MicroRNA expressing profiles in A53T mutant alpha-synuclein transgenic mice and Parkinsonian

α-synuclein gene mutations can cause α-synuclein protein aggregation in the midbrain of Parkinson's disease (PD) patients. MicroRNAs (miRNAs) play a key role in the metabolism of α-synuclein but the mechanism involved in synucleinopathy remains unclear. In this study, we investigated the miRNA profiles in A53T-α-synuclein transgenic mice and analyzed the candidate miRNAs in the cerebrospinal fluid (CSF) of PD patients. The 12-month A53T-transgenic mouse displayed hyperactive movement and anxiolytic-like behaviors with α-synuclein aggregation in midbrain. A total of 317,759 total and 289,207 unique small RNA sequences in the midbrain of mice were identified by high-throughput deep sequencing. We found 644 miRNAs were significantly changed in the transgenic mice. Based on the conserved characteristic of miRNAs, we selected 11 candidates from the 40 remarkably expressed miRNAs and explored their expression in 44 CSF samples collected from PD patients. The results revealed that 11 microRNAs were differently expressed in CSF, emphatically as miR-144-5p, miR-200a-3p and miR-542-3p, which were dramatically up-regulated in both A53T-transgenic mice and PD patients, and had a helpful accuracy for the PD prediction. The ordered logistic regression analysis showed that the severity of PD has strong correlation with an up-expression of miR-144-5p, miR-200a-3p and miR-542-3p in CSF. Taken together, our data suggested that miRNAs in CSF, such as miR-144-5p, miR-200a-3p and miR-542-3p, may be useful to the PD diagnosis as potential biomarkers.

Non-coding RNA (ncRNA) was found to be involved in α-synuclein pathogenesis [21].MicroRNAs (miRNAs), or short ncRNAs, regulate the translation or degradation of target messenger RNAs (mRNAs) at the post-transcriptional stage [22].For example, miR-7, -433 and -153 bind to the 3'-untranslated region of α-synuclein to inhibit the gene expression in DA neurons [23,24].The mutant α-synuclein is more difficult to be degraded than wild-type by the ubiquitin-proteasome system [25].Thus, it should be more important to explore the miRNA profiles in mutant α-synuclein than wild-type to evaluate protein aggregation in PD [26].Recent reports found that some miRNAs can be packaged into lipid-based carriers and stable in the plasma, cerebrospinal fluid (CSF) and urine [27,28].Down regulation of miR-16-2-3p and -1294, up regulation of miR-338-3p, -30e-3p, and -30a-3p were found in the plasma or CSF of PD patients [29,30].These miRNAs may be novel biomarkers for PD diagnosis and prognosis.However, the miRNA signatures of PD remain unclear to date.In this study, we attempted to screen the miRNAs profiles in A53T-transgenic mice and evaluate their value for the clinical diagnosis of PD.

