Alzheimer's-associated PU.1 expression levels regulate microglial inflammatory response

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

In the 5XFAD mouse model of Alzheimer's disease, the disease-associated microglia signature represses homeostatic microglial genes and increases genes involved in phagocytosis and lipid metabolism.

“The molecular signature of disease-associated microglia (DAM), described in the 5XFAD mouse model of AD, shows repression of microglial homeostatic genes that are expressed in microglia within a healthy brain and upregulation of genes with roles in phagocytosis and lipid metabolism (Keren-Shaul et al., 2017).”

• cites Keren-Shaul et al., 2017 sourceSupported

May combine two claims.

In microglia and macrophages, PU.1 binds myeloid-specific enhancers and sustains the cellular response to injury induced by stimulus-dependent transcription factors.

“In microglia and macrophages PU.1 is bound to myeloid-specific enhancers sustaining cell response to injury induced by stimulus-dependent transcription factors (Holtman et al., 2017).”

• cites Holtman et al., 2017 sourcePartly supported

  The PU.1 section identifies PU.1 as a myeloid lineage-determining factor that binds most microglial enhancers. The Introduction explains that stimulus-dependent transcription factors act at enhancers established by lineage-determining factors to produce cell-specific responses. It does not specifically establish that PU.1 sustains a response to injury in both microglia and macrophages. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

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

The disease-associated microglia response depends on TREM2 and APOE in mouse models of Alzheimer's disease and metabolic disease.

“The DAM response is dependent on TREM2 and APOE, two major AD risk genes, in mouse models of AD and metabolic disease (Krasemann et al., 2017; Pimenova et al., 2017a).”

• cites Krasemann et al., 2017 sourcePartly supported

  The Discussion says the disease-associated microglial signature depends on the TREM2–APOE pathway, and the Results demonstrate a TREM2-mediated phenotype switch in APP-PS1 Alzheimer’s-model mice. The Results also find the signature in Mfp2-deficient mice, identified in Methods as a lipid-disorder model, but do not establish that the response in that model depends on both TREM2 and APOE. Page numbers are unavailable in our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites Pimenova et al., 2017aNot matched

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

May combine two claims.

SPI1/PU.1 is a master regulator of microglial development and homeostasis and of several Alzheimer's disease genetic risk genes, and small changes in PU.1 expression can significantly affect cell function.

“SPI1/PU.1 is a master regulator of microglial development and homeostasis and the expression of several AD genetic risk genes, e.g. ABCA7, CD33, MS4A4A, MS4A6A, TREM2, APOE and CLU, and small changes in PU.1 expression can significantly affect cell function (Huang et al., 2017; Satoh et al., 2014).”

• cites Huang et al., 2017 sourcePartly supported

  The Results section on the PU.1 cistrome reports binding at several AD-associated genes, and its functional experiments show that altering PU.1 changes gene expression and phagocytosis in mouse microglial cells. The Discussion describes PU.1 as required for myeloid-cell development and function, but the paper does not establish that *small* expression changes significantly affect cell function or specifically characterize PU.1 as a master regulator of microglial homeostasis. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites Satoh et al., 2014 sourcePartly supported

  The Introduction and Discussion (pages unavailable) support PU.1’s importance for microglial development and function: PU.1-deficient mice lack microglia, and PU.1 knockdown reduces human microglial viability and phagocytosis. The paper identifies Trem2 as a Spi1 target, but does not establish regulation of the other listed Alzheimer’s disease risk genes or show that small changes in PU.1 expression significantly affect cell function. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

The mouse disease-associated microglia transcriptome profile may not be fully present in Alzheimer's disease brain, because human microglia show a distinct Alzheimer's-related gene signature.

“although the mouse DAM transcriptome profile may not be fully present in AD brain, as human microglia show a distinct AD-related gene signature (Jaitin et al., 2019; Mathys et al., 2019).”

• cites Jaitin et al., 2019 sourceCould not verify

  The Results and Discussion (page not provided) describe a DAM-like signature shared across macrophages and say it is highly similar to signatures in the brains of mice and humans with neurodegenerative disease. Our copy does not assess whether the mouse DAM transcriptome is fully present in Alzheimer’s disease brain or report a distinct Alzheimer’s-related signature in human microglia (searched for those comparisons). High similarity does not rule out differences, so this is not a contradiction. (LLM, conf=0.73)

  Experimental — accuracy not yet measured
• cites Mathys et al., 2019 sourceSupported

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

SPI1/PU.1 is a myeloid lineage-determining transcription factor that, together with MEF2C, shapes the enhancer landscape specific to microglia in the brain.

“SPI1/PU.1 is a myeloid lineage-determining transcription factor that together with MEF2C shapes the enhancer landscape specific to microglia in the brain (Lavin et al., 2014).”

• cites Lavin et al., 2014 sourcePartly supported

  The Results (‘Tissue Regulators Define Distinct Sets of Macrophage Enhancers’; page not provided) report MEF2 motif enrichment at microglia-specific enhancers and identify Mef2c as the most differentially expressed MEF2 family member in microglia. The Discussion describes PU.1 as a master regulator but calls Mef2c a candidate regulator; it does not establish that PU.1 and MEF2C together shape the microglial enhancer landscape. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

Lower PU.1 expression is protective against Alzheimer's disease, whereas higher expression is associated with increased risk.

“Higher expression of SPI1 is associated with increased risk for AD, while lower expression is protective.”

• Also stated in: § Introduction · plain

  “Reduced PU.1 expression confers protection, while increased expression is associated with higher AD risk.”

• cites Huang et al., 2017 sourceSupported

May combine two claims.

Modulation of PU.1 significantly affected the microglial transcriptome, with knock-down showing opposing effects to overexpression.

“Modulation of PU.1 significantly affected the microglial transcriptome with knock-down of PU.1 showing opposing effects to overexpression (Fig. 1), which we further compared to the previously described DAM signature to elaborate on the possible significance of these changes in vivo (Keren-Shaul et al., 2017).”

• cites Keren-Shaul et al., 2017 sourceSupported

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

Microglial phagocytic capability is affected by AD risk factors such as TREM2 and CD33.

