EMBO/soda-nel huggingface dataset:
Neuron type-specific increase in lamin B1 contributes to nuclear dysfunction in Huntington's disease (2020)
Figure 1

Figure 1. Lamin B1 and B2 are differentially increased in R6/1 mouse brain.

Lamin B1 and B2 were analysed by Western blot in R6/1 mice at different stages of the disease progression (w: weeks) and their corresponding controls (WT: wild-type littermates).

A Quantification and representative immunoblots of lamin B1 in the striatum (8w, 12w and 20w; N = 6 for both genotypes; 30w; N = 4 and 5 for WT and R6/1 mice, respectively), hippocampus (8w; N = 8 and 6 for WT and R6/1 mice, respectively; 12w; N = 7 and 6 for WT and R6/1 mice, respectively; 20w N = 5 and 6 for WT and R6/1 mice, respectively; 30w; N = 8 and 5 for WT and R6/1 mice, respectively) and cortex (8w; N = 5 and 4 for WT and R6/1 mice, respectively; 12w and 20w; N = 6 for both genotypes; 30w; N = 6 and 5 for WT and R6/1 mice, respectively).

B Quantification and representative immunoblots of lamin B2 in the striatum (8w, 12w and 20w; N = 6 for both genotypes, 30w; N = 5 for both genotypes), hippocampus (8w; N = 8 and 6 for WT and R6/1 mice, respectively; 12w; N = 6 and 7 for WT and R6/1 mice, respectively; 20w; N = 5 for both genotypes; 30w; N = 8 and 5 for WT and R6/1 mice, respectively) and cortex (8w and 12w; N = 6 for both genotypes; 20w; N = 5 for both genotypes; 30w; N = 8 and 5 for WT and R6/1 mice, respectively).

Data information: Data is expressed as a percentage of controls. Each point corresponds to the value from an individual sample. Bars represent the mean ± S.E.M. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with WT mice (two-tailed unpaired Student's t test). Tubulin was used as loading control. Exact P values are reported in Appendix Table S3.
Neuron type-specific increase in lamin B1 contributes to nuclear dysfunction in Huntington's disease (2020)
Figure 2

Figure 2. Lamin B1 distribution in the striatum of R6/1 mouse and in the putamen of HD patients.

A Mouse brain tissue was processed for immunohistochemistry by combining anti-lamin B1 (red) and anti-NeuN (green) antibodies. Representative images (maximal Z-projections) showing the distribution of lamin B1 in the striatum of 30-week-old wild-type (WT) and R6/1 mice. Yellow arrowheads show co-localization between lamin B1 and NeuN, and white arrowheads show NeuN negative lamin B1 positive nuclei. Scale bar 50 μm and 25 μm for low and high magnification, respectively.

B Lamin B1 distribution in human putamen was analysed by immunohistochemistry. Antibody against lamin B1 (red) was combined with DAPI-Fluoromount G (blue) to label nuclei. Representative images show the distribution of lamin B1 in the putamen of non-affected individuals (CTL) and HD patients at different stages of the disease (VS II-IV: Vonsattel grades). Yellow and white arrowheads indicate MSNs and glial cells, respectively. Scale bar 50 μm and 20 μm for low and high magnification, respectively.

C The distribution of lamin B1 in the putamen of HD patients was analysed by immunohistochemistry. Anti-lamin B1 antibody (red) was combined with anti-GFAP antibody (green) and nuclei were labelled with DAPI-Fluoromount G (blue). Representative images show the distribution of lamin B1 at Vonsattel grade III. Yellow arrowheads indicate MSNs and white arrowheads indicate GFAP-positive cells. Scale bar 10 μm.
Neuron type-specific increase in lamin B1 contributes to nuclear dysfunction in Huntington's disease (2020)
Figure 3

Figure 3. Lamin B1 distribution in the hippocampus of R6/1 mouse.

Hippocampal sections from 30-week-old wild-type (WT) and R6/1 mice were labelled with anti-lamin B1 antibody (red), anti-GFAP antibody (green) and nuclei were labelled with DAPI-Fluoromount G (blue).

