matsui M, Mizushima N (2007) LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization. Autophagy 3:323–328. https://doi.org/10.4161/auto.4012 sourceNot checked yet

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May combine two claims.

Cells with critically short telomeres obtained from the 4th generation of Tert and Terc deficient mice show impaired autophagy, and autophagic flux reduction has been observed in senescent neurons both in vitro and in vivo.

“Additionally, some reports have demonstrated impaired autophagy in cells with critically short telomeres obtained from the 4th generation of Tert and Terc deficient mice [20], and an autophagic flux reduction has been observed in senescent neurons both in vitro and in vivo [80].”

• cites [20] Cheng H, Fan X, Lawson WE, Paueksakon P, Harris RC (2015) Telomerase deficiency delays renal recovery in mice after ischemia-reperfusion injury by impairing autophagy. Kidney Int 88:85–94. https://doi.org/10.1038/ki.2015.69 sourceNot checked yet

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• cites [80] Moreno-Blas D, Gorostieta-Salas E, Pommer-Alba A, Muciño-Hernández G, Gerónimo-Olvera C, Maciel-Barón LA et al (2019) Cortical neurons develop a senescence-like phenotype promoted by dysfunctional autophagy. Aging (Albany NY) 11:6175–6198. https://doi.org/10.18632/aging.102181 sourceNot checked yet

  Not checked in the free run.

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

A similar pattern of autophagic marker alteration is observed in vulnerable brain regions of AD patients.

“A similar pattern of autophagic marker alteration is observed in vulnerable brain regions of AD patients.”

May combine two claims.

Telomere-induced senescence enhances early intraneuronal Aβ accumulation and causes neuronal loss in specific brain areas of FAD mice, while reducing Aβ deposition at later disease stages.

“In this study, we provide first evidence that telomere-induced senescence enhances early intraneuronal Aβ accumulation and causes neuronal loss in specific brain areas of FAD mice, while reducing Aβ deposition at later disease stages.”

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

LC3 is converted from LC3-I to LC3-II during autophagosome formation, and p62 is an autophagy cargo that binds to LC3.

“This can be achieved by measuring the levels of proteins associated with autophagosomes, such as the microtubule‐associated protein 1A/1B‐light chain 3 (LC3), which is converted from LC3-I to LC3-II during autophagosome formation, and p62 (also called sequestosome 1 or SQSTM1), an autophagy cargo that binds to LC3 [119].”

• cites [119] Yoshii SR, Mizushima N (2017) Monitoring and measuring autophagy. Int J Mol Sci. https://doi.org/10.3390/ijms18091865 sourceNot checked yet

  Not checked in the free run.

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

Neurons are terminally differentiated cells with high and continuous energy requirements and a predominant use of oxidative phosphorylation, and are prone to accumulate cellular damage due to oxidative stress over extended periods.

“Since neurons are terminally differentiated cells with high and continuous energy requirements and a predominant use of oxidative phosphorylation (OXPHOS) as energy supply, they are prone to accumulate cellular damage due to oxidative stress over extended periods of time.”

May combine two claims.

Senescent neurons display impaired autophagy, which might be involved in an aberrant accumulation of intraneuronal Aβ42.

“Taken together, our results indicate that senescent neurons display impaired autophagy, which might be involved in an aberrant accumulation of intraneuronal Aβ42.”

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

Intraneuronal Aβ accumulation has been found in the brains of patients presenting AD or mild cognitive impairment and is suggested to represent an early step in AD neuropathology.

“Intraneuronal Aβ accumulation has been found in the brains of patients presenting AD or mild cognitive impairment [3, 21, 41, 47, 78], and studies suggest that this phenomenon represents an early step in AD neuropathology [40].”

