Epigenetic regulation of brain region-specific microglia clearance activity

May combine two claims.

PRC2 loss in microglia leads to impaired motor responses, decreased learning and memory, and development of anxiety and seizures in mice.

“leading to impaired motor responses, to decreased learning and memory, and to the development of anxiety and seizures in mice.”

May combine two claims.

Cerebellar microglia show increased expression of genes characteristic of immature microglia and genes expressed in neurodegenerative disease-associated microglia.

“Furthermore, cbMg show an increase in the expression of genes characteristic of immature microglia9,10, as well as genes that are expressed in neurodegenerative disease-associated microglia (such as primed microglia, MGnD, and DAMs)11–13 (Supplementary Fig. 5c,d, Supplementary Table 4).”

• cites [9] Mass, E. et al. Specification of tissue-resident macrophages during organogenesis. Science 353, aaf4238 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [10] Matcovitch-Natan, O. et al. Microglia development follows a stepwise program to regulate brain homeostasis. Science 353, aad8670 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [11] Mathys, H. et al. Temporal tracking of microglia activation in neurodegeneration at single-cell resolution. Cell Rep. 21, 366–380 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [12] Wang, Y. et al. TREM2 lipid sensing sustains the microglial response in an Alzheimer’s disease model. Cell 160, 1061–1071 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [13] 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

  Not checked in the free run.

May combine two claims.

Adult mouse cerebellar microglia show a primed/phagocytic phenotype characterized by less ramified morphology, reduced branching sites, decreased cell volume, and increased CD68+ lysosome content.

“cbMg in the adult mouse brain show a primed/phagocytic microglia phenotype11–13 characterized by a less ramified morphology, reduced branching sites (Supplementary Fig. 1a), decreased cell volume (Supplementary Fig. 1b,c), and an increase in CD68+ lysosome content (Fig. 1a, Supplementary Fig. 1b,c).”

• cites [11] Mathys, H. et al. Temporal tracking of microglia activation in neurodegeneration at single-cell resolution. Cell Rep. 21, 366–380 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [12] Wang, Y. et al. TREM2 lipid sensing sustains the microglial response in an Alzheimer’s disease model. Cell 160, 1061–1071 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [13] 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

  Not checked in the free run.

May combine two claims.

A large fraction of cerebellar microglia-enriched genes encode proteins involved in apoptotic cell detection, engulfment, clearance of cell debris, and lipid/protein catabolism, and have been shown to support clearance activity in phagocytic macrophages in peripheral tissues.

“A large fraction of the cbMg-enriched genes, including Apoe, Axl, Colec12, Cd74, Lilrb4, Mrc1, and Ms4a7 (Fig. 1d,e, Supplementary Fig. 5a,b, Supplementary Table 3), encode proteins involved in apoptotic cell detection, engulfment, clearance of cell debris, and lipid/protein catabolism and have been shown to support clearance activity in phagocytic macrophages in peripheral tissues21.”

• cites [21] Blander, J. M. The many ways tissue phagocytes respond to dying cells. Immunol. Rev. 277, 158–173 (2017). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Expression of the clearance-associated proteins AXL, ApoE, CD74, MHCII, and MRC1 is significantly higher in cerebellar microglia, while FSCN1 protein expression is significantly enriched in striatal microglia.

“Expression of the clearance-associated proteins AXL, ApoE, CD74, MHCII, and MRC1 is significantly higher in cbMg, while actin polymerization-regulating FSCN1 protein expression shows a significant enrichment in stMg (Fig. 2c, Supplementary Fig. 7d).”

May combine two claims.

Changes in PRC2-deficient cerebellar microglia gene expression patterns are associated with enhanced cerebellar microglia ramification, a reduction in cerebellar microglia lysosomal content, and an increased number of cCASP3+ cells in the cerebellum.

“The changes in PRC2-deficient cbMg gene expression patterns are associated with enhanced cbMg-ramification, a reduction in cbMg lysosomal content (Fig. 6f), and an increased number of cCASP3+ cells in the cerebellum (Fig. 6g).”

May combine two claims.

Anxiety and seizure phenotypes are not observed in one-year-old wild-type mice living in chronic absence of microglia (continuous PLX treatment starting at 1 month of age).

“Notably, the anxiety and seizure phenotypes are not observed in one-year-old wild-type mice that live in the chronic absence of microglia (continuous PLX treatment starting 1 month of age, Supplementary Fig. 13h-j)”

May combine two claims.

Most of the down-regulated genes in Eed-deficient striatal microglia are H3K27me3-negative and are therefore not directly controlled by PRC2 in striatal microglia.

“Most of these down-regulated genes are H3K27me3-negative and are, therefore, not directly controlled by PRC2 in stMg (Fig. 5e).”

May combine two claims.

PRC2 deficiency in cerebellar microglia has no significant impact on cerebellar Purkinje cell spine numbers and cerebellum-dependent behaviors.

