mTORC1 drives cell-autonomous astrocyte reactivity in tuberous sclerosis

May combine two claims.

Gene-expression analysis of tuber tissue has revealed increased expression of astrocyte-enriched genes and prominent neuroinflammation.

“Indeed, gene-expression analysis of tuber tissue has revealed increased expression of astrocyte-enriched genes and prominent neuroinflammation14,15.”

• cites [14] Martin, K. R. et al. The genomic landscape of tuberous sclerosis complex. Nat. Commun. 8, 15816 https://doi.org/10.1038/ncomms15816 (2017). sourcePartly supported

  The Results section, “Brain lesions show evidence of significant neuroinflammation” (page unavailable), reports increased expression of immune- and inflammation-related genes in cortical tubers. It does not report increased expression of astrocyte-enriched genes in tubers; its cell-type analysis instead estimates an astrocyte proportion similar to normal brain tissue. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [15] Boer, K. et al. Gene expression analysis of tuberous sclerosis complex cortical tubers reveals increased expression of adhesion and inflammatory factors. Brain Pathol. 20, 704–719 https://doi.org/10.1111/j.1750-3639.2009.00341.x (2009). sourcePartly supported

  The Results (Gene ontology, pathway, and expression analysis; page not provided) report increased expression of immune and inflammatory-response genes in cortical tubers, supporting the neuroinflammation portion of the claim. The passages do not establish increased expression of astrocyte-enriched genes; the reported glial glutamate transporter SLC1A2 was instead lower in tubers. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

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

Tubers contain heterogeneous populations of dysmorphic neurons, dysplastic and gliotic astrocytes, activated microglia and giant or balloon cells within a background of normal-appearing cells.

“On a cellular level, tubers contain heterogeneous populations of dysmorphic neurons, dysplastic and gliotic astrocytes, activated microglia and giant or balloon cells within a background of normal-appearing cells7,8,9.”

• cites [7] Talos, D. M. et al. Cell-specific alterations of glutamate receptor expression in tuberous sclerosis complex cortical tubers. Ann. Neurol. 63, 454–465 https://doi.org/10.1002/ana.21342 (2008). sourceNot checked — no full text of the source
• cites [8] Zhang, J. et al. Iconography of abnormal non-neuronal cells in pediatric focal cortical dysplasia type IIb and tuberous sclerosis complex. Front. Cell. Neurosci. 18, 1486315 https://doi.org/10.3389/fncel.2024.1486315 (2024). sourcePartly supported

  The 'Electrophysiological and morphological findings in ex vivo slices' section (page unavailable) describes TSC tissue as containing a heterogeneous range of dysplastic neurons, giant cells, and dysplastic or reactive astrocytes. The nomenclature section says microglia are affected in TSC but does not say they are activated; the cited paper also does not establish that these cells occur within a background of normal-appearing cells. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [9] Muhlebner, A. et al. Novel histopathological patterns in cortical tubers of epilepsy surgery patients with tuberous sclerosis complex. PLoS ONE 11, e0157396 https://doi.org/10.1371/journal.pone.0157396 (2016). sourcePartly supported

  The Introduction and Results (Patterns of cortical tubers; Inflammation and small vessel density; mTORC1 activation, neuronal quantity and gliosis; pages unavailable) support dysmorphic neurons, giant cells, microglial activation and gliosis in tubers. They do not establish dysplastic astrocytes, balloon cells as a distinct category, or normal-appearing cells within tubers; the histologically normal cortex examined was perituberal. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

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

mTORC1 is a key signalling node that regulates cellular anabolic and catabolic processes.

“Loss of the TSC1–TSC2 protein complex leads to hyperactivation of mTOR complex 1 (mTORC1)18, a key signalling node that regulates cellular anabolic and catabolic processes19.”

• cites [19] Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 168, 960–976 https://doi.org/10.1016/j.cell.2017.02.004 (2017). sourceSupported

May combine two claims.

Autophagy genes are upregulated in TSC2−/− astrocytes, including SQSTM1, and a module score related to the initiation of autophagy is increased.