“Phagocytic capability of microglia has been shown to be affected by risk factors for AD, such as TREM2 and CD33 (Griciuc et al., 2013; Kleinberger et al., 2014).”

• cites Griciuc et al., 2013 sourceSupported
• cites Kleinberger et al., 2014 sourceNot checked — no full text of the source

May combine two claims.

Activated microglia propagate the inflammatory response to astrocytes, which become reactive and neurotoxic.

“Activated microglia propagate the inflammatory response to astrocytes, which become reactive and neurotoxic (Liddelow et al., 2017).”

• cites Liddelow et al., 2017 sourceSupported

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

Amyloid and inflammation trigger and exacerbate pathological changes in the AD brain.

“Next we tested the effect of Aβ42 and LPS on oxidative stress response in microglial cells as amyloid and inflammation trigger and exacerbate pathological changes in AD brain (Wendeln et al., 2018).”

• cites Wendeln et al., 2018 sourcePartly supported

  In “Immune memory shapes neuropathology” (page not provided), the paper reports that amyloid plaques activate microglia and that a single LPS exposure increased amyloid burden in an AD mouse model. The claim is too broad about inflammation exacerbating pathology: four LPS exposures instead decreased amyloid burden. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

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

The SPI1 locus is one of the few AD risk loci for which a mechanism of action based on modulation of gene expression by common disease risk alleles has been delineated.

“The SPI1 locus is one of the few AD risk loci for which a mechanism of action based on the modulation of gene expression by common disease risk alleles has been delineated (Huang et al., 2017).”

• cites Huang et al., 2017 sourcePartly supported

  The Results (Cis-eQTL associations and colocalization analysis; page unavailable) link a protective allele to lower SPI1 expression in myeloid cells, and the Discussion proposes expression changes as a route by which the locus may affect AD risk. But the Discussion calls this a hypothesis, and the Results (Conditional and SMR analysis; page unavailable) say the functional variant has not been definitively identified. Saying the mechanism has been delineated is therefore stronger than the paper's conclusion. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

In the transcriptomic comparison, both DAM and PU.1 overexpression upregulated TREM1 pathways, whereas PU.1 knock-down showed decreased activation of those pathways.

“Interestingly, DAM and PU.1 overexpression both upregulated TREM1 pathways, while PU.1 knock-down showed decreased activation, which aligns with reduced uptake capacity in BV2 cells after PU.1 downregulation (Huang et al., 2017).”

• cites Huang et al., 2017 sourceSupported

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

The iPSC lines used had confirmed normal karyotyping.

“F11349, F12455 and 75.11-IW1A12 (Karch et al., 2019) iPSC lines were used with confirmed normal karyotyping (WiCell).”

• cites Karch et al., 2019 sourcePartly supported

  In Results, “Generation and Characterization of iPSCs” (page unavailable), the paper says chromosomal stability was assessed by karyotyping and all reported iPSC lines met its quality-control criteria. Table 4 identifies 75.11-IW1A12, but the copy does not identify F11349 or F12455 or establish their karyotyping results. It also does not attribute testing to WiCell. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

Oxidative stress is part of AD pathology as Aβ42 neurotoxicity leads to induction of reactive oxygen species and other free radicals, which may escalate production and deposition of Aβ and phosphorylation and polymerization of tau.

“Oxidative stress is part of AD pathology as Aβ42 neurotoxicity leads to induction of reactive oxygen species and other free radicals, which in turn may escalate production and deposition of Aβ and phosphorylation and polymerization of tau (Kim et al., 2015).”

• cites Kim et al., 2015 sourcePartly supported

  The Alzheimer’s disease section says Aβ accumulation may increase oxidative stress and ROS, while oxidative stress can increase Aβ production and deposition and promote tau phosphorylation. It does not specifically identify Aβ42 as the cause or say that oxidative stress promotes tau polymerization. Page numbers were not provided in our copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

Under normal conditions microglia sculpt neuronal brain connections, and increased phagocytic activity by PU.1 overexpression may result in elevated removal of dendritic spines contributing to neuronal loss in disease.

“On the other hand, under normal conditions microglia sculpt neuronal brain connections and increased phagocytic activity by PU.1 overexpression may result in elevated removal of dendritic spines contributing to neuronal loss in disease based on studies of microglia depletion in mouse models (Elmore et al., 2018).”

• cites Elmore et al., 2018 sourcePartly supported

  The Results section (page unavailable) reports that microglial elimination increased dendritic spine density in aged mice and that replacement microglia sculpted the synaptic landscape. The paper does not connect PU.1 overexpression to increased phagocytosis, excess spine removal, or neuronal loss in disease. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

The neurodegeneration-related state is controlled by APOE, and agonists of LXR promoted this anti-inflammatory state and microglial phagocytosis of Aβ42 in vitro.

“The neurodegeneration-related state is controlled by APOE, and agonists of LXR promoted this anti-inflammatory state and microglial phagocytosis of Aβ42 in vitro (Rangaraju et al., 2018).”

• cites Rangaraju et al., 2018 sourcePartly supported

  The Discussion identifies Apoe as a hub gene in the anti-inflammatory/phagocytic disease-associated microglia module and describes evidence for its role in regulating these responses. The Results report that LXR agonist treatment increased anti-inflammatory microglia and Aβ42 phagocytosis, but the mice were treated in vivo; phagocytosis was then measured ex vivo, not following LXR treatment in vitro. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

In mouse and human brain, turnover of microglia depends on high proliferation rates spatially coupled to efficient apoptosis to maintain steady state cell numbers in each brain region.

“In mouse and human brain turnover of microglia depends on high proliferation rates spatially coupled to efficient apoptosis in order to maintain steady state cell numbers in each brain region (Askew et al., 2017; Füger et al., 2017).”

• cites Askew et al., 2017 sourcePartly supported

  The Introduction and Results report high microglial proliferation in mouse and human brains and spatially coupled apoptosis that helps maintain relatively stable cell numbers. However, the Results examined only selected regions and found that microglial numbers increased with age in the mouse thalamus; they do not support steady numbers in each brain region. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Füger et al., 2017 sourceNot checked — no full text of the source

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

Another possibility explaining the protective effect of reduced phagocytic uptake is that microglial cells with PU.1 knock-down may continue to maintain a barrier around amyloid plaques, which insulates toxic Aβ42 and improves cognition in mice independent of phagocytic activity.