• cites [3] Aoki M, Volkmann I, Tjernberg LO, Winblad B, Bogdanovic N (2008) Amyloid beta-peptide levels in laser capture microdissected cornu ammonis 1 pyramidal neurons of Alzheimer’s brain. NeuroReport 19:1085–1089. https://doi.org/10.1097/WNR.0b013e328302c858 sourceNot checked yet

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• cites [21] Christensen DZ, Schneider-Axmann T, Lucassen PJ, Bayer TA, Wirths O (2010) Accumulation of intraneuronal Abeta correlates with ApoE4 genotype. Acta Neuropathol 119:555–566. https://doi.org/10.1007/s00401-010-0666-1 sourceNot checked yet

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• cites [41] Gouras GK, Tsai J, Naslund J, Vincent B, Edgar M, Checler F et al (2000) Intraneuronal Abeta42 accumulation in human brain. Am J Pathol 156:15–20. https://doi.org/10.1016/s0002-9440(10)64700-1 sourceNot checked yet

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• cites [47] Hashimoto M, Bogdanovic N, Volkmann I, Aoki M, Winblad B, Tjernberg LO (2010) Analysis of microdissected human neurons by a sensitive ELISA reveals a correlation between elevated intracellular concentrations of Abeta42 and Alzheimer’s disease neuropathology. Acta Neuropathol 119:543–554. https://doi.org/10.1007/s00401-010-0661-6 sourceNot checked yet

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• cites [78] Mochizuki A, Tamaoka A, Shimohata A, Komatsuzaki Y, Shoji S (2000) Abeta42-positive non-pyramidal neurons around amyloid plaques in Alzheimer’s disease. Lancet 355:42–43. https://doi.org/10.1016/s0140-6736(99)04937-5 sourceNot checked yet

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• cites [40] Gouras GK, Tampellini D, Takahashi RH, Capetillo-Zarate E (2010) Intraneuronal beta-amyloid accumulation and synapse pathology in Alzheimer’s disease. Acta Neuropathol 119:523–541. https://doi.org/10.1007/s00401-010-0679-9 sourceNot checked yet

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

Telomeres of various non-proliferative tissues, including the brain, are particularly resistant to DNA repair and can undergo persistent DNA damage response even without being critically short.

“Still, it has been shown that telomeres of various non-proliferative tissues, including the brain, are particularly resistant to DNA repair and can undergo persistent DNA damage response (DDR) even without being critically short [35].”

• cites [35] Fumagalli M, Rossiello F, Clerici M, Barozzi S, Cittaro D, Kaplunov JM et al (2012) Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation. Nat Cell Biol 14:355–365. https://doi.org/10.1038/ncb2466 sourceNot checked yet

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

Insoluble extracellular deposits made up of the Aβ peptide are a defining pathological hallmark of AD, but their pathological properties seem restricted.

“Insoluble extracellular deposits made up of the Aβ peptide were initially discovered in the brains of all AD patients and are considered as a defining pathological hallmark, but their pathological properties seem restricted.”

May combine two claims.

Extracellular amyloid deposition correlates poorly with the degree of cognitive decline in AD, and Aβ-targeted immunotherapies removing Aβ deposition do not result in cognitive improvement.

“Supporting this idea, studies have shown that extracellular amyloid deposition correlates poorly with the degree of cognitive decline in AD [10], in agreement with the results obtained in clinical trials showing that Aβ-targeted immunotherapies removing Aβ deposition in the brain do not result in cognitive improvement [99].”

• cites [10] Braak H, Braak E (1991) Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82:239–259. https://doi.org/10.1007/bf00308809 sourceNot checked yet

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• cites [99] Salloway S, Sperling R, Fox NC, Blennow K, Klunk W, Raskind M et al (2014) Two phase 3 trials of bapineuzumab in mild-to-moderate Alzheimer’s disease. N Engl J Med 370:322–333. https://doi.org/10.1056/NEJMoa1304839 sourceNot checked yet

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Classified as probably not a substantial claim.

Accumulation of LC3-II and p62 proteins is a well-known feature of autophagy-deficient tissues.

“Indeed, accumulation of LC3-II and p62 proteins is a well-known feature of autophagy-deficient tissues [32, 64, 80].”