A53T-transgenic mice display hyperactive behavior with increased α-synuclein deposition in the degenerating DA neurons
The behavior of mice at 12 months of age was tested using the open field test.The A53T-transgenic mice displayed hyperactivity, as indicated by a longer distance traveled in the center region (Figure 1A).The distances moved within 25 min by A53T mice and wild-type mice were 3,721.73± 238.81 cm and 2,181.74± 290.50 cm (p < 0.05) respectively.The inner distances moved by A53T mice and wild-type mice were 2,138.37 ± 365.92 cm and 975.01 ± 184.93 cm (p < 0.05, Figure 1B) respectively.Thus, the ratio of inner/total distances in A53T was significantly higher than that of in wild-type mice (0.58 ± 0.17 vs. 0.34 ± 0.08, p < 0.01, Figure 1C).It suggested that A53T-transgenic mice display anxiolytic-like and hyperactive behaviors.The immunofluorescence analysis indicated that a dense distribution of mutant α-synuclein particles was commonly observed at the DA neurons of A53T-transgenic mice but rarely found in wild-type (Figure 1D).The quantitative cell analyses revealed a slight decrease without significance in DA count in the SN of A53T mice (1741 ± 94.18 cells in wild-type vs.1560 ± 213.72 cells in A53T, p = 0.064, Figure 1E).Western blot analysis of midbrain revealed the total and phosphorylated ɑ-synuclein increased significantly in the mutant mice compared to the wild-type (p < 0.001, Figure 1F-1H).Small RNA (sRNA) libraries from the midbrain of A53T-transgenic mice were analyzed using the Illumina HiSeq2500 platform.A total of 12,334,900 and 10,916,235 clean reads were monitored in the A53T-transgenic and wild-type mice, respectively.We excluded the sequences of null or poor-quality 3' insert nucleotides with sequence lengths > 30 or < 18 nt.The majority of sRNAs were 21-24 nt, which contained the miRNAs (Supplementary Figure .2).These RNAs were divided into several sub-categories, including rRNAs, scRNAs, snRNAs, snoRNAs, tRNAs, repbase, and unannotated RNAs (Supplementary Table 2).By high-throughput sequencing, 317,759 (2.92%) of the total sRNA sequences or 289,207 (34.80%) of the unique sRNA sequences were uniquely detected in the A53T-trangenic mice, 452,631 (4.16%) of the total sRNA sequences and 388,626 (46.76%) of the unique sRNA sequences were detected in wild-type mice.There was an overlap in 10,107,585 (92.02%) of the total sequences and 153,195 (18.43%) of the unique sequences (Figure 2A and 2B).Six hundred and forty four unique miRNAs were significantly different between A53T- The numbers of genes annotated to the term in the background and input data are shown in the "Annotated" and "Significant" columns, respectively.The number in the "Expected" column represents the genes expected in the input data by chance.The p-value was used to evaluate the term enrichment after adjusting the false discovery rate.
The A53T mutation of α-synuclein gene was reported in Greek familial PD patients [34].Then a series of A53T transgenetic models were developed for PD study.These models showed different symptoms and pathologies with different promoters, genotypes and mouse lines [4,9,16,[35][36][37].The A53T transgenic mice, applied Thy-1 promoter, were found the amorphous aggregates of human α-synuclein accompanied with paralysis-like motor dysfunction, but no DA neurons lost in SNc [38,39].Another transgenic B6 mice, applied the prion protein (Prp) promoter, were used to promote α-synuclein expression and show an increased neurotoxicity in nervous system [40,41].As to the genotype factor, the homozygous offspring of A53T mice develop some paralysis-like symptoms and die approximately at 16-month old as reported by Jackson Laboratories, but the heterozygous offspring rarely show the similar pathology signs and the onset time will has 22-28 months delay [9,42,43].To mimic the impaired SN system on PD, other Prp-A53T model, used C3H mice mixed with C57BL line, was constructed.These transgenic mice showed that TH-positive cells decreased significantly in SN accompanied with α-synuclein deposition without the paralysis-like symptoms [4,5].Here, we used the hemizygous Prp-A53T offspring derived in the C57BL/ B6 background and confirmed these transgenetic mice developed some abnormalities included the mutantα-synuclein aggregation, increased p-α-synuclein and hyperactive movements.Their behavior abnormalities, such as hyperactive movements and anxiolytic, were token as non-motor symptoms in the preclinical stage of PD [4,18,39].Our mice had the similar background as Paumier's and Wills' study, but their DA neurons in SN are not dramatically decreased as report [4,5].It may be related with the preclinical or early stage of disease and young age on 12-month-old A53T mice.
