Targeted differential gene expression profiling of skeletal muscles isolated from Ts1Cje mouse model for Down syndrome

Introduction: Down Syndrome (DS) is a chromosomal condition characterised by human chromosome 21 (HSA21) with DS critical region located on the chromosome 21q22. Owing to its polygenic basis, the phenotypes of DS are associated with delays in development of motor function including muscle hypotoria, joint hyperextensibility, delayed acquisition of postural control and poor balance. Numerous studies have revealed alterations of numerous pathways at the transcript level in muscles from other DS models such as rat and Ts65Dn mice. The mouse model used in this study is the Ts1Cje mouse model, trisomic for a region of MMU16 which encompasses a higher number of HSA21 orthologous genes. Thus, Ts1Cje model has developmental, behavioural and physical characteristics similar to that of individuals with Down syndrome. Method: In this study, soleus and extensor digitorum longus (EDL) muscles were harvested from both male and female Ts1Cje and disomic control mice. The gene expressions were profiled using the quantitative reverse transcription PCR (RT-qPCR) and analysed with the relative expression software tool (REST). We focused on genes in pathways involved in muscle structural stability, force production as well as neuromuscular signalling pathways. Results: The gene studied were Lamc1, Leprel1, My16b, Msn, Pgm5, Tmod1, Istn, Synj and Rcan. Results showed significant differential expression on My16b and Itsn1 in male soleus muscles while there was no significant differential expression of the other genes in both male and female soleus and EDL muscles. Further investigation on the role of My16b and Istn at the protein level may provide insight on the underlying mechanism responsible for hypotonia in Ts1Cje as well as Down syndrome individuals

In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts

SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1] and [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1

Transcriptomic and protein expression analyses of skeletal muscles isolated from Ts1cje mouse model for Down syndrome

Down Syndrome (DS) is caused by an additional copy of human chromosome 21 (HSA21). It is characterised by several clinical phenotypes such as intellectual disability, facial features, early onset dementia, weak immune system and hypotonia. Deficits in motor functions happens throughout development among DS individuals leading to muscle weakness, joint hyperextensibility, delayed acquisition of postural control and poor balance. To date, the underlying cause of hypotonia in DS individuals remains unknown and limited studies on the pathophysiological and molecular characterisation of hypotonia in DS could be found. Ts1Cje is a DS mouse model with partial trisomy of mouse chromosome 16 (MMU16), which encompasses a high number of HSA21 orthologous genes. This mouse model has been well reported to exhibit various typical neuropathological features as well characterised with muscle weakness that is similar to individuals with DS. The hypothesis of this study is that the trisomic genes in MMU16 are over-expressed in Ts1Cje mice and disrupts the functional molecular networks, leading to muscle weakness in Ts1Cje. To test this hypothesis, the study was divided into two major parts namely the targeted approach to study selected important markers in muscle development and function, and global transcriptomic gene expression study to identify novel genes involved in Ts1Cje muscle weakness. In this study, the soleus and extensor digitorum longus (EDL) skeletal muscles were harvested from both Ts1Cje mice and disomic control mice. Reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) analysis of nine selected genes Lamc1, Leprel1, Myl6b, Msn, Pgm5, Tmod1, Istn1, Synj1 and Rcan1 showed an upregulation of the triplicated genes Itsn1, Synj1 and Rcan1 in both soleus and EDL muscles of the Ts1Cje mice. The disomic genes Lamc1, Leprel1, Msn, Pgm5 and Tmod1 did not show any significant dysregulation in expression while Myl6b was the only disomic gene found to be significantly upregulated in the soleus muscles of Ts1Cje mice. Western blot analysis on three proteins of the myogenic regulatory factors (MRFs) family namely MyoD, Myf5 and Myg showed no significant difference in expression in both muscles. Following the targeted study, transcriptomic profiling of the soleus and EDL muscles of the Ts1Cje and wild-type disomic mice using microarray was performed. Results showed a total of 166 coding DEGs found in soleus muscles with 37 of them located on MMU16 and a total of 262 coding differentially expressed genes (DEGs) found in EDL muscles with 13 of them located on MMU16 (p-value ≤ 0.05, absolute fold change (abs FC) ≥ 1.5). Functional annotation clustering of these DEGs showed 5 significant functional clusters for soleus (signal transduction; development of reproductive system; nucleic acid biosynthesis; protein modification and metabolism and regulation of gene expression) and 3 significant functional clusters for EDL (neuron and cell development; protein modification and metabolic processes; and ion transport). Validation of nine selected genes which were found to be differentially expressed in both soleus muscles and EDL muscles was performed using RT-qPCR. The genes were Cdk13, Mansc1, Ifnar1, Ifnar2, Donson, Dyrk1a, Runx1, Sod1, and Tmem50b with the later 7 the trisomic genes. The analysis showed that all genes were upregulated in Ts1Cje soleus muscles by ≥ 1.5 fold while only Don, Ifnar2 and Tmem50bwere upregulated in Ts1Cje EDL muscles by ≥ 1.5 fold. Western blot analysis of two of the trisomic genes at the protein level showed a downregulation of Ifnar1 in Ts1Cje soleus muscles and downregulation of Ifnar2 in both soleus and EDL muscles of Ts1Cje mice as oppose to being upregulated in microarray and RT-qPCR analysis. Collectively, these findings showed an overexpression of the trisomic genes in Ts1Cje skeletal muscles, validating the hypothesis that the dysregulation of the genes caused by the triplicated region of MMU16 leads to muscle weakness in Ts1Cje. However, further investigation on the role of these genes and the protein expression levels may provide further insight on the underlying mechanism responsible for muscle weakness in Ts1Cj

