Synaptic changes contribute to persistent extra-motor behaviour deficits in amyotrophic lateral sclerosis methods
Aim. Evidence-backed execution summary for Synaptic changes contribute to persistent extra-motor behaviour deficits in amyotrophic lateral sclerosis methods from Synaptic changes contribute to persistent extra-motor behaviour deficits in amyotrophic lateral sclerosis.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
RNA extraction and analysis
reagent used in the protocol.
- Use
- RNA was extracted with Qiazol (Qiagen #79,306) using the Qiagen RNeasy Mini Kit (Qiagen, #74,104) and Precellys tissue homogeniser (Bertin Instruments, Montigny-le-Bretonneux, France). On-column DNase I digestion was conducted using RNase-free DNase I (Qiagen #79,254). The concentration of extracted RNA was determin...
Early and persistent differential exon usage in neuronal genes correlates with behavioural abnormalities in rNLS8 mice
reagent used in the protocol.
- Use
- At disease onset, n = 321 genes were significantly differentially expressed in the cortex (log fold change > 1, P < 0.05) including n = 106 downregulated and n = 215 upregulated (Fig. A, Supplementary Table ). In contrast, after 6 weeks...
Open field test
Open Field Assessment of general exploratory locomotion in a novel clear Plexiglas (30 cm × 30 cm × 50 cm) arena with white floor divided by two zones: periphery and centre (comprising 50% of the total area cantered) as previously described [ ]. The activities were as...
- Use
- Open Field Assessment of general exploratory locomotion in a novel clear Plexiglas (30 cm × 30 cm × 50 cm) arena with white floor divided by two zones: periphery and centre (comprising 50% of the total area cantered) as previously described [ ]. The activities were as...
Social interaction test
Social interaction was assessed using a social approach test in a three chamber apparatus with identical Plexiglas chambers (40 × 40 × 40 cm) as previously described [ ]. Before testing, the animal was allowed to explore freely for 5 min in the testing arena which served t...
- Use
- Social interaction was assessed using a social approach test in a three chamber apparatus with identical Plexiglas chambers (40 × 40 × 40 cm) as previously described [ ]. Before testing, the animal was allowed to explore freely for 5 min in the testing arena which served t...
Y-maze test
A Y-maze with three identical arms made of transparent Plexiglas (25 cm × 25 cm × 25 cm) placed at 120º angles to each other was used and placed in a room with clues to allow for visual orientation with illumination 30 lx as previously described [ ]. Each mo...
- Use
- A Y-maze with three identical arms made of transparent Plexiglas (25 cm × 25 cm × 25 cm) placed at 120º angles to each other was used and placed in a room with clues to allow for visual orientation with illumination 30 lx as previously described [ ]. Each mo...
RNA extraction and analysis
RNA was extracted with Qiazol (Qiagen #79,306) using the Qiagen RNeasy Mini Kit (Qiagen, #74,104) and Precellys tissue homogeniser (Bertin Instruments, Montigny-le-Bretonneux, France). On-column DNase I digestion was conducted using RNase-free DNase I (Qiagen #79,254). The concentration of extracted RNA was determin...
- Use
- RNA was extracted with Qiazol (Qiagen #79,306) using the Qiagen RNeasy Mini Kit (Qiagen, #74,104) and Precellys tissue homogeniser (Bertin Instruments, Montigny-le-Bretonneux, France). On-column DNase I digestion was conducted using RNase-free DNase I (Qiagen #79,254). The concentration of extracted RNA was determin...
Transcriptomic analysis
RNA was purified from the right rostral cortex, hippocampus, and lumbar spinal cord of control and rNLS8 mice ( n = 4/group, 2 male and 2 female) at disease onset (2 WOD) and recovery (2 WOD followed by 6 weeks on dox). The rostral cortex was chosen as it contains the frontal and temporal-parietal...
- Use
- RNA was purified from the right rostral cortex, hippocampus, and lumbar spinal cord of control and rNLS8 mice ( n = 4/group, 2 male and 2 female) at disease onset (2 WOD) and recovery (2 WOD followed by 6 weeks on dox). The rostral cortex was chosen as it contains the frontal and temporal-parietal...
POSTAR3 analysis of TDP-43 binding sites
Transcripts with DEU events identified in the disease onset (2 WOD) and recovery (2 WOD followed by 6 weeks on dox) were analysed for TDP-43 binding capacity using the publicly available CLIP-seq database accessible via POSTAR3 [ ]. The CLIPdb module of POSTAR3 was used to identify known binding sites of human...
- Use
- Transcripts with DEU events identified in the disease onset (2 WOD) and recovery (2 WOD followed by 6 weeks on dox) were analysed for TDP-43 binding capacity using the publicly available CLIP-seq database accessible via POSTAR3 [ ]. The CLIPdb module of POSTAR3 was used to identify known binding sites of human...