• cites [32] Ferguson CJ, Lenk GM, Meisler MH (2009) Defective autophagy in neurons and astrocytes from mice deficient in PI(3,5)P2. Hum Mol Genet 18:4868–4878. https://doi.org/10.1093/hmg/ddp460 sourceNot checked yet

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• cites [64] Komatsu M, Waguri S, Koike M, Sou YS, Ueno T, Hara T et al (2007) Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 131:1149–1163. https://doi.org/10.1016/j.cell.2007.10.035 sourceNot checked yet

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• cites [80] Moreno-Blas D, Gorostieta-Salas E, Pommer-Alba A, Muciño-Hernández G, Gerónimo-Olvera C, Maciel-Barón LA et al (2019) Cortical neurons develop a senescence-like phenotype promoted by dysfunctional autophagy. Aging (Albany NY) 11:6175–6198. https://doi.org/10.18632/aging.102181 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Telomere-induced senescence triggers neurodegeneration per se in specific brain regions, but this is aggravated in regions where early intraneuronal Aβ accumulation occurs.

“Our data indicate that telomere-induced senescence triggers neurodegeneration per se in specific brain regions, but that this is aggravated in regions where early intraneuronal Aβ accumulation occurs.”

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

Crossing the Terc−/− model with APP23 mice causes an unexpected reduction in Aβ deposition.

“A previous study demonstrated that crossing the Terc−/− model with APP23 mice (which express human APP bearing the Swedish mutation [109]) causes an unexpected reduction in Aβ deposition [95], which the authors linked to telomere-dependent effects on microglia activation.”

• cites [95] Rolyan H, Scheffold A, Heinrich A, Begus-Nahrmann Y, Langkopf BH, Hölter SM et al (2011) Telomere shortening reduces Alzheimer’s disease amyloid pathology in mice. Brain 134:2044–2056. https://doi.org/10.1093/brain/awr133 sourceNot checked yet

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Probably not a substantial claim: may not pass S2 (checkable).

Extrapolation of the study's results from mouse models to human pathology must be done with caution.

“Therefore, the extrapolation of our results obtained in mouse models to human pathology must be done with caution.”

• AssumptionRests on an assumption: Human AD brains do not overexpress APP and intracellular Aβ levels are expected to be very low.

May combine two claims.

APP23+ mice with dysfunctional autophagy due to conditional knockout of Atg7 exhibit a substantial reduction of amyloid plaques but increased intraneuronal Aβ early in pathology, which might trigger neurodegeneration and memory impairment.

“The role of autophagy on intraneuronal Aβ is supported by a previous study showing that an AD-related amyloid mouse model (APP23+) presenting dysfunctional autophagy due to conditional knockout of Atg7 exhibits a substantial reduction of amyloid plaques but increased intraneuronal Aβ early in the pathology, which might trigger neurodegeneration and memory impairment [85].”

• cites [85] Nilsson P, Loganathan K, Sekiguchi M, Matsuba Y, Hui K, Tsubuki S et al (2013) Aβ secretion and plaque formation depend on autophagy. Cell Rep 5:61–69. https://doi.org/10.1016/j.celrep.2013.08.042 sourceNot checked yet

  Not checked in the free run.

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

Long postmortem intervals could be a confounding factor in the evaluation of intracellular Aβ in human material.

“Long postmortem intervals could also be another confounding factor in the evaluation of this feature in human material, as compared to its detection in mice, in which samples are collected and rapidly fixed by transcardial perfusion in a well-controlled manner [6].”

• cites [6] Bayer TA, Wirths O (2010) Intracellular accumulation of amyloid-Beta - a predictor for synaptic dysfunction and neuron loss in Alzheimer’s disease. Front Aging Neurosci 2:8. https://doi.org/10.3389/fnagi.2010.00008 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Intracellular Aβ accumulation in neurons that are particularly vulnerable in AD precedes plaque formation but decreases later on with the progression of Aβ deposition in both mice and humans.

“Indeed, intracellular Aβ accumulation in neurons that are particularly vulnerable in AD precedes plaque formation but decreases later on with the progression of Aβ deposition in both mice [89, 117] and humans [41, 81].”

