1. Computational and Systems Biology
  2. Genetics and Genomics

Downregulation of glial genes involved in synaptic function mitigates Huntington's disease pathogenesis

  1. Tarik Seref Onur
  2. Andrew Laitman
  3. He Zhao
  4. Ryan Keyho
  5. Hyemin Kim
  6. Jennifer Wang
  7. Megan Mair
  8. Huilan Wang
  9. Lifang Li
  10. Alma Perez
  11. Maria de Haro
  12. Ying-Wooi Wan
  13. Genevera Allen
  14. Boxun Lu
  15. Ismael Al-Ramahi
  16. Zhandong Liu
  17. Juan Botas  Is a corresponding author
  1. Department of Molecular and Human Genetics, Baylor College of Medicine, United States
  2. Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, United States
  3. Genetics & Genomics Graduate Program, Baylor College of Medicine, United States
  4. Quantitative & Computational Biosciences, Baylor College of Medicine, United States
  5. Department of Pediatrics, Baylor College of Medicine, United States
  6. State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, China
  7. Departments of Electrical & Computer Engineering, Statistics and Computer Science, Rice University, United States
https://doi.org/10.7554/eLife.64564
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Cite this article
  1. Tarik Seref Onur
  2. Andrew Laitman
  3. He Zhao
  4. Ryan Keyho
  5. Hyemin Kim
  6. Jennifer Wang
  7. Megan Mair
  8. Huilan Wang
  9. Lifang Li
  10. Alma Perez
  11. Maria de Haro
  12. Ying-Wooi Wan
  13. Genevera Allen
  14. Boxun Lu
  15. Ismael Al-Ramahi
  16. Zhandong Liu
  17. Juan Botas
(2021)
Downregulation of glial genes involved in synaptic function mitigates Huntington's disease pathogenesis
eLife 10:e64564.
https://doi.org/10.7554/eLife.64564
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6 figures, 1 table and 16 additional files

Figures

Figure 1 with 1 supplement
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Differentially expressed genes (DEGs) in Huntington's disease (HD) human striatal tissue are concordantly altered in mouse and Drosophila HD models.

(A) Our approach to identifying orthologous genes in tissues from humans, mice, and Drosophila with concordant expression changes (i.e., upregulated or downregulated in all three systems) following …

Figure 1—source data 1

List of up- and down-regulated differentially expressed genes (DEGs) in humans, mice, and Drosophila affected by mutant Huntingtin (mHTT).

https://cdn.elifesciences.org/articles/64564/elife-64564-fig1-data1-v2.xlsx
Figure 1—source data 2

Lists of up- and down-regulated differentially expressed genes (DEGs) in humans, mice, and Drosophila affected by mutant Huntingtin (mHTT) grouped by homology for each Drosophila Huntington's disease (HD) model.

https://cdn.elifesciences.org/articles/64564/elife-64564-fig1-data2-v2.zip
Figure 1—figure supplement 1
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Expressing mutant Huntingtin (mHTT) in Drosophila glia or neurons leads to distinct gene expression profiles.

In contrast, the full-length and N-terminal models show relatively similar gene expression profiles. Principal component analysis (PCA) plot of RNA-sequencing samples. Circles represent samples with …

Figure 2 with 2 supplements
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Clusters of concordant differentially expressed genes (DEGs) between human and mouse Huntington's disease (HD) striata and Drosophila expressing mutant Huntingtin (mHTT) in glia.

Clustered protein-protein interaction (PPI) networks of DEGs (STRING-db) that have higher (A) or lower (B) concordant expression in HD human tissue, an allelic series of knock-in HD mouse models, …

Figure 2—source data 1

Lists of human Huntington's disease (HD) differentially expressed genes (DEGs) (Entrez IDs) concordantly dysregulated in mouse and Drosophila HD models.

https://cdn.elifesciences.org/articles/64564/elife-64564-fig2-data1-v2.xlsx
Figure 2—source data 2

List of proteins expressed in the human striata.

https://cdn.elifesciences.org/articles/64564/elife-64564-fig2-data2-v2.zip
Figure 2—figure supplement 1
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Network of differentially expressed genes (DEGs) responding concordantly to mutant Huntingtin (mHTT) expression in glia or neurons.

(A) Heatmap representing all pairwise comparisons between clusters of upregulated (left) and downregulated (right) DEGs in response to glial (x-axis) or neuronal (y-axis) mHTT expression. Clusters …

Figure 2—figure supplement 2
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Network of differentially expressed genes (DEGs) concordantly altered in human Huntington's disease (HD) tissue and HD mouse models, but not in Drosophila HD models.