A Representative images showing the distribution of lamin B1. On the left, images showing maximal Z-projection. Scale bar 150 μm. Small images correspond to CA1 and DG magnified images showing independent DAPI, GFAP and Lamin B1 channels, and merge, of representative confocal Z-stack images. Scale bar 50 μm.

B Left, representative images showing the distribution of lamin B1 in the nuclei of CA-1 hippocampal neurons from WT and R6/1 mice along different Z axis planes (1-6). On the right, a 3D reconstruction of the Z-stack confocal images generated using ImageJ. Scale bar 20 μm.
Neuron type-specific increase in lamin B1 contributes to nuclear dysfunction in Huntington's disease (2020)
Figure 4

Figure 4. Increase in lamin B1 levels and altered nuclear morphology occur in striatal MSNs and hippocampal CA-1 neurons from R6/1 mice.

Lamin B1 levels and morphology were analysed in specific neuronal nuclei from the striatum and hippocampus of 30-week-old wild-type (WT) and R6/1 mice, and in the putamen of HD patients by FANSI and immunohistochemistry, respectively.

A Graphs show the quantification of different parameters in mice striatal MSNs nuclei (Ctip2+/NeuN+). N = 4 for both genotypes. An average of 5,000 nuclei was analysed for each sample. Representative images are shown. Scale bar 7 μm.

B Graphs show the quantification of different parameters in mice striatal interneurons (Ctip2-/NeuN+). N = 4 for both genotypes. An average of 5,000 nuclei was analysed for each sample. Representative images are shown. Scale bar 7 μm.

C Graphs show the quantification of lamin B1 intensity and circularity in 30-week-old R6/1 mouse striatal neurons nuclei with (mHtt +) or without (mHtt -) nuclear inclusions. N = 6. EM48 antibody was used to label mHtt inclusions. Representative images are shown. Scale bar 7 μm.

D Graphs showing lamin B1 intensity and circularity in MSNs nuclei from the putamen of HD patients at different stages of the disease (VS: Vonsattel grade) and corresponding controls (CTL: non-affected individuals). N = 6 for CTL, N = 3 for VS I-II and N = 4 for VS III-IV. An average of 50 nuclei was examined for each sample. Representative images (maximal Z-projections) are shown. Scale bar 7μm.

E Graphs show the quantification of different parameters in hippocampal CA1 ( Ctip2+/Prox1-) and DG (Ctip2+/Prox1+) neurons nuclei. N = 4 for each genotype. Representative images are shown. Scale bar 7 μm.

Data information: Each point corresponds to the value from an individual sample. Bars represent the mean ± S.E.M. Data in (A-C) and (E) were analysed by two-tailed unpaired Student's t test. *P < 0.05, ** P < 0.01, ***P < 0.001 as compared with corresponding controls. Data in (D) was analysed by one-way ANOVA followed by Turkey's post hoc test. *P < 0.05 as compared with CTL; #P < 0.05 as compared with VS I-II. Exact P values are reported in Appendix Table S3.
Neuron type-specific increase in lamin B1 contributes to nuclear dysfunction in Huntington's disease (2020)
Figure 5

Figure 5. Lamin B1 overexpression in primary striatal neurons alters nuclear morphology.

Primary striatal neurons were transfected with a vector to overexpress lamin B1 (mApple-LB1) or with an empty vector (mApple-C1) and lamin B1 intensity and nuclear circularity were examined by immunocytochemistry against lamin B1 24 h after transfection.

A Graphs show the quantification of lamin B1 intensity and circularity. N = 3. An average of 12 nuclei was examined in each culture. Data are expressed as a percentage of controls. Bars represent the mean ± S.E.M. *P < 0.05 and **P < 0.01 as compared to mApple-C1 control neurons (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3.

