Meningeal lymphatics affect microglia responses and anti-Aβ immunotherapy

May combine two claims.

In 5xFAD mice with impaired meningeal lymphatic drainage, IBA1+ cell coverage, peri-plaque IBA1+ cell counts, CD68 levels on IBA1+ cells, and fibrinogen coverage indicate aberrant myeloid activation and elevated brain fibrinogen.

“Accordingly, measurements of IBA1+ cell coverage, number of peri-Aβ plaque IBA1+ cells, levels of CD68 on IBA1+ cells and fibrinogen coverage revealed an aberrant activation of myeloid cells and increased fibrinogen levels in the brain of mice with impaired meningeal lymphatic drainage (Fig. 1f–j and Extended Data Fig. 3m–q).”

May combine two claims.

Neither mAducanumab nor mAb158 changes the average size of amyloid-beta plaques in 5xFAD mice.

“Neither mAducanumab nor mAb158 affected the average size of Aβ plaques (Extended Data Fig. 3i, j).”

May combine two claims.

Treatment with mAducanumab or mAb158 reduces amyloid-beta plaque density in 5xFAD mice.

“Treatments with mAducanumab or mAb158 proved to be efficient in reducing the density of Aβ plaques (Fig. 1b, c and Extended Data Fig. 3g, h).”

May combine two claims.

mAducanumab treatment lowers expression of disease-related genes in microglia and significantly raises expression of the homeostatic genes P2ry12, Tmem119, and Selplg.

“Interestingly, treatments with mAducanumab reduced the expression of the aforementioned disease-related genes in microglia, and significantly increased the expression of the homeostatic P2ry12, Tmem119 and Selplg (Extended Data Fig. 6f and Supplementary Table 2).”

May combine two claims.

Mice with impaired meningeal lymphatics spend less time in the open field center and take longer to locate the submerged platform during Morris water maze acquisition than controls.

“Notably, mice with impaired meningeal lymphatics spent less time in the center of the open field arena (Extended Data Fig. 4a–d and h–k) and took more time to find the submerged platform in the acquisition of the Morris water maze test (Extended Data Fig. 4e–g and l–n), when compared to their control counterparts.”

May combine two claims.

In 5xFAD mice with ablated meningeal lymphatics, less mAb158 colocalizes with brain parenchymal amyloid-beta aggregates, while vascular amyloid-beta localization is unchanged.

“Interestingly, although there were no differences in the amount of mAb158 localized to brain vascular Aβ (Extended Data Fig. 5j, k), significantly less mAb158 was found to be colocalized with brain parenchymal Aβ aggregates in 5xFAD mice with ablated meningeal lymphatics (Extended Data Fig. 5j, l).”

May combine two claims.

Prolonged meningeal lymphatic ablation increases amyloid-beta burden in the meninges and brain parenchyma, impairs amyloid-beta plaque clearance by cerebrospinal fluid-injected mAb158, and raises ferric iron deposits.

“Prolonged ablation of meningeal lymphatics led to significantly higher Aβ burden in the meninges and brain parenchyma, interfered with Aβ plaque clearance by CSF-injected mAb158 (Extended Data Fig. 5b–g) and resulted in increased ferric iron deposits (Prussian blue staining) (Extended Data Fig. 5h, i).”

May combine two claims.

Mice treated with mVEGF-C and mAb158 show significantly less peri-amyloid-beta microgliosis and lower CD68 levels in IBA1+ cells.

“Mice treated with mVEGF-C and mAb158 also presented significantly less peri-Aβ microgliosis and decreased levels of CD68 in IBA1+ cells (Extended Data Fig. 7d–g).”

May combine two claims.

In 5xFAD mice, lymphatic vessel coverage along the superior sagittal sinus, transverse sinus, and confluence of sinuses declines significantly by 13-14 months but not at 5-6 months.

“No changes were observed at 5–6 months (Extended Data Fig. 1a–e), but a significant decrease in lymphatic vessel coverage along the superior sagittal sinus (SSS), transverse sinus (TS) and the confluence of sinuses (COS) was evident at 13–14 months in the meninges of 5xFAD mice (Extended Data Fig. 1c, d).”