Statistical analysis
For data from two groups, statistical analyses were conducted using a two-tailed t-test after confirming normality assumptions. For data from four groups, Two-way ANOVA was used for analyses of datasets with Bonferroni's post hoc test using Prism-GraphPad software (version 9). Statistical signif...
- Use
- For data from two groups, statistical analyses were conducted using a two-tailed t-test after confirming normality assumptions. For data from four groups, Two-way ANOVA was used for analyses of datasets with Bonferroni's post hoc test using Prism-GraphPad software (version 9). Statistical signif...
Early and persistent differential exon usage in neuronal genes correlates with behavioural abnormalities in rNLS8 mice
To identify molecular changes associated with the enduring hyperlocomotion phenotype in the rNLS8 mice, we conducted bulk RNA sequencing of cortex tissue. Given that TDP-43 acts as an RNA-binding protein regulating both expression and splicing, we assessed changes to gene expression and differential exon usage (DEU;...
- Use
- To identify molecular changes associated with the enduring hyperlocomotion phenotype in the rNLS8 mice, we conducted bulk RNA sequencing of cortex tissue. Given that TDP-43 acts as an RNA-binding protein regulating both expression and splicing, we assessed changes to gene expression and differential exon usage (DEU;...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- For data from two groups, statistical analyses were conducted using a two-tailed t-test after confirming normality assumptions. For data from four groups, Two-way ANOVA was used for analyses of datasets with Bonferroni's post hoc test using Prism-GraphPad software (version 9). Statistical signif...
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Methods
rNLS8 mice were produced from the intercross of >10 generation back-crossed C57BL/6JAusb background homozygous tetO -hTDP-43 ΔNLS line 4 mice (B6.C3-Tg(tetO-TARDBP*)4Vle/JAusb, derived from Jackson Laboratories stock #014650) with hemizygous NEFH -tTA line 8 mice (B6.C3-Tg(NEFH-tTA)8Vle/JAusb, derived from Jackson stock #025397), constantly fed with dox-containing chow (200 mg/kg, Specialty Feeds, Australia) [, ]. The number of experimental mice per sex in individual tests can be seen in individual figure legends. Experiments were conducted with approval from the Animal Ethics Committee of The University of Queensland (#QBI/131/18 and 2022/ AE000578 ). During experiments, male and female mice were switched to normal chow (SF00-100, Specialty Feeds, Australia) to induce expression of hTDP-43 ∆NLS at approximately ten weeks of age. All mice were housed in tempera...
Transcriptomic analysis
RNA was purified from the right rostral cortex, hippocampus, and lumbar spinal cord of control and rNLS8 mice ( n = 4/group, 2 male and 2 female) at disease onset (2 WOD) and recovery (2 WOD followed by 6 weeks on dox). The rostral cortex was chosen as it contains the frontal and temporal-parietal regions relevant to ALS and FTD. The quality of total RNA samples was determined by Agilent Bioanalyzer. Samples with RNA Integrity Number (RIN) > 8.0 were then used for library construction. RNA was analysed by RNA-seq (polyA enriched, strand-specific, Illumina paired-end 150 bp, 20 M reads) with Azenta Life Science. Quality assessment of the data was determined (FastQC, v0.10.1), low quality reads and adapter sequences were removed (Cutadapt, v1.9.1), and aligned (Hisat2, v2.0.1) to the reference genome ( Mus musculus, ensembl, GRCm39.107). Transcri...
Data availability statement
The transcriptomic data has been deposited to the Sequence Research Archive (PRJNA1298393). Previously published mouse proteomics [ ], human proteomics [ ] and human transcriptomics [ ] datasets used in this work for comparative analyses are available from the respective references. Supplementary Data are provided with this paper and any additional information required to re-analyse the data reported in this paper is available from the corresponding authors upon request.
Early and persistent differential exon usage in neuronal genes correlates with behavioural abnormalities in rNLS8 mice
To identify molecular changes associated with the enduring hyperlocomotion phenotype in the rNLS8 mice, we conducted bulk RNA sequencing of cortex tissue. Given that TDP-43 acts as an RNA-binding protein regulating both expression and splicing, we assessed changes to gene expression and differential exon usage (DEU; a measure of alternative splicing) in the cortex of mice at disease onset (2 WOD) and in the "short-term disease" recovery experimental paradigm (2 WOD + 6 weeks, Fig. ). Fig. 2 The cortical transcriptome at disease onset is strongly associated with neuroinflammation and this is completely normalised in recovery in rNLS8 mice. A, B Volcano plots of transcript mean log fold change (LFC; rNLS8/Con) and significance level [-log10(Pvalue)] of cortex from A disease onset (2 WOD) and B recovery (2 WOD + 6 weeks back on do...
rNLS8 mice develop hyperlocomotion, hyperactivity, and habituation deficits, concomitant with impaired motor function...
