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Mutant Huntingtin promotes autonomous microglia activation via myeloid lineage-determining factors

Abstract

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an extended polyglutamine repeat in the N terminus of the Huntingtin protein (HTT). Reactive microglia and elevated cytokine levels are observed in the brains of HD patients, but the extent to which neuroinflammation results from extrinsic or cell-autonomous mechanisms in microglia is unknown. Using genome-wide approaches, we found that expression of mutant Huntingtin (mHTT) in microglia promoted cell-autonomous pro-inflammatory transcriptional activation by increasing the expression and transcriptional activities of the myeloid lineage-determining factors PU.1 and C/EBPs. We observed elevated levels of PU.1 and its target genes in the brains of mouse models and individuals with HD. Moreover, mHTT-expressing microglia exhibited an increased capacity to induce neuronal death ex vivo and in vivo in the presence of sterile inflammation. These findings suggest a cell-autonomous basis for enhanced microglia reactivity that may influence non-cell-autonomous HD pathogenesis.

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Figure 1: RNA-Seq analysis reveals that mHTT N terminus expression triggers pro-inflammatory gene expression in BV2 microglia.
Figure 2: mHTT promotes pro-inflammatory gene expression via PU.1 and C/EBPs.
Figure 3: PU.1 and 1-C/EBPs target genes are upregulated in primary microglia from R6/2 mice.
Figure 4: PU.1 and 1-C/EBPs target genes are upregulated in primary microglia from Hdh175/175 knock-in mice.
Figure 5: Inflammation in vivo in HD individuals.
Figure 6: Effect of mHTT-expressing microglia on primary neurons ex vivo and in vivo.
Figure 7: Effect of mHTT-expressing microglia on wild-type neurons in vivo.

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Acknowledgements

We thank E. Mejia for Fluoro-Jade B staining, H. Kordasiewicz (ISIS Pharmaceuticals) for providing R6/2 mice, M. McAlonis, J. Artates and J. Boubaker for stereotaxic injection, University of California at San Diego (UCSD) Histology Core for PU.1 staining, the UCSD Human Embryonic Stem Cell Core Facility at Sanford Consortium for Regenerative Medicine for assistance with cell sorting, J. Corey-Bloom (Shiley-Marcos Alzheimer's Disease Research Center) for providing blood samples from HD patients, M. Hayden (University of British Columbia) for providing HTT and mHTT N548 aa original cDNA, S. Georges for assistance in quantification of TUNNEL assay, the Harvard Brain Tissue Resource Center (supported in part by PHS R24 MH068855), New York Brain Bank at Columbia University and Massachusetts General Hospital for HD post-mortem samples. C.L.T. and D.W.C. are supported by the CHDI Foundation. These studies were supported by US National Institutes of Health grants DK091183, DK063491, GM 069338 and CA17390 to C.K.G.

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Authors and Affiliations

Authors

Contributions

C.K.G. and A.C. developed the study, conceived the experimental plans and analyzed the data. C.B. analyzed the genome-wide data. A.C. performed most of the biological, biochemical and molecular experiments. B.E.K. conceived and performed the TUNNEL assay experiment. D.G. performed microglia purification from adult mice brains. C.L.-T. provided Hdh175/175 SOD1G37R tissues, mice and performed the in vivo experiment. E.C. and C.Z. participated in the elaboration of the project and provided original constructs and mRNA from post-mortem human samples. D.W.C. and F.H.G. participated in experimental design and provided essential resources and reagents. C.K.G. and A.C. interpreted the data and wrote the manuscript. All of the authors read and edited the manuscript. C.K.G. supervised the entire work, directed the strategies, provided financial support and gave final approval of the manuscript.

Corresponding author

Correspondence to Christopher K Glass.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 PU.1 and PU.1-C/EBPs target genes are upregulated in primary microglia but not in BMDM from R6/2 mice.

qRT-PCR analysis for Sfpi1 (a), Il6 (b) and Tnfα (c) mRNAs expression in primary microglia (mean±sd, n= 6 biological replicates, two-tailed paired student't test) and bone marrow derived macrophages (mean±sd, n= 5 biological replicates, two-tailed paired student't test) purified from non-transgenic littemates and R6/2 mice.