The non-motor symptoms, such as dysautonomia, sensory dysfunctions and behavioral abnormalities, appear earlier than motor symptoms at the early stage of PD [44,45].The symptoms like anxiety and depressive disorders at the preclinical phase are taken as risk factors for PD [46].The anxiety and depressive behaviors are commonly evaluated by open field test in animal models [47].In our study, the open filed test was also used and the result showed the heterozygous A53T mice took more time and higher ratio in the center region with hyperactive motor, which was also found in the homozygous mice on Katrina L's study [4].These data supported that A53T mice may develop an anxiolytic phenotype [4,16,18,39,41,48].Although anxiolytic is not like the signs and symptoms in PD, it should be emphasized that A53T mutant is only one of PD-related factors and anxiety is a complex disorder involved with multiple factors [49].A study suggested this hyperactivity acts as an abrupt appearance, but not a progressive impairment in motor ability [4].It may be involved with the increased sensitivity of DA neurons in midbrain, which is caused by a series of functional disorders included the decreased DA transporter expression, impaired striatal DA uptake and elevated D1 receptor expression [16,36,37,50].In a word, the anxiolytic-like phenotype reflect the abnormality of dopaminergic nigrostriatal system after A53T mutant.Other non-motor symptoms, such as olfactory dysfunction, were also found in the A53T mice 6 months before the motor deficits and DA neurons impairment [10,14,41].These evidences suggested that A53T model may share some similar symptoms and mechanism of the preclinical stage of PD.And the A53T mice may provide meaningful diagnostic markers at early stage before the motor symptoms.
The bioinformatics analyses further indicated that the miRNA signatures in A53T mice may contribute to the predicted functional network, including the positive regulation of apoptotic processes, ATP binding biological processes, protein binding, protein-related functions and metabolism, neuroactive ligand-receptor interactions and aging.Among them, 99 target genes were found to be related to PD. Due to miRNA binding sites are short, a little difference in the algorithms will render the dramatic diversity, the bioinformatics prediction of miRNA targets is hard to avoid some disadvantages, such as false positives and false negatives, or inconsistent results using different algorithms, and the predicted targets might not be expressed in specific conditions [65,71].To avoid these drawbacks, we integrated the prediction by a combination with our founding and other experimental results and reports.The bioinformatics result first was partly supported by the pathology of α-synuclein inclusions and decreased DA neurons in PD (Figure 1).The α-synuclein inclusions impaired the ubiquitin-proteasome system as major protein degradation pathways [72], aggregated at endoplasmic reticulum (ER) and induced ER stress [73], accompanied with ATP depletion and oxidative stress related with neuron apoptosis [74,75].These PD-related molecular mechanisms were mostly overlap with the www.impactjournals.com/oncotargetpredicted functional of miRNA signature in A53T mice.
Several miRNAs, embedded in lipid or as lipoprotein complexes, were reported to be detectable and stable in CSF and serum [31].Seventy-three percent of the distinct miRNAs in Alzheimer's disease (AD) brain can be detected in CSF [29].Seventeen miRNAs in the CSF and five miRNAs in the serum were found in PD patients at abnormal level [30], suggesting that miRNAs in CSF or serum may be potential biomarkers of neurodegenerative diseases.In this study, we determined the CSF miRNA signature from A53T-transgenic mice and PD patients with same primers based on their highly conserved sequence.The miR-144-5p, -200a-3p and -542-3p were found to be significantly up-regulated in both A53T-transgenic mice and the CSF samples of PD patients.Similarly, it has been reported that miR-144-5p changed in the brain of Huntington disease and blood of AD [76,77].The miR-200a-3p was involved in the regulation of neuronal differentiation and proliferation and miR-542-3p upregulated in blood after ischemic stroke, intracerebral hemorrhage, and kainate seizures [78,79].In our results, miR-144-5p, -200a-3p and -542-3p were further selected by their differential expression of high-abundance in sequencing.The ROC analysis confirmed the sensitivity and specificity of these CSF markers are enough to distinguish the PD from healthy control.The ordinal regression analysis found their expression significant increased across pathologic severity of PD.And only the raised tendency of miR-542-3p was influenced by different smoking habit.These results supported these miRNA as independent factors may be the ideal biomarker-assisted diagnosis of PD.However, this topic still requires further investigation.In our study, we screened the similar miRNAs from PD patients and animal model to improve the repeatability, and some miRNAs were also reported in other study enrolled the patient from difference ethnicity, geography and had different inclusion and exclusion criteria.