“Another possibility explaining the protective effect of a reduction in phagocytic uptake is that microglial cells with PU.1 knock-down may continue to maintain a barrier around the amyloid plaques, which has been shown to insulate toxic Aβ42 and improve cognition in mice independent of microglial phagocytic activity based on studies of Trem2−/−mice (Condello et al., 2015; Yuan et al., 2016).”

• cites Condello et al., 2015 sourcePartly supported

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

  Experimental — accuracy not yet measured
• cites Yuan et al., 2016 sourceNot checked — no full text of the source

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

Proliferation of microglia depends on CSF1R signaling through PU.1 and C/EBPα.

“Proliferation of microglia depends on CSF1R signaling through PU.1 and C/EBPα (Gomez-Nicola et al., 2013), and we now show that microglial apoptosis is also modulated by PU.1 (Fig. 3), which in vivo leads to regeneration of white matter after death of pro-inflammatory microglia following demyelination (Lloyd et al., 2019).”

• cites Gomez-Nicola et al., 2013 sourceSupported

May combine two claims.

In mouse models of AD, genetic or pharmacological depletion of microglia prevented microglia association with amyloid plaques and neuronal loss, leading to improved learning and memory without affecting amyloid plaque load.

“In mouse models of AD genetic or pharmacological depletion of microglia prevented microglia association with amyloid plaques and neuronal loss, which led to improved learning and memory without affecting amyloid plaque load in mouse brain (Dagher et al., 2015; Rice et al., 2015; Spangenberg et al., 2016).”

• cites Dagher et al., 2015 sourcePartly supported

  The Results and Discussion report that pharmacological CSF1R inhibition in aged 3xTg-AD mice reduced microglial association with plaques and improved learning and memory without changing Aβ levels or plaque load. This paper does not report that its treatment prevented neuronal loss, and its AD-mouse experiment used pharmacological inhibition rather than genetic depletion. The indexed passages have section labels but no page numbers. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites Rice et al., 2015 sourcePartly supported

  The Abstract reports improved behavioral outcomes after pharmacological microglial elimination following a genetically induced hippocampal lesion in mice. This was not an amyloid-plaque AD model, and the paper does not report plaque association or plaque load. The Results state the opposite of the neuronal-loss claim: eliminating microglia during lesioning exacerbated neuronal loss. Page numbers were not available in our copy. (LLM, conf=0.90)

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

  The Introduction and the section on contextual memory report that pharmacological microglial depletion in 5xfAD mice prevented neuronal loss and reduced plaque-associated microglia; the amyloid-pathology section reports no change in plaque load. This paper does not report genetic depletion, and plaque association was reduced rather than prevented. Contextual memory improved only as a statistical trend (P = 0.0813), so the claim overstates that result. (LLM, conf=0.89)

  Experimental — accuracy not yet measured

May combine two claims.

Microglial repopulation after depletion reversed age-related deficits in long-term potentiation as initial elimination of microglia increased neurogenesis and dendritic spine densities.

“Moreover, microglial repopulation after depletion reversed age-related deficits in long-term potentiation as initial elimination of microglia increased neurogenesis and dendritic spine densities (Elmore et al., 2018).”

• cites Elmore et al., 2018 sourceSupported

May combine two claims.

The AD-associated risk allele of CD33 likely produces the longer more active spliced form of this inhibitory receptor, which correlates with increased cell surface expression and reduced phagocytic capacity observed in human monocytes.

“The AD-associated risk allele of CD33 likely produces the longer more active spliced form of this inhibitory receptor (Pimenova et al., 2017b), which correlates with increased cell surface expression and reduced phagocytic capacity observed in human monocytes (Bradshaw et al., 2013).”

• cites Pimenova et al., 2017bNot matched

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

• cites Bradshaw et al., 2013 sourceSupported

May combine two claims.

Activation of the DAM signature is dependent on TREM2, suggesting DAM have increased phagocytic clearance ability.

“Moreover, activation of the DAM signature is dependent on TREM2 suggesting DAM have increased phagocytic clearance ability (Keren-Shaul et al., 2017).”

• cites Keren-Shaul et al., 2017 sourcePartly supported

  The Results and Discussion show that full DAM activation, including phagocytic pathways, depends on TREM2. The Results also identify DAM markers in microglia containing Aβ particles, supporting the suggested phagocytic ability. However, the paper explicitly says initial DAM activation is TREM2-independent, so the claim is too broad without specifying full activation. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

Genetic variants in several targets of LXR/RXR transcription factors have been associated with risk for AD, including APOE, CLU, ABCA1, ABCA7, ABCG1, and TREM2.

“Indeed, genetic variants in several targets of LXR/RXR transcription factors have been associated with risk for AD, e.g. APOE, CLU, ABCA1, ABCA7, ABCG1, TREM2 (A-González and Castrillo, 2011; Pimenova et al., 2017b; Savage et al., 2015).”

• cites A-González and Castrillo, 2011 sourcePartly supported

  Section 4 identifies ABCA1 as a direct LXR target and reports LXR-dependent expression of ABCG1 and ApoE. Section 8 discusses LXR activity in models of Alzheimer’s disease, but the paper does not report Alzheimer’s-risk genetic variants in these genes or establish CLU, ABCA7, and TREM2 as LXR/RXR targets. Pages are not specified in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Pimenova et al., 2017bNot matched

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

• cites Savage et al., 2015 sourcePartly supported

  The Introduction states that TREM2 variants confer elevated AD risk and that TREM2 expression is regulated by nuclear receptor agonists. The References also identify a study associating ABCA7 variants with AD. Our copy does not establish the claimed AD-risk associations and LXR/RXR-target status for the full list, particularly APOE, CLU, ABCA1, and ABCG1. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

Co-expression network analysis of transcriptomic changes in mouse models of AD revealed distinct states of DAM with pro- and anti-inflammatory profiles.

“Indeed, co-expression network analysis of transcriptomic changes in mouse models of AD revealed distinct states of DAM with pro- and anti-inflammatory profiles (Rangaraju et al., 2018).”

