Complement C3aR Inactivation Attenuates Tau Pathology and Reverses an Immune Network Deregulated in Tauopathy Models and Alzheimer’s Disease

May combine two claims.

In PS19 tau transgenic mice, brain C3ar1 mRNA levels are comparable to wild-type at 4 months, significantly upregulated at 6 months, and further elevated at 9 months.

“Although the expression levels of C3 and C3ar1 were comparable between PS19 mice and littermate wild-type (WT) controls at 4 months, their levels were significantly upregulated at 6 months and were further elevated at 9 months (Figures 1G and 1H).”

May combine two claims.

In 9-month PS19 mice, C3aR co-localizes with Iba1 and shows stronger intensity in hippocampus compared to wild-type controls.

“Double immunostaining using anti-C3aR and anti-Ibal antibodies documented a high degree of co-localization and stronger intensity in the hippocampus of 9 month-old PS19 mice compared to WT controls (Figures 1J and 1K), indicating that increased C3aR expression in PS19 mice predominantly originates from microglia.”

May combine two claims.

In PS19 tau transgenic mice, brain C3 mRNA levels are comparable to wild-type at 4 months, significantly upregulated at 6 months, and further elevated at 9 months.

“Although the expression levels of C3 and C3ar1 were comparable between PS19 mice and littermate wild-type (WT) controls at 4 months, their levels were significantly upregulated at 6 months and were further elevated at 9 months (Figures 1G and 1H).”

May combine two claims.

In PS19 mice, astrocytes and microglia show increased volumes and reduced branching and complexity, and these phenotypes are corrected by C3aR knockout.

“Quantitative analysis of astrocyte and microglia morphologies using the Imaris software showed increased volumes and reduced branching and complexity in both cell types of PS19 mice, and these phenotypes were corrected by C3aR KO (Figure S2).”

May combine two claims.

In PS19 mice, brain TNFα levels are elevated, and C3ar1 inactivation significantly reduces them.

“All three cytokines were elevated in brain tissues of PS19 mice, where inactivation of C3ar1 resulted in a significant dampening of the cytokine levels.”

May combine two claims.

C3ar1 ablation in PS19 mice leads to robust rescue of neurodegeneration, with insignificant differences between PS19/C3aR KO and WT controls.

“C3ar1 ablation in PS19 mice led to a robust rescue of neurodegeneration, evidenced by the insignificant differences between PS19/C3aR KO and WT controls (Figure 4H).”

May combine two claims.

In PS19 mice, pre-, post-, and double-positive synaptic puncta are reduced, and synapse loss is significantly restored by C3aR KO.

“Similarly, co-immunostaining of Syp with postsynaptic marker PSD-95 revealed reduced pre-, post-, and double-positive synaptic puncta in PS19 mice, and the synapse loss was significantly restored by C3aR KO (Figures 4C and 4D; Figure S3D-S3G).”

May combine two claims.

In 9-month PS19 mice, C3 intensity in GFAP-positive astrocytes is drastically increased and almost completely abolished by C3ar1 deletion.

“Consistent with the elevated C3 mRNA and protein levels in 9 month-old PS19 mice (Figures 1G and 1I), we detected a drastic increase of C3 intensity in GFAP-positive astrocytes in PS19 mice that was almost completely abolished by C3ar1 deletion (Figures 2D and 2E).”

May combine two claims.

Comparison of PS19/C3aR KO to WT identified only 317 DEGs (137 up, 180 down), implying these two groups are closer in expression profiles than PS19 vs WT.

“Indeed, comparison of PS19/C3aR KO to WT mice identified only 317 DEGs (137 up and 180 down, Figure S4B), implying that these two groups of animals are much closer in expression profiles than PS19 vs WT.”

May combine two claims.

PS19 mice show a reduced contextual fear response, and C3aR KO significantly rescues it.

“We observed a reduction in contextual, but not cued fear response in PS19 mice, and a significant rescue in PS19/C3aR KO animals (Figure 3E), suggesting that C3aR KO is protective against the hippocampal-dependent memory impairment.”