May combine two claims.

Mouse microglial phenotypes show enrichment for homeostatic microglia in cluster 1, activated microglia in cluster 2 with a disease-associated signature, and alternatively activated microglia in cluster 3.

“Concerning mouse microglial phenotypes, there was an obvious enrichment for homeostatic in cluster 1, activated in cluster 2 (showing a DAM signature) and alternatively activated in cluster 3 (Extended Data Fig. 10d).”

May combine two claims.

The microglia gene set shows significant connections with age at onset (P = 4.82E-12), CSF soluble TREM2 (P = 3.59E-07), Aβ42 (P = 0.021), phospho-Tau (P = 0.029), and Tau (P = 0.005) levels.

“Significant connections were observed between the microglia gene-set and age at onset (P = 4.82E-12), and CSF soluble TREM2 (P = 3.59E-07), Aβ42 (P = 0.021), phospho-Tau (P = 0.029) and Tau (P = 0.005) levels.”

May combine two claims.

More than half of microglial nuclei from non-AD patients show a resting-state phenotype, while presymptomatic AD samples are enriched in alternatively activated cluster 3 microglia.

“More than half of the microglial nuclei from the brains of non-AD patients presented a resting-state phenotype, whereas the samples from presymptomatic AD patients (patients showing characteristic AD brain pathology but a clinical dementia rating of 0.5 or less) were enriched in alternatively activated cluster 3 microglia (Fig. 3e).”

May combine two claims.

The brain blood endothelial cell gene set is strongly associated with amyloid imaging (P = 0.0089) and age at onset (P = 1.04E-10).

“A strong association was observed between the brain BECs’ gene-set, amyloid imaging (P = 0.0089) and age at onset (P = 1.04E-10).”

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

Principal component analysis separated meningeal LEC transcriptomes of WT and 5xFAD mice.

“Principal component analysis (PCA) plot showing segregation between WT (blue) and 5xFAD (purple) meningeal LEC transcriptomes.”

May combine two claims.

Brains of 5xFAD mice with ablated meningeal lymphatics have a significantly lower fraction of cluster 1 resting-state microglia (P = 0.00026) and higher fractions of clusters 2 (P = 0.00191) and 3 (P = 0.03535) activated microglia than mice with intact meningeal lymphatics.

“Noticeably, the brains of 5xFAD mice with ablated meningeal lymphatics showed a significantly lower fraction of cluster 1 resting-state microglia (P = 0.00026) and higher fractions of clusters 2 (P = 0.00191) and 3 (P = 0.03535) activated microglia, when compared to mice with intact meningeal lymphatics (Fig. 3e).”

May combine two claims.

Thirty-nine Alzheimer's disease-associated genes lie within the 10th percentile of highly expressed genes in meningeal lymphatic endothelial cells, including Apoe, which is among the few highly expressed genes shared by all three cell types.

“In fact, 39 AD-associated genes were found within the 10th percentile of highly expressed genes in meningeal LECs, including Apoe, which is intrinsically linked to altered risk for late onset AD15,31 and was one of the few highly expressed genes found at the intersection between all three cell types (Extended Data Fig. 9o).”

• cites [15] Yamazaki, Y., Zhao, N., Caulfield, T. R., Liu, C. C. & Bu, G. Apolipoprotein E and Alzheimer disease: pathobiology and targeting strategies. Nat. Rev. Neurol. 15, 501–518 (2019). sourceNot checked yet

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• cites [31] Corder, E. H. et al. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 261, 921–923 (1993). sourceNot checked yet

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May combine two claims.

In 5xFAD mouse meninges at 11-12 months, several immune cell populations increase significantly: B cells, CD4+ T cells 1, CD8+ T cells, ILC3s, and undefined cells.

“Numbers of different meningeal immune cells showing a statistically significant increase in B cells, CD4+ T cells 1, CD8+ T cells, type 3 innate lymphoid cells (ILC3s) and undefined cells in the meninges of 5xFAD mice at 11-12 months of age.”

May combine two claims.

In the learning or memory pathway, gene upregulation is amplified when mVEGF-C is given alone and sometimes counteracted when combined with mAducanumab.