(A) Clustered, annotated protein-protein interaction (PPI) network (STRING-db) of DEGs concordantly upregulated (top) and downregulated (down) in human HD striatal tissue collected post-mortem (Hodge…

Figure 3 with 1 supplement
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Reducing the expression of Synapse Assembly cluster genes in glia mitigates mutant Huntingtin (mHTT)-induced behavioral impairments.

(A) Overlaps between concordant differentially expressed genes (DEGs) from the cross-species analysis defined as responding to mHTT expression in glia and DEGs identified in Huntington's disease (HD)…

Figure 3—source data 1

Raw behavioral data for Drosophila expressing mutant Huntingtin (mHTT) in glia following reduced expression of synaptic genes.

https://cdn.elifesciences.org/articles/64564/elife-64564-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
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Suppressors of glial mutant Huntingtin (mHTT)-induced behavioral impairments among differentially expressed genes (DEGs) in the Synapse Assembly cluster.

(A) Representative graphs of behavioral assays (climbing speed as a function of time) of Drosophila expressing mHTT in glia (repo >HTTNTNT231Q128) and alleles knocking down genes in the Synapse …

Figure 3—figure supplement 1—source data 1

Raw behavioral data for Drosophila expressing mutant Huntingtin (mHTT) in glia following reduced expression of synaptic genes, expanded number of genes, and alleles.

https://cdn.elifesciences.org/articles/64564/elife-64564-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
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Glia-specific dNRXN3 knockdown mitigates impairments caused by mutant Huntingtin (mHTT) expression.

(A) Behavioral assays (climbing speed as a function of age) showing that dNRXN3 heterozygous loss of function (LOF) ameliorates behavioral impairments caused by expression of mHTT in both neurons …

Figure 4—source data 1

Raw behavioral data for Drosophila expressing mutant Huntingtin (mHTT) following reduced expression of dNRXN.

https://cdn.elifesciences.org/articles/64564/elife-64564-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
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Additional images of astrocytes expressing Nrxn3 in the striatum of HdhzQ175/+ mice and quantification.

Astrocytes in the striatum of 6-month-old knock-in Huntington's disease (HD) mice (HdhzQ175/+) expressing Nrxn3. In situ probe for Nrxn3 mRNA is in red (appears magenta when overlapping with the …

Figure 5 with 1 supplement
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Compensatory and pathogenic gene expression changes shared by neurons and glia in response to mutant Huntingtin (mHTT) expression.

(A) Our approach for identifying modifiers of mHTT-induced behavioral impairments common to both neurons and glia. Genes that were central to their respective clusters were prioritized and …

Figure 5—source data 1

Numerical data for bar charts summarizing the improvement in behavior in Drosophila expressing mutant Huntingtin (mHTT) in neurons and glia by manipulating common pathogenic and compensatory alterations.

https://cdn.elifesciences.org/articles/64564/elife-64564-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
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Genetic modifiers suppress behavioral impairments caused by mutant Huntingtin (mHTT) expression in neurons and glia.

(A) Representative graphs of the climbing speed of animals expressing mutant HTT in neurons and glia in combination with common modifiers as a function of time. ***p<0.001 between the positive …

Figure 5—figure supplement 1—source data 1

Raw behavioral data for Drosophila expressing mutant Huntingtin (mHTT) in neurons and glia by manipulating common pathogenic and compensatory alterations.

https://cdn.elifesciences.org/articles/64564/elife-64564-fig5-figsupp1-data1-v2.xlsx
Figure 6 with 2 supplements
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Antagonizing the pathogenic overexpression of SERPINA1 in neurons and glia mitigates mutant Huntingtin (mHTT)-induced behavioral impairments and lowers mHTT protein levels in Drosophila and Huntington's disease (HD) mouse striatal cells.

(A) Behavioral assays following knockdown of dSERPINA1 in Drosophila expressing mHTT in neurons and glia. *** indicates p<0.001 by linear mixed effects model and post-hoc pairwise comparison between …

Figure 6—source data 1

Raw behavioral data for Drosophila expressing mutant Huntingtin (mHTT) in neurons and glia following knockdown of Spn42De.

https://cdn.elifesciences.org/articles/64564/elife-64564-fig6-data1-v2.xlsx
Figure 6—source data 2

Summary of numerical and raw data for western blots of protein lysates from Drosophila heads expressing mutant Huntingtin (mHTT) in neurons and glia with knockdown of Spn42De.

https://cdn.elifesciences.org/articles/64564/elife-64564-fig6-data2-v2.xlsx
Figure 6—source data 3

Summary of numerical and raw data for homogenous time-resolved fluorescence (HTRF) for protein lysates of STHdhQ111/Q7 treated with pooled small interfering RNAs (siRNAs) against Serpina1a.

https://cdn.elifesciences.org/articles/64564/elife-64564-fig6-data3-v2.xlsx
Figure 6—figure supplement 1
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dSERPINA1 knockdown does not reduce the expression of the GAL4-UAS system.

