Molecular pathway analysis towards understanding tissue vulnerability in spinocerebellar ataxia type 1

  1. Terri M Driessen
  2. Paul J Lee
  3. Janghoo Lim  Is a corresponding author
  1. Yale School of Medicine, Unites States
8 figures, 4 tables and 7 additional files

Figures

Schematic illustrating the cross-tissue and cross-model comparisons conducted to identify common and unique molecular alterations across SCA1 affected tissues.

Transcriptomics data from Atxn1154Q/2Q KI inferior olive and cerebellum were first analyzed individually (1-2) before comparing the two tissues (3). Using the ATXN1-82Q Tg inferior olive, comparison …

https://doi.org/10.7554/eLife.39981.002
Figure 2 with 4 supplements
Defense Response-related genes are significantly enriched in the 12 week old Atxn1154Q/2Q KI inferior olive.

(A) PolyQ-expanded Atxn1 is expressed in the inferior olive of Atxn1154Q/2Q KI mice (n = 2 animals per genotype). * marks Atxn1-2Q and ** marks Atxn1-154Q. (B) Illustration of the brain region …

https://doi.org/10.7554/eLife.39981.003
Figure 2—source data 1

5 week old Atxn1154Q/2Q KI inferior olive functional annotation clustering enrichment analysis.

https://doi.org/10.7554/eLife.39981.008
Figure 2—source data 2

Functional annotation enrichment analysis for the 12 week old Atxn1154Q/2Q KI inferior olive.

https://doi.org/10.7554/eLife.39981.009
Figure 2—source data 3

Upstream regulators for differentially regulated 12 week old Atxn1154Q/2Q KI inferior olive genes.

https://doi.org/10.7554/eLife.39981.010
Figure 2—source data 4

5 week old Atxn1154Q/2Q KI inferior olive enriched pathways for cross time-point overlap with EnrichmentMap (enrichment FDR p-value < 0.05).

https://doi.org/10.7554/eLife.39981.011
Figure 2—source data 5

12 week old Atxn1154Q/2Q KI Inferior Olive enriched patwhays for cross-time point overlap with EnrichmentMap.

https://doi.org/10.7554/eLife.39981.012
Figure 2—figure supplement 1
Response to Organic Substance and Hormone Metabolic Process genes are differentially regulated in the 5 week old Atxn1154Q/2Q KI inferior olive.

(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in Atxn1154Q/2Q KI inferior olive at 5 weeks of age (FDR p-value < 0.05; n = 3 males per genotype). (C) …

https://doi.org/10.7554/eLife.39981.004
Figure 2—figure supplement 2
Features of the Defense Response, including Interferon Signaling and IRF Activation, are predicted to have increased activity in the 12 week old Atxn1154Q/2Q KI inferior olive.

(A) Biological pathway enrichment using Qiagen’s IPA in the 12 week old Atxn1154Q/2Q KI inferior olive differentially expressed genes. X-axis marks the enrichment score using a Benjamini-Hochberg …

https://doi.org/10.7554/eLife.39981.005
Figure 2—figure supplement 3
Subset of genes associated with the enriched biological pathway Neurological System Process are down-regulated in the 12 week old Atxn1154Q/2Q KI inferior olive.

(A) Down-regulated genes within the Neurological System Process pathway (** FDR p-value < 0.01; n = 3 males per genotype used in RNA-seq analysis). (B) Validation of Calb1 down-regulation using …

https://doi.org/10.7554/eLife.39981.006
Figure 2—figure supplement 4
Divergent gene expression changes and minimal biological pathway overlap in the temporal comparison of the Atxn1154Q/2Q KI inferior olive.

(A) Schematic of the time-point comparison in Atxn1154Q/2Q KI inferior olive between 5 and 12 weeks. (B) Total number of differentially regulated genes in the Atxn1154Q/2Q KI inferior olive relative …

https://doi.org/10.7554/eLife.39981.007
Figure 3 with 4 supplements
Chemical Synaptic Transmission genes, including GABAergic and glutamatergic genes, are differentially regulated in the 12 week old Atxn1154Q/2Q KI cerebellum.