• cites [89] Oddo S, Caccamo A, Smith IF, Green KN, LaFerla FM (2006) A dynamic relationship between intracellular and extracellular pools of Abeta. Am J Pathol 168:184–194. https://doi.org/10.2353/ajpath.2006.050593 sourceNot checked yet

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• cites [117] Wirths O, Multhaup G, Czech C, Blanchard V, Moussaoui S, Tremp G et al (2001) Intraneuronal Abeta accumulation precedes plaque formation in beta-amyloid precursor protein and presenilin-1 double-transgenic mice. Neurosci Lett 306:116–120. https://doi.org/10.1016/s0304-3940(01)01876-6 sourceNot checked yet

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• cites [41] Gouras GK, Tsai J, Naslund J, Vincent B, Edgar M, Checler F et al (2000) Intraneuronal Abeta42 accumulation in human brain. Am J Pathol 156:15–20. https://doi.org/10.1016/s0002-9440(10)64700-1 sourceNot checked yet

  Not checked in the free run.

• cites [81] Mori C, Spooner ET, Wisniewsk KE, Wisniewski TM, Yamaguch H, Saido TC et al (2002) Intraneuronal Abeta42 accumulation in Down syndrome brain. Amyloid 9:88–102 sourceNot checked yet

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

Tert transcriptional activation in transgenic 3xTg-AD mice reduces abnormal intracellular Aβ staining in the CA1 region and upregulates genes involved in synaptic signaling and neuronal survival.

“In line with our data, a very recent study has shown that Tert transcriptional activation in transgenic 3xTg-AD mice (which carry two FAD mutations together with a mutation of the MAPT gene [88]) reduces abnormal intracellular Aβ staining in the CA1 region and upregulates genes involved in synaptic signaling and neuronal survival [106].”

• cites [106] Shim HS, Horner JW, Wu CJ, Li J, Lan ZD, Jiang S et al (2021) Telomerase reverse transcriptase preserves neuron survival and cognition in Alzheimer’s disease models. Nat Aging 1:1162–1174. https://doi.org/10.1038/s43587-021-00146-z sourceNot checked yet

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

Abnormal increases in lipofuscin are found in AD.

“abnormal increases in lipofuscin are found in AD [29]”

• cites [29] Dowson JH, Mountjoy CQ, Cairns MR, Wilton-Cox H (1992) Changes in intraneuronal lipopigment in Alzheimer’s disease. Neurobiol Aging 13:493–500. https://doi.org/10.1016/0197-4580(92)90077-b sourceNot checked yet

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

A transgenic mouse model bearing APP with a recently described AD-associated mutation exhibits intraneuronal Aβ accumulation in the absence of plaques and still shows signs of AD-related neurodegeneration.

“This idea is confirmed by studies reporting a transgenic mouse model bearing APP with a recently described AD-associated mutation that exhibits intraneuronal Aβ accumulation in the absence of plaques and still shows signs of AD-related neurodegeneration [113].”

• cites [113] Tomiyama T, Matsuyama S, Iso H, Umeda T, Takuma H, Ohnishi K et al (2010) A mouse model of amyloid beta oligomers: their contribution to synaptic alteration, abnormal tau phosphorylation, glial activation, and neuronal loss in vivo. J Neurosci 30:4845–4856. https://doi.org/10.1523/jneurosci.5825-09.2010 sourceNot checked yet

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

A limitation of the study is the lack of intracellular Aβ evaluation in AD samples.

“A limitation of our study is the lack of intracellular Aβ evaluation in AD samples.”

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

The CA1 region is one of the earliest and more affected brain regions in AD patients.

“In contrast to 5xFAD, other FAD mouse models show clear intraneuronal Aβ accumulation and neuronal loss in the CA1 region [7, 16, 88], which has also been shown to be one of the earliest and more affected brain regions in AD patients [4, 27, 68, 82].”