• cites Rangaraju et al., 2018 sourceSupported

May combine two claims.

This effect was propagated to primary astrocytes inducing a neurotoxic reactive gene signature, which was reduced by PU.1 knock-down in LPS-stimulated microglial cells.

“This effect was propagated to primary astrocytes inducing a neurotoxic reactive gene signature, which was reduced by PU.1 knock-down in LPS-stimulated microglial cells (Fig. 5g), suggesting reduced PU.1 expression converts microglial cells to a neuroprotective phenotype (Joshi et al., 2019; Yun et al., 2018).”

• cites Joshi et al., 2019 sourcePartly supported

  The Results section, “Microglia propagate pro-inflammatory response to activate astrocytes” (page unavailable), reports that signals from activated microglia induced an A1 proinflammatory state in primary astrocytes. It reports that P110 treatment of microglia blunted this response, but does not support the attributed reduction by PU.1 knock-down or establish a neurotoxic reactive gene signature. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Yun et al., 2018 sourcePartly supported

  The passage on astrocyte experiments (section labeled Abstract; no page given) reports that conditioned medium from α-syn PFF-treated microglia induces neurotoxic A1 markers in astrocytes. It reports that NLY01 prevents this conversion, not that PU.1 knock-down reduces a reactive gene signature in LPS-stimulated microglia. The latter, more specific part of the attributed claim is unsupported by this paper. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

Single-cell RNA sequencing data identified interferon-related and neurodegeneration-related states of microglia activation present in aging and several models of neurodegeneration and inflammation in mice.

“Further analyses of single-cell RNA sequencing data identified interferon-related and neurodegeneration-related states of microglia activation that are present in aging and several models of neurodegeneration and inflammation in mice (Friedman et al., 2018; Sala Frigerio et al., 2019).”

• cites Friedman et al., 2018 sourcePartly supported

  The Discussion (page not provided) says the authors used single-cell RNA-seq data to distinguish interferon-related and core neurodegeneration-related microglial activation states. It also says the gene modules were derived from profiles spanning aging and multiple mouse disease and inflammatory models, but the single-cell analysis was of an Alzheimer’s disease model; it does not establish that the distinct cell states were present across all those settings. (LLM, conf=0.85)

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

  The Results (‘Aging and Aβ Deposition Induce Similar Responses in Microglia’; page not provided) identify interferon-response and activated-response microglial states that increase with aging and Aβ deposition. Another Results passage finds similar states in a second amyloid mouse model, APP/PS1. The paper does not establish their presence across several models of neurodegeneration and inflammation; its Discussion says that interferon-responsive cells reported in the CK-p25 neurodegeneration model look different from the cells identified here. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

The concept of microglial priming is related to the idea of trained immunity that is controlled by epigenetic mechanisms imprinted after first exposure to a pro-inflammatory stimulus.

“The concept of microglial priming is related to the idea of trained immunity that is controlled by epigenetic mechanisms imprinted after first exposure to a pro-inflammatory stimulus (Haley et al., 2019).”

• cites Haley et al., 2019 sourceSupported

May combine two claims.

Myelin uptake was accelerated in PU.1 overexpressing cells and delayed in PU.1 knock-down cells, but both lines reached a similar plateau in 60-80 hours.

“Myelin uptake was accelerated in PU.1 overexpressing cells and delayed in PU.1 knock-down cells, however, both lines with differential PU.1 expression show a similar capacity to internalize myelin over time illustrated by the plateau that is reached by both lines in 60 – 80 hours (Fig. 2b, f).”

May combine two claims.

PU.1 overexpression reduced and PU.1 knock-down increased the population of Annexin V+/DRAQ7− viable early apoptotic cells after staurosporine treatment.

“PU.1 overexpression reduced and PU.1 knock-down increased the population of Annexin V+/DRAQ7− cells describing viable early apoptotic cells positive for phosphatidylserine staining with Annexin V and negative for DRAQ7, which stains nuclei in dead and permeabilized cells (Fig. 3e–f).”

May combine two claims.

Increased PU.1 expression makes cells more resistant to cell death and increases inflammatory response, which may harm other cell types in the diseased brain.

“this report is the first to show that increased PU.1 expression renders cells more resistant to cell death and increased inflammatory response, which may be detrimental for other cell types of the diseased brain.”

• AssumptionRests on an assumption: The BV2 immortalized mouse microglial cell line is a valid model for microglial functions relevant to Alzheimer's disease.
• AssumptionRests on an assumption: Stable overexpression and knock-down of PU.1 in BV2 cells mimic the risk and protective SPI1 genotypes.

May combine two claims.

Staurosporine treatment of BV2 cells with PU.1 overexpression reduced PU.1 expression levels, but had no effect on PU.1 in control CTL cells.

“Interestingly, staurosporine treatment of BV2 cells with PU.1 overexpression reduced PU.1 expression levels, but had no effect on PU.1 in control CTL cells (Supplementary Fig. S4e).”

May combine two claims.

BV2 cells with transient PU.1 overexpression increased and PU.1 knock-down decreased phagocytic uptake of all tested bioparticles independent of their nature, as measured by FACS 3 hours after treatment.

“BV2 cells with transient overexpression of PU.1 increased and knock-down of PU.1 decreased phagocytic uptake of all bioparticles independent of their nature as measured by FACS 3 hours after treatment (Supplementary Fig. S3a–e).”

• AssumptionRests on an assumption: BV2 cells are a valid model of microglia for studying PU.1-dependent functions relevant to AD.

May combine two claims.

Viability of PU.1 knock-down cells was affected to the same extent as control SCR cells, with similar levels of early apoptotic cells, rate of cell death, and caspase-3 activation.

“On the contrary, viability of PU.1 knock-down cells was affected to the same extent as in control SCR cells showing similar levels of early apoptotic cells (Fig. 4f), rate of cell death (Supplementary Fig. S6f) and caspase-3 activation (Fig. 4g–h).”

May combine two claims.

PU.1 overexpression affected 191 genes with Spi1 upregulated by 60%, whereas PU.1 knock-down produced 3892 differentially expressed genes with Spi1 downregulated by 71%.