(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in the 12 week old Atxn1154Q/2Q KI cerebellum (FDR p-value < 0.05; n = 3 males per genotype for RNA-seq). (C)…

https://doi.org/10.7554/eLife.39981.014
Figure 3—source data 1

5 week old Atxn1154Q/2Q KI cerebellum functional annotation clustering (differentially expressed genes FDR p-value < 0.05).

https://doi.org/10.7554/eLife.39981.019
Figure 3—source data 2

12 week old Atxn1154Q/2Q KI cerebellum functional annotation enrichment analysis.

https://doi.org/10.7554/eLife.39981.020
Figure 3—source data 3

5 week old Atxn1154Q/2Q KI cerebellum functional annotation enrichment analysis (using nominal p-value < 0.01 for genes).

https://doi.org/10.7554/eLife.39981.021
Figure 3—source data 4

Atxn1154Q/2Q KI Cerebellum 5 weeks enrichment chart edited for EnrichmentMap (nominal p-value < 0.01).

https://doi.org/10.7554/eLife.39981.022
Figure 3—source data 5

12 week old Atxn1154Q/2Q KI cerebellum enrichment chart edited for EnrichmentMap (FDR p-value < 0.05).

https://doi.org/10.7554/eLife.39981.023
Figure 3—figure supplement 1
Few genes and pathways are significantly enriched in the 5 week Atxn1154Q/2Q KI cerebellum.

(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in Atxn1154Q/2Q KI cerebellum at 5 weeks of age (FDR p-value < 0.05; n = 3 males per genotype for RNA-seq). …

https://doi.org/10.7554/eLife.39981.015
Figure 3—figure supplement 2
Consistent gene expression changes in the Atxn1154Q/2Q KI cerebellum across the 5 and 12 week time-points.

(A) Schematic of the time-point comparison in Atxn1154Q/2Q KI cerebellum. (B) Total number of differentially regulated genes in the Atxn1154Q/2Q KI cerebellum that are commonly altered in both the 5 …

https://doi.org/10.7554/eLife.39981.016
Figure 3—figure supplement 3
Nominally significant differentially regulated genes in the 5 week old Atxn1154Q/2Q KI cerebellum overlapped with previously published microarray datasets and revealed significantly enriched pathways.

(A) Subset of genes with a nominal p-value < 0.01 that overlapped with a previously published dataset (* nominal p-value < 0.01 but FDR p-value > 0.05; n = 3 males per genotype used in RNA-seq). (B) …

https://doi.org/10.7554/eLife.39981.017
Figure 3—figure supplement 4
Consistent gene expression changes and pathway enrichment in the 5 week and 12 week Atxn1154Q/2Q KI cerebellum with an expanded 5 week p-value cutoff.

(A) Schematic of the time-point comparison in Atxn1154Q/2Q KI cerebellum. (B) Total number of differentially regulated genes in the Atxn1154Q/2Q KI cerebellum relative to appropriate WT controls …

https://doi.org/10.7554/eLife.39981.018
Figure 4 with 1 supplement
Cross-tissue comparison of pathway enrichment reveals common and unique features of the 12 week old Atxn1154Q/2Q KI inferior olive and cerebellum.