Perturbed metabolic profiles associated with muscle weakness seen in adult Ts1Cje mouse model of Down syndrome

Down syndrome (DS) is a genetic condition resulting from a partial or full triplication of human chromosome 21. Besides intellectual disability, DS is frequently associated with hypotonia. Ts1Cje, mouse model of DS, displays the muscle weakness characteristic. The metabolic profiles of the skeletal muscle was characterised using 1H nuclear magnetic resonance spectroscopy and multivariate data analysis. Ts1Cje muscle had significantly decreased levels of glutamine, guanidinoacetate, adenosine monophosphate, and histidine, suggesting perturbation of energy, glutamate, and histidine metabolic pathways. Glycine amidinotransferase/arginine glycine amidinotransferase enzyme-linked immunosorbent assay indicated this mitochondrial enzyme was 74% and 50% lower in Ts1Cje kidney and liver than the wildtype respectively. In conclusion, our findings suggest that perturbed metabolite profiles contribute to muscle weakness in Ts1Cje skeletal muscle

Defects in nerve conduction velocity and different muscle fibre-type specificity contribute to muscle weakness in Ts1Cje Down syndrome mouse model.

BACKGROUND:Down syndrome (DS) is a genetic disorder caused by presence of extra copy of human chromosome 21. It is characterised by several clinical phenotypes. Motor dysfunction due to hypotonia is commonly seen in individuals with DS and its etiology is yet unknown. Ts1Cje, which has a partial trisomy (Mmu16) homologous to Hsa21, is well reported to exhibit various typical neuropathological features seen in individuals with DS. This study investigated the role of skeletal muscles and peripheral nerve defects in contributing to muscle weakness in Ts1Cje mice. RESULTS:Assessment of the motor performance showed that, the forelimb grip strength was significantly (P<0.0001) greater in the WT mice compared to Ts1Cje mice regardless of gender. The average survival time of the WT mice during the hanging wire test was significantly (P<0.0001) greater compared to the Ts1Cje mice. Also, the WT mice performed significantly (P<0.05) better than the Ts1Cje mice in the latency to maintain a coordinated motor movement against the rotating rod. Adult Ts1Cje mice exhibited significantly (P<0.001) lower nerve conduction velocity compared with their aged matched WT mice. Further analysis showed a significantly (P<0.001) higher population of type I fibres in WT compared to Ts1Cje mice. Also, there was significantly (P<0.01) higher population of COX deficient fibres in Ts1Cje mice. Expression of Myf5 was significantly (P<0.05) reduced in triceps of Ts1Cje mice while MyoD expression was significantly (P<0.05) increased in quadriceps of Ts1Cje mice. CONCLUSION:Ts1Cje mice exhibited weaker muscle strength. The lower population of the type I fibres and higher population of COX deficient fibres in Ts1Cje mice may contribute to the muscle weakness seen in this mouse model for DS