“The result of this PCA for the overexpression condition reflects the small number of genes affected by PU.1 overexpression – 191 (Spi1 was upregulated by 60%), while there were 3892 genes differentially expressed after PU.1 knock-down (Spi1 was downregulated by 71%) (Fig. 1a–b, Supplementary Tables S2–3).”

May combine two claims.

PU.1 overexpression reduced staurosporine-induced cell death as measured by MTS assay, and increased viability observed with LDH assay.

“PU.1 overexpression reduced staurosporine-induced cell death as measured by an MTS assay assessing cell proliferation potential, which was enhanced by increased PU.1 (Fig.3a) likely due to increased viability observed with an LDH assay assessing the rate of cell death after staurosporine treatment (Fig. 3a–b).”

May combine two claims.

Knock-down of PU.1 did not affect caspase activation, only showing a trend towards increased activation.

“Knock-down of PU.1 also did not affect caspase activation only showing a trend towards increased activation of caspases (Fig. 3g–i).”

May combine two claims.

Longitudinal experiments with stable PU.1 overexpression or knock-down showed that increased PU.1 expression led to increased uptake and decreased PU.1 expression inhibited bioparticle uptake, based on uptake half-life.

“Longitudinal experiments tracking internalization of pHrodo-labeled myelin and zymosan in BV2 cells with stable overexpression or knock-down of PU.1 showed similar results with increased PU.1 expression leading to increased uptake and decreased PU.1 expression inhibiting bioparticle uptake, based on the quantification of uptake half-life (Fig. 2d, h).”

• AssumptionRests on an assumption: BV2 cells are a valid model of microglia for studying PU.1-dependent functions relevant to AD.

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

Proliferation of PU.1 overexpression and knock-down cells was comparable to control cell lines.

“Interestingly, live imaging of myelin and zymosan internalization showed different kinetics of uptake, while proliferation of PU.1 overexpression and knock-down cells was comparable to control cell lines (Fig. 2a, e).”

May combine two claims.

Populations of nonviable phosphatidylserine-negative late apoptotic and necrotic cells seemed unaffected by PU.1 expression, whereas Z-VAD-FMK reduced cells in all three states.

“Populations of nonviable phosphatidylserine-negative late apoptotic (Annexin V+/DRAQ7 +) and necrotic (Annexin V−/DRAQ7+) cells seem to be unaffected by PU.1 expression when compared to Z-VAD-FMK, which reduced the number of cells in all three states.”

May combine two claims.

The effect on NO was partially reduced by NAC antioxidant pre-treatment and was due to PU.1-mediated regulation of iNOS expression, which was increased in PU.1 overexpressing cells and reduced in PU.1 knock-down cells.

“This effect was specific to induction of oxidative stress as it could be partially reduced by N-acetyl cysteine (NAC) antioxidant pre-treatment and was due to PU.1-mediated regulation of inducible nitric oxide synthase (iNOS) expression which showed increased levels in PU.1 overexpressing cells and reduced levels in cells with PU.1 knock-down (Fig. 5c–d).”

May combine two claims.

Expression levels of PU.1 were unchanged under stimulated conditions compared to baseline, contrary to data after staurosporine treatment.

“Indeed, expression levels of PU.1 were unchanged under stimulated conditions compared to baseline, contrary to data after staurosporine treatment (Supplementary Fig. S6g).”

May combine two claims.

Treatment of primary astrocytes with conditioned medium from LPS-stimulated microglial cells with PU.1 overexpression resulted in increased expression of the astrocytes reactive signature, while PU.1 knock-down negated induction of A1 reactive astrocytes after LPS treatment.

“Treatment of primary astrocytes with conditioned medium from LPS-stimulated microglial cells with PU.1 overexpression resulted in increased expression of the astrocytes reactive signature, while PU.1 knock-down negated induction of A1 reactive astrocytes after LPS treatment (Fig. 5g, Supplementary Fig. S7d).”

May combine two claims.

PU.1 overexpressing cells showed increased zymosan internalization compared to control CTL cells, while PU.1 knock-down cells displayed limited capability to internalize zymosan compared to control SCR cells.

“PU.1 overexpressing cells showed increased zymosan internalization compared to control CTL cells, while PU.1 knock-down cells displayed limited capability to internalize zymosan in comparison to control SCR cells (Fig. 2c, g).”

May combine two claims.

PU.1 overexpression potentiated NO production after LPS treatment compared to control cells, while PU.1 knock-down repressed NO production.

“PU.1 overexpression further potentiated NO production after treatment with LPS compared to control cells, while PU.1 knock-down repressed NO production (Fig. 5a–b).”

May combine two claims.

Levels of PU.1 were equal under staurosporine treatment in overexpression and knock-down conditions, due to decreased PU.1 expression in control SCR cells but not in PU.1 knock-down cells.

“In fact, levels of PU.1 were equal under staurosporine treatment in overexpression and knock-down conditions, however, the latter was due to decreased PU.1 expression in control SCR cells, but not in PU.1 knock-down cells (Supplementary Fig. S4f).”

May combine two claims.

PU.1 overexpression delayed the onset of BV2 cell death after staurosporine treatment, while PU.1 knock-down cells died at the same rate as control cells.

“Live-imaging of the DRAQ7+ necrotic cells revealed that PU.1 overexpression delayed the onset of BV2 cell death after staurosporine treatment, while PU.1 knock-down cells died at the same rate as control cells (Supplementary Fig. S4c–d).”

May combine two claims.

PU.1 knock-down activates protein translation, antioxidant action and cholesterol/lipid metabolism pathways while decreasing pro-inflammatory gene expression.

“Moreover, PU.1 knock-down resulted in activation of protein translation, antioxidant action and cholesterol/lipid metabolism pathways with a concomitant decrease of pro-inflammatory gene expression.”

• Also stated in: § Knock-down of PU.1 repressed homeostatic microglial gene expression and activated disease-associated microglial signature pathways · hedged

  “Pathway analysis showed activation of LXR/RXR and EIF2 signaling and increased antioxidant action of vitamin C in cells with PU.1 knock-down suggesting a sustained level of oxidative stress response, cholesterol/lipid metabolism and protein translation (Fig. 1e).”