“In addition to astrocytic genes, autophagy genes were upregulated in TSC2−/− astrocytes, including SQSTM1, which encodes p62. A module score related to the initiation of autophagy33 showed that TSC2−/− cells had increased expression of this module (Fig. 1j and Supplementary Table 2).”

• cites [33] Bordi, M. et al. A gene toolbox for monitoring autophagy transcription. Cell Death Dis. 12, 1044 https://doi.org/10.1038/s41419-021-04121-9 (2021). sourceSupported

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

Somatic mutations in mTOR pathway regulators are often found in focal cortical dysplasia type II.

“In addition, somatic mutations in mTOR pathway regulators are often found in focal cortical dysplasia type II, which has overlapping histological and clinical features with TSC24,25.”

• cites [24] Sim, N. S. et al. Precise detection of low-level somatic mutation in resected epilepsy brain tissue. Acta Neuropathol. 138, 901–912 https://doi.org/10.1007/s00401-019-02052-6 (2019). sourceSupported
• cites [25] Baldassari, S. et al. Dissecting the genetic basis of focal cortical dysplasia: a large cohort study. Acta Neuropathol. 138, 885–900 https://doi.org/10.1007/s00401-019-02061-5 (2019). sourceSupported

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

Second-hit mutations are frequently observed in TSC-related hamartomas and have been identified in some resected tubers from patients.

“Second-hit mutations are frequently observed in TSC-related hamartomas and have been identified in some resected tubers from patients14,21,22,23.”

• cites [14] Martin, K. R. et al. The genomic landscape of tuberous sclerosis complex. Nat. Commun. 8, 15816 https://doi.org/10.1038/ncomms15816 (2017). sourceSupported
• cites [21] Henske, E. P. et al. Allelic loss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions. Am. J. Hum. Genet. 59, 400–406 (1996).Not matched

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

• cites [22] Crino, P. B., Aronica, E., Baltuch, G. & Nathanson, K. L. Biallelic TSC gene inactivation in tuberous sclerosis complex. Neurology 74, 1716–1723 https://doi.org/10.1212/WNL.0b013e3181e04325 (2010). sourceSupported

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

In other contexts, such as acquired epilepsy, the presence of reactive astrocytes alone is sufficient to drive epileptogenesis.

“In other contexts, such as acquired epilepsy, the presence of reactive astrocytes alone is sufficient to drive epileptogenesis54.”

• cites [54] Vezzani, A. et al. Astrocytes in the initiation and progression of epilepsy. Nat. Rev. Neurol. 18, 707–722 https://doi.org/10.1038/s41582-022-00727-5 (2022). sourcePartly supported

  The Introduction (page unavailable) says reactive astrocytes might contribute to the onset and progression of acquired epilepsy, and reports that experimentally inducing reactive astrocytes in mice elicits spontaneous seizures. It does not say that their mere presence is sufficient to drive epileptogenesis in acquired epilepsy; it distinguishes that setting from a genetic disease in which astrocytes can initiate epilepsy. (LLM, conf=0.89)

  Experimental — accuracy not yet measured

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

Genetic variants of many differentially expressed genes identified in this study, such as CLU and APOE, have been associated with increased Alzheimer's disease risk.

“Genetic variants of many of the differentially expressed genes identified in this study, such as CLU and APOE, have been associated with increased Alzheimer’s disease risk58,59.”

• cites [58] Foster, E. M., Dangla-Valls, A., Lovestone, S., Ribe, E. M. & Buckley, N. J. Clusterin in Alzheimer’s disease: mechanisms, genetics, and lessons from other pathologies. Front. Neurosci. 13, 164 https://doi.org/10.3389/fnins.2019.00164 (2019). sourceSupported
• cites [59] Raulin, A.-C. et al. ApoE in Alzheimer’s disease: pathophysiology and therapeutic strategies. Mol. Neurodegener. 17, 72 https://doi.org/10.1186/s13024-022-00574-4 (2022). sourcePartly supported

  The section ‘ApoE isoform is a risk determinant for AD and related dementias’ states that APOE4 increases Alzheimer’s disease risk. The passages from this paper do not establish the corresponding risk association for CLU or for the broader group of genes named in the citing sentence. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

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

TSC1-TSC2 loss affects neuronal development and properties.