(A) Schematic of the cross-tissue comparison conducted. (B) Clustering of GO terms from the 12 week old inferior olive and cerebellum differentially expressed genes lists. In this and all following …

https://doi.org/10.7554/eLife.39981.025
Figure 4—source data 1

5 week old Atxn1154Q/2Q KI Inferior Olive enrichment chart for EnrichmentMap (FDR p < 0.05).

https://doi.org/10.7554/eLife.39981.027
Figure 4—source data 2

5 week old Atxn1154Q/2Q KI cerebellum enrichment chart edited for EnrichmentMap (nominal p-value < 0.01).

https://doi.org/10.7554/eLife.39981.028
Figure 4—source data 3

12 week old Atxn1154Q/2Q KI inferior olive enrichment chart for EnrichmentMap.

https://doi.org/10.7554/eLife.39981.029
Figure 4—source data 4

12 week old Atxn1154Q/2Q KI cerebellum enrichment chart for EnrichmentMap.

https://doi.org/10.7554/eLife.39981.030
Figure 4—figure supplement 1
Minimal gene overlap in the 5 week old Atxn1154Q/2Q KI cerebellum and inferior olive in the cross-tissue comparison.

(A) Schematic of the cross-tissue comparison in the 5 week old Atxn1154Q/2Q KI inferior olive and cerebellum. (B) Total number of differentially regulated genes in the Atxn1154Q/2Q KI inferior olive …

https://doi.org/10.7554/eLife.39981.026
Figure 5 with 3 supplements
The Defense Response is enriched in both the 12 week old Atxn1154Q/2Q KI inferior olive and ATXN1-82Q Tg inferior olive.

(A) ATXN1 mRNA expression in the 12 week old ATXN1-82Q Tg cerebellum and inferior olive (***p < 0.001, t-test; n = 3 males per genotype). (B) PolyQ-expanded ATXN1 is present in the cerebellum, but …

https://doi.org/10.7554/eLife.39981.031
Figure 5—source data 1

5 week old ATXN1-82Q Tg inferior olive functional annotation clustering analysis.

https://doi.org/10.7554/eLife.39981.035
Figure 5—source data 2

12 week old ATXN1-82Q Tg inferior olive functional annotation clustering.

https://doi.org/10.7554/eLife.39981.036
Figure 5—source data 3

5 week old ATXN1-82Q Tg inferior olive enrichment chart for EnrichmentMap.

https://doi.org/10.7554/eLife.39981.037
Figure 5—source data 4

12 week old ATXN1-82Q Tg inferior olive enrichment chart for EnrichmentMap.

https://doi.org/10.7554/eLife.39981.038
Figure 5—figure supplement 1
The majority of genes significantly altered in the 5 week ATXN1-82Q Tg inferior olive are up-regulated.

(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in ATXN1-82Q Tg inferior olive at 5 weeks of age (FDR p-value < 0.05; n = 3 males per genotype for RNA-seq). …

https://doi.org/10.7554/eLife.39981.032
Figure 5—figure supplement 2
Differentially regulated genes identified in the 12 week ATXN1-82Q Tg inferior olive are associated with Blood Vessel Morphogenesis, Response to Organic Substance, and Defense Response-related pathways.

(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in the 12 week old ATXN1-82Q Tg inferior olive (FDR p-value < 0.05; n = 3 males per genotype for RNA-seq). (C

https://doi.org/10.7554/eLife.39981.033
Figure 5—figure supplement 3
Divergent gene expression changes and minimal biological pathway overlap in the temporal comparison of the ATXN1-82Q Tg inferior olive.

(A) Schematic of the time-point comparison in ATXN1-82Q Tg inferior olive. (B) Total number of differentially regulated genes in the ATXN1-82Q Tg inferior olive relative to appropriate WT controls …

https://doi.org/10.7554/eLife.39981.034
Figure 6 with 4 supplements
Divergent gene expression changes and low biological pathway overlap between 12 week old ATXN1-82Q Tg inferior olive and cerebellum.

(A) Schematic of the cross-tissue comparison in ATXN1-82Q Tg mice. (B) Total number of differentially regulated genes that are common, and uniquely altered, in the inferior olive and cerebellum of …

https://doi.org/10.7554/eLife.39981.039
Figure 6—source data 1

5 week old ATXN1-82Q Tg cerebellum functional annotation enrichment analysis.

https://doi.org/10.7554/eLife.39981.044
Figure 6—source data 2

12 week old ATXN1-82Q Tg functional annotation clustering enrichment analysis.

https://doi.org/10.7554/eLife.39981.045
Figure 6—figure supplement 1
Chemical Synaptic Transmission and Regulation of Cell Communication are the most enriched pathways in the 5 week ATXN1-82Q Tg cerebellum.