(A) Representative immunoblot performed on Drosophila protein lysates assessing mCD8::GFP protein levels as a proxy for GAL4-UAS expression. mCD8::GFP was expressed in neurons using the elav-GAL4 …

Figure 6—figure supplement 1—source data 1

Summary of numerical and raw data for western blots of protein lysates from Drosophila heads expressing mCD8::GFP in neurons with knockdown of Spn42De.

https://cdn.elifesciences.org/articles/64564/elife-64564-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
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Serpina1a protein accumulates in the striata of HdhzQ175/+ mice compared to wildtype littermates.

(A) Representative images of fixed, paraffin-embedded coronal brain slices at the striatum of HdhzQ175/+ and wildtype mice, stained with an antibody against Serpina1a (green). Images were taken on a …

Figure 6—figure supplement 2—source data 1

Summary of numerical and raw data for immunohistochemistry analysis of Serpina1 staining in 8.5-month-old mouse striata from wildtype and zQ175 mice.

https://cdn.elifesciences.org/articles/64564/elife-64564-fig6-figsupp2-data1-v2.xlsx

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional
information
AntibodyAnti-HTT (mouse monoclonal)EMD MilliporemAb5490,
RRID:AB_2233522		WB (1:500)
AntibodyAnti-GFP (rabbit polyclonal)ThermoFisherA-11122,
RRID:AB_221569		WB (1:1000)
AntibodyAnti-HTT (mouse monoclonal)Novartis2B7HTRF (0.023 μg/mL)
AntibodyAnti-laminC (mouse monoclonal)Hybridoma BankLC28.26,
RRID:AB_528339		WB (1:1000)
AntibodyAnti-GFAP (rabbit polyclonal)DAKOZ0334, RRID:AB_10013382IF (1:500)
AntibodyAlpha-tubulin (rabbit polyclonal)AbcamEP1332Y,
RRID:AB_922700		WB (1:1000)
AntibodyAnti-HTT (mouse monoclonal)SigmaAldrichmAb2166, RRID:AB_11213141HTRF (1.4 μg/mL)
AntibodyAnti-rabbit IgG Alexa 488 (goat polyclonal)InvitrogenA-11008,
RRID:AB_143165		IF (1:500)
AntibodyAnti-Serpina1a (rabbit polyclonal)InvitrogenPA5-16661, RRID:AB_10985745IF (1:250)
AntibodyRDye 680RD anti-Rabbit IgG (goat polyclonal)LI-COR Biosciences925-68071,
RRID:AB_2721181		WB (1:5000)
AntibodyIRDye 800CW anti-Mouse IgG (goat polyclonal)LI-COR Biosciences925-32210,
RRID:AB_2687825		WB (1:5000)
Chemical compound, drugLipofectamine 2000Life Technologies11668
Chemical compound, drugEDTA-free protease inhibitorCalbiochem539134
Commercial assay, kitmiRNeasy Mini KitQiagen217004
Commercial assay, kitIllumina TruSeq Stranded mRNAIllumina20020595
Commercial assay, kitTyramide-Cy3 Plus kitPerkin ElmerNEL744001KT
Commercial assay, kitPicoGreenQuant-iT PicoGreen dsDNA Assay KitP7589
Cell line (Mus musculus)STHdhQ111/Q7 CellsCoriell Cell RepositoriesCH00096
Strain, strain background (Drosophila)White mutant, background genotypeBloomington Drosophila Stock Center3605w1118
Genetic reagent (Drosophila)Non-targeting hpRNAVienna Drosophila Resource Center13974
Strain, strain background (Drosophila)repo-Gal4Bloomington Drosophila Stock Center7415w1118; P{w+m*=GAL4}repo/TM3, Sb1
Strain, strain background (Drosophila)elav-Gal4Bloomington Drosophila Stock Center458P{GawB}elavC155
Genetic reagent (Drosophila)N-terminal HD modelBotas LaboratoryBranco et al., 2008UAS-HTTNT231Q128/TM6B, tubulin-GAL80 (N-terminal)
Genetic reagent (Drosophila)Full-length HD modelBotas LaboratoryThis paperUAS-HTTFL200Q/CyO (full-length)
Genetic reagent (Drosophila)siRNA against human mutant HTTBotas Laboratory
Kaltenbach et al., 2007		UAS-siHTT
Genetic reagent (Drosophila)Classic CenG1A loss-of-function alleleBloomington Drosophila Stock Center44301CenG1ALOF or y1w*;Mi{MIC}CenG1AMI06024 (Figure 3D)
Genetic reagent (Drosophila)Classic vlc loss-of-function alleleBloomington Drosophila Stock Center10366vlcLOF or y1w67c23;P{w+mc = lacW}vlck01109/CyO (Figure 3D)
Genetic reagent (Drosophila)Classic trn loss-of-function alleleBloomington Drosophila Stock Center4550trnLOF or y1w67c23;P{w+mc = lacW}trnS064117/TM3, Sb1 Ser1 (Figure 3D)