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Unsupervised clustering showed WT and C3aR KO cluster together while PS19/C3aR KO group between PS19 and WT/C3aR KO, indicating partial rescue of PS19 DEGs by C3ar1 deletion.

“Using unsupervised clustering analysis, we next examined the expression profiles of the 1726 DEGs across the four genotypes and found that the WT and C3aR KO are clustered together while the PS19/C3aR KO mice grouped in between PS19 and WT/C3aR KO animals (Figure 5A), indicating a partial rescue of PS19 DEGs by C3ar1 deletion.”

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Of these, 301 genes positively correlated with C3aR1 in human AD datasets were downregulated in PS19/C3aR KO; seven were negatively correlated and upregulated; three were negatively correlated but not rescued.

“Of these, 301 genes positively correlated with C3aR1 expression in the human AD datasets were found downregulated in PS19/C3aR KO mice (red circle in the network), seven were negatively correlated with C3AR1 human AD database and upregulated in PS19/C3aR KO (blue circles in network), and only three were negatively correlated but not rescued in PS19/C3aR KO (pink circles in the network).”

May combine two claims.

LTP induction in the Schaffer collateral pathway is significantly reduced in PS19 mice compared to WT and C3aR KO, and this phenotype is significantly improved in PS19/C3aR KO mice.

“The LTP induction was significantly reduced in PS19 mice compared to WT and C3aR KO groups, and this phenotype was significantly improved in PS19/C3aR KO mice (Figure 3F).”

May combine two claims.

In contrast to bulk brain, no significant changes in Aif1 or Cd68 expression were observed in purified microglia from PS19 mice, indicating elevated Aif1 in bulk brain may be attributed to increased microglia numbers.

“In contrast to the bulk brain result, we did not observe significant changes in Aif1 or Cd68 expression in purified microglia from PS19 mice (Figure 6D), indicating that elevated Aif1 expression in the bulk brain may be attributed by increased microglia numbers.”

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In PS19 mice, brain IL1β levels are elevated, and C3ar1 inactivation significantly reduces them.

“All three cytokines were elevated in brain tissues of PS19 mice, where inactivation of C3ar1 resulted in a significant dampening of the cytokine levels.”

May combine two claims.

In PS19 mice, brain IL6 levels are elevated, and C3ar1 inactivation significantly reduces them.

“All three cytokines were elevated in brain tissues of PS19 mice, where inactivation of C3ar1 resulted in a significant dampening of the cytokine levels.”

May combine two claims.

RNAseq identified 1726 differentially expressed genes in PS19 mice compared to WT controls, of which 1155 were upregulated and 571 downregulated.

“After removal of outliers identified by principal component analysis (PCA) (Figure S4A), we identified 1726 differentially expressed genes (DEGs, adjusted-p<0.01) in PS19 mice compared to WT controls, of which 1155 genes were upregulated and 571 downregulated (Figure S4B).”

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

Of the 27 downregulated genes rescued by C3aR KO, GO term analysis failed to reveal significant enrichment of any particular pathways.

“Of the 27 downregulated genes that were rescued by C3aR KO, GO term analysis failed to reveal a significant enrichment of any particular pathways.”

May combine two claims.

GSEA showed microglia sensome and reactive astrocyte gene sets were highly enriched in PS19 mice and rescued almost to the same degree in PS19/C3aR KO animals.

“We found that both gene sets were highly enriched in PS19 mice (Figures 6A and 6B, PS19 vs WT), and rescued almost to the same degree in PS19/C3aR KO animals (Figures 6A and 6B, PS19/C3aR KO vs PS19).”

May combine two claims.

RPPA identified total- and phospho-STAT3 (pY705) as upregulated in PS19 mice and rescued by C3aR KO.

“Indeed, independent proteomics analysis using the reverse-phase protein array (RPPA) platform identified total- and phospho-STAT3 (pY705) as a candidate upregulated in PS19 mice and subsequently rescued by C3aR KO (Figures 7A and 7B).”