(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in ATXN1-82Q Tg cerebellum at 5 weeks of age (FDR p-value < 0.05; n = 3 males per genotype for RNA-seq). (C) …

https://doi.org/10.7554/eLife.39981.040
Figure 6—figure supplement 2
Pathways related to Synaptic Signaling are enriched in the 12 week ATXN1-82Q Tg cerebellum.

(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in ATXN1-82Q Tg cerebellum at 12 weeks of age (FDR p-value < 0.05; n = 3 males per genotype for RNA-seq). (C)…

https://doi.org/10.7554/eLife.39981.041
Figure 6—figure supplement 3
Common gene expression changes and pathway enrichment in the 5 and 12 week ATXN1-82Q Tg cerebellum.

(A) Schematic of the time-point comparison in ATXN1-82Q Tg cerebellum. (B) Total number of differentially regulated genes in the ATXN1-82Q Tg cerebellum relative to appropriate WT controls that are …

https://doi.org/10.7554/eLife.39981.042
Figure 6—figure supplement 4
Divergent gene expression changes and pathway enrichment in the 5 week ATXN1-82Q Tg inferior olive and cerebellum.

(A) Schematic of the cross-tissue comparison in ATXN1-82Q Tg inferior olive and cerebellum at 5 weeks of age. (B) Total number of differentially regulated genes in the ATXN1-82Q Tg cerebellum and …

https://doi.org/10.7554/eLife.39981.043
Genes commonly regulated in the cerebellum across 12 week old SCA1 mouse models are altered in opposing directions.

(A) Schematic of the cross-model comparison in 12 week old Atxn1154Q/2Q KI and ATXN1-82Q Tg cerebellum. (B) Number and directionality of shared differentially regulated genes in the Atxn1154Q/2Q KI …

https://doi.org/10.7554/eLife.39981.047
Summary of biological and molecular pathway enrichment across SCA1 mouse models in each brain region over time.

Differentially regulated genes in the 5 week old inferior olive and cerebellum from both SCA1 mouse models are associated with specific biological and molecular pathways, a subset of which are …

https://doi.org/10.7554/eLife.39981.048

Tables

Table 1
Enrichment for Irf7 binding site DNA motifs in up-regulated 12 week old Atxn1154Q/2Q inferior olive genes.
https://doi.org/10.7554/eLife.39981.013
Brain regionMotif occurrenceDown-RegulatedShuffledp-valueq-value
Inferior OlivePresent32260.4680.123
Absent128134
Brain RegionMotif OccurrenceUp-RegulatedShuffledp-valueq-value
Inferior OlivePresent2728.8e-99.2e-9
Absent1742
  1. FDR p-value calculated using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (Q 5%).

Table 2
Enrichment for Cic binding site DNA motifs in down- and up-regulated 12 week old Atxn1154Q/2Q inferior olive and cerebellum.
https://doi.org/10.7554/eLife.39981.024
Cic DNA motifBrain regionMotif occurrenceDown-RegulatedShuffledp-valueq-value
TGAATGAAInferior OlivePresent31190.0900.095
Absent129141
CerebellumPresent126575.6e-82.9e-7
Absent604673
TGAATGGAInferior OlivePresent35250.1970.148
Absent125135
CerebellumPresent1571043.7e-49.7e-4
Absent573626
Cic DNA MotifBrain RegionMotif OccurrenceUp-RegulatedShuffledp-valueq-value
TGAATGAAInferior OlivePresent1040.1430.125
Absent3440
CerebellumPresent32190.0730.095
Absent181194
TGAATGGAInferior OlivePresent1320.0030.005
Absent3142
CerebellumPresent38290.2870.188
Absent175184
  1. FDR p-value calculated using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (Q 5%).