“However, in the learning or memory pathway (Extended Data Fig. 8l), gene upregulation was amplified when mVEGF-C was administered alone and, in some instances, counteracted when combined with mAducanumab.”

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

In reanalysis of GSE98816, five of seven BEC clusters matched the arterial, capillary, and venous BEC categories from Vanlandewijck et al.

“Five out of the seven identified clusters matched a similar transcriptional profile to the arterial, capillary, and venous BECs identified by Vanlandewijck et al25 and were subset and re-clustered.”

• cites Vanlandewijck et al25Not checked yet

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

Reclustering produced four brain BEC groups: capillary 1, capillary 2, arterial, and venous.

“This analysis resulted in 4 clusters of brain BECs: capillary 1, capillary 2, arterial and venous (in Extended Data Fig. 9j).”

May combine two claims.

Gene-set analyses show significant correlations between up-regulated genes in microglia from 5xFAD mice with dysfunctional meningeal lymphatics and amyloid imaging (P = 0.017), CSF Tau levels (P = 0.036), age at onset (P = 0.027), and AD risk (P = 0.011).

“Furthermore, gene-set based analyses for genome-wide associations of AD-related phenotypes and risk revealed significant correlations between the set of up-regulated genes in microglia isolated from 5xFAD mice with dysfunctional meningeal lymphatics and amyloid imaging (P = 0.017), CSF Tau levels (P = 0.036), age at onset (P = 0.027) and AD risk (P = 0.011; Supplementary Table 6).”

Classified as probably not a substantial claim.

The meningeal lymphatic dysfunction microglial signature comprised 54 genes with log2 fold change greater than 1 and false discovery rate q-value less than 0.05 that were overexpressed in the Vis./photo. plus mIgG group compared with the Vis. plus mIgG group and mapped to human orthologs.

“A microglial gene signature of meningeal lymphatic dysfunction (Supplementary Table 5) was created by selecting the 54 genes that showed a log2 (fold change) > 1 and were significantly overexpressed (false discovery rate q-value < 0.05) in the Vis./photo. plus mIgG group, when compared to the Vis. plus mIgG group (Fig. 1l), and mapped to a human gene ortholog.”

May combine two claims.

Impaired meningeal lymphatic drainage in 5xFAD mice reduces mAb158 influx from cerebrospinal fluid into the brain, leading to less targeting of amyloid-beta plaques for clearance.

“These findings suggest that impairment of meningeal lymphatic drainage in 5xFAD mice leads to decreased influx of mAb158 from the CSF into the brain, resulting in less targeting of Aβ plaques for clearance.”

May combine two claims.

Reduced meningeal lymphatic drainage has a harmful effect on brain fibrinogen levels and neuroinflammation in 5xFAD mice, affecting neuronal function and leading to greater anxiety and faster cognitive decline.

“Altogether, this data is indicative of a deleterious effect of reduced meningeal lymphatic drainage on brain fibrinogen levels and neuroinflammation in 5xFAD mice, which impact neuronal function, as previously shown19, and translate into heightened anxiousness and accelerated cognitive decline.”

• cites [19] Ryu, J. K. et al. Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration. Nat. Immunol. 19, 1212–1223 (2018). sourceNot checked yet

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

Alzheimer's disease involves accumulation of toxic amyloid-beta aggregates in the brain, partly because cleansing mechanisms progressively fail.

“Alzheimer’s disease (AD) is characterized by accumulation of toxic Aβ aggregates in the brain1, in part, due to progressive impairment of cleansing mechanisms2–6.”

