Spinocerebellar ataxia type 3 (SCA3) or Machado-Joseph disease (MJD) is the most common autosomal dominant spinocerebellar ataxia in China with highly clinical heterogeneity, such as progressive cerebellar ataxia, dysarthria, pyramidal signs, external ophthalmoplegia, dysphagia, and distal muscle atrophy. It is caused by the abnormal expansion of CAG repeats in a coding region of
PolyQ diseases is a group of disorders caused by CAG repeat expansions within the, respectively, responsible genes, including Huntington disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), spinocerebellar ataxias (SCA1, SCA2, SCA3/Machado-Joseph disease, SCA6, SCA7, and SCA17) [
The SCA3/MJD, the most common case, accounts for 62.64% of autosomal dominant spinocerebellar ataxia in China [
Long noncoding RNA (lncRNA) is defined the nontranslatable RNA with the length of 200 nucleotides or above. The lncRNAs used to be regarded as the transcriptional “noise,” the products of RNA polymerase II transcription, and did not have the biological function. However, the emerging evidence has proved their significant roles in the regulation of gene transcription, posttranscriptional regulation, and epigenetic regulation [
In recent years, accumulating studies have found that lncRNAs are associated with neurodegenerative diseases. Spinocerebellar ataxia type 8 (SCA8), a kind of slowly progressive ataxia, is caused by the abnormal expansion of (CTG)n within the responsible gene ATXN8. A study proposes that the pathogenesis of SCA8 involves both protein and RNA gain-of-function mechanisms. (CTG)n-expanded ATXN8 encodes a pathogenic protein, and the antisense strand encodes CUG-enriched lncRNA ATXN8OS which is deposited in the nucleus and activates alternative splicing, resulting in an alternation of the expression of GABA-A transport factor 4 (GAT4/Gabt4) and finally loss of the GABAergic inhibition [
In a separate study, the expression of lncRNA was compared between the normal brain tissue and the brain tissue of patients with Huntington disease. A total of 35 upregulated and 146 downregulated lncRNA molecules were identified, and NEAT1 was selected by Bioinformatics. Based on the cell-level experiments, it was found that overexpression of NEAT1 was significantly resistant to H2O2-induced cellular damage, providing a new potential strategy for clinical treatment of the Huntington’s disease [
To further explore the pathogenesis of SC3/MJD at RNA level, the lncRNAs specifically expressed in SCA3/MJD mice were investigated in this study.
SCA3/MJD mouse model (B6; CBA-Tg (ATXN3
Validation of genotype was conducted in the second generation. Polymerase chain reaction (PCR), agarose gel electrophoresis, and capillary electrophoresis sequencing were used for genotype validation. Genomic DNA was extracted from mice tails. CAG repeats were amplified using a pair of primers 5
The phenotype was validated using the footprint and rotating tests. For footprint pattern analysis, the hind paws of mice were painted with black ink and the forepaws were painted with red ink. The mice walked along a narrow corridor paved with white paper. Pretraining was conducted for one week before the formal test. Mice were tested three times with 5-minute intervals. Stride length, hind paw width, front paw width, and front/hind footprint overlap were measured. For rotation, mice were placed on a rotating rod and must maintain its balance. The interval from the start of the rod rotating to the mice falling from the rotating rod was recorded. Mice were tested on separate trials at fixed speeds including 10 r/min and 20 r/min.
lncRNA-Seq, a high-throughput sequencing, was performed in BGI. After extracting the total RNA from mice cerebellum (three SCA3/MJD mice versus three wild-type mice), mRNA and noncoding RNAs are enriched by removing rRNA from the total RNA. By using the fragmentation buffer, the mRNAs and noncoding RNAs are fragmented into short fragments (about 200~500 nt), then the first-strand cDNA is synthesized by random hexamer-primer using the fragments as templates, and dTTP is substituted by dUTP during the synthesis of the second strand. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters, and then the second strand is degraded using UNG (uracil-N-glycosylase) finally [
The total RNA from mice cerebellum (six SCA3/MJD mice versus six wild-type mice) was reversely transcribed to cDNA with a kit (Thermo Scientific, RevertAid First Strand cDNA #K1622). The 44 most differentially expressed lncRNAs were screened for further validation by qRT-PCR assays (Maxima SYBR Green qPCR Master Mix, CFX96, Bio-Rad, USA).
We conducted some biological analysis of the differentially expressed lncRNAs by search and comparison of databases such as NONCODE v5 (
The SCA3/MJD mice that carry the ATXN3 were genotyped and phenotyped. As a result, the ATXN3 carrying rate was about 50% in the second generation of SCA3/MJD mice, by Jackson Laboratory’s report. The number of CAG repeats was 84 or 83 in the second generation SCA3/MJD mice. In rotation test, the interval of mice keeping balance on the rotating rod was significantly different between the SCA3/MJD and wild-type mice no matter the rotation speed was 10 r/min or 20 r/min (see Table
The results of rotation test.
Rotation speed | Time (seconds) | Wild-type mice ( |
SCA3/MJD mice ( |
|
---|---|---|---|---|
10 r/min | Mean ± SD | 250.067 ± 9.487 | 104.400 ± 8.902 | 0.023 |
20 r/min | Mean ± SD | 196.467 ± 8.126 | 34.600 ± 6.710 | 0.002 |
The wild-type mice performed much better than the SCA3/MJD mice in the rotation test.