Supplementary Figure 2 Effect of siRNA knockdown of PU.1, C/EBPα and C/EBPβ.

Efficiency of siRNA knockdown in primary microglia derived from R6/2 mice and nontransgenic littermates as determined by qRT-PCR (mean±sd, n= 3 biological replicates, p values determined by two-tailed paired student t-test).

Supplementary Figure 3 Differential gene expression between wild-type microglia and wild-type macrophages.

Scatter Plot representing the differential gene expression observed in microglia from wild-type Hdh7/7 versus BMDM from wild-type Hdh7/7 mice.

Supplementary Figure 4 PU.1 and PU.1-C/EBPs target genes are upregulated in the striatum from R6/2 mice.

qRT-PCR analysis for Sfpi1 (a), Il6 (b), Tnfα (c), Irf1 (d) and Tlr2 (e) mRNAs expression in striatum from nontransgenic littermates, pre-symptomatic (5 weeks-old) and symptomatic (10 weeks-old) R6/2 mice. Each dot is representative of one mouse (unpaired student's test).

Supplementary Figure 5 PU.1 and PU.1-C/EBPs target genes are not differentially expressed in SOD1G37R mouse model of ALS.

qRT-PCR analysis for Sfpi1 (a), Il6 (b), Tnfα (c), Irf1 (d) and Tlr2 (e) mRNAs expression in striatum, cortex and spinal cord from nontransgenic littermates and SOD1G37R mice (8-12 months old). Each dot is representative of one mouse (unpaired student's test).

Supplementary Figure 6 Inflammation in vivo in HD individuals.

qRT-PCR analysis for IRF1 (a) and TNFα (b) mRNAs expression in striatum (first column, n= 9 individual per group), cortex (second column, n= 9 individual per group) and monocytes (third column, n= 5 individual per group) from controls and HD individuals. Each dot is representative of one individual. All p values were determined by unpaired student t-test. (c) IHC controls: brain section in presence of rabbit IgG (negative control) (i); spleen section in presence of rabbit IgG (negative control) (ii); spleen section in presence of diluting buffer (BSA, 1% bovine serum albumin in PBS phosphate buffered saline) (iii); H&E staining on spleen section (iv): PU.1 IHC staining on spleen section (positive control) (v); Von Willebrand factor IHC staining on spleen section (positive control for endothelial cells/blood vessels) (vi). Scale bar: 100μm.

Supplementary Figure 7 A model for mechanisms by which mutant Huntingtin influences the selection and activation of microglia enhancers.

Left: PU.1 and C/EBPs function in a collaborative manner to select microglia enhancers from inactive chromatin in basal conditions. Pro-inflammatory signals that activate transcription factors such as the p65 component of NFκB lead to inflammatory response. Right: mutant Huntingtin expression enhances this process by increasing PU.1 expression and PU.1-C/EBPs promoter binding, leading to increased enhancer activity under basal conditions that results in increased basal pro-inflammatory and neurotoxic genes expression. This phenomenon increases the sensitivity to pro-inflammatory signals. In fact, under conditions of sterile inflammation mutant Huntingtin-expressing microglia appears to be more efficient in inducing neuronal death.

Supplementary Figure 8 Full-length pictures of the blots presented in the main figures.

To examine proteins of interest on the same samples, blots were cut first and then probed with indicated antibodies.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 25393 kb)

Supplementary Table 1

Complete gene list for BV2 EV-, HTT N548-and mHTT N548-expressing cell lines. (XLSX 300 kb)

Supplementary Table 2

Complete gene list for microglia and BMDM from Hdh7/7 and Hdh175/175 mice. (XLSX 5194 kb)

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Crotti, A., Benner, C., Kerman, B. et al. Mutant Huntingtin promotes autonomous microglia activation via myeloid lineage-determining factors. Nat Neurosci 17, 513–521 (2014). https://doi.org/10.1038/nn.3668

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