B Representative images showing primary striatal neurons transfected with mApple-C1 or with mApple-LB1, both in red. Neuronal nuclei were stained with DAPI-Fluoromount G (blue). Lamin B1 is shown in white. Scale bar 10 μm.
Neuron type-specific increase in lamin B1 contributes to nuclear dysfunction in Huntington's disease (2020)
Figure 7

Figure 7. Lamin B1 chromatin binding in wild-type and R6/1 mice hippocampus.

A UCSC genome browser capture of lamin B1 ChIP-seq signal (log(LB1/Input)) and LADs discovered in wild-type (WT LAD) and R6/1 (R6/1 LADs) mice in combination with NPC lamin B1 DamID (top). Black arrows highlight differences in identified LADs between WT and R6/1 mice ChIP-seq data. Boxplots of LADs size (log10(LADs size + 1)) and of LADs score (log10(LADs score + 1)) obtained by EDD (bottom) from WT (N=3) and R6/1 (N=3) lamin B1 ChIP-seq data. Hipp, hippocampus. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.

B Venn diagram of overlapping LADs between wild-type (WT, N=3) and R6/1 (N=3) mice hippocampus (top). Metaprofile of lamin B1 and Input datasets mean read density within common LADs for WT and R6/1 mice (bottom).

C UCSC genome browser capture of lamin B1 ChIP-seq signal (log(LB1/Input)) LADs discovered in wild-type (WT LADs) and R6/1 (R6/1 LADs) mice; and common (green), wild-type (WT) specific (WT spec. LADs, red) and R6/1 specific (R6/1 spec. LADs, blue) LADs identified by EDD (top, WT (N=3), R6/1 (N=3)). Black arrows highlight WT specific LADs not identified in R6/1 mice. Boxplot of average size (log10(LADs size + 1)) for common, WT specific and R6/1 specific LADs (right). The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.

D Lamin B1 enrichment in common, wild-type (WT, N=3) specific (spec) and R6/1 (N=3) specific LADs (log10(LB1/Input reads +1) in hippocampus (Hipp; left). Bar graphs of significant (Benjamini's adjusted P-value < 0.05) Biological Processes terms from DAVID for genes within common and WT specific LADs (right). Gene-term enrichment was estimated by DAVID using a modified Fisher's exact test and Benjamini multiple correction test. Bars represents the -log10 (Benjamini's adjusted P-value). Exact P values are reported in Appendix Table S3.

E Representaive immunoblot showing lamin B1 levels in the nucleoplasm (Nucleop) and chromatin (Chrom) in the hippocampus of 30-week-old wild-type (WT, N = 7) and R6/1 (N = 7) mice. TBP, TATA binding protein.
Neuron type-specific increase in lamin B1 contributes to nuclear dysfunction in Huntington's disease (2020)
Figure 8

Figure 8. Hippocampal chromatin accessibility and gene expression analysis in R6/1 mice hippocampus.

A Scheme showing major steps of ATAC-seq technique.

B UCSC genome browser capture of wild-type (WT, N=3) and R6/1 (N=3) mice hippocampus ATAC-seq data, hippocampal H3K9ac and unchanged, closed in R6/1 and open in R6/1 accessible detected regions (left) in Tuba1α, Gabra2α and Dlx2 gene locus. Arrows in blue (closed in R6/1) and red (open in R6/1) indicate differential accessible regions. Bar graphs of significant (Benjamini's adjusted P-value < 0.05) Biological Processes terms from DAVID for genes associated with decreased (right top) or increased (right bottom) chromatin accessibility regions. Gene-term enrichment westimated by DAVID using a modified Fisher's exact test and Benjamini multiple correction test. Bars represents the -log10 (Benjamini's adjusted P-value). Exact P values are reported in Appendix Table S3.