Abnormal gait was observed by analyzing the footprint pattern. In contrast to the control mice’s straight line with regular alternating gait, the SCA3/MJD mice showed unstable movements in a way that weaved from side to side when walked along the narrow corridor. Altogether suggest that the SCA3/MJD mice have similar clinical manifestations with the SCA3/MJD patients.
By using RNA-seq, 10,443 of novel and 13,395 of known lncRNAs were detected in 3 of the SCA3/MJD and 3 of the WT mice. One by one differential expression analysis showed total 2964 upregulated and 4376 downregulated lncRNAs, respectively, in the three experimental groups. More specifically, there were 745, 776, and 1250 of the upregulated lncRNAs in three groups, in contrast to the significantly increased numbers of 1285, 1065, and 1600, the downregulated lncRNAs, respectively, in three groups. Further group differential expression analysis found 193 of the upregulated and 467 of the downregulated lncRNAs in the SCA3/MJD mice. The lncRNAs with differential expression identified by the one by one differential expression analysis in two or three groups were chosen for further validation. The lncRNAs were sorted based on their FPKM and
lncRNA number TCONS_00031478 was excluded due to Ct > 35. In the rest 43 of the chosen lncRNAs, 3 of the lncRNAs were validated to be differential expression between SCA3/MJD and WT mice cerebellum. It turned out that two of the three are the known lncRNAs number n297477 (
The expression level of the three lncRNAs.The expression level of three lncRNAs was different between wild-type and SCA3/MJD mice with statistical significance. The two known lncRNAs, n297477 and n297609, were upregulated in the cerebellum of SCA3/MJD mice. The expression level was increased by 3.329 times (
In this study, 2964 upregulated lncRNAs and 4376 downregulated lncRNAs were identified specifically in the SCA3/MJD mice using lncRNA-Seq analysis. Additionally, differentially expressed three lncRNAs, including one novel lncRNA and two known lncRNAs, were further characterized.
The lncRNA n297477 is transcribed from the antisense strand of the chr11: 6270375–6271530, which is highly expressed in mouse heart, hippocampus, liver, lung, spleen, and thymus. According to the records in database FANTOM5, n297477 is considered one of the transcripts of the
The summary of genetic association.
Gene name | Associated gene name | Proteins of associated gene |
---|---|---|
ATXN3 | VCP | Valosin containing protein |
UBC | Ubiquitin C | |
KCTD10 | Potassium channel tetramerisation domain containing 10 | |
RAD23A | RAD23 homolog A | |
RAD23B | RAD23 homolog B | |
PARK2 | Parkinson protein 2 | |
USP13 | Ubiquitin specific peptidase 13 | |
UBE4B | Ubiquitination factor E4B | |
STUB1 | STIP1 homology and U-box containing protein 1 | |
SERPINC1 | Serpin peptidase inhibitor, clade C (antithrombin), member 1 | |
UBC | PSMD4 | Proteasome (prosome, macropain) 26S subunit, non-ATPase 4 |
HSP90AA1 | Heat shock protein 90 kDa alpha (cytosolic), class A member 1 | |
HGS | Hepatocyte growth factor-regulated tyrosine kinase substrate | |
PSMC2 | Proteasome (prosome, macropain) 26S subunit, ATPase 2 | |
TSG101 | Tumor susceptibility gene 101 | |
UBE2D2 | Ubiquitin-conjugating enzyme E2D 2 | |
PSMD14 | Proteasome (prosome, macropain) 26S subunit, non-ATPase 14 | |
CUL1 | Cullin 1 | |
RPS27A | Ribosomal protein S27a (156 aa) |
Given that the ubiquitin-proteasome system (UPS) is involved in the pathogenesis of SCA3/MJD [
The lncRNA n297609 is transcribed from the antisense strand of the chr9: 44047488–43856016, which is highly expressed in mouse heart, hippocampus, liver, lung, spleen, and thymus. According to the records in database FANTOM5, n297609 is considered one of the transcripts of the
Moreover, we also found a novel lncRNA TCONS_00072962 with the genomic location at chr7:119737479–119737966, contributing to the expansion of lncRNA expression profiles of the mouse.
In summary, we used lncRNA-Seq to profile cerebellar expression in SCA3/MJD mice and identified three potential lncRNAs significantly associated with the disease. These identified lncRNAs will be beneficial for the further understanding of cerebellum gene coexpression network correlating with disease progression. Furthermore, investigation on the lncRNA-associated neuroprotective factors remains to be necessary for the elucidation of the therapeutic target implication.
The location of all lncRNAs is from the mm10 database.
The authors declare no conflict of interest.
Zhe Long and Tianjiao Li contributed equally to this work.
This study was supported by the National Natural Science Foundation of China (nos. 81471156 and 81771231 to Hong Jiang; nos. 81430023 and 81130021 to Beisha Tang; no. 31401135 to Rong Qiu), National Key Research and Development Program of China (nos. 2016YFC0901504 and 2016YFC0905100 to Hong Jiang; no. 2016YFC1306000 to Beisha Tang), Clinical Research Funds of Xiangya Hospital (no. 2014L03 to Hong Jiang), the Clinical and Rehabilitation Fund of Peking University Weiming Biotech Group (no. xywm2015I10 to Hong Jiang), Youth Foundation of Xiangya Hospital (no. 2017Q03 to Zhao Chen), and Independent Exploration and Innovation Project of Graduate Student of Central South University (no. 1053320170177 to Tianjiao Li). The authors want to give the sincerest thanks to all those who helped in the study.
Table S1: the supplementary material included 44 lncRNA locations, dysregulation (upregulated/downregulated), and primers used for qPCR amplification.