rNLS8 mice also displayed a dramatic increase of rearing number in the open field test at 1 and 2 WOD (Fig. D) which was less evident at later timepoints when hindlimb function was impaired. Importantly, the rNLS8 mice re-displayed the increased rearing behaviour by 2 weeks back on dox (Fig. D and Supplementary Fig. 6E), indicating persistence of this phenotype that had been masked by motor impairment in the later disease stages. We also observed significant decreases in the relative central travel (%) in the rNLS8 mice after motor onset at 3 WOD, which persisted to the recovery phase through 6 WOD + 6 weeks, implying an anxiety-like phenotype (Fig. E). However, we did not observe significant space-related anxiety-like behaviours in the commonly used elevated plus maze paradigm (Supplementary Fig. 7). Nevertheless, we detected slightly...
Measurement outputs
What raw and processed outputs should exist?
RNLS8 mice were produced from the intercross of >10 generation back-crossed C57BL/6JAusb background homozygous tetO -hTDP-43 ΔNLS line 4 mice (B6.C3-Tg(tetO-TARDBP*)...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
RNA was extracted with Qiazol (Qiagen #79,306) using the Qiagen RNeasy Mini Kit (Qiagen, #74,104) and Precellys tissue homogeniser (Bertin Instruments, Montigny-le-Bretonneux, F...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
RNA was purified from the right rostral cortex, hippocampus, and lumbar spinal cord of control and rNLS8 mice ( n = 4/group, 2 male and 2 female) at disease onset (...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Transcripts with DEU events identified in the disease onset (2 WOD) and recovery (2 WOD followed by 6 weeks on dox) were analysed for TDP-43 binding capacity using the publ...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
For data from two groups, statistical analyses were conducted using a two-tailed t-test after confirming normality assumptions.
from paperScoring or quantification
Quantify the primary readouts for this experiment: RNLS8 mice were produced from the intercross of >10 generation back-crossed C57BL/6JAusb background homozygous tetO -hTDP-43 ΔNLS line 4 mice (B6.C3-Tg(tetO-TARDBP*)...; RNA was extracted with Qiazol (Qiagen #79,306) using the Qiagen RNeasy Mini Kit (Qiagen, #74,104) and Precellys tissue homogeniser (Bertin Instruments, Montigny-le-Bretonneux, F...; RNA was purified from the right rostral cortex, hippocampus, and lumbar spinal cord of control and rNLS8 mice ( n = 4/group, 2 male and 2 female) at disease onset (...; Transcripts with DEU events identified in the disease onset (2 WOD) and recovery (2 WOD followed by 6 weeks on dox) were analysed for TDP-43 binding capacity using the publ....
from paperStatistical comparison
For data from two groups, statistical analyses were conducted using a two-tailed t-test after confirming normality assumptions. For data from four groups, Two-way ANOVA was...; We then hypothesised that alternative splicing changes due to TDP-43 dysfunction may persist from disease onset to recovery to drive enduring behavioural changes in the rNLS8 mi...; In the recovery cortex (2 WOD + 6 weeks), n = 175 genes showed significant DEU (199 DEU events, P < 0.05), which were enriched for neu...; Cell type enrichment analysis of genes with DEU events in recovery rNLS8 cortex (2 WOD + 6 weeks on dox) revealed pronounced CNS specificity (Supplementary Fig....
from paperReporting output
Report representative outputs alongside summary comparisons for RNLS8 mice were produced from the intercross of >10 generation back-crossed C57BL/6JAusb background homozygous tetO -hTDP-43 ΔNLS line 4 mice (B6.C3-Tg(tetO-TARDBP*)..., RNA was extracted with Qiazol (Qiagen #79,306) using the Qiagen RNeasy Mini Kit (Qiagen, #74,104) and Precellys tissue homogeniser (Bertin Instruments, Montigny-le-Bretonneux, F..., RNA was purified from the right rostral cortex, hippocampus, and lumbar spinal cord of control and rNLS8 mice ( n = 4/group, 2 male and 2 female) at disease onset (..., Transcripts with DEU events identified in the disease onset (2 WOD) and recovery (2 WOD followed by 6 weeks on dox) were analysed for TDP-43 binding capacity using the publ....
inferred from protocolStructured statistical methods
For data from two groups, statistical analyses were conducted using a two-tailed t-test after confirming normality assumptions. For data from four groups, Two-way ANOVA was...; We then hypothesised that alternative splicing changes due to TDP-43 dysfunction may persist from disease onset to recovery to drive enduring behavioural changes in the rNLS8 mi...; In the recovery cortex (2 WOD + 6 weeks), n = 175 genes showed significant DEU (199 DEU events, P < 0.05), which were enriched for neu...; Cell type enrichment analysis of genes with DEU events in recovery rNLS8 cortex (2 WOD + 6 weeks on dox) revealed pronounced CNS specificity (Supplementary Fig....