C Boxplots showing average gene expression (log10(FPKMs +1) for genes up or down-regulated (adjusted P-value < 0.001, |FC|>2) in R6/1 (N=9) versus WT (N=9) mice (left). The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. Heatmap showing expression profile (log10 (FPKMs +1)) of genes up- or down-regulated (adjusted P-value<0.001, |FC|>2, N=9) in R6/1 mice (mid). Genes are ranked by the degree of expression. Numbers in colour scale show the correspondence between gene expression values and colours. Boxplots showing average TSS chromatin accessibility (log10(ATAC reads +1), N=3) for genes up or down-regulated (adjusted P-value < 0.001, |FC|>2, N=9) in R6/1 versus WT mice hippocampus (right). Exact P values are reported in Appendix Table S3. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.

D Average gene expression (closed or open regions FPKMs/ unchanged regions FPKMs) for genes associated with differential accessible regions in R6/1 (N=9) versus WT (N=9) mice (adjusted P-value < 0.05, N=3). Each point corresponds to the value from an individual sample. Data is shown as the mean ± S.E.M. *P < 0.05 as compared with WT mice (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3.
Neuron type-specific increase in lamin B1 contributes to nuclear dysfunction in Huntington's disease (2020)
Figure 9

Figure 9. Chromatin accessibility, gene expression and lamin B1 chromatin binding interconnexion.

A Boxplots of average expression (log10(FPKMs +1)) for genes sublist 1-5 (lowest to highest expression) for wild-type (WT, N=3) mice (left). Pie charts of gene distribution among generated sublists (1-5) for all genes (middle) and genes in LADs (right) in WT mice. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.

B Average gene expression (FPKMs) of genes found in common and wild-type (WT, N=3) specific LADs for WT (N=9) and R6/1 (N=9) mice. Each point corresponds to the value from an individual sample. Data is shown as the mean ± S.E.M. * P < 0.05 as compared to WT mice (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3.

C Average chromatin accessibility (normalized read counts) of genes found in common and wild-type (WT, N=3) specific LADs for WT (N=3) and R6/1 (N=3) mice. Each point corresponds to the value from an individual sample. Data is shown as the mean ± S.E.M. Wilcoxon Mann Whitney test was used for statistical analysis. Exact P values are reported in Appendix Table S3.

D Venn diagram showing the total number of genes found in common and wild-type (WT) specific LADs regions (left). Bar graph showing the number of up- and down-regulated genes found in common and WT specific LADs regions filtering only according to adjusted P-value (adjusted P-value <0.001) or additionally with fold change (adjusted P-value <0.001, |FC| > 2) obtained from deseq2 differential expression analysis (see methods).

E UCSC genome browser capture of wild-type (WT, N=3) and R6/1 (N=3) mice hippocampus ATAC-seq data, lamin B1 ChIP-seq (log(Lb1 ChIP/Input)) for WT (N=3) and R6/1 (N=3) mice hippocampus, hippocampal H3K9ac, CTCF and H3K9me3 for WT mice hippocampus in Fos locus (left). Enhancer regions (E1-E5) are indicated with arrows. Boxplot of lamin B1 enrichment (log10(Lb1 ChIP/Input)) for regions with unchanged, decreased (closed in R6/1) and increased (opened in R6/1) chromatin accessibility in R6/1 (N=3) mice hippocampus. * P < 0.05 as compared to WT (N=3) mice. (Wilcoxon Mann Whitney test). Exact P values are reported in Appendix Table S3. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.
Neuron type-specific increase in lamin B1 contributes to nuclear dysfunction in Huntington's disease (2020)
Figure 10

Figure 10. Chronic treatment with betulinic acid improves cognitive function and modulates lamin B1 levels in the hippocampus and cortex of R6/1 mice.

A Timeline of the behavioral, biochemical and histopathological analyses performed in wild-type (WT) and R6/1 mice to assess the effect of betulinic acid (BA) administration. w, weeks; AR, accelerating rotarod.

B,C Graphs show the percentage of time exploring each object with respect to the total exploration time in the (B) NOLT and (C) NORT, 5 weeks after treatment (Veh, vehicle; BA, betulinic acid; WT, wild-type). ****P < 0.0001 compared to the corresponding old location/object. WT veh N = 13; R6/1 veh N = 10; R6/1+BA N = 10.