“In addition, whereas this work highlights astrocytes, we also observed transcriptomic changes in neurons, and considerable previous work has shown that TSC1–TSC2 loss affects neuronal development and properties11,13,35,55,56,57.”

• cites [11] Meikle, L. et al. A mouse model of tuberous sclerosis: neuronal loss of Tsc1 causes dysplastic and ectopic neurons, reduced myelination, seizure activity, and limited survival. J. Neurosci. 27, 5546–5558 https://doi.org/10.1523/JNEUROSCI.5540-06.2007 (2007). sourceSupported
• cites [13] Karalis, V., Caval-Holme, F. & Bateup, H. S. Raptor downregulation rescues neuronal phenotypes in mouse models of tuberous sclerosis complex. Nat. Commun. 13, 4665 https://doi.org/10.1038/s41467-022-31961-6 (2022). sourceSupported
• cites [35] Winden, K. D. et al. Biallelic mutations in TSC2 lead to abnormalities associated with cortical tubers in human iPSC-derived neurons. J. Neurosci. 39, 9294–9305 https://doi.org/10.1523/JNEUROSCI.0642-19.2019 (2019). sourceSupported

May combine two claims.

TSC2−/− cluster 7 cells have striking upregulation of astrocyte genes including S100B, GFAP, AQP4, APOE, SPARCL1, CLU and PTGDS, and reduced expression of oligodendrocyte-lineage genes OLIG1, OLIG2 and PDGFRA.

“Compared with TSC2c/− cells, TSC2−/− cluster 7 cells had striking upregulation of astrocyte genes including S100B, GFAP, AQP4, APOE, SPARCL1, CLU and PTGDS, together with reduced expression of oligodendrocyte-lineage genes (OLIG1, OLIG2 and PDGFRA) (Fig. 1h).”

May combine two claims.

TSC2−/− astrocytes are highly enlarged with bigger somas, notably thicker processes and fewer fine branches across all morphological types.

“Across all morphological types, TSC2−/− astrocytes were highly enlarged with bigger somas, notably thicker processes and fewer fine branches (Fig. 3n and Supplementary Video 3).”

May combine two claims.

TSC2−/− cells produce disproportionately more astrocytes than TSC2c/− cells in the same organoid, and these astrocytes express higher levels of genes associated with reactivity.

“In summary, our scRNA-seq analysis across several genetic backgrounds demonstrates that TSC2−/− cells produce disproportionately more astrocytes than TSC2c/− cells in the same organoid, and that these astrocytes express higher levels of genes associated with reactivity.”

May combine two claims.

The astrocytes generated from TSC2-deficient neural progenitors are enlarged, pro-inflammatory and reactive.

“Here we show, using single-cell transcriptomics and cyclic immunostaining across human brain organoids and resected tuber tissue from patients, that loss of TSC2 biases neural progenitors to differentiate into enlarged, pro-inflammatory reactive astrocytes in a cell autonomous manner.”

May combine two claims.

Bulk RNA-seq of TSC2−/− organoids at around day 200 shows marked increase in GFAP, CLU, CRYAB, APOE and SQSTM1 expression and strongly decreased expression of neuronal mRNAs including MAP2, RBFOX3, NEUROD1 and STMN2.

“We performed bulk RNA-seq of these organoids at around day 200 and observed a marked increase in GFAP, CLU, CRYAB, APOE and SQSTM1 expression in TSC2−/− organoids, together with strongly decreased expression of neuronal mRNAs including MAP2, RBFOX3, NEUROD1 and STMN2 (Extended Data Fig. 5c–p).”

May combine two claims.

TSC2−/− neurons show transcriptional changes related to synaptic transmission and excitability, oxidative stress, upregulation of glycolysis and mitochondrial dysfunction.