• cites [1] Jack, C. R. Jr et al. NIA-AA Research Framework: toward a biological definition of Alzheimer’s disease. Alzheimers Dement. 14, 535–562 (2018). sourceNot checked yet

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• cites [2] Logovinsky, V. et al. Safety and tolerability of BAN2401—a clinical study in Alzheimer’s disease with a protofibril selective Aβ antibody. Alzheimers Res. Ther. 8, 14 (2016). sourceNot checked yet

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• cites [3] Sevigny, J. et al. The antibody aducanumab reduces Aβ plaques in Alzheimer’s disease. Nature 537, 50–56 (2016). sourceNot checked yet

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• cites [4] Da Mesquita, S. et al. Functional aspects of meningeal lymphatics in ageing and Alzheimer’s disease. Nature 560, 185–191 (2018). sourceNot checked yet

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• cites [5] Mawuenyega, K. G. et al. Decreased clearance of CNS beta-amyloid in Alzheimer’s disease. Science 330, 1774 (2010). sourceNot checked yet

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• cites [6] Tarasoff-Conway, J. M. et al. Clearance systems in the brain—implications for Alzheimer diseaser. Nat. Rev. Neurol. 12, 248 (2016). sourceNot checked yet

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May combine two claims.

Meningeal lymphatic ablation causes loss of homeostatic microglia and acquisition of a disease-associated microglial signature, effects that passive immunotherapy counteracts.

“These observations suggest that meningeal lymphatic ablation leads to a loss of homeostatic microglia, and acquisition of the DAM signature (and/or the neurodegenerative microglial signature)21,22,26, all of which is counteracted by passive immunotherapy.”

• cites [21] Keren-Shaul, H. et al. A unique microglia type associated with restricting development of Alzheimer’s disease. Cell 169, 1276–1290.e17 (2017). sourceNot checked yet

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• cites [22] Krasemann, S. et al. The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity 47, 566–581.e9 (2017). sourceNot checked yet

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• cites [26] Zhou, Y. et al. Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer’s disease. Nat. Med. 26, 131–142 (2020). sourceNot checked yet

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

The Morris water maze test was administered by an experimenter who was blinded.

“All MWM testing was performed between 10 a.m. and 6 p.m., during the lights-on phase, by a blinded experimenter.”

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

Sample sizes were generally not recalculated or predetermined by statistical methods.

“In general, statistical methods were not used to re-calculate or predetermine sample sizes.”

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

Dispersion was estimated using estimateGLMRobustDisp to make the analysis more robust to outliers.

“Dispersion was estimated with estimateGLMRobustDisp to increase robustness against outliers and a quasi-likelihood model was fit with glmQLFit.”

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

Brain single-nucleus RNA-seq data were annotated using a marker gene list curated previously.

“We employed a list of marker genes that we had previously curated64 to annotate brain single-nucleus RNA-seq data.”

• cites [64] Del-Aguila, J. L. et al. A single-nuclei RNA sequencing study of Mendelian and sporadic AD in the human brain. Alzheimers Res. Ther. 11, 71 (2019). sourceNot checked yet

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

Low-quality cells were removed based on unique molecular counts, unique gene counts, and mitochondrial gene percentage.

“The resulting gene by count matrices were then read into R and filtered to remove low quality cells based on unique molecular counts, unique genes, and percent mitochondrial gene expression.”

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

Multiple testing in the eQTL analysis was corrected while accounting for linkage disequilibrium, following an approach used in prior bulk RNA-seq eQTL studies.

“We followed an approach previously employed in bulk RNA-seq expression quantitative trait loci (eQTL) analyses66–69 to correct for multiple testing while accounting for linkage disequilibrium.”

• cites [66] Sul, J. H. et al. Accurate and fast multiple-testing correction in eQTL studies. Am. J. Hum. Genet. 96, 857–868 (2015). sourceNot checked yet

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• cites [67] Duong, D. et al. Using genomic annotations increases statistical power to detect eGenes. Bioinformatics 32, i156–i163 (2016). sourceNot checked yet

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• cites [68] Darnell, G., Duong, D., Han, B. & Eskin, E. Incorporating prior information into association studies. Bioinformatics 28, i147–i153 (2012). sourceNot checked yet

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• cites [69] Eskin, E. Increasing power in association studies by using linkage disequilibrium structure and molecular function as prior information. Genome Res. 18, 653–660 (2008). sourceNot checked yet

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May combine two claims.

In 5xFAD mice, meningeal lymphatic dysfunction produces anxious-like behavior and poorer spatial learning and memory.

“Extended Data Figure 4 | Meningeal lymphatic dysfunction leads to anxious-like behavior and worsened spatial learning and memory in 5xFAD mice.”