Table 3
Enrichment for Cic binding site DNA motifs in down- and up-regulated genes from 12 week old ATXN1-82Q Tg inferior olive and cerebellum.
https://doi.org/10.7554/eLife.39981.046
Cic DNA motifBrain regionMotif occurrenceDown-RegulatedShuffledp-valueq-value
TGAATGAAInferior OlivePresent1210.0020.002
Absent5667
CerebellumPresent90481.9e-44.0e-4
Absent510552
TGAATGGAInferior OlivePresent19130.3120.187
Absent4955
CerebellumPresent110860.0730.061
Absent490514
Cic DNA MotifBrain RegionMotif OccurrenceUp-RegulatedShuffledp-valueq-value
TGAATGAAInferior OlivePresent1070.6060.318
Absent6063
CerebellumPresent97514.9e-52.1e-4
Absent366412
TGAATGGAInferior OlivePresent1570.1030.072
Absent5563
CerebellumPresent96590.0020.002
Absent367404
  1. FDR p-value calculated using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (Q 5%).

Key resources table
Reagent typeDesignationSource or referenceIdentifiersAdditional

information
Strain, or strain
background (Mus
musculus, C57BL/6J)
Atxn1154Q/2Q KIPMID:12086639RRID:MGI:2429435, backcrossed in house on C57BL/6J background
Strain, or strain
background (Mus
musculus, FVB/NJ)
ATXN1-82Q TgPMID:7553854RRID:MGI:2447854
backcrossed in house on FVB/NJ background
Strain, or strain
background
(Mus
musculus,
C57BL/6J)
C57BL/6JThe Jackson LabStock: 000664
Strain, or
strain background
(Mus musculus,
FVB/NJ)
FVB/NJThe Jackson LabStock: 001800
Cell line
(Mus musculus)
BV2A kind gift from Dr. Katerina Akassoglou (Adams et al., 2007)RRID: CVCL_0182
Transfected
construct
human Flag-ATXN1 82QA kind gift from Dr. Huda ZoghbiAddgene Plasmid #33237
Transfected
construct
mammalian expression vector pcDNA3.1+Invitrogen (ThermoFisher Scientific)Cat. # V790-20
antibodyrabbit polyclonal anti-Atxn111750a kind gift from Huda Zoghbi (Servadio et al., 1995)RRID:AB_2721278(1:1,000) WB
Antibodymouse monoclonal anti-CalbindinSigmaCat. #: C9848
RRID:AB_476894
(1:2,000) IHC
Antibodymouse monoclonal anti-GapdhSigmaCat. #G8795
RRID:AB_1078991
(1:10,000) WB
Antibodychicken polyclonal anti-GfapAbcamCat. # ab4674
RRID:AB_304558
(1:1,000) IHC
Antibodyrabbit polyclonal anti-Iba1WakoCat. # 019–19741
RRID:AB_839504
(1:2,000) IHC
Antibodygoat polyclonal anti-chicken Alexa555ThermoFisherCat. #A21437
RRID:AB_2535858
(1:500) IHC
Antibodygoat polyclonal anti-mouse Alexa488ThermoFisherCat. # A11001
RRID:AB_2534069
(1:500) IHC
Antibodygoat polyclonal anti-rabbit Alexa568ThermoFisherCat. #A11011
RRID:AB_143157
(1:500) IHC
Antibodydonkey anti-rabbitGE HealthcareCat. #NA934
RRID:AB_772206
(1:4,000) WB
Antibodyanti-mouseGE HealthcareCat. #NXA931
RRID:AB_772209
(1:4,000) WB
Commercial assay or kitRNeasy Mini KitQiagenCat #74106
Commercial assay or kitiScript cDNA Synthesis KitBio-RadCat. #1708891
Commercial assay or kitiTaq Universal Probes SupermixBio-RadCat. #1725131
Commercial assay or kitPierce BCA Protein AssayThermoFisherCat. #23225