“We also observed transcriptional changes in TSC2−/− neurons that were related to synaptic transmission and excitability, oxidative stress, upregulation of glycolysis and mitochondrial dysfunction (Extended Data Fig. 2 and Supplementary Table 3).”

May combine two claims.

Both the number and magnitude of differentially expressed genes in TSC2−/− astrocytes increase at later time points.

“Both the number of differentially expressed genes and the magnitude of the differential expression increased at later time points (Fig. 1h and Supplementary Table 1).”

May combine two claims.

TSC2−/− astrocytes show upregulation of neuroinflammation and immune signalling, antioxidant response, extracellular matrix remodelling and lipid metabolism and transport.

“In addition to autophagy, several other biological processes were altered at the transcriptional level in TSC2−/− astrocytes including upregulation of neuroinflammation and immune signalling, antioxidant response, extracellular matrix remodelling and lipid metabolism and transport (Extended Data Fig. 2 and Supplementary Table 3).”

May combine two claims.

TSC2−/− GLCs have higher maturity scores and lower immaturity scores than corresponding TSC2c/− GLCs at each time point.

“We found that TSC2−/− GLCs had higher maturity scores and lower immaturity scores than corresponding TSC2c/− GLCs at each time point (Extended Data Fig. 4c,d,g,h).”

May combine two claims.

TSC2 deficiency cell-autonomously induces the formation of reactive astrocytes showing distinct molecular and morphological alterations, including upregulation of genes associated with neurodegeneration.

“We demonstrate that TSC2 deficiency cell-autonomously induces the formation of reactive astrocytes showing distinct molecular and morphological alterations, including upregulation of genes associated with neurodegeneration.”

May combine two claims.

TSC2+/− GLCs show mild activation of the reactive astrocyte gene module, less pronounced than in TSC2−/− cells.

“Although TSC2+/− GLCs did show mild activation of the reactive astrocyte gene module (Extended Data Fig. 3i), this was less pronounced than in TSC2−/− cells.”

May combine two claims.

TdTomato-positive cells in TSC2c/+ organoids do not have dysmorphic morphology, a glial differentiation bias or increased phosphorylation of the mTORC1 pathway target S6 (p-S6).

“TdTomato-positive cells in TSC2c/+;LSL-TdTom organoids did not have dysmorphic morphology, a glial differentiation bias or increased phosphorylation of the mTORC1 pathway target S6 (p-S6) (Extended Data Fig. 3a,b).”

May combine two claims.

In BJ TSC2c/− organoids, there is a pronounced GLC differentiation bias, generation of cell-autonomously reactive astrocytes and activation of the autophagy induction module.

“In BJ TSC2c/−;LSL-TdTom organoids, we again observed a pronounced GLC differentiation bias, the generation of cell-autonomously reactive astrocytes and activation of the autophagy induction module (Fig. 2e–i, Supplementary Tables 6 and 7 and Extended Data Fig. 2c,d).”

May combine two claims.

The glial-lineage cell bias of TSC2−/− cells is observable as early as day 50 and grows more prominent over time.

“The GLC bias of TSC2−/− cells was observable as early as day 50 and grew more prominent over time (Fig. 1f).”

May combine two claims.

Treatment of TSC2−/− astrocytes with mTOR inhibitors does not fully restore the wild-type state but shifts them to an intermediate state, with rapamycin and Torin having largely similar effects.

“Treatment of TSC2−/− astrocytes with mTOR inhibitors did not fully restore the wild-type state but rather shifted them to an intermediate state, with rapamycin and Torin having largely similar effects (Extended Data Fig. 9c).”

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

High p-S6 cells expressing neuronal markers are present in TSC patient tubers.

“Two sets of example images of high p-S6 cells (white circles) expressing neuronal markers from different tubers.”

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

tdTomato-positive TSC2+/− cells are present in immunostained WIBR3 TSC2c/+;LSL-TdTom organoids.

“Example image of an immunostained WIBR3 TSC2c/+;LSL-TdTom organoid showing tdTomato-positive TSC2+/− cells.”