May combine two claims.

Similar upregulation of the TFs was observed in human tauopathy brain samples, indicating inflammatory and glial differentiation transcriptional programs are conserved in mouse and human tauopathy conditions.

“Interestingly, we observed similar upregulation of the TFs in human tauopathy brain samples (Figure S7C), indicating that the inflammatory and glial differentiation transcriptional programs are conserved in mouse and human tauopathy conditions.”

May combine two claims.

Western blot analysis confirmed these results, and nuclear pSTAT3 staining appeared in astrocytes and microglia of aged PS19 mice.

“These results were further confirmed by Western blot analysis of brain lysates (Figures 7C-7E), and corroborated by the appearance of nuclear pSTAT3 staining in astrocytes and microglia of aged PS19 mice (Figure S8A).”

May combine two claims.

SH treatment in PS19 mice significantly reduced reactive astrogliosis and microgliosis as well as diminished pSTAT3 staining in hippocampus.

“Immunostaining analysis using antibodies against pSTAT3, GFAP, and Iba1 demonstrated a significant reduction of reactive astrogliosis and microgliosis as well as diminished pSTAT3 staining in the hippocampus of SH-treated PS19 mice (Figures 8E-8G).”

May combine two claims.

In primary microglial cultures, IL6-induced pSTAT3 and SOCS3 expression occurred in both WT and C3aR KO microglia, while C3-mediated pSTAT3 and SOCS3 were only observed in WT, not in C3ar1-deficient microglia.

“Specifically, while IL6-induced pSTAT3 and SOCS3 expression in both WT and C3aR KO microglia (Figures 7K-7N, IL6), the C3-mediated pSTAT3 and SOCS3 were only observed in WT (Figures 7K and 7M, C3), but not in C3ar1-deficient microglia (Figures 7L and 7N, C3).”

May combine two claims.

Expression of all 11 TFs upregulated in PS19 mice can be readily detected in microglia preparations, while only a subset (Stat3, Stat5b, Nfkb1, Nfe2I2, Fli1, Pax6) were expressed in sorted astrocytes, suggesting microglia play a primary role in mediating transcriptional changes downstream of C3aR.

“We found that the expression of all 11 TFs upregulated in PS19 mice can be readily detected in microglia preparations (Figure 6G), while only a subset (Stat3, Stat5b, Nfkb1, Nfe2I2, Fli1 and Pax6) were expressed in sorted astrocytes (Figure 6H), suggesting that microglia play a primary role in mediating the transcriptional changes in PS19 mice downstream of C3aR.”

May combine two claims.

Of these, only Stat3 expression was rescued by C3aR KO in bulk brain samples as well as in purified microglia and astrocytes, indicating STAT3 may be a specific target of C3aR.

“Of these only Stat3 expression was rescued by C3aR KO in bulk brain samples as well as in purified microglia (Figure 6G) and astrocytes (Figure 6H), indicating that STAT3 may be a specific target of C3aR.”

May combine two claims.

SH treatment in PS19 mice reduced PHF1- and CP13-positive phospho-tau proteins and PHF1 and MC1 immunoreactivities in hippocampus and cortex.

“This is accompanied by reduced PHF1- and CP13-positive phospho-tau proteins (Figures 8A and 8B) and PHF1 (Figures 8C and 8D) and MC1 (Figures S8J and S8K) immunoreactivities in the hippocampus (HPC) and cortex (CTX) of PS19 mice.”

May combine two claims.

Increased luciferase reporter activity and SOCS3 mRNA levels were observed in both IL6- and C3-treated cells, and C3 effects were blocked by C3aRA.

“Consistent with the elevated pSTAT3 protein levels and its nuclear staining, we observed increased luciferase reporter activity (Figure 7I) and SOCS3 mRNA levels (Figure 7J) in both IL6- and C3-treated cells, and the C3 effects were blocked by C3aRA.”