• cites [4] Apostolova LG, Dutton RA, Dinov ID, Hayashi KM, Toga AW, Cummings JL et al (2006) Conversion of mild cognitive impairment to Alzheimer disease predicted by hippocampal atrophy maps. Arch Neurol 63:693–699. https://doi.org/10.1001/archneur.63.5.693 sourceNot checked yet

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• cites [27] Davies DC, Horwood N, Isaacs SL, Mann DM (1992) The effect of age and Alzheimer’s disease on pyramidal neuron density in the individual fields of the hippocampal formation. Acta Neuropathol 83:510–517. https://doi.org/10.1007/bf00310028 sourceNot checked yet

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• cites [68] La Joie R, Perrotin A, de La Sayette V, Egret S, Doeuvre L, Belliard S et al (2013) Hippocampal subfield volumetry in mild cognitive impairment, Alzheimer’s disease and semantic dementia. Neuroimage Clin 3:155–162. https://doi.org/10.1016/j.nicl.2013.08.007 sourceNot checked yet

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• cites [82] Mueller SG, Weiner MW (2009) Selective effect of age, Apo e4, and Alzheimer’s disease on hippocampal subfields. Hippocampus 19:558–564. https://doi.org/10.1002/hipo.20614 sourceNot checked yet

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Probably not a substantial claim: may not pass S1 (asserted).

Autophagy plays a key role in the balance between Aβ secretion and intracellular accumulation.

“The authors hypothesized that autophagy plays a key role in the balance between Aβ secretion and intracellular accumulation.”

• cites [85] Nilsson P, Loganathan K, Sekiguchi M, Matsuba Y, Hui K, Tsubuki S et al (2013) Aβ secretion and plaque formation depend on autophagy. Cell Rep 5:61–69. https://doi.org/10.1016/j.celrep.2013.08.042 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Intraneuronal Aβ correlates with the apolipoprotein E4 (ApoE4) genotype, which has been described to downregulate autophagy.

“Another piece of evidence for the role of autophagy in intraneuronal Aβ accumulation is the finding that intraneuronal Aβ correlates with the apolipoprotein E4 (ApoE4) genotype [21], the main known genetic risk factor for AD, which has been described to downregulate autophagy [108].”

• cites [21] Christensen DZ, Schneider-Axmann T, Lucassen PJ, Bayer TA, Wirths O (2010) Accumulation of intraneuronal Abeta correlates with ApoE4 genotype. Acta Neuropathol 119:555–566. https://doi.org/10.1007/s00401-010-0666-1 sourceNot checked yet

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• cites [108] Sohn HY, Kim SI, Park JY, Park SH, Koh YH, Kim J et al (2021) ApoE4 attenuates autophagy via FoxO3a repression in the brain. Sci Rep 11:17604. https://doi.org/10.1038/s41598-021-97117-6 sourceNot checked yet

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

Aβ staining could colocalize with or nonspecifically bind to lipofuscin.

“Aβ staining could colocalize with or nonspecifically bind to lipofuscin [33, 38]”

• cites [33] Fernández-Vizarra P, Fernández AP, Castro-Blanco S, Serrano J, Bentura ML, Martínez-Murillo R et al (2004) Intra- and extracellular Abeta and PHF in clinically evaluated cases of Alzheimer’s disease. Histol Histopathol 19:823–844. https://doi.org/10.14670/hh-19.823 sourceNot checked yet

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• cites [38] Gómez-Ramos P, Asunción Morán M (2007) Ultrastructural localization of intraneuronal Abeta-peptide in Alzheimer disease brains. J Alzheimers Dis 11:53–59. https://doi.org/10.3233/jad-2007-11109 sourceNot checked yet

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May combine two claims.

Other FAD mouse models show clear intraneuronal Aβ accumulation and neuronal loss in the CA1 region.

“In contrast to 5xFAD, other FAD mouse models show clear intraneuronal Aβ accumulation and neuronal loss in the CA1 region [7, 16, 88], which has also been shown to be one of the earliest and more affected brain regions in AD patients [4, 27, 68, 82].”