“PRC2 deficiency in cbMg, on the other hand, has no significant impact on cerebellar Purkinje cell spine numbers (Supplementary Fig. 12e) and cerebellum-dependent behaviors (Supplementary Fig. 12f).”

May combine two claims.

The shift of striatal microglia from a surveillance state toward a more clearance-promoting cerebellar microglia-like state, while not affecting overall microglia numbers or morphology, is associated with a significant increase in striatal microglia lysosomal content.

“The shift of stMg from a surveillance state toward a more clearance-promoting cbMg-like state (Fig. 6a,b), while not affecting overall microglia numbers or morphology (Fig. 6c, Supplementary Fig. 11i), is associated with a significant increase in the stMg lysosomal content (Fig. 6c).”

May combine two claims.

Loss of PRC2 activity in cortical microglia is associated with a switch towards a microglia clearance phenotype, including an increase in cortical microglia lysosomal content.

“Like in the striatum, we found that loss of PRC2 activity in cortical microglia (cxMg) is associated with a similar switch towards a microglia clearance phenotype (Supplementary Fig. 12g), including an increase in cxMg lysosomal content (Supplementary Fig. 12h).”

May combine two claims.

Deficiency in Eed has no effect on overall size and morphology of the striatum, medium spiny neuron density, or survival as defined by presence of cCASP3+ cells.

“Deficiency in Eed has no effect on the overall size and morphology of the striatum (data not shown), MSN density (Supplementary Fig. 12a), or survival as defined by the presence of cCASP3+ cells (Supplementary Fig. 12b).”

May combine two claims.

The majority of apoptotic cells that accumulate after microglia depletion are of neuronal origin and are distributed within all layers of the cerebellum.

“Co-labeling with different cell type-specific markers revealed that the majority of the apoptotic cells are of neuronal origin and are distributed within all layers of the cerebellum (Supplementary Fig. 9a,b).”

May combine two claims.

Deficiency in Eed and ensuing loss of H3K27me3 had no immediate impact on microglia activation but leads to moderate and progressive changes in striatal microglia gene expression between 3-9 months of age.

“We found that deficiency in Eed and the ensuing loss of H3K27me3 had no immediate impact on microglia activation but leads to rather moderate and progressive changes in stMg gene expression between 3-9 months of age (Fig. 5e,f).”

May combine two claims.

During an elevated plus maze paradigm, mutant mice spend significantly more time in the closed compartment and significantly less time in the open compartment compared to littermate controls.

“Likewise, during an elevated plus maze paradigm, mutant mice spend significantly more time in the closed compartment and significantly less time in the more stressful open compartment as compared to their littermate controls (Fig. 7d, Supplementary Fig. 13b,c).”

May combine two claims.

Young Cx3cr1CreErt2/+;Eedfl/fl and control mice did not display notable seizure events prior to 10 months of age, while aged mutant mice show increased frequency of handling-induced tonic-clonic seizure episodes.

“While young Cx3cr1CreErt2/+;Eedfl/fl and control mice did not display any notable seizure events prior to 10 months of age, we detected an increased frequency of handling-induced tonic-clonic seizure episodes in aged mutant mice (Fig. 7e, Supplementary Fig. 13f).”

May combine two claims.

Exposure to dying cells is associated with time-dependent induction of cerebellar microglia-enriched transcription factors and down-regulation of striatal microglia-enriched homeostatic surveillance genes in the absence of pro-inflammatory gene activation.

“This exposure to dying cells was associated with the time-dependent induction of cbMg-enriched transcription factors (Fig. 3c, Supplementary Fig. 10c) as well as the down-regulation of stMg enriched homeostatic surveillance genes (Fig. 3c, Supplementary Fig. 10d,e) in the absence of pro-inflammatory gene activation (Supplementary Fig. 10f).”

May combine two claims.

Deficiency in Eed and loss of H3K27me3 led to up-regulation of known PRC2 targets such as Tbx15 and Hoxd8, but did not affect expression of genes defining microglia lineage identity or genes characteristic of pro-inflammatory activation.

“The deficiency in Eed and the ensuing loss of H3K27me3 led to the up-regulation of several known PRC2 targets, such as Tbx15 and Hoxd830,31 (Fig. 5g,h, Supplementary Fig. 11f,g, Supplementary Table 8,9), but did not affect the expression of genes that define microglia lineage identity (Supplementary Fig. 11f,g, Supplementary Table 8,9) or genes characteristic of microglia pro-inflammatory”

• cites [30] Jadhav, U. et al. Acquired tissue-specific promoter bivalency is a basis for PRC2 necessity in adult cells. Cell 165, 1389–1400 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [31] von Schimmelmann, M. et al. Polycomb repressive complex 2 (PRC2) silences genes responsible for neurodegeneration. Nat. Neurosci. 19, 1321–1330 (2016). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Loss of PRC2 in cerebellar microglia, while following a similar temporal pattern of progressive PRC2 target gene induction, is associated with up-regulation of selected striatal microglia-enriched genes and down-regulation of cerebellar microglia-enriched genes.