Genetic reagent (Drosophila)Classic cora loss-of-function alleleBloomington Drosophila Stock Center9099coraLOF or P{ryt7.2=neoFRT}43D cora14/CyO (Figure 3D)
Genetic reagent (Drosophila)RNAi against SytbetaVienna Drosophila Resource Center106559UAS-SytbetahpRNA (Figure 3D)
Genetic reagent (Drosophila)RNAi against mGluRNational Institute of Genetics, Japan11144 R-3UAS-mGluRRNAi (Figure 3D)
Genetic reagent (Drosophila)Neuronal mCD8::GFP reporter lineBloomington Drosophila Stock Center5146P{w+mW.hs=GawB}elavC155, P{w+mC = UAS-mCD8::GFP.L}Ptp4E[LL4], P{ry[+t7.2]=hsFLP}1, w*
Genetic reagent (Drosophila)Classical loss of function and overexpression alleles in DrosophilaBloomington Drosophila Stock CenterProvided in Supplementary files 4 and 5
Genetic reagent (Drosophila)RNAi alleles in DrosophilaVienna Drosophila Resource CenterProvided in Supplementary files 4 and 5
Genetic reagent (Drosophila)Cytological duplication alleles in DrosophilaGenetiVisionProvided in Supplementary files 4 and 5
Genetic reagent (M. musculus)HdhzQ175 MiceJackson Laboratories027410B6J.129S1-Htttm1Mfc/190ChdiJ
Recombinant DNA reagentpMF3 VectorDrosophila Genome Resource Center1237
Software, algorithmAdept DesktopOmronN/A
Software, algorithmVideo SavantIO IndustriesN/A
Software, algorithmMatLab with Image Processing Toolkit and Statistics ToolkitMathWorkshttps://www.mathworks.com/products/matlab.html
Software, algorithmRSLogixRockewell AutomationN/A
Software, algorithmUltrawareRockewell AutomationN/A
Software, algorithmAssay ControlSRI InternationalN/A
Software, algorithmFastPhenoTrack Vision ProcessingSRI InternationalN/A
Software, algorithmTrackingServer Data ManagementSRI InternationalN/A
Software, algorithmScoringServer Behavioral ScoringSRI InternationalN/A
Software, algorithmTrackviewer Visual Tracking and ViewingSRI InternationalN/A
Software, algorithmIllustrator CCAdobehttps://www.adobe.com
Software, algorithmRR Project for Statistical Computinghttps://www.r-project.org/
Software, algorithmFijiThe Fiji Teamhttps://fiji.sc/
Software, algorithmImage Studio LiteLI-COR Bioscienceshttps://www.licor.com/bio/image-studio-lite/
Software, algorithmBowtieLangmead and Salzberg, 2012http://bowtie-bio.sourceforge.net/index.shtml
Software, algorithmRSEMLi and Dewey, 2011https://github.com/deweylab/RSEM
Software, algorithmDESeq2Love et al., 2014https://bioconductor.org/packages/release/bioc/html/DESeq2.html
Software, algorithmDIOPTHu et al., 2011https://www.flyrnai.org/cgi-bin/DRSC_orthologs.pl
Software, algorithmMGIThe Mouse Genome Databasehttp://www.informatics.jax.org/genes.shtml
Software, algorithmSTRINGSzklarczyk et al., 2015https://string-db.org/
Software, algorithmInfoMapRosvall and Bergstrom, 2008https://cran.r-project.org/web/packages/igraph/index.html
Software, algorithmCytoscapeThe Cytoscape Consortiumhttps://cytoscape.org
Transfected construct (M. musculus)AllStars Negative Control siRNA (Scramble)Qiagen1027280
Transfected construct (M. musculus)Htt SMARTPool siRNAsHorizon Discovery LimitedL-040632-01-00055′- GAAAUUAAGGUUCUGUUGA-3′
5′- CCACUCACGCCAACUAUAA-3′
5′- GAUGAAGGCUUUCGAGUCG-3′
5′- UAACAUGGCUCAUUGUGAA-3′
Transfected construct (M. musculus)Serpina1a SMARTPool siRNAsHorizon Discovery LimitedL-043380-01-00055′- GAAUAUAACUUGAAGACAC-3′
5′-GGGCUGACCUCUCCGGAAU-3′
5′- UGGUAGAUCCCACACAUAA-3′
5′- GAAAGAUAGCUGAGGCGGU-3′
Sequence-based reagentPrimers for cloning human HTTThis paperSee experimental model detailForward 5′-gaattcGCACCGACCAAAGAAAGAAC-3′
Reverse 5′-tctagaGGCAGAAGGTTCACCAGGTA-3′
Sequence-based reagentPrimers for generating in situ probes for mouse Nrxn3 including RNA polymerase promoter sequences for T3 (forward) and T7 (reverse)Allen Brain Atlashttps://portal.brain-map.org/Forward: 5′- GCGAATTAACCCTCACTAAAGGGTCCTTCCCCTTTCCTCCTAA-3′
Reverse:
5′-GCGTAATACGACTCACTATAGGGCAGGCATGCTCTGTACTCCA-3′