May combine two claims.

These effects were effectively blocked by C3aR antagonist, demonstrating direct regulation of STAT3 pathway by C3aR.

“These effects were effectively blocked by treating the cells with a C3aR antagonist (C3aRA) (Figures 7F-7H), demonstrating a direct regulation of the STAT3 pathway by C3aR.”

May combine two claims.

Compared to PS19-VC, SH-treated PS19 mice exhibited significantly lower pSTAT3 protein levels and SOCS3 mRNA levels.

“Compared to the PS19-VC group, SH-treated PS19 mice exhibited significantly lower pSTAT3 protein levels (Figure 8A and quantified in 8B) as well as SOCS3 mRNA levels (Figure S8I).”

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

STAT5a was not altered across genotypes.

“In contrast, STAT5a was not altered across genotypes (Figure S8E).”

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C3ar1 deficiency and STAT3 inhibitor treatment reverse the elevated Stat3 mRNA and pSTAT3 staining in PS19 mice.

“these phenotypes are reversed by C3ar1 deficiency and by STAT3 inhibitor treatment.”

May combine two claims.

C3 induces STAT3 phosphorylation, nuclear translocation, and SOCS3 transcription in BV2 cells and primary microglial cultures; these effects, but not IL6-induced STAT3 pathway, are abolished by C3aR antagonist treatment and in C3aR KO microglia.

“Although such an indirect mechanism could mediate C3aR-STAT3 interaction, here we establish a direct link between C3-C3aR signaling and STAT3 activation: C3 induces STAT3 phosphorylation, nuclear translocation, and SOCS3 transcription in both BV2 cells and primary microglial cultures; these effects, but not IL6- induced STAT3 pathway, are abolished by C3aR antagonist treatment and in C3aR KO micro”

May combine two claims.

C3 is highly expressed in reactive astrocytes where it marks the A1 neurotoxic subtype implicated in AD and other neurodegenerative disorders.

“Complement C3 is highly expressed in reactive astrocytes where it marks the A1 neurotoxic sub-type implicated in AD and other neurodegenerative disorders (Lian et al., 2016; Liddelow et al., 2017).”

• cites Lian et al., 2016 sourceNot checked yet

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• cites Liddelow et al., 2017 sourceNot checked yet

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

The complement pathway contributes to amyloid pathology in Alzheimer's disease.

“Strong evidence implicates the complement pathway as an important contributor to amyloid pathology in Alzheimer’s disease (AD); however, the role of complement in tau modulation remains unclear.”

• cites Hong et al., 2016 sourceNot checked yet

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• cites Shi et al., 2017 sourceNot checked yet

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May combine two claims.

Genetic deletion of C3ar1 exerts beneficial effects on synaptic plasticity and cognitive function in PS19 mice.

“Combined, these data demonstrate that genetic deletion of C3ar1 exerts beneficial effects on synaptic plasticity and cognitive function in PS19 mice.”

May combine two claims.

These results support conserved changes in C3aR-regulated gene signatures between mice and humans and indicate elevated C3-C3aR signaling may contribute to AD pathogenesis through regulating inflammatory response and immune cell activation.

“Together, these results provide support for conserved changes in the expression of C3aR-regulated gene signatures between mice and humans and indicate that the elevated C3-C3aR signaling may contribute to AD pathogenesis through regulating inflammatory response and immune cell activation.”

May combine two claims.

C3aR ablation ameliorates pathological tau but not total tau.

“Thus, C3aR ablation results in the amelioration of pathological tau but not total tau.”

May combine two claims.

Changes in microglia gene expression in PS19 mice are likely caused by increased cell number and activation status, phenotypes mitigated by C3ar1 ablation.

“Thus, the changes in microglia gene expression in PS19 mice are likely caused by increased cell number and activation status, phenotypes that are mitigated by C3ar1 ablation.”

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

Canonical STAT3 signaling involves cytokine-induced, JAK-dependent STAT3 phosphorylation and nuclear translocation.