“Accordingly, loss of PRC2 in cbMg, while following a similar temporal pattern of progressive PRC2 target gene induction (Fig. 5f, Supplementary Fig. 11e), is associated with the up-regulation of selected stMg-enriched genes and the down-regulation of cbMg-enriched genes (Fig. 6d,e).”

May combine two claims.

These transcription factors are also induced upon exposure to apoptotic cells as well as in Eed-deficient striatal microglia, which acquire an aberrant clearance phenotype.

“These transcription factors are also induced upon exposure to apoptotic cells as well as in the Eed-deficient stMg, which acquire an aberrant clearance phenotype (Supplementary Fig. 14b).”

May combine two claims.

Both deletion of microglia and attenuation of their cell clearance capability are associated with accumulation of dying cells in the cerebellum but not in the striatum or cortex.

“Accordingly, we found that both the deletion of microglia as well as the attenuation of their cell clearance capability are associated with the accumulation of dying cells in the cerebellum but not in the striatum or the cortex.”

May combine two claims.

When forebrain microglia are exposed to dying cells in culture, expression of the H3K27me3 demethylases Kdm6a/b rises and cbMg-enriched clearance genes become progressively activated.

“Moreover, exposure of forebrain microglia to dying cells in vitro increases the expression of the H3K27me3-specific demethylases, Kdm6a/b, and is associated with the progressive activation of cbMg-enriched clearance genes.”

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

All single cells expressed at least one of Cx3cr1, Csf1r, Hexb, or Tmem119.

“All cells were positive for Cx3cr1, Csf1r, Hexb, or Tmem119.”

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

After staurosporine treatment, about 70% of Jurkat cells stained positive for phosphatidylserine and about 95% were negative for propidium iodide, marking most as early apoptotic.

“Staurosporine-treated Jurkat cells were ~70% positive for phosphatidylserine staining and ~95% negative for propidium iodide staining identifying the major population as early apoptotic (Supplementary Fig. 7e).”

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

The Shapiro-Wilk test gave a p-value below 0.0001 for every sample.

“SW p-value was < 0.0001 for all samples.”

May combine two claims.

Microglia clearance activity in the adult brain is regionally regulated and depends on the rate of neuronal attrition.

“Here we show that microglia clearance activity in the adult brain is regionally regulated and depends on the rate of neuronal attrition.”

May combine two claims.

The cerebellar microglia phenotype resembles microglia during early brain development and microglia associated with neurodegenerative disease.

“The cerebellar microglia phenotype is reminiscent of microglia during early brain development9,10 as well as microglia associated with neurodegenerative disease11–13.”

• cites [9] Mass, E. et al. Specification of tissue-resident macrophages during organogenesis. Science 353, aaf4238 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [10] Matcovitch-Natan, O. et al. Microglia development follows a stepwise program to regulate brain homeostasis. Science 353, aad8670 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [11] Mathys, H. et al. Temporal tracking of microglia activation in neurodegeneration at single-cell resolution. Cell Rep. 21, 366–380 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [12] Wang, Y. et al. TREM2 lipid sensing sustains the microglial response in an Alzheimer’s disease model. Cell 160, 1061–1071 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [13] 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

  Not checked in the free run.

Classified as probably not a substantial claim.

Development and ageing of the brain are associated with attrition and loss of neurons.

“Development and ageing of the brain are associated with the attrition and loss of neurons1.”

• cites [1] Dickstein, D. L., Weaver, C. M., Luebke, J. I. & Hof, P. R. Dendritic spine changes associated with normal aging. Neuroscience 251, 21–32 (2013). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The cerebellum displays the most pronounced age-dependent neuronal loss, with neuronal numbers starting to decline at the end of adolescence.

“One of the brain regions displaying the most pronounced age-dependent neuronal loss is the cerebellum, where neuronal numbers start declining at the end of adolescence3,4.”

• cites [3] Woodruff-Pak, D. S. et al. Differential effects and rates of normal aging in cerebellum and hippocampus. Proc. Natl Acad. Sci. USA 107, 1624–1629 (2010). sourceNot checked yet

  Not checked in the free run.

• cites [4] Morterá, P. & Herculano-Houzel, S. Age-related neuronal loss in the rat brain starts at the end of adolescence. Front. Neuroanat. 6, 45 (2012). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The microglia-specific TRAP approach allows region-specific analysis of microglia-enriched mRNA expression and precludes nonspecific microglia activation and concurrent up-regulation of immediate early and pro-inflammatory genes that occur during commonly used microglia isolation approaches.