The quote only partly states this claim.

p-S6 intensity in cells differs between TSC1+/+ and TSC1−/− organoids.

“Quantification (mean ± SD) of p-S6 intensity in cells from TSC1+/+ and TSC1−/− organoids.”

• AssumptionRests on an assumption: p-S6 immunoreactivity is a read-out for mTORC1 activity.

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

High p-S6 astrocytes and high p-S6 neurons are present in TSC patient tubers.

“Example images of a high p-S6 astrocyte (yellow arrow & circle) and a high p-S6 neuron (white arrow & circle) across all cyclic staining antibodies.”

May combine two claims.

There is an abundance of reactive astrocyte markers and a paucity of neuronal markers in hyperactive mTORC1 cells in tubers from patients with TSC.

“In summary, we find an abundance of reactive astrocyte markers and a paucity of neuronal markers in the hyperactive mTORC1 cells in tubers from patients with TSC.”

The quote only partly states this claim.

The glia-to-neuron ratio, determined as the ratio of S100β- and HuC/D-positive cells, differs between TSC1+/+ and TSC1−/− organoids at two time points.

“Quantification (mean ± SD) of the glia to neuron ratio, as determined by the ratio of S100β and HuC/D positive cells in TSC1+/+ and TSC1−/− organoids at two time points.”

May combine two claims.

A small, unique population of abnormal cells in tubers has hyperactive mTORC1 signalling and expression profiles consistent with reactive astrocytes.

“Our image UMAP approach identifies a small, unique population of abnormal cells that have hyperactive mTORC1 signalling and expression profiles consistent with reactive astrocytes.”

May combine two claims.

In cyclic staining of ten tubers from eight patients, cells with high p-S6 are more likely to be glial cells and overexpress candidate differentially expressed proteins compared with low p-S6 cells in the same tuber.

“Cyclic staining of ten tubers from eight different patients with TSC (Supplementary Table 9) revealed that cells with high p-S6 were more likely to be glial cells and overexpress candidate differentially expressed proteins compared with low p-S6 cells in the same tuber.”

• AssumptionRests on an assumption: High p-S6 cells in tuber tissue correspond to cells with biallelic TSC1/TSC2 loss (or TSC2 deficiency).

May combine two claims.

Immunopanned TSC2−/− astrocytes do not show increased proliferation, as the percentage of Ki67+ cells is not different from controls.

“Immunopanned TSC2−/− astrocytes did not show increased proliferation, as the percentage of Ki67+ cells was not different from controls (Extended Data Fig. 7g,h).”

May combine two claims.

TSC2−/− astrocytes in 2D culture are highly enlarged and dysmorphic compared with TSC2+/+ astrocytes, with significantly larger cell bodies.

“Whereas two-dimensional (2D) culture substantially changes astrocyte morphology compared with the native three-dimensional (3D) organoid environment (Fig. 3n versus Fig. 4f), we again observed that TSC2−/− astrocytes in culture were highly enlarged and dysmorphic compared with TSC2+/+ astrocytes (Fig. 4f); with significantly larger cell bodies (Fig. 4g,h).”

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

TdTomato-positive cells are present in immunostained sections of WIBR3 and BJ TSC2c/−;LSL-TdTom organoids at day 79 (WIBR3) and day 85 (BJ).

“Example images of immunostained organoid sections showing TdTomato-positive cells in WIBR3 and BJ TSC2c/−;LSL-TdTom organoids at day 79 (WIBR3) and day 85 (BJ).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims. The quote only partly states this claim.

Immature and mature astrocyte gene module scores differ by time point and genotype (p < 0.01, p < 0.0001, or not significant).

“Violin plots show module scores calculated for immature (g) and mature (h) astrocyte gene modules, divided by time point and genotype.”

May combine two claims.

Reactive astrocyte gene module scores differ significantly by genotype and time point in WIBR3 TSC2c/+;LSL-TdTom organoids (p < 0.001, p < 0.0001).