Gene and protein expression profiles of JAK-STAT signalling pathway in the developing brain of the Ts1Cje down syndrome mouse model

Aims: The JAK-STAT signalling pathway is one of the key regulators of pro-gliogenesis process during brain development. Down syndrome (DS) individuals, as well as DS mouse models, exhibit an increased number of astrocytes, suggesting an imbalance of neurogenic-to-gliogenic shift attributed to dysregulated JAK-STAT signalling pathway. The gene and protein expression profiles of JAK-STAT pathway members have not been characterised in the DS models. Therefore, we aimed to profile the expression of Jak1, Jak2, Stat1, Stat3 and Stat6 at different stages of brain development in the Ts1Cje mouse model of DS. Methods: Whole brain samples from Ts1Cje and wild-type mice at embryonic day (E)10.5, E15, postnatal day (P)1.5; and embryonic cortex-derived neurospheres were collected for gene and protein expression analysis. Gene expression profiles of three brain regions (cerebral cortex, cerebellum and hippocampus) from Ts1Cje and wild-type mice across four time-points (P1.5, P15, P30 and P84) were also analysed. Results: In the developing mouse brain, none of the Jak/Stat genes were differentially expressed in the Ts1Cje model compared to wild-type mice. However, Western blot analyses indicated that phosphorylated (p)-Jak2, p-Stat3 and p-Stat6 were downregulated in the Ts1Cje model. During the postnatal brain development, Jak/Stat genes showed complex expression patterns, as most of the members were downregulated at different selected time-points. Notably, embryonic cortex-derived neurospheres from Ts1Cje mouse brain expressed lower Stat3 and Stat6 protein compared to the wild-type group. Conclusion: The comprehensive expression profiling of Jak/Stat candidates provides insights on the potential role of the JAK-STAT signalling pathway during abnormal development of the Ts1Cje mouse brains

Defects in nerve conduction velocity and different muscle fibre-type specificity contribute to muscle weakness in Ts1Cje Down syndrome mouse model - Fig 4

<p><b>The protein expression of myogenic regulatory factors (MRFs) markers in quadriceps and triceps (A) Soleus and EDL (B).</b> MyoD expression was found to be significantly (<i>P</i><0.05) upregulated in Ts1Cje quadriceps <b>(C).</b> There was a reduction trend of the expression levels of myogenin in the muscles screened in Ts1Cje male mice but it was statistically insignificant <b>(D).</b> Myf5 was significantly (<i>P</i><0.05) downregulated in the Ts1Cje triceps <b>(E).</b></p

Behavioural assessment of muscle weakness in Ts1Cje mouse.

<p>Forelimb grip strength was significantly (<i>P</i><0.0001) greater in the WT mice compared to the Ts1Cje mice for both genders (male: <i>P</i> = 0.0016; female: <i>P =</i> 0.0021) <b>(A).</b> For the hanging wire test, the survival proportion of the WT mice was significantly (<i>P</i><0.01) greater than that of the Ts1Cje mice <b>(B).</b> Ts1Cje mice had a significantly (<i>P</i><0.0001) greater number of falls compared to the WT mice for both genders <b>(C)</b>. At an accelerated speed of 4–64 rpm, the motor coordination of the WT mice was significantly (<i>P</i><0.05) greater than that of the Ts1Cje group <b>(D).</b> Asterisks *, **, *** and **** denote p <0.05, 0.005, 0.0005 and 0.0001 respectively.</p