“Importantly, we show that the TRAP approach not only allows for the region-specific analysis of microglia-enriched mRNA expression (Supplementary Fig. 3, Supplementary Table 1) but also precludes nonspecific microglia activation and concurrent up-regulation of immediate early and pro-inflammatory genes that occur during commonly used microglia isolation approaches (Supplementary Fig. 4, Supplement”

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

LC3 has a known role in autophagy and has been recently identified as a regulator of rapid apoptotic cell clearance in macrophages in LC3-associated phagocytosis, which occurs in the absence of immune activation.

“In addition to its known role in autophagy22, LC3 has been recently identified as a novel regulator of rapid apoptotic cell clearance in macrophages in a process termed LC3-associated phagocytosis (LAP), which occurs in the absence of immune activation23.”

• cites [22] Galluzzi, L. et al. Molecular definitions of autophagy and related processes. EMBO J. 36, 1811–1836 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [23] Heckmann, B. L., Boada-Romero, E., Cunha, L. D., Magne, J. & Green, D. R. LC3-associated phagocytosis and inflammation. J. Mol. Biol. 429, 3561–3576 (2017). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Age-dependent loss of cerebellar mass and neuronal numbers implies ongoing cell death.

“While the age-dependent loss of cerebellar mass and neuronal numbers3,4 implies ongoing cell death, we could not detect dying cells in the cerebellum of adult wild-type mice.”

• cites [3] Woodruff-Pak, D. S. et al. Differential effects and rates of normal aging in cerebellum and hippocampus. Proc. Natl Acad. Sci. USA 107, 1624–1629 (2010). sourceNot checked yet

  Not checked in the free run.

• cites [4] Morterá, P. & Herculano-Houzel, S. Age-related neuronal loss in the rat brain starts at the end of adolescence. Front. Neuroanat. 6, 45 (2012). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The behavioral changes in mutant mice are unlikely due to a general loss of microglia function but rather reflect aberrant microglia clearance activity in the striatum and cortex.

“which indicates that the behavioral changes are unlikely due to a general loss of microglia function but rather reflect the aberrant microglia clearance activity in the striatum and cortex.”

Classified as probably not a substantial claim.

Microglia-specific Eed ablation was induced by tamoxifen treatment between 4-6 weeks of age to exclude any impact of PRC2 inactivation on microglia development.

“Microglia-specific Eed ablation was induced by tamoxifen treatment between 4-6 weeks of age in order to exclude any impact of PRC2 inactivation on microglia development (Fig. 5a).”

Classified as probably not a substantial claim.

KDM6A/B mediate removal of the suppressive chromatin modification H3K27me3, which is associated with silenced genes.

“KDM6A/B mediate the removal of the suppressive chromatin modification H3K27me3, which is associated with silenced genes26.”

• cites [26] Swigut, T. & Wysocka, J. H3K27 demethylases, at long last. Cell 131, 29–32 (2007). sourceNot checked yet

  Not checked in the free run.

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

Induction of behavioral locomotor sensitization in response to cocaine depends on structural plasticity of medium spiny neurons and is associated with lasting changes in medium spiny neuron spine density.

“The induction of behavioral locomotor sensitization in response to cocaine depends on the structural plasticity of MSNs and is associated with lasting changes in MSN spine density32.”

• cites [32] Nestler, E. J. The neurobiology of cocaine addiction. Sci. Pract. Perspect. 3, 4–10 (2005). sourceNot checked yet

  Not checked in the free run.

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

Disorders accompanied by alterations in spine numbers, including neurodegenerative and neuropsychiatric disorders, are frequently associated with development of anxiety and seizures and cognitive decline.

“Disorders accompanied by alterations in spine numbers, including neurodegenerative and neuropsychiatric disorders, are frequently associated with the development of anxiety and seizures33 and cognitive decline34.”

• cites [33] Friedman, D., Honig, L. S. & Scarmeas, N. Seizures and epilepsy in Alzheimer’s disease. CNS Neurosci. Ther. 18, 285–294 (2012). sourceNot checked yet

  Not checked in the free run.

• cites [34] Hong, S. et al. Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science 352, 712–716 (2016). sourceNot checked yet

  Not checked in the free run.

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

H3K27me3 is mediated by PRC2, which has a well-established role in regulation of cell differentiation during development.

“H3K27me3 is mediated by PRC2, which has a well-established role in the regulation of cell differentiation during development16.”

• cites [16] Margueron, R. & Reinberg, D. The polycomb complex PRC2 and its mark in life. Nature 469, 343–349 (2011). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Enrichment of mRNAs encoding KDM6A/B in cerebellar microglia and up-regulation of Kdm6a/b upon exposure to dying cells in vitro point to a possible role of PRC2/H3K27me3 in suppressing clearance-promoting genes in striatal microglia.