D Accelerating rotarod was assessed after 7 weeks of treatment. **P < 0.01 and ***P < 0.001 compared to vehicle-treated wild-type (WT) mice; #P < 0.05 compared to vehicle-treated R6/1 mice. WT veh N = 13; R6/1 veh N = 9; R6/1+BA N = 9.

E Lamin B1 levels were analysed by Western blot in the striatum (WT veh N = 10; R6/1 veh N = 6; R6/1+BA N = 7), hippocampus (WT veh N = 12; R6/1 veh N = 8; R6/1+BA N = 7) and cortex (WT veh N = 11; R6/1 veh N = 6; R6/1+BA N = 7) after 12 weeks of treatment. *P < 0.05, **P < 0.05 compared to vehicle-treated wild-type (WT) mice and #P < 0.05 compared to vehicle-treated R6/1 mice. Representative immunoblots of lamin B1 and α-tubulin (as loading control) for each treatment group are shown.

F Lamin B1 intensity and nuclear morphology were analysed by FANSI in hippocampal CA1 neurons nuclei from wild-type (WT) and R6/1 mice after 12 weeks of treatment. *P < 0.05 compared to vehicle-treated WT mice. *P < 0.05 as compared to vehicle-treated WT mice. Lamin B1 intensity: WT veh N = 6; R6/1 veh N = 5; R6/1+BA N = 4; Lamin B1 circularity: WT veh N = 6; R6/1 veh N = 5; R6/1+BA N = 5.

G Nuclear permeability was analysed by FRAP in hippocampal CA1 neurons nuclei after 12 weeks of treatment. *p < 0.05 compared to vehicle-treated wild-type (WT) mice. WT veh N = 4; R6/1 veh N = 5; R6/1+BA N = 6.

Data information: In all graphs, bars represent the mean ± S.E.M and each point corresponds to the value from an individual sample Statistical analysis was performed by one-way ANOVA followed by Bonferroni's post hoc test except in (D) where data was analyzed by two-way ANOVA followed by Bonferroni's post hoc test. Exact P values are reported in Appendix Table S3.

Expanded View Figure Legends
SCA8 RAN polySer protein preferentially accumulates in white matter regions and regulated by eIF3F (2018)
Figure 1

Figure 1 - Novel polySer RAN protein encoded by the higher expressing ATXN8 transcript in SCA8

A Schematic diagram of ATXN8OS (top strand) and ATXN8 (bottom strand). RNA-seq read coverage visualized by Integrative Genomic Viewer across the ATXN8/ATXN8OS locus is depicted.

B Bar graph showing relative mRNA levels of ATXN8 and ATXN8OS. FPKM values for each transcript are normalized to FPKM value for ATXN8OS. (****; p<0.0001; mean ± SEM; unpaired t test) FPKM= fragments per kilo base of transcript per million mapped reads.

C Known and predicted homopolymeric protein expressed across SCA8 CAG expansion (ATXN8) transcripts.

D Immunohistochemistry of 10-12 month old end-stage SCA8 BAC mice brain tissue shows accumulation of novel polySer RAN protein (detected by unique antibody to the polySer protein C-terminus, α-SerCT) in cerebellar white matter, brainstem, hippocampus and layer II of the frontal cortex. Representative polySer aggregates are indicated by black arrows. No aggregates are found in age matched non-transgenic (NT) littermates (n=6 for each cohort).

E Schematic of a sagittal section of mouse brain shows the distribution of polySer RAN protein in SCA8 BAC expansion mice. Red circles represent variable size and frequency of polySer aggregates throughout different brain regions.