• AssumptionRests on an assumption: The BV2 immortalized mouse microglial cell line is a valid model for microglial functions relevant to Alzheimer's disease.

May combine two claims.

PU.1 overexpression increases and PU.1 knock-down decreases phagocytic uptake in BV2 cells regardless of the material being engulfed.

“PU.1 overexpression upregulated and knock-down downregulated phagocytic uptake in BV2 cells independent of the nature of the engulfed material.”

• AssumptionRests on an assumption: The BV2 immortalized mouse microglial cell line is a valid model for microglial functions relevant to Alzheimer's disease.

May combine two claims.

Knock-down of PU.1 decreased the number of ROS+ cells and ROS production per cell, while baseline ROS levels were unaffected.

“In contrast, knock-down of PU.1 decreased the number of ROS+ cells and ROS production per cell as measured by FACS, while baseline levels of ROS were unaffected (Fig. 4c–d, Supplementary Fig. S6b).”

May combine two claims.

Pathway analysis showed activation of LXR/RXR and EIF2 signaling and increased antioxidant action of vitamin C in PU.1 knock-down cells, suggesting sustained oxidative stress response, cholesterol/lipid metabolism and protein translation.

“Pathway analysis showed activation of LXR/RXR and EIF2 signaling and increased antioxidant action of vitamin C in cells with PU.1 knock-down suggesting a sustained level of oxidative stress response, cholesterol/lipid metabolism and protein translation (Fig. 1e).”

May combine two claims.

BV2 cells with reduced PU.1 expression retain the ability to internalize myelin similarly to controls, but with a delay, which is consistent with their anti-inflammatory profile.

“However, cells with reduced PU.1 expression retained their ability to internalize myelin similar to control albeit with a delay, which aligns with their anti-inflammatory profile.”

• AssumptionRests on an assumption: The BV2 immortalized mouse microglial cell line is a valid model for microglial functions relevant to Alzheimer's disease.

May combine two claims.

Rotenone treatment affected cell viability similarly to staurosporine, with PU.1 overexpression showing increased survival and reduced early apoptotic cells.

“Rotenone treatment of BV2 cells affected cell viability in a similar manner to staurosporine, with PU.1 overexpression showing increased survival upon rotenone treatment (Supplementary Fig. S6e) and a reduced number of early apoptotic cells (Fig. 4e, Supplementary Fig. S6c).”

May combine two claims.

Rotenone-treated BV2 cells showed an increase in ROS with increased number of responding cells and increased ROS production per cell.

“Rotenone-treated BV2 cells showed an increase in ROS with increased number of cells responding to rotenone treatment and increased production of ROS per cell.”

May combine two claims.

Treatment of control CTL cells with Aβ42 or LPS did not produce a robust ROS response, but PU.1 overexpression amplified ROS production in cells exposed to these stimuli.

“Treatment of control CTL cells with Aβ42 or LPS did not produce a robust ROS response, nevertheless, PU.1 overexpression amplified production of ROS in cells exposed to these stimuli (Fig. 4i–j).”

May combine two claims.

PU.1 overexpression led to increased resistance and knock-down to increased susceptibility of microglial cells to staurosporine- and rotenone-induced cell death.

“While baseline cell viability was not affected by modulation of PU.1, likely because of in vitro cell culture conditions, PU.1 overexpression led to increased resistance and knock-down to increased susceptibility of microglial cells to staurosporine- and rotenone-induced cell death (Figs. 3a–d and 4e–f).”

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

Baseline cell viability was not affected by modulation of PU.1, likely because of in vitro cell culture conditions.

“While baseline cell viability was not affected by modulation of PU.1, likely because of in vitro cell culture conditions, PU.1 overexpression led to increased resistance and knock-down to increased susceptibility of microglial cells to staurosporine- and rotenone-induced cell death (Figs. 3a–d and 4e–f).”

May combine two claims.

Microglia with PU.1 knock-down specifically showed preserved capacity for storing internalized myelin albeit with delayed uptake, which was not observed for zymosan.

“Interestingly, PU.1 may modulate specificity of microglial phagocytosis towards pro- and anti-inflammatory bioparticles, e.g. amyloid β and zymosan versus myelin, as microglia with PU.1 knock-down specifically showed preserved capacity for storing internalized myelin albeit with a delayed uptake, which was not observed for zymosan (Fig. 2f–g).”

May combine two claims.

PU.1 overexpression potentiated and knock-down repressed production of ROS, NO and inflammatory cytokines after treatment with rotenone, Aβ42 or LPS.

“Indeed, we show here that PU.1 overexpression potentiated and knock-down repressed production of ROS, NO and inflammatory cytokines after treatment with rotenone, Aβ42 or LPS (Figs. 4 and 5).”

May combine two claims.

PU.1 overexpression reduced and knock-down increased caspase-activated apoptosis of microglia, corroborated by PU.1 regulation of organismal death and morbidity and mortality functions.

“Specifically, PU.1 overexpression reduced and knock-down increased caspase-activated apoptosis of microglia (Fig. 3e–i), which was corroborated by PU.1 regulation of “organismal death” and “morbidity and mortality” functions (Fig. 1c).”

May combine two claims.

Low PU.1 expression increases cell death under cytotoxic conditions and reduces pro-inflammatory signaling, which decreases the A1 reactive astrocyte signature and supports the protective effect of the SPI1 genotype in Alzheimer's disease.

“In contrast, low expression of PU.1 leads to increased cell death under cytotoxic conditions accompanied by reduced pro-inflammatory signaling that decreased A1 reactive astrocytes signature supporting the protective effect of SPI1 genotype in AD.”

• Also stated in: § Introduction · plain

  “PU.1 knock-down makes microglia more susceptible to cell death, but also less pro-inflammatory, which was beneficial in reducing A1 reactive astrocytes signature relevant to diseased brain milieu.”

• AssumptionRests on an assumption: The BV2 immortalized mouse microglial cell line is a valid model for microglial functions relevant to Alzheimer's disease.
• AssumptionRests on an assumption: Stable overexpression and knock-down of PU.1 in BV2 cells mimic the risk and protective SPI1 genotypes.
• AssumptionRests on an assumption: In vitro microglial responses to PU.1 modulation reflect in vivo microglial biology and Alzheimer's disease pathogenesis.
• AssumptionRests on an assumption: The A1 reactive astrocyte signature induced by conditioned medium from PU.1 knock-down BV2 cells is relevant to the diseased brain milieu.