“Enrichment of mRNAs encoding the H3K27me3-specific demethylases, KDM6A/B26 in cbMg (Supplementary Fig. 5b, Supplementary Table 3) as well as the up-regulation of Kdm6a/b in microglia upon exposure to dying cells in vitro (Fig. 3c, Supplementary Fig. 10g) point to the possible role of PRC2/H3K27me3 in suppressing clearance-promoting genes in stMg.”

• cites [26] Swigut, T. & Wysocka, J. H3K27 demethylases, at long last. Cell 131, 29–32 (2007). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

PRC2 has a largely selective role in maintenance of regional microglia specification in the adult brain, where it controls cell-intrinsic suppression of an aberrant pro-phagocytic/clearance-like phenotype in striatal microglia in vivo.

“Collectively, our data point to a largely selective role of PRC2 in the maintenance of regional microglia specification in the adult brain, where it controls the cell-intrinsic suppression of an aberrant pro-phagocytic/clearance-like phenotype in stMg in vivo.”

May combine two claims.

H3K27me3 may causally repress genes that promote clearance in microglia, with support from multiple lines of evidence.

“The possible causal role of H3K27me3 in suppressing clearance-promoting genes in microglia is supported at several levels.”

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

Microglia can sense and clear apoptotic cells at the earliest stages of apoptosis.

“Recent evidence suggests that microglia can sense and clear apoptotic cells at the earliest stages of apoptosis25.”

• cites [25] Fourgeaud, L. et al. TAM receptors regulate multiple features of microglial physiology. Nature 532, 240–244 (2016). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Higher MEK-ERK pathway activity has been associated with overexpression of Ezh2.

“On the other hand, increased activation of the MEK-ERK pathway has been linked to Ezh2 overexpression48.”

• cites [48] Fujii, S. et al. RAS oncogenic signal upregulates EZH2 in pancreatic cancer. Biochem. Biophys. Res. Commun. 417, 1074–1079 (2012). sourceNot checked yet

  Not checked in the free run.

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

Efferocytosis is a multi-step process comprising recognition of eat-me signals, engulfment of cells/debris, degradation of engulfed material, and excretion of metabolic products.

“This process, often referred to as efferocytosis, is a multi-step process: (i) the recognition of “eat-me” signals presented by apoptotic cells, (ii) engulfment of cells/debris, (iii) degradation of engulfed material, and (iv) excretion of metabolic products21.”

• cites [21] Blander, J. M. The many ways tissue phagocytes respond to dying cells. Immunol. Rev. 277, 158–173 (2017). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

In ES cells, reduced PRC2 activity accompanies lower MEK-ERK activation, while blocking MEK-ERK weakens PRC2 binding to its target genes.

“While the loss of PRC2 activity is associated with reduced MEK-ERK activation in ES cells47, the inhibition of MEK-ERK activity leads to attenuated PRC2 chromatin association on PRC2 target genes in these cells47.”

• cites [47] Tee, W.-W., Shen, S. S., Oksuz, O., Narendra, V. & Reinberg, D. Erk1/2 activity promotes chromatin features and RNAPII phosphorylation at developmental promoters in mouse ESCs. Cell 156, 678–690 (2014). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Although clearance by microglia is needed to remove dying cells, it requires tight regulation in other brain areas so that healthy neurons are not damaged by microglia.

“Our data also show that microglia clearance function, while important for the removal of dying cells, must be tightly controlled in other brain regions to avoid microglia-mediated damage of healthy neurons.”

May combine two claims.

Because cbMg show little inflammatory gene activation, their clearance activity may oppose or suppress inflammatory gene activation, similar to peripheral macrophages.

“The lack of inflammatory gene activation in cbMg suggests that the microglia clearance activity may antagonize/suppress the activation of inflammatory genes in a fashion that has been observed in peripheral macrophages21,23.”

• cites [21] Blander, J. M. The many ways tissue phagocytes respond to dying cells. Immunol. Rev. 277, 158–173 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [23] Heckmann, B. L., Boada-Romero, E., Cunha, L. D., Magne, J. & Green, D. R. LC3-associated phagocytosis and inflammation. J. Mol. Biol. 429, 3561–3576 (2017). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

When the RAF-MEK-ERK pathway is activated cell-autonomously in microglia, synapses are lost and a severe late-onset neurodegenerative disorder develops.

“The ability of aberrant intrinsically activated microglia to cause neuronal damage is further supported by recent findings showing that cell-autonomous activation of the RAF–MEK–ERK pathway in microglia leads to the loss of synapses and the development of a severe late-onset neurodegenerative disorder7.”

• cites [7] Mass, E. et al. A somatic mutation in erythro-myeloid progenitors causes neurodegenerative disease. Nature 549, 389–393 (2017). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Most newly generated neurons/T-cells undergo apoptotic cell death, and dying cells cannot be readily detected due to rapid removal by resident macrophages/microglia.

“While most of the newly generated neurons/T-cells undergo apoptotic cell death, the dying cells cannot be readily detected due to their rapid removal by resident macrophages/microglia25,39.”