F Immunohistochemistry of human SCA8 autopsy tissue (n=4-7) shows accumulation of novel polySer RAN protein in cerebellar white matter, brainstem and frontal cortex.
SCA8 RAN polySer protein preferentially accumulates in white matter regions and regulated by eIF3F (2018)
Figure 4

Figure 4 - SCA8 BAC mice and human autopsy tissue show white matter abnormalities at sites of polySer accumulation

A Disease-specific sensitivity to vacuolization after prolonged storage in ethanol shown by H&E in cerebellar white matter and brainstem of 10-12 month old end-stage SCA8 BAC mice but not in age matched NT (n=3) (panels 1&2). Demyelination shown by luxol fast blue staining (LFB) (panel 3) and axonal degeneration shown by α-SMI-32 (panel 4; degenerated axons are indicated by black arrows) observed in sites of polySer accumulation shown by α-SerCT (panel 5; representative aggregates are indicated by black arrows) in deep cerebellar white matter and brainstem in SCA8 BAC mice (lower panels) but not in NT mice (upper panels) (n=3).

B Demyelination shown by luxol fast blue staining (LFB) (panel 1) and axonal degeneration shown by α-SMI-32 (panel 2) observed sites of polySer accumulation shown by a-SerCT2 (panel 3) in deep cerebellar white matter was found in SCA8 autopsy tissue but not in control brains (n=3).

C Immunohistochemistry using CC1 (α- APC) antibody shows significantly lower numbers of mature oligodendrocytes in SCA8 BAC mice compared to NT mice (NT n=5, SCA8 BAC n=5; **** p<0.0001; mean ± SEM; unpaired t test).

D Immunofluorescence using α-GFAP antibody shows significant increase in astrogliosis in SCA8 BAC mice compared to NT mice (NT n=3, SCA8 BAC n=3, ** p<0.01; mean ± SEM; unpaired t test).

E Cell death measured by lactase dehydrogenase (LDH) assay in T98 cells transfected with codon replacement constructs expressing polyGln (light blue) or polySer (dark blue). NT=nontransfected, EV=empty vector (n=5, ** p<0.003; *** p< 0.0003; mean ± SEM; unpaired t test).
SCA8 RAN polySer protein preferentially accumulates in white matter regions and regulated by eIF3F (2018)
Figure 5

Figure 5 - Mammalian translation factor eIF3F is upregulated in symptomatic SCA8 BAC mice and can regulate RAN translation

A qRT-PCR showing Eif3f expression levels are increased 2-fold in SCA8 cerebellar white matter compared to SCA8 cerebellar grey matter (n=3, **p<0.01; mean ± SEM; unpaired t test).

B Schematic diagram showing constructs used for eIF3F knockdown experiments. All constructs have a tag in each reading frame. Red M indicates methionine (ATG) initiated reading frames.

C Dot blot detection of polySer expressionin using α-FLAG antibody showing decrease in RAN polySer but not ATG-polySer when HEK293 cells are co-transfected with eIF3F siRNA.

D Quantification of polySer detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test).

E Detection of polyAla expression using α-HA antibody showing decrease in RAN polyAla but not ATG-polyAla when HEK293 cells are co-transfected with eIF3F siRNA.

F Detection of polyGP expression using α-FLAG antibody showing a decrease in RAN polyGP when HEK293 cells are co-transfected with eIF3F siRNA..

G Quantification of polyAla and polyGP detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test).

Table 1: Summary of polySer Staining in SCA8 and Control Autopsy Tissue

RAN staining: --, negative; +, low-intensity staining; ++, moderate intensity; +++, frequent and intense; ++++, highly frequent positive cells with high intensity staining.

SCA8, spinocerebellar ataxia type 8; CTRL, control; HD, Huntington disease; HDL2, Huntington disease like-2; SCA2, Spinocerebellar ataxia type 2; SCA5, Spinocerebellar ataxia type 5; SCA7, spinocerebellar ataxia type 7; DM1, myotonic dystrophy type 1; n.d, not determined; M, male; F, female; PMI, post-mortem interval

Table 2: Gait parameters from DigiGait in SCA8 BAC (forelimb only unless noted) are indicated along with corresponding P-value

NT Average

± SEM

SCA8 Average

± SEM

NT vs SCA8

Difference

SCA8 BAC

Tg Vs. NT

#, number; ↑, upregulated; ↓, downregulated; CV, coefficient of variation; ns, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001; NT n=13; SCA8 BAC n=16