“The canonical STAT3 signaling involves cytokine-induced, Janus kinase (JAK)-dependent, STAT3 phosphorylation and nuclear translocation (Shuai and Liu, 2003).”

• cites Shuai and Liu, 2003Not checked yet

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Genetic deletion of C3ar1 leads to attenuation of neuroinflammation, rescue of tau pathology, and amelioration of synaptic impairment and neuronal loss in PS19 mice.

“Genetic deletion of C3ar1 leads to the attenuation of neuroinflammation, rescue of tau pathology, and amelioration of synaptic impairment and neuronal loss in PS19 mice.”

May combine two claims.

This assessment is consistent with microglial C3aR expression pattern and robust rescue of microglia sensome gene expression in PS19/C3aR KO mice.

“This assessment is consistent with the microglial C3aR expression pattern in mouse brains (Figure 1J) and a robust rescue of microglia sensome gene expression in PS19/C3aR KO mice (Figure 6A; Figure S5A).”

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

C3aR promotes viral-induced synapse loss and cognitive decline.

“This idea gained further support by a recent report showing a detrimental effect of C3aR in promoting viral-induced synapse loss and cognitive decline (Vasek et al., 2016).”

• cites Vasek et al., 2016 sourceNot checked yet

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Tau is secreted from neurons and can be taken up by microglia for degradation or transfer to other neurons.

“Second, recent reports indicate that tau is secreted from neurons and can be taken up by microglia for degradation or transfer to other neurons (Asai et al., 2015; Bolos et al., 2016).”

• cites Asai et al., 2015 sourceNot checked yet

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• cites Bolos et al., 2016 sourceNot checked yet

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STATs represent a class of transcriptional factors involved in multiple cellular processes, particularly immune response.

“The signal transducers and activators of transcription (STATs) represent a class of transcriptional factors involved in multiple cellular processes, particularly immune response (Shuai and Liu, 2003).”

• cites Shuai and Liu, 2003Not checked yet

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SH-4-54 is a commercially available STAT3 phosphorylation site inhibitor reported to cross the blood-brain barrier.

“To test this possibility, we resorted to a commercially available STAT3 phosphorylation site inhibitor SH-4-54 that was reported to cross the blood-brain barrier (Haftchenary et al., 2013).”

• cites Haftchenary et al., 2013 sourceNot checked yet

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Blocking STAT3 pathway with its phosphorylation inhibitor reduced neuroinflammation and partially rescued tau pathology in PS19 mice.

“Mechanistically, we identify STAT3 as a downstream effector of C3aR; blocking this pathway with its phosphorylation inhibitor reduced neuroinflammation and partially rescued tau pathology in PS19 mice.”

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The C3aR-regulated network is highly conserved between humans and mice and constitutes immune and microglia factors implicated in late-onset AD.

“Further, the C3aR-regulated network is highly conserved between humans and mice and constitutes immune and microglia factors implicated in late-onset AD.”

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

Complement molecules C3a and C5a activate canonical JAK-STAT3 signaling via cytokine intermediates in models of retinal and liver regeneration.

“The complement molecules C3a and C5a have been shown to activate the canonical JAK-STAT3 signaling via cytokine intermediates in models of retinal and liver regeneration (Haynes et al., 2013; He et al., 2009; Strey et al., 2003).”

• cites Haynes et al., 2013 sourceNot checked yet

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• cites He et al., 2009Not checked yet

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• cites Strey et al., 2003 sourceNot checked yet

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

These TF results were validated by qPCR analysis in an independent group of animals.

“These results were validated by qPCR analysis in an independent group of animals (Figure S7B).”

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

Activation of STAT pathway involves phosphorylation and translocation to nucleus where it mediates transcription of target genes.

“The activation of the STAT pathway involves its phosphorylation and translocation to the nucleus where it mediates the transcription of target genes.”