• cites [7] Billings LM, Oddo S, Green KN, McGaugh JL, LaFerla FM (2005) Intraneuronal Abeta causes the onset of early Alzheimer’s disease-related cognitive deficits in transgenic mice. Neuron 45:675–688. https://doi.org/10.1016/j.neuron.2005.01.040 sourceNot checked yet

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• cites [16] Casas C, Sergeant N, Itier JM, Blanchard V, Wirths O, van der Kolk N et al (2004) Massive CA1/2 neuronal loss with intraneuronal and N-terminal truncated Abeta42 accumulation in a novel Alzheimer transgenic model. Am J Pathol 165:1289–1300. https://doi.org/10.1016/s0002-9440(10)63388-3 sourceNot checked yet

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• cites [88] Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R et al (2003) Triple-transgenic model of Alzheimer’s disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron 39:409–421. https://doi.org/10.1016/s0896-6273(03)00434-3 sourceNot checked yet

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

Lesioning the dorsal subiculum of a mouse model of AD amyloidosis reduces the spread of amyloid pathology in brain regions projecting to and receiving connections from this region, such as the CA1 and retrosplenial cortex.

“Supporting the importance of the subiculum in the spread of AD pathology, a study showed that lesioning the dorsal subiculum of a mouse model of AD amyloidosis reduces the spread of amyloid pathology in brain regions projecting to and receiving connections from this region, such as the CA1 and retrosplenial cortex [37].”

• cites [37] George S, Rönnbäck A, Gouras GK, Petit GH, Grueninger F, Winblad B et al (2014) Lesion of the subiculum reduces the spread of amyloid beta pathology to interconnected brain regions in a mouse model of Alzheimer’s disease. Acta Neuropathol Commun 2:17. https://doi.org/10.1186/2051-5960-2-17 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Accelerated senescence induces neurotoxic intraneuronal Aβ accumulation, probably through autophagy dysfunction.

“Overall, our data demonstrated that accelerated senescence induces neurotoxic intraneuronal Aβ accumulation, probably through autophagy dysfunction, shedding light on the causative role of pathological aging in AD.”

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

Amyloid pathology might be gradually transmitted through connected brain structures.

“Some studies have indicated that amyloid pathology might be gradually transmitted through connected brain structures [10, 69, 84, 96], by mechanisms that are still under investigation.”

• cites [10] Braak H, Braak E (1991) Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82:239–259. https://doi.org/10.1007/bf00308809 sourceNot checked yet

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• cites [69] Lazarov O, Lee M, Peterson DA, Sisodia SS (2002) Evidence that synaptically released beta-amyloid accumulates as extracellular deposits in the hippocampus of transgenic mice. J Neurosci 22:9785–9793. https://doi.org/10.1523/jneurosci.22-22-09785.2002 sourceNot checked yet

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• cites [84] Nath S, Agholme L, Kurudenkandy FR, Granseth B, Marcusson J, Hallbeck M (2012) Spreading of neurodegenerative pathology via neuron-to-neuron transmission of β-amyloid. J Neurosci 32:8767–8777. https://doi.org/10.1523/jneurosci.0615-12.2012 sourceNot checked yet

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• cites [96] Rönnbäck A, Sagelius H, Bergstedt KD, Näslund J, Westermark GT, Winblad B et al (2012) Amyloid neuropathology in the single Arctic APP transgenic model affects interconnected brain regions. Neurobiol Aging 33:831.e811-839. https://doi.org/10.1016/j.neurobiolaging.2011.07.012 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

There is a dynamic correlation between extra- and intracellular pools of Aβ, the latter being the source for some of the extracellular Aβ plaques.

“Indeed, there is a dynamic correlation between extra- and intracellular pools of Aβ, the latter being the source for some of the extracellular Aβ plaques [89].”

• cites [89] Oddo S, Caccamo A, Smith IF, Green KN, LaFerla FM (2006) A dynamic relationship between intracellular and extracellular pools of Abeta. Am J Pathol 168:184–194. https://doi.org/10.2353/ajpath.2006.050593 sourceNot checked yet

  Not checked in the free run.