Classified as probably not a substantial claim.

Maturation of induced microglial-like cells was confirmed by immunostaining for PU.1 and CD11b after three to five days in mature microglial medium.

“iMGLs maturation was confirmed with immunostaining for PU.1 and CD11b after 3–5 days of culture in mature microglial medium.”

May combine two claims.

PU.1 overexpression and knock-down have opposing effects on microglial function, supporting a mechanism for SPI1 contribution to Alzheimer's disease pathogenesis based on differential expression.

“Interestingly, the effect of PU.1 overexpression on microglial function shows opposing effects to PU.1 knock-down, which supports the mechanism of action for SPI1 contribution to AD pathogenesis based on differential expression.”

• AssumptionRests on an assumption: Stable overexpression and knock-down of PU.1 in BV2 cells mimic the risk and protective SPI1 genotypes.

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

Changes in PU.1 expression in the stable BV2 lines were confirmed at the mRNA level by qPCR and at the protein level by western blotting.

“Changes in PU.1 expression were confirmed on the mRNA level using qPCR and protein level using western blotting of polyclonal selected lines.”

May combine two claims.

PU.1 overexpression potentiates microglial oxidative stress through a direct effect on ROS production and increased survival after cytotoxic stimuli, while PU.1 knock-down represses the oxidative stress response after rotenone, Aβ42 and LPS treatment.

“In conclusion, PU.1 overexpression potentiates microglial oxidative stress through a direct effect on ROS production and increased survival after treatment with cytotoxic stimuli, while PU.1 knock-down represses the microglial oxidative stress response after rotenone, Aβ42 and LPS treatment.”

• AssumptionRests on an assumption: BV2 cells are a valid model of microglia for studying PU.1-dependent functions relevant to AD.

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

RNA sequencing samples passed quality control with Qubit and BioAnalyzer and had an RNA integrity number of 10.

“Samples passed quality control with Qubit and BioAnalyzer showing RIN = 10.”

May combine two claims.

The generated BV2 cell lines achieved 2-fold overexpression of PU.1 and 75% knock-down of PU.1 at the protein level relative to their respective controls.

“Analysis of mRNA and protein expression of PU.1 showed that we were able to obtain 2-fold overexpression of PU.1 (OE cell line) and 75% knock-down of PU.1 (KD cell line) at the protein level (Supplementary Fig. S2a–c) relative to their respective controls (CTL line for overexpression and SCR line for knock-down).”

• AssumptionRests on an assumption: The achieved 2-fold overexpression and 75% knock-down of PU.1 are sufficient to model the range of PU.1 expression relevant to human AD risk.

May combine two claims.

BV2 cells with PU.1 knock-down exhibit lower activity and inflammatory response in contrast to DAM.

“Along with reduced phagocytosis we detect reduced activation of IRF by cytosolic pattern recognition receptors in BV2 cells with PU.1 knock-down, suggesting these cells exhibit lower activity and inflammatory response in contrast to DAM.”

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

Routine testing with MycoAlert showed that the cell lines used were negative for mycoplasma contamination.

“Routine testing of cell lines using MycoAlert PLUS mycoplasma detection kit (Lonza) showed that all cells were negative for mycoplasma contamination.”

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

PU.1 knock-down reduces Aβ42 uptake in human microglial cells.

“We have previously reported that PU.1 regulates zymosan phagocytosis (Huang et al., 2017) and others reported that PU.1 knock-down reduces Aβ42 uptake in human microglial cells (Smith et al., 2013).”

• cites Smith et al., 2013 sourceNot checked — no full text of the source

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

RNA sequencing results were validated by qPCR on 75 genes using Pearson correlation analysis.

“RNA sequencing results were validated using qPCR on 75 genes with Pearson correlation analysis.”

• Also stated in: § Knock-down of PU.1 repressed homeostatic microglial gene expression and activated disease-associated microglial signature pathways · plain

  “Pearson correlation of 75 genes showed good overlap of DEGs from RNA sequencing with qPCR results in BV2 cells (Supplementary Fig. S1b, Supplementary Tables S2–3).”

May combine two claims.

PU.1 overexpression renders microglial cells resistant to caspase-mediated apoptosis, while PU.1 knock-down cells become more susceptible to caspase-induced cell death.

“In conclusion, PU.1 overexpression renders microglial cells resistant to caspase-mediated apoptosis, while PU.1 knock-down cells become more susceptible to caspase-induced cell death.”

• AssumptionRests on an assumption: BV2 cells are a valid model of microglia for studying PU.1-dependent functions relevant to AD.

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

The researcher was not blinded to sample collection.

“The researcher was not blinded to sample collection.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The difference in response of respective control cells may be due to the transformation procedure rendering SCR cells more inflammatory prone than the CTL cell line.

“The difference in response of respective control cells may be due to the transformation procedure rendering SCR cells more inflammatory prone than CTL cell line.”

May combine two claims.

PU.1 regulates the inflammatory response of microglial cells to pathogenic stimuli; increased PU.1 expression would amplify and propagate it, while decreased PU.1 expression will mitigate microglia-mediated pro-inflammatory responses.

“These data suggest that PU.1 regulates the inflammatory response of microglial cells to pathogenic stimuli and that increased PU.1 expression would amplify and propagate it, while decreased PU.1 expression will mitigate microglia-mediated pro-inflammatory responses.”

• AssumptionRests on an assumption: BV2 cells are a valid model of microglia for studying PU.1-dependent functions relevant to AD.

May combine two claims.

PU.1 overexpression amplified cytokine secretion, suggesting an increased pro-inflammatory response.

“As we have previously seen with ROS and NO, PU.1 overexpression amplified cytokine secretion suggesting an increased pro-inflammatory response (Fig. 5e).”

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

Each analysis used three to five independent experiments, with three to five biological replicates.

“In each analysis three to five independent experiments were performed (n = 3 – 5 biological replicates).”

May combine two claims.