• cites [25] Fourgeaud, L. et al. TAM receptors regulate multiple features of microglial physiology. Nature 532, 240–244 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [39] Dzhagalov, I. L., Chen, K. G., Herzmark, P. & Robey, E. A. Elimination of self-reactive T cells in the thymus: a timeline for negative selection. PLoS Biol. 11, e1001566 (2013). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

Work with a small-molecule inhibitor of KDM6A/B demethylase activity indicated that H3K27 demethylation, specifically KDM6A/B catalysis, is required for pro-inflammatory gene activation in human primary macrophages.

“recent studies using a small molecule-based inhibitor specifically targeting KDM6A/B demethylase activity revealed that H3K27 demethylation, and more specifically KDM6A/B catalytic activity, are critical determinants of pro-inflammatory gene activation in human primary macrophages28.”

• cites [28] Kruidenier, L. et al. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature 488, 404–408 (2012). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

It is plausible that microglial clearance gene expression depends on KDM6A/B and H3K27me3, and that turning on these genes involves local decreases in H3K27me3 at their loci in vivo.

“Based on these data, it is plausible that clearance gene expression in microglia is controlled in a KDM6A/B/H3K27me3-dependent fashion and that the induction of clearance genes is associated with a targeted reduction in H3K27me3 levels at specific clearance gene loci in vivo.”

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

All behavioral subjects were within two standard deviations of the sample mean, and none were excluded.

“All subjects correspond to data points within two standard deviations from the sample mean, and no subjects were excluded from the behavioral analyses.”

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

ChIP-seq data quality was checked using FastQC metrics.

“The ChIP-seq data was first checked for quality using the various metrics generated by FastQC (v0.11.2; http://www.bioinformatics.babraham.ac.uk/projects/fastqc).”

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

No statistical method was used to pre-determine the sample sizes.

“No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications19,31,95,99,108.”

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

Behavioral experiments used sex- and age-matched littermate controls.

“For all behavioral experiments, a mixed population of males and females with sex- and age-matched corresponding littermate control animals were used.”

May combine two claims.

The Cx3cr1CreErt2/+(Jung) line was dropped because it targeted microglia poorly, while Vav1-iCre, Csf1r-iCre, and Csf1r-Cre/Esr1* lines were dropped because they targeted neurons.

“Tg(Vav1-iCre), Tg(Csf1r-iCre), and Tg(Csf1r-Cre/Esr1*) lines were excluded due to neuronal targeting, while the Cx3cr1CreErt2/+(Jung) line was excluded due to insufficient targeting of microglia.”

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

Outliers were further justified using quality control metrics and experimental metadata.

“and the outliers were further justified by the aforementioned quality control metrics as well as experimental metadata.”

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

No randomization protocol was used in the behavioral experiments.

“No randomization protocol was used.”

Classified as probably not a substantial claim.

The authors targeted exon 6 because a prior report showed that a one-base mutation there prevents EED from interacting with EZH1/2 and produces an Eed-null phenotype.

“In brief, exon 6 was chosen for gene deletion as a single base pair mutation in this exon was previously shown to disrupt the interaction of EED with EZH1/2 proteins, thus resulting in an Eed-null phenotype61,62.”

• cites [61] Schumacher, A., Faust, C. & Magnuson, T. Positional cloning of a global regulator of anterior-posterior patterning in mice. Nature 384, 648 (1996). sourceNot checked yet

  Not checked in the free run.

• cites [62] Denisenko, O., Shnyreva, M., Suzuki, H. & Bomsztyk, K. Point mutations in the WD40 domain of Eed block its interaction with Ezh2. Mol. Cell. Biol. 18, 5634–5642 (1998). sourceNot checked yet

  Not checked in the free run.

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

Single-nuclei library pool quality was checked with a 2200 TapeStation.

“The quality of the library pools was assessed by 2200 TapeStation (Agilent).”

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

Normality of the samples was tested with the Shapiro-Wilk test.

“Normal distribution was assessed by the Shapiro-Wilk (SW) normality test.”

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

Plates were included as covariates in the generalized linear model to account for individual confounding.

“We included plates as covariates to account for confounding effect of individuals in the generalized linear model”

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

Samples with RIN above 9 were the only ones used in downstream analysis.

“only samples with RIN>9 were considered for subsequent analysis.”

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

Every experimental mouse had been backcrossed onto C57Bl/6 for five or more generations.

“All mice used for experiments were backcrossed to the C57Bl/6 background for ≥ 5 generations.”

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

Data collection was randomized for all experiments.

“The data collection was randomized for all experiments.”

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

The sample sizes used in the study were similar to those reported in previous publications.

“No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications19,31,95,99,108.”

• cites [19] Heiman, M. et al. A translational profiling approach for the molecular characterization of CNS cell types. Cell 135, 738–748 (2008). sourceNot checked yet

  Not checked in the free run.