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
rNLS8 mice were produced from the intercross of >10 generation back-crossed C57BL/6JAusb background homozygous tetO -hTDP-43 ΔNLS line 4 mice (B6.C3-Tg(tetO-TARDBP*)4Vle/JAusb, derived from Jackson Laboratories stock #014650) with hemizygous NEFH -tTA line 8 mice (B6.C3-Tg(NEFH-tTA)8Vle/JAusb, derived from Jackson stock #025397), constantly fed with dox-containing chow (200 mg/kg, Specialty Feeds, Australia) [, ]. The number of experimental mice per sex in individual tests can be seen in individual figure legends. Experiments were conducted with approval from the Animal Ethics Committee of The University of Queensland (#QBI/131/18 and 2022/ AE000578 ). During experiments, male and female mice were switched to normal chow (SF00-100, Specialty Feeds, Australia) to induce expression of hTDP-43 ∆NLS at approximately ten weeks of age. All mice were housed in temperature- and humidity-controlled conditions (21 ± 1 °C, 55 ± 5%) with a 12-h light/dark cycle (lights on at 06:00 h).
RNA was purified from the right rostral cortex, hippocampus, and lumbar spinal cord of control and rNLS8 mice ( n = 4/group, 2 male and 2 female) at disease onset (2 WOD) and recovery (2 WOD followed by 6 weeks on dox). The rostral cortex was chosen as it contains the frontal and temporal-parietal regions relevant to ALS and FTD. The quality of total RNA samples was determined by Agilent Bioanalyzer. Samples with RNA Integrity Number (RIN) > 8.0 were then used for library construction. RNA was analysed by RNA-seq (polyA enriched, strand-specific, Illumina paired-end 150 bp, 20 M reads) with Azenta Life Science. Quality assessment of the data was determined (FastQC, v0.10.1), low quality reads and adapter sequences were removed (Cutadapt, v1.9.1), and aligned (Hisat2, v2.0.1) to the reference genome ( Mus musculus, ensembl, GRCm39.107). Transcriptomic data was analysed for differential gene expression (DESeq2 [ ], v1.6.3) and differential exon usage (DEXSeq [ ], v1.18.4) as a measure of alternative splicing.
The transcriptomic data has been deposited to the Sequence Research Archive (PRJNA1298393). Previously published mouse proteomics [ ], human proteomics [ ] and human transcriptomics [ ] datasets used in this work for comparative analyses are available from the respective references. Supplementary Data are provided with this paper and any additional information required to re-analyse the data reported in this paper is available from the corresponding authors upon request.
To identify molecular changes associated with the enduring hyperlocomotion phenotype in the rNLS8 mice, we conducted bulk RNA sequencing of cortex tissue. Given that TDP-43 acts as an RNA-binding protein regulating both expression and splicing, we assessed changes to gene expression and differential exon usage (DEU; a measure of alternative splicing) in the cortex of mice at disease onset (2 WOD) and in the "short-term disease" recovery experimental paradigm (2 WOD + 6 weeks, Fig. ). Fig. 2 The cortical transcriptome at disease onset is strongly associated with neuroinflammation and this is completely normalised in recovery in rNLS8 mice. A, B Volcano plots of transcript mean log fold change (LFC; rNLS8/Con) and significance level [-log10(Pvalue)] of cortex from A disease onset (2 WOD) and B recovery (2 WOD + 6 weeks back on dox). Significantly upregulated genes are in red and downregulated in blue. C Gene ontology analysis of biological process enrichment at disease onset (2 WOD) of significantly upregulated (top) and downregulated (bottom) genes. Size of the data points are relative to the number of genes in the term. D...
rNLS8 mice also displayed a dramatic increase of rearing number in the open field test at 1 and 2 WOD (Fig. D) which was less evident at later timepoints when hindlimb function was impaired. Importantly, the rNLS8 mice re-displayed the increased rearing behaviour by 2 weeks back on dox (Fig. D and Supplementary Fig. 6E), indicating persistence of this phenotype that had been masked by motor impairment in the later disease stages. We also observed significant decreases in the relative central travel (%) in the rNLS8 mice after motor onset at 3 WOD, which persisted to the recovery phase through 6 WOD + 6 weeks, implying an anxiety-like phenotype (Fig. E). However, we did not observe significant space-related anxiety-like behaviours in the commonly used elevated plus maze paradigm (Supplementary Fig. 7). Nevertheless, we detected slightly decreased anxiety-like behaviour in rNLS8 mice in the light-dark transition test, with significantly decreased duration (%) in the dark chamber (Supplementary Fig. 8), in line with our previous finding in the CAMKII -hTDP-43 ΔNLS mice [ ]. These data suggest the decreases of relativ...
Machine-readable layer
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