The pathways activated by PU.1 knock-down are similarly activated in disease-associated microglia and oppositely inhibited in PU.1 overexpression associated with increased Alzheimer's disease risk, suggesting these pathways may be central to the phenotype.

“These pathways are similarly activated in DAM and oppositely inhibited in PU.1 overexpression that is associated with increased risk for AD based on human genetics, suggesting these pathways may be central to”

May combine two claims.

Changes in immune response and lipid metabolism genes or direct PU.1 targets in human induced microglial-like cells overlapped with BV2 cells, suggesting PU.1 affects similar aspects of microglial biology in human and mouse cells.

“Changes in the expression of genes mediating immune response and lipid metabolism or direct targets of PU.1 in iMGLs overlapped with BV2 cells suggesting PU.1 affects similar aspects of microglial biology in human and mouse cells (Figure 1c).”

• AssumptionRests on an assumption: The BV2 immortalized mouse microglial cell line is a valid model for microglial functions relevant to Alzheimer's disease.

May combine two claims.

This effect may be beneficial in AD considering how microglial uptake of Aβ or lipids may hinder effective debris clearance and result in microglial cell death contributing to seeding of the amyloid plaque core.

“This effect may be beneficial in AD considering how microglial uptake of Aβ or lipids may hinder effective debris clearance and result in microglial cell death contributing to seeding of the amyloid plaque core (Baik et al., 2016).”

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

May combine two claims.

SPI1 is a master regulator of this gene network, and its upregulation suggests clearance of lipids and debris may be preserved with PU.1 knock-down.

“SPI1 is a master regulator of this gene network, upregulation of which suggests clearance of lipids and debris may be preserved with PU.1 knock-down.”

May combine two claims.

Based on these observations and our data, PU.1 knock-down may be beneficial in slowing overt neurodegeneration at the onset of disease due to decreased ability of microglia to seed amyloid plaques, possibly without compromising clearance and maintenance of brain tissue homeostasis at later stages.

“Based on these observations and our data we propose that PU.1 knock-down may be beneficial in slowing overt neurodegeneration at the onset of disease due to decreased ability of microglia to seed amyloid plaques, possibly without compromising on clearance and maintenance of brain tissue homeostasis at later stages.”

Classified as probably not a substantial claim.

This dampened response is generally considered protective in neurodegenerative conditions.

“PU.1 knock-down dampened stimulus-dependent pro-inflammatory response, which is generally considered protective in neurodegenerative conditions.”

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

TREM2 rare genetic variants associated with AD risk impair microglial lipid sensing and phagocytosis.

“TREM2 rare genetic variants associated with AD risk impair microglial lipid sensing and phagocytosis (Kleinberger et al., 2014; Yeh et al., 2016).”

• cites Kleinberger et al., 2014 sourceNot checked — no full text of the source
• cites Yeh et al., 2016 sourceNot checked — no full text of the source

May combine two claims.

The protective effect of reduced PU.1 expression in AD is mediated through pathways of LXR/RXR signaling, antioxidant action of vitamin C and/or EIF2 protein translation, similar to the protective effect of DAM.

“We propose that the protective effect of reduced PU.1 expression in AD is mediated through pathways of LXR/RXR signaling, antioxidant action of vitamin C and/or EIF2 protein translation similar to the protective effect of DAM.”

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

Mutations in EIF2B1-5 genes lead to leukoencephalopathy with vanishing white matter characterized by progressive degeneration of white matter due to myelination failure and impairments in oligodendrocyte function.

“Mutations in EIF2B1-5 genes, encoding subunits of eIF2b, responsible for protein translation, lead to leukoencephalopathy with vanishing white matter characterized by progressive degeneration of white matter due to myelination failure and impairments in oligodendrocyte function (Bugiani et al., 2010).”

• cites Bugiani et al., 2010 sourceNot checked — no full text of the source

May combine two claims.

Since impairments in these pathways contribute to AD progression, counteracting consequences of defects in these pathways will be beneficial in AD, supporting the protective effect of reduced PU.1 expression in microglia.

“Since impairments in these pathways contribute to AD progression, counteracting consequences of defects in these pathways will be beneficial in AD, supporting the protective effect of reduced PU.1 expression in microglia.”

May combine two claims.

Deletion of PU.1 results in ablation of myeloid lineage cell types in mouse, e.g. macrophages and B cells, suggesting modulation of PU.1 levels in adult brain may affect microglial viability.

“Deletion of PU.1 results in ablation of myeloid lineage cell types in mouse, e.g. macrophages and B cells, suggesting modulation of PU.1 levels in adult brain may affect microglial viability (Kierdorf et al., 2013; McKercher et al., 1996).”

• cites Kierdorf et al., 2013 sourceNot checked — no full text of the source
• cites McKercher et al., 1996 sourceNot checked — no full text of the source

May combine two claims.

PU.1 downregulation reduced phagocytosis, increased apoptotic cell death and inhibited immune response, which promoted the anti-inflammatory state of microglia that is likely protective in AD.

“PU.1 downregulation reduced phagocytosis, increased apoptotic cell death and inhibited immune response, which promoted the anti-inflammatory state of microglia that is likely protective in AD.”

May combine two claims.

Downregulation of PU.1 expression seems to transform microglial cells into an anti-inflammatory state with activated LXR/RXR and reduced IRF signaling that is protective in AD, albeit distinct from previously described DAM.

“Overall, downregulation of PU.1 expression seems to transform microglial cells into an anti-inflammatory state with activated LXR/RXR and reduced IRF signaling that is protective in AD (Fig. 1), albeit distinct from previously described DAM.”

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

These studies represent the first step toward investigating PU.1 inhibition or lower expression as a possible therapeutic target for AD treatment.

“These studies represent the first step toward investigating PU.1 inhibition/lower expression as a possible therapeutic target for AD treatment.”

Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

As PU.1 is a master regulator of microglial function and several genetic risk factors for AD, it may be possible to modulate several phenotypes at once by targeting a single gene for treatment of AD.

“As PU.1 is a master regulator of microglial function and several genetic risk factors for AD (APOE, CLU, MS4A4A/MS4A6A, PILRA), it may be possible to modulate several phenotypes at once by targeting a single gene for treatment of AD.”