• cites [31] von Schimmelmann, M. et al. Polycomb repressive complex 2 (PRC2) silences genes responsible for neurodegeneration. Nat. Neurosci. 19, 1321–1330 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [95] Schaefer, A. et al. Control of cognition and adaptive behavior by the GLP/G9a epigenetic suppressor complex. Neuron 64, 678–691 (2009). sourceNot checked yet

  Not checked in the free run.

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

The quasi-likelihood framework in edgeR was used to handle the pronounced variability of single-cell expression states.

“and the quasi-likelihood framework within edgeR to handle the pronounced variability of the single cell expression states.”

• cites [79] Robinson, M. D. & Smyth, G. K. Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics 9, 321–332 (2008). sourceNot checked yet

  Not checked in the free run.

• cites [80] Robinson, M. D. & Smyth, G. K. Moderated statistical tests for assessing differences in tag abundance. Bioinformatics 23, 2881–2887 (2007). sourceNot checked yet

  Not checked in the free run.

• cites [81] Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010). sourceNot checked yet

  Not checked in the free run.

• cites [82] McCarthy, D. J., Chen, Y. & Smyth, G. K. Differential expression analysis of multifactor RNA-seq experiments with respect to biological variation. Nucleic Acids Res. 40, 4288–4297 (2012). sourceNot checked yet

  Not checked in the free run.

• cites [83] Zhou, X., Lindsay, H. & Robinson, M. D. Robustly detecting differential expression in RNA sequencing data using observation weights. Nucleic Acids Res. 42, e91 (2014). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

The Prox1-Cre line served as a second TRAP line to check that regional expression differences did not arise from Cx3cr1 haploinsufficiency in the Cx3cr1CreErt2/+(Litt) mice.

“This line was used as an alternative TRAP line to ensure that region-specific gene expression changes are not caused by Cx3cr1 haplo-insufficiency in the Cx3cr1CreErt2/+(Litt) mice.”

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

For ChIP-seq alignments, only uniquely mapped reads were kept and duplicate reads were removed.

“Only uniquely mapped reads were retained, and the alignments were subsequently filtered using the SAMtools package (v0.1.19)88 to remove duplicate reads.”

• cites [88] Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009). sourceNot checked yet

  Not checked in the free run.

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

Jurkat cell cultures were routinely confirmed mycoplasma-free.

“Cultures were regularly tested negative for mycoplasma using Lonza kit according to manufacturer’s instructions (MycoAlert LT-07).”

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

Single-nuclei cells with fewer than 100,000 mapped reads were excluded.

“Cells with fewer than 100,000 mapped reads were removed.”

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

During imaging and behavioral experiments, the experimenters were blinded.

“Experimenters were blinded during imaging and behavioral experiments.”

May combine two claims.

The Cx3cr1CreErt2/+(Litt) line was chosen for later experiments because its Cre expression is microglia-specific and highly efficient (>99% of microglia) across the brain.

“The Cx3cr1CreErt2/+(Litt)52 line was selected for all subsequent experiments due to its highly efficient (>99% of microglia) and microglia-specific Cre expression pattern throughout the brain.”

• cites [52] Parkhurst, C. N. et al. Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor. Cell 155, 1596–1609 (2013). sourceNot checked yet

  Not checked in the free run.

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

Cytochalasin D-pretreated cells served as a negative control in the phagocytosis assay.

“Cells pretreated with 2 μM Cytochalasin D (Sigma-Aldrich, C8273) for 30 min before and during the incubation of microglia with pHrodo-EAJ were used as a negative control.”

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

In imaging data, gender ratios and ages were matched between control and treatment groups.

“Gender ratios and ages were matched within control and treatment groups.”

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

A negative binomial model in edgeR was used to account for the sparse single-cell count matrix.

“We used the negative binomial (NB) model implemented in edgeR (3.10.0)79–83 for our single cell differential expression analysis to explicitly account for the sparse count matrix”

• cites [79] Robinson, M. D. & Smyth, G. K. Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics 9, 321–332 (2008). sourceNot checked yet

  Not checked in the free run.

• cites [80] Robinson, M. D. & Smyth, G. K. Moderated statistical tests for assessing differences in tag abundance. Bioinformatics 23, 2881–2887 (2007). sourceNot checked yet

  Not checked in the free run.

• cites [81] Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010). sourceNot checked yet

  Not checked in the free run.

• cites [82] McCarthy, D. J., Chen, Y. & Smyth, G. K. Differential expression analysis of multifactor RNA-seq experiments with respect to biological variation. Nucleic Acids Res. 40, 4288–4297 (2012). sourceNot checked yet

  Not checked in the free run.

• cites [83] Zhou, X., Lindsay, H. & Robinson, M. D. Robustly detecting differential expression in RNA sequencing data using observation weights. Nucleic Acids Res. 42, e91 (2014). sourceNot checked yet

  Not checked in the free run.