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

To ensure randomization, animals were selected from different cages within each experimental group.

“Animals from different cages, but within the same experimental group, were selected to assure randomization.”

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

Benjamini-Hochberg correction was applied to differential expression P-values across all datasets, and adjusted P-values below 0.05 were treated as significant.

“The Benjamini-Hochberg correction was used to adjust the associated P-values for differentially expressed genes in all datasets and those with adjusted P-values below 0.05 were considered to be significant.”

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

For all independent experiments, experimenters did not know group identities from euthanasia until data collection and analysis ended.

“Experimenters were blinded to the identity of experimental groups from the time of euthanasia until the end of data collection and analysis for all the independent experiments.”

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

All quantitative measurements were made by an experimenter who was blinded.

“All measurements were performed by a blinded experimenter, Microsoft Excel was used to calculate average values in each experiment and statistical analysis performed using Prism 8.3.4.”

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

Within and between independent groups of the same experiment, variance was comparable.

“Variance was similar within independent groups of the same experiment and between groups from independent experiments.”

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

Neuropathological changes of AD were evaluated with National Institute on Aging-Alzheimer's Association criteria.

“AD neuropathological changes were assessed according to the criteria of the National Institute on Aging-Alzheimer’s Association (NIA-AA).”

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

The results shown in panels a-i were reproduced in two separate experiments.

“Data in a-i are representative of 2 independent experiments”

May combine two claims.

In 5xFAD mice, impairing meningeal lymphatic function restricts mAb158-mediated brain Aβ clearance and changes neuritic dystrophy, microglial activation, and fibrinogen levels.

“Extended Data Figure 3 | Compromising meningeal lymphatic function in 5xFAD mice limits brain Aβ clearance by mAb158 and modulates neuritic dystrophy, microglial activation and fibrinogen levels.”

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

Live lymphatic endothelial cells were gated as DAPI-negative, CD45-negative, CD31-positive, and PDPN-positive using the same gating strategy as a prior publication.

“The live LECs were then gated as DAPI−CD45−CD31+PDPN + (using the exact same gating strategy as described in a previous publication5) and sorted into a 96-well plate containing 100 μL of RNA extraction lysis buffer using the Influx™ Cell Sorter (BD Biosciences) that is available at the University of Virginia Flow Cytometry Core Facility.”

• cites [5] Mawuenyega, K. G. et al. Decreased clearance of CNS beta-amyloid in Alzheimer’s disease. Science 330, 1774 (2010). sourceNot checked yet

  Not checked in the free run.

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

A previously proposed dynamic model guided removal of nuclei with high mitochondrial gene expression.

“We removed nuclei with high mitochondria gene expression following a previously proposed dynamic model61.”

• cites [61] Mathys, H. et al. Single-cell transcriptomic analysis of Alzheimer’s disease. Nature 570, 332–337 (2019). sourceNot checked yet

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

Sequencing depth per cell, number of unique features, and mitochondrial gene percentage were regressed out.

“The resulting normalized counts were then transformed from log2 scale to the natural log scale for compatibility with Seurat v355 and the effects of sequencing depth per cell, number of unique features, and percentage of mitochondrial genes were regressed out.”

• cites [55] Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902.e21 (2019). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The advanced disease stage or advanced age at which antibody therapies are given might explain part of their marginal benefits and potential harmful side effects, which could be due to compromised meningeal lymphatic function.

“The advanced stage of disease (or simply the advanced age) at which antibody-based therapies are administered, might explain part of their marginal beneficial effects (and potential deleterious side effects), which could be attributed to a compromised meningeal lymphatic function.”

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

The open field test was conducted by a blinded experimenter.

“Individual mice were then placed into the open field arena (made of opaque white plastic material, 35 cm × 35 cm) by a blinded experimenter and allowed to explore it for 15 min.”

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

Samples were barcoded with six palladium isotopes to reduce variability between tubes.

“Individual samples were barcoded using six palladium metal isotopes according to the manufacturer’s instructions (Cell-ID 20-plex Pd barcoding kit, Fluidigm) to reduce tube-to-tube variability.”

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

No exclusion criteria were established before the study.

“No exclusion criteria were pre-established.”