“Violin plots show module scores for a reactive astrocyte gene module separated by genotype and time point. Violins show overall distributions and dots represent values for individual cells. ***p < 0.001, ****p < 0.0001.”

May combine two claims.

High p-S6 cells are more likely to be positive for reactive astrocyte proteins S100β, nestin and vimentin, but not more likely to be positive for neuronal markers NeuN or HuC/D.

“Specifically, high p-S6 cells were more likely to be positive for the reactive astrocyte proteins S100β, nestin and vimentin, but were not more likely to be positive for the neuronal markers NeuN or HuC/D (Fig. 5k–o).”

• AssumptionRests on an assumption: High p-S6 cells in tuber tissue correspond to cells with biallelic TSC1/TSC2 loss (or TSC2 deficiency).

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

TSC is a multisystem developmental disorder caused by mutations in TSC1 or TSC2.

“Tuberous sclerosis complex (TSC) is a multisystem developmental disorder caused by mutations in TSC1 or TSC24.”

• cites [4] Salussolia, C. L., Klonowska, K., Kwiatkowski, D. J. & Sahin, M. Genetic etiologies, diagnosis, and treatment of tuberous sclerosis complex. Annu. Rev. Genomics Hum. Genet. 20, 217–240 https://doi.org/10.1146/annurev-genom-083118-015354 (2019). sourceNot checked — no full text of the source

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

Malformations of cortical development, known as cortical tubers, are a hallmark of TSC.

“Malformations of cortical development, known as cortical tubers, are a hallmark of TSC5.”

• cites [5] Mizuguchi, M. & Takashima, S. Neuropathology of tuberous sclerosis. Brain Dev. 23, 508–515 https://doi.org/10.1016/s0387-7604(01)00304-7 (2001). sourceNot checked — no full text of the source

May combine two claims.

These changes are cell autonomous and triggered by loss of TSC2 alone, in the absence of signals from microglia or other immune cells.

“These changes are cell autonomous and triggered by loss of TSC2 alone, in the absence of signals from microglia or other immune cells.”

• AssumptionRests on an assumption: The cell-autonomous astrocyte phenotype observed in organoids is not an artifact of the absence of microglia, vascular cells or peripheral immune cells.

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

TSC is a genetic neurodevelopmental disorder characterized by focal brain malformations called cortical tubers, which are associated with severe, intractable epilepsy.

“Tuberous sclerosis complex (TSC) is a genetic neurodevelopmental disorder characterized by focal brain malformations called cortical tubers, which are associated with severe, intractable epilepsy1.”

• cites [1] Crino, P. B., Nathanson, K. L. & Henske, E. P. The tuberous sclerosis complex. N. Engl. J. Med. 355, 1345–1356 https://doi.org/10.1056/NEJMra055323 (2006). sourceNot checked — no full text of the source

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

TSC leads to hamartomas in several organs and a constellation of neurological and psychiatric conditions, including severe, early-onset epilepsy.

“TSC leads to hamartomas in several organs and a constellation of neurological and psychiatric conditions, including severe, early-onset epilepsy1.”

• cites [1] Crino, P. B., Nathanson, K. L. & Henske, E. P. The tuberous sclerosis complex. N. Engl. J. Med. 355, 1345–1356 https://doi.org/10.1056/NEJMra055323 (2006). sourceNot checked — no full text of the source

May combine two claims.

TSC2−/− cells preferentially differentiate into astrocytes and not other glial fates such as oligodendrocyte progenitor cells.

“This shift demonstrates that TSC2−/− cells preferentially differentiate into astrocytes and not other glial fates such as oligodendrocyte progenitor cells.”

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

A subset of tubers and surrounding regions can become seizure foci, and surgical removal can be beneficial for individuals with intractable epilepsy.

“A subset of tubers and surrounding regions can become seizure foci, and surgical removal can be beneficial for individuals with intractable epilepsy6.”

