Synaptic changes contribute to persistent extra-motor behaviour deficits in amyotrophic lateral sclerosis methods
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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...
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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...
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