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

Antibody staining controls omitted the primary antibody incubation.

“For antibody staining controls, primary antibody incubation was omitted, and instead slides were incubated with Alexa Fluor-conjugated secondary antibodies”

Classified as probably not a substantial claim.

In TRAP experiments on Eed-deficient microglia, the fl/fl mice were compared with sex- and age-matched Eedfl/+ littermates.

“For TRAP from Eed-deficient microglia, Cx3cr1CreErt2/+(Litt);Eef1a1LSL.eGFPL10a/+;Eedfl/fl mice were compared to Cx3cr1CreErt2/+(Litt);Eef1a1LSL.eGFPL10a/+;Eedfl/+ sex- and age-matched littermate controls.”

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

EZH2 that is abnormally active can cooperate with hyperactive BRAF to promote tumorigenesis.

“Furthermore, aberrantly activated EZH2 was recently found to synergistically cooperate with hyperactive BRAF in the regulation of tumorigenesis49.”

• cites [49] Souroullas, G. P. et al. An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation. Nat. Med. 22, 632–640 (2016). sourceNot checked yet

  Not checked in the free run.

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

Animals were allocated to treatment groups so that ages and sexes were uniformly distributed.

“Animals were allocated to treatment groups to ensure uniform distribution of ages and sexes in each group.”

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

Hierarchical clustering was also used to check outliers, guarding against PCA false positives or negatives.

“Additionally, hierarchical clustering was used to assess the outliers once again to protect against false positives/negatives from PCA”

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

IRES-YFP expression did not reduce the efficiency of microglial TRAP.

“The expression of IRES-YFP did not interfere with microglia TRAP efficiency.”

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

Principal component analysis of the 500 most variable genes was used to visually identify outliers.

“Principal component analysis (PCA) was performed on the top 500 most variable genes across all samples based on the VST data to visually assess if there were any outliers.”

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

Cx3cr1+ perivascular/meningeal macrophages, identified by low Tmem119, low Hexb, and high Mrc1, were excluded from single-cell analysis.

“Cx3cr1+ perivascular/meningeal macrophages were identified by low Tmem119, low Hexb, and high Mrc1 expression35,84 and excluded from single cell analysis (n=1-5 per plate).”

• cites [35] Goldmann, T. et al. Origin, fate and dynamics of macrophages at central nervous system interfaces. Nat. Immunol. 17, 797–805 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [84] Bennett, M. L. et al. New tools for studying microglia in the mouse and human CNS. Proc. Natl. Acad. Sci. USA 113, E1738–E1746 (2016). sourceNot checked yet

  Not checked in the free run.

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

The analyses of gene expression and protein were not blinded to the experimental conditions.

“Gene expression and protein analyses were not performed blind to the conditions of the experiments.”

Classified as probably not a substantial claim.

Eed-deficient microglia comparisons used age- and sex-matched control mice.

“Comparisons for Cx3cr1CreErt2/+(Litt);Eef1a1LSL.eGFPL10a/+;Eedfl/fl mice were carried out with age- and sex-matched control Cx3cr1CreErt2/+(Litt);Eef1a1LSL.eGFPL10a/+;Eedfl/+ mice.”

May combine two claims.

More EZH2 expression or activity accompanies a genome-wide shift in H3K27me3, with H3K27me3 loss and activation of many PRC2-repressed genes.

“Increased EZH2 expression/activity is associated with a genome-wide redistribution of H3K27me3 that includes the loss of H3K27me3 and the transcriptional activation of numerous PRC2 suppressed genes in these cells49.”

• cites [49] Souroullas, G. P. et al. An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation. Nat. Med. 22, 632–640 (2016). sourceNot checked yet

  Not checked in the free run.

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

Wild-type C57Bl/6J mice served to assess nonspecific mRNA enrichment in TRAP.

“C57Bl/6J wild-type mice were used to test for nonspecific mRNA enrichment.”

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

Quality assessment of bulk RNA-seq data included total mapping rate, mitochondrial mapping rate, and ribosomal mapping rate.

“The read alignment, read counting as well as quality assessment using metrics such as total mapping rate, mitochondrial and ribosomal mapping rates were done in parallel using an in-house workflow pipeline called SPEctRA70.”

• cites [70] Purushothaman, I. & Shen, L. SPEctRA: First stable release. https://zenodo.org/record/60547#.WyweGlVKipp (2016).Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

A cohort of Cx3cr1CreErt2/+ and wild-type littermate controls was tested to rule out behavioral effects from Cx3cr1 haploinsufficiency.

“A cohort of Cx3cr1CreErt2/+ and their respective Cx3cr1+/+ wild-type littermate controls were tested on selected behavior paradigms to exclude potential behavioral alterations due to Cx3cr1 haploinsufficiency in Cx3cr1CreErt2/+; Eedfl/fl mice.”