• cites [6] Mohamed, A. R. et al. Intrinsic epileptogenicity of cortical tubers revealed by intracranial EEG monitoring. Neurology 79, 2249–2257 https://doi.org/10.1212/WNL.0b013e3182768923 (2012). sourceNot checked — no full text of the source

May combine two claims.

The TSC2−/− phenotype is cell autonomous and does not depend on signals from microglia, vascular cells or peripheral immune cells.

“Although their absence may influence astrocyte development, it also demonstrates that the TSC2−/− phenotype is cell autonomous and does not depend on signals from these populations.”

• AssumptionRests on an assumption: The cell-autonomous astrocyte phenotype observed in organoids is not an artifact of the absence of microglia, vascular cells or peripheral immune cells.

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

Experimenters were not blind to genotype during experiments.

“Experimenters were not blind to genotype during experiments, however analysis pipelines were automated and the same settings were applied to all conditions.”

May combine two claims.

TSC2−/− astrocytes show morphological and protein changes consistent with a reactive state.

“Together, these data show that, in addition to a reactive transcriptomic signature, TSC2−/− astrocytes show morphological and protein changes consistent with a reactive state.”

May combine two claims.

Under these conditions and time frame, mTOR inhibition partially rescues the observed reactivity phenotype of TSC2−/− astrocytes but does not fully restore a homeostatic state.

“These results indicate that under these conditions and time frame, mTOR inhibition partially rescues the observed reactivity phenotype of TSC2−/− astrocytes but does not fully restore a homeostatic state.”

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

The pluripotency marker staining results are from one round of staining.

“Results are from one round of staining.”

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

Randomization is not relevant to this study.

“Randomization is not relevant to this study.”

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

Pluripotency status was confirmed by immunostaining with OCT4 and NANOG.

“Pluripotency status was confirmed by immunostaining with OCT4 and NANOG (Extended Data Fig. 1d).”

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

Genomic integrity of the stem cell lines was verified after gene editing using array comparative genomic hybridization.

“Genomic integrity of the stem cell lines was verified after gene editing using array comparative genomic hybridization (Cell Line Genetics) (Supplementary Table 11).”

Classified as probably not a substantial claim.

Adjusted P values for gene expression changes were calculated using DESeq2 differential expression analysis from 3 batches of 3 organoids each.

“Adjusted P values are shown, calculated using DESeq2 differential expression analysis from 3 batches of 3 organoids each.”

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

The organoid images shown are representative of at least 3 differentiation batches.

“Representative images from at least 3 differentiation batches.”

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

Antibodies and cycles were excluded from the analysis if residual signal was still present.

“Antibodies and cycles were excluded from the analysis if residual signal was still present.”

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

The elution validation images shown are representative of 13 elution cycles.

“Representative images from 13 elution cycles.”

May combine two claims.

mTORC1 activation is both pro-gliogenic and promotes the transition to a reactive state.

“Together with our work, these studies provide a compelling case for the idea that mTORC1 activation is both pro-gliogenic and promotes the transition to a reactive state.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims. The quote only partly states this claim.

Western blots of mTOR pathway proteins and of whole-organoid proteins were run on three independent samples per genotype.

“Three independent samples per genotype are shown.”

• Also stated in: § Competing interests · plain

  “Three independent samples per genotype are shown.”

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

For statistical comparisons, sample sizes were determined based on pilot experiments and prior publications in the field.

“For statistical comparisons, sample sizes were determined based on pilot experiments and prior publications in the field.”

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

Antibody elution across immunostaining cycles leaves no residual signal, confirmed by Hoechst staining and re-imaging at the same exposure settings as the previous cycle.

“After elution, samples were stained with Hoechst and re-imaged using the same exposure settings as the previous cycle to confirm that no residual signal remained.”

Classified as probably not a substantial claim.

TSC2 loss-of-function gene editing was validated in our laboratory as previously reported in ref. 27.

“Gene editing of hPS cells to generate TSC2 loss of-function was performed using CRISPR–Cas9 editing and validated in our laboratory as previously reported in ref. 27.”

• cites ref. 27Not matched

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

Classified as probably not a substantial claim.

On-target gene editing was confirmed by PCR.