Additional files

Source code 1

R script for prefiltering differentially expressed genes (DEGs) in Drosophila Huntington's disease (HD) models.

https://cdn.elifesciences.org/articles/64564/elife-64564-code1-v2.zip
Source code 2

R script for identification of differentially expressed genes (DEGs) in Drosophila Huntington's disease (HD) models.

https://cdn.elifesciences.org/articles/64564/elife-64564-code2-v2.zip
Source code 3

Python code for identification of differentially expressed genes (DEGs) in postmortem human Huntington's disease (HD) striata.

https://cdn.elifesciences.org/articles/64564/elife-64564-code3-v2.zip
Source code 4

Python code for identification of differentially expressed genes (DEGs) from Huntington's disease (HD) mouse models.

https://cdn.elifesciences.org/articles/64564/elife-64564-code4-v2.zip
Source code 5

Python code for homology mappings across humans, mice, and Drosophila.

https://cdn.elifesciences.org/articles/64564/elife-64564-code5-v2.zip
Source code 6

Python code for comparing Huntington's disease (HD) differentially expressed genes (DEGs) across species.

https://cdn.elifesciences.org/articles/64564/elife-64564-code6-v2.zip
Source code 7

Python code for clustering networks.

https://cdn.elifesciences.org/articles/64564/elife-64564-code7-v2.zip
Source code 8

R script for network analysis in a striatal background summarized in Supplementary file 2.

https://cdn.elifesciences.org/articles/64564/elife-64564-code8-v2.zip
Source code 9

R script for analyzing Drosophila behavioral assays.

https://cdn.elifesciences.org/articles/64564/elife-64564-code9-v2.zip
Supplementary file 1

Mappings of orthologous Huntington's disease (HD) human, mouse, and Drosophila differentially expressed genes (DEGs).

https://cdn.elifesciences.org/articles/64564/elife-64564-supp1-v2.xlsx
Supplementary file 2

Network connectivity of differentially expressed genes (DEGs) responding to glial or neuronal mutant Huntingtin (mHTT) expression.

https://cdn.elifesciences.org/articles/64564/elife-64564-supp2-v2.xlsx
Supplementary file 3

Cluster membership and summary of cluster annotations for differentially expressed genes (DEGs) responding to glial or neuronal mutant Huntingtin (mHTT) expression.

https://cdn.elifesciences.org/articles/64564/elife-64564-supp3-v2.xlsx
Supplementary file 4

Summary of statistical analysis for alleles in the Synapse Assembly cluster that modify glial mutant Huntingtin (mHTT)-induced behavioral impairments.

https://cdn.elifesciences.org/articles/64564/elife-64564-supp4-v2.xlsx
Supplementary file 5

Summary of alleles screened and results for common modifiers of mutant Huntingtin (mHTT)-induced behavioral impairments in neurons and glia.

https://cdn.elifesciences.org/articles/64564/elife-64564-supp5-v2.xlsx
Supplementary file 6

Summary of statistics for alleles that are common suppressors of neuronal and glial mutant Huntingtin (mHTT)-induced behavioral impairments.

https://cdn.elifesciences.org/articles/64564/elife-64564-supp6-v2.xlsx
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https://cdn.elifesciences.org/articles/64564/elife-64564-transrepform-v2.docx

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