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Combined, direct activation of pSTAT3 by C3-C3aR in vitro and similar effects by blockade of C3aR or pSTAT3 in vivo support a mechanistic and functional crosstalk between complement and STAT3 signaling pathways in mediating tau pathogenic process.

“Combined, the direct activation of pSTAT3 signaling by C3-C3aR in vitro and the similar effects exerted by the blockade of C3aR or pSTAT3 in vivo provide support for a mechanistic and functional crosstalk between the complement and STAT3 signaling pathways in mediating tau pathogenic process.”

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

STAT3 can also be phosphorylated by other kinases including Src and MAPK through activation of growth factor receptors and GPCR.

“Besides the canonical JAK-dependent STAT phosphorylation, STAT3 can also be phosphorylated by other kinases, including Src and MAPK, through the activation of growth factor receptors and G-protein-coupled receptors (GPCR) (Mertens and Darnell, 2007; Ram and Iyengar, 2001).”

• cites Mertens and Darnell, 2007Not checked yet

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• cites Ram and Iyengar, 2001Not checked yet

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Strong evidence supports a pivotal role for STAT3 in controlling astrogliosis and astrocyte reactivity in Alzheimer's disease.

“strong evidence implicates a pivotal role of STAT3 in regulating astrogliosis and astrocyte reactivity in AD (Ceyzeriat et al., 2016).”

• cites Ceyzeriat et al., 2016 sourceNot checked yet

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Experimenters were blinded to genotype and treatment group.

“The genotypes and treatment groups were blinded to the experimenter.”

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

Unless stated otherwise, every sample or animal was kept in the statistical analysis.

“All samples or animals were included in the statistical analysis unless otherwise specified.”

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In Aβ mouse models, SOCS3 expression effectively reduced reactive astrogliosis and microgliosis.

“where the expression of SOCS3 effectively reduced reactive astrogliosis and microgliosis (Ben Haim et al., 2015).”

• cites Ben Haim et al., 2015 sourceNot checked yet

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Sex and gender identity effects were not examined, owing to the small sample size.

“Influence of sex, gender identity or both on the study results was not the objective of the study. It was not analyzed due to small sample size.”

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

After PCA, three samples (AL2, AL15, AL16) were excluded as outliers.

“3 outliers (AL2, AL15 and AL16) were removed after PCA analysis (Figure S2A)”

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

In the RPPA analysis, three samples from the PS19 and PS19/C3aR KO groups were excluded because of low detection signal.

“3 outliers from PS19 and PS19/C3aR KO group were removed from the analysis due to low detection signal.”

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

An independent study documented that the IL10-STAT3 pathway mediates microglial Aβ phagocytosis.

“an independent study documented a role for the IL10-STAT3 pathway in mediating microglial Aβ phagocytosis (Guillot-Sestier et al., 2015).”

• cites Guillot-Sestier et al., 2015 sourceNot checked yet

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May combine two claims.

The C3-C3aR pathway and STAT3 signaling interact mechanistically and functionally.

“We unveil a mechanistic and functional interaction between the C3-C3aR pathway and STAT3 signaling”

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The analysis was restricted to resident immune cells of the CNS.

“our analysis is restricted to the resident immune cells in the CNS.”

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The STAT3 pathway is elevated in reactive astrocytes of Aβ mouse models.

“the STAT3 pathway is shown to be elevated in reactive astrocytes of Aβ mouse models”

• cites Ben Haim et al., 2015 sourceNot checked yet

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

The C3aR KO mice were backcrossed to C57BL/6J for five generations and then crossed with PS19 mice to produce littermate controls of four genotypes: WT, C3aR KO, PS19, and PS19/C3aR KO.

“The C3aR KO mice were backcrossed to C57BL/6J for 5 generations prior to crossing with PS19 mice to obtain littermate WT, C3aR KO, PS19, and PS19/C3aR KO mice.”

May combine two claims.

C3aR is essential for CNS immune homeostasis and tau pathology.

“our studies establish a critical role of C3aR in mediating CNS immune homeostasis and tau pathology”