“On-target gene editing was confirmed by PCR (Extended Data Fig. 1b).”

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

Alterations in SQSTM1 expression and autophagy have been implicated broadly in neurodegenerative disorders.

“Alterations in SQSTM1 expression and autophagy have also been implicated broadly in neurodegenerative disorders60.”

• cites [60] Nixon, R. A. The role of autophagy in neurodegenerative disease. Nat. Med. 19, 983–997 https://doi.org/10.1038/nm.3232 (2013). sourceNot checked — no full text of the source

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

mTORC1 dysregulation has been reported in Alzheimer's disease.

“Moreover, mTORC1 dysregulation has been reported in Alzheimer’s disease2.”

• cites [2] Crino, P. B. The mTOR signalling cascade: paving new roads to cure neurological disease. Nat. Rev. Neurol. 12, 379–392 https://doi.org/10.1038/nrneurol.2016.81 (2016). sourceNot checked — no full text of the source

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

All antibodies were validated by the manufacturer for use in human samples for the specific assays used in this study.

“All antibodies were used in accordance with manufacturer guidelines and were validated by the manufacturer for use in human samples for the specific assays used in this study.”

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

The cyclic staining images shown are representative of one cyclic staining experiment per tuber.

“Representative images from one cyclic staining experiment per tuber (see Fig. 5).”

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

Human pluripotent stem cell colonies in feeder-free cultures express the pluripotency markers OCT4 and NANOG.

“Images of human pluripotent stem cell colonies immunostained for the pluripotency markers OCT4 and NANOG in feeder-free cultures.”

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

PCR genotyping of the TSC2 exon 5 deletion yields allele-specific products: 2000 bp for the WT allele, 2070 bp for the floxed exon 5 allele, and 1500 bp for the exon 5 deleted allele.

“The WT allele produces a 2000 bp product, the floxed exon 5 allele produces a 2070 bp product, and the exon 5 deleted allele produces a 1500 bp product.”

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

All cell lines were tested regularly for Mycoplasma contamination.

“All cell lines were tested regularly for Mycoplasma contamination.”

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

Analysis pipelines were automated and the same settings were applied to all conditions.

“Experimenters were not blind to genotype during experiments, however analysis pipelines were automated and the same settings were applied to all conditions.”

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

Each analysis was validated by visualizing positive and negative cells using Napari to confirm that cells were correctly classified.

“Each analysis was validated by visualizing positive and negative cells using Napari v.0.6.6 (ref. 79) to confirm that cells were correctly classified.”

• cites ref. 79Not 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).

The reactive astrocyte gene module score was calculated from 3 batches of 3 organoids each.

“Quantification (mean +/- SEM) of the reactive astrocyte gene module score calculated from 3 batches of 3 organoids each.”

May combine two claims.

A small population of cortical progenitor cells with activated mTORC1 signalling are biased to become astrocytes, which are dysmorphic and express proteins indicative of a reactive state.

“Taken together with our data in organoids, this suggests a model whereby a small population of cortical progenitor cells with activated mTORC1 signalling are biased to become astrocytes, which are dysmorphic and express proteins indicative of a reactive state.”

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

For in vitro cultures, all comparisons were made within isogenic controls.

“For in vitro cultures, all comparisons were made within isogenic controls, so group membership was determined by genotype, rather than allocation.”

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

Because these cells emerge early in cortical development, they are poised to be primary drivers of neuropathophysiology in TSC.

“Because these cells emerge early in cortical development, they are poised to be primary drivers of neuropathophysiology in TSC.”

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

Individual antibody stains were repeated in at least one single-cycle pilot experiment before inclusion in cyclic staining.

“Individual antibody stains were repeated in at least one single-cycle pilot experiment before inclusion in cyclic staining.”

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

The immunostaining experiment on WIBR3 TSC2c/+;LSL-TdTom organoids was performed once.

“This experiment was performed once.”

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

Genotyping was performed at least 3 times per cell line.

“Representative gel from at least 3 genotyping experiments per line.”