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

One outlier in the Visudyne group was identified and excluded in Extended Data Fig. 3e.

“An outlier was identified and excluded in Extended Data Fig. 3e, in the Visudyne group (the outlier is indicated in the correspondent source data file).”

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

Potentially contaminating oligodendrocyte precursor cells, oligodendrocytes, neurons, and astrocytes were identified and removed using Cspg4, Mbp, Cam2ka, and Gfap expression, respectively.

“In the cortical myeloid single-cell dataset obtained using 5xFAD mice, small populations of potentially contaminating oligodendrocyte precursor cells, oligodendrocytes, neurons and astrocytes were identified and removed based on the expression levels of Cspg4, Mbp, Cam2ka, and Gfap genes, respectively.”

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

All experiments were approved by the University of Virginia Institutional Animal Care and Use Committee.

“All experiments were approved by the Institutional Animal Care and Use Committee of the University of Virginia.”

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

For viSNE visualization, an equal number of cells was randomly sampled from each replicate, totaling 12,000 cells per condition.

“A subset of cells was selected for viSNE visualization by randomly sampling an equal number of cells from each replicate, totaling 12,000 cells from each condition.”

May combine two claims.

Microglia are unlikely to respond directly to mVEGF-C because few express Flt4, though a direct effect on brain blood endothelial cells cannot be excluded.

“Although we cannot rule out a direct effect of mVEGF-C on brain BECs, which show some expression of Flt4, microglia are unlikely to respond to mVEGF-C due to the low frequency of Flt4-expressing cells (Extended Data Fig. 8t).”

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

Potential outliers were identified and discarded using the ROUT test.

“The ROUT test was used to identify and discard potential outliers.”

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

Genes with fewer than five counts in at least two samples, or with high within-sample standard deviations, were removed.

“Genes were filtered to remove those with fewer than five counts in at least two samples as well as those with high intra-sample standard deviations.”

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

Raw mass cytometry data were normalized for detector sensitivity by adding five element beads.

“Raw data were normalized for detector sensitivity by adding five element beads to the sample and processed as described previously42.”

• cites [42] Finck, R. et al. Normalization of mass cytometry data with bead standards. Cytometry A 83, 483–494 (2013). sourceNot checked yet

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

Fluorescent microsphere drainage was counted by a blinded experimenter.

“Fluorescent microsphere drainage was analyzed in FIJI software by drawing a line demarcating the draining lymphatic vessel afferent to the dCLN and manually counting the microspheres passing the line by a blinded experimenter.”

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

Doublets were detected and discarded with Doublet Finder, which removes nuclei whose expression profiles resemble synthetically mixed nuclei.

“To detect and discard doublets, we used Doublet Finder62, which removes nuclei with expression profiles that resemble synthetically mixed nuclei from the dataset.”

• cites [62] McGinnis, C. S., Murrow, L. M. & Gartner, Z. J. DoubletFinder: doublet detection in single-cell RNA sequencing data using artificial nearest neighbors. Cell Syst. 8, 329–337.e4 (2019). sourceNot checked yet

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

Power calculations (α = 0.05, power = 0.8) based on similar previously published experiments guided the choice of sample sizes.

“Sample sizes were chosen on the basis of standard power calculations (with α = 0.05 and power of 0.8) performed for similar experiments that were previously published5,12.”

• cites [5] Mawuenyega, K. G. et al. Decreased clearance of CNS beta-amyloid in Alzheimer’s disease. Science 330, 1774 (2010). sourceNot checked yet

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• cites [12] Louveau, A. et al. CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature. Nat. Neurosci. 21, 1380–1391 (2018). sourceNot checked yet

  Not checked in the free run.

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

Genes were retained only if they were expressed in more than five cells.

“Additionally, genes were filtered to retain only those which were expressed more than five cells.”

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

For hippocampal bulk RNA sequencing, differential expression results were filtered using a Benjamini-Hochberg adjusted P-value threshold of 0.05.

“Expression differential was tested with empirical Bayes quasi-likelihood F-test and results were then filtered using as threshold a Benjamini-Hochberg adjusted P-value < 0.05 (statistically significant).”