Commercial assay or kitAmaxa Cell Line Nucleofector Kit TLonzaCat. #VCA-1002
OtherDAPIVector LaboratoriesCat. #H-1500
RRID:AB_2336788
OtherActb probeApplied BiosystemsCat. #4352932E
OtherATXN1 probeApplied BiosystemsCat. # 4331182
Hs00165656_m1
OtherCalb1 probeApplied BiosystemsCat. #4331182
Mm00486647_m1
OtherGapdh probeApplied BiosystemsCat. #4352933E
OtherIfitm3 probeApplied BiosystemsCat. # 4331182
Mm00847057_s1
OtherIfr7 probeApplied BiosystemsCat. #4331182
Mm00516793_g1
OtherOasl2 probeApplied BiosystemsCat. # 4331182
Mm01201449_m1
OtherStat1 probeApplied BiosystemsCat. # 4331182
Mm01257286_m1
Software,
algorithm
Ingenuity Pathway Analysis (IPA) (Spring Release 2018)http://www.ingenuity.com/products/pathways_analysis.htmlRRID:SCR_008653
Software,
algorithm
NIH DAVID v6.8http://david.abcc.ncifcrf.gov/RRID:SCR_001881
Software, algorithmTopHat v2.1.0http://tophat.cbcb.umd.edu/RRID:SCR_013035
Software, algorithmCufflinks v2.2.1http://cole-trapnell-lab.github.io/cufflinks/RRID:SCR_014597
Software, algorithmBowtie2 v2.1.0http://bowtie-bio.sourceforge.net/bowtie2/index.shtmlRRID:SCR_016368
Software, algorithmFastQC v0.11.3http://www.bioinformatics.babraham.ac.uk/projects/fastqc/RRID:SCR_014583
Software, algorithmCummerbundhttp://compbio.mit.edu/cummeRbund/index.htmlRRID:SCR_014568
Software, algorithmBioconductor R with heatmap.2 gplots packagehttps://www.rdocumentation.org/packages/gplots/versions/3.0.1/topics/heatmap.2
Software, algorithmR project for statistical computing v3.3.3http://www.r-project.org/RRID:SCR_001905
Software, algorithmMEME Suite - Motif-based sequence analysis toolshttp://meme-suite.org/RRID:SCR_001783
Software, algorithmEnrichmentMaphttp://baderlab.org/Software/EnrichmentMapRRID:SCR_016052
Software, algorithmCytoscape v3.5.1http://cytoscape.orgRRID:SCR_003032
Software,
algorithm
GraphPad Prismhttp://www.graphpad.com/RRID:SCR_002798
Software, algorithmImageJhttps://imagej.net/RRID:SCR_003070

Additional files

Supplementary file 1

5 week old Atxn1154Q/2Q KI inferior olive IPA canonical pathway enrichment.

https://doi.org/10.7554/eLife.39981.049
Supplementary file 2

5 week old Atxn1154Q/2Q KI inferior olive IPA predicted upstream regulators.

https://doi.org/10.7554/eLife.39981.050
Supplementary file 3

Upstream regulators for differentially regulated 12 week old Atxn1154Q/2Q KI cerebellum genes.

https://doi.org/10.7554/eLife.39981.051
Supplementary file 4

5 week old ATXN1-82Q Tg inferior olive IPA canonical pathways.

https://doi.org/10.7554/eLife.39981.052
Supplementary file 5

12 week old ATXN1-82Q Tg inferior olive upstream regulators predicted in IPA.

https://doi.org/10.7554/eLife.39981.053
Supplementary file 6

RNA integrity numbers (RIN) for samples used in RNA-seq analysis.

https://doi.org/10.7554/eLife.39981.054
Transparent reporting form
https://doi.org/10.7554/eLife.39981.055

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