Animal and clinical studies
All mice were used in accordance with the Animal Ethics Guidelines of the Institutional Animal Care Committee of Sun Yat-sen University (No. 20120112178).The transgenic mice B6; C3-Tg (Prnp-SNCA*A53T) 83Vle/J expressing A53T human α-synuclein, were originally obtained in Jackson Laboratory (JAX004479, USA).The breeding pairs were kindly offered by Dr. Ben Wu in the state key laboratory of medical genetics of Central South University.The hemizygous A53T mice on a mixed C57BL/6J × B6 background provided the transgenic and non-transgenic litter mates for study.DNA was purified from the tail by a DNA extraction kit (Beyotime, Beijing, China) with genotype identification (5'-TGTAGGCTCCAAAACCAAGG-3', 5'-TGTCAGGATCC ACAGGCATA-3').PD patient tissues were collected from the PD center of the First Affiliated Hospital of Sun Yat-Sen University and the Department of Neurology of Guangdong Brain Hospital between 2014 and 2015.All patients were diagnosed according to the Criteria of United Kingdom's Parkinson's disease Society [49].The CSF samples of forty-four PD patients and forty-two healthy controls were collected from South China.The patient information was showed in Table .
Mice brains were perfused with 4% paraformaldehyde and dehydrated by a sequential sucrose gradient from 10% to 30%.The brain coronal section was blocked with 10% goat serum in 0.01 M phosphatebuffered saline (PBS) containing 0.1% Triton X-100 for 1 h.Slices were incubated with human ɑ-synuclein (1:1000, Millipore, MABN826, USA) antibody and rabbit tyrosine hydroxylase (TH) antibody (1:400, Millipore, AB6211, USA) overnight at 4°C, washed three times with 0.01 M PBS, and then incubated with Alexa Fluor 488 (green) and 594 (red) conjugated secondary antibodies.The positive staining were visualized by the EVOS® FLoid® Cell Imaging Station (Thermo Scientific, USA).According to the study before [81], the TH positive cells in SN were counted in 15 µm coronary midbrain sections.Every sixth section from AP: -2.70 to AP: -3.88 mm and a total number of 11 sections was collected.The TH positive cells on each side were counted and combined under the blinded genotype to the counter used the Stereo Investigator software (MBF Bioscience, Williston, VT, USA) [48].

Deep sequencing
The 12-month-old mice were decapitated after deeply anesthetized with isoflurane (Baxter Healthcare, Deerfield, IL, USA).Total RNA was purified from midbrain tissues of one-year-old mice using TRIzol reagent (Invitrogen), concentrated by Qiagen RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA, USA), quantified (Nanodrop; Thermo Fisher, Uppsala, Sweden) and evaluated by RNA electrophoresis.RNA absorbance was measured between the range of 1.90 to 2.02 using an Agilent 2100 Bioanalyzer, with an RNA integrity number (RIN) ≥ 2.6 and RNA concentrations ≥ 3.0 mg/l.Small RNA libraries were constructed following the instruction of TruSeq Small RNA Sample Preparation Kits (Illumina, Inc., Hayward, CA, USA) and purified for deep sequencing with single-end reads of 36 bases on the Illumina HiSeq 2500 (Huada, Shenzhen, China, Supplementary Figure 1) [82,83].The annotated and unannotated reads from RNA sequencing were detected by Bowtie (v0.12.7) and the miRDeep2 software to analyze the mapped reads and predict novel miRNAs.The miRNAs from miRDeep2 were further analyzed by the Bioconductor DESeq v. 2.0 package using a p value < 0.05 and fold change (FC) > 2 [84].The prediction analysis of miRNA-targeted genes was based on COG (Cluster of Orthologous Groups of proteins), GO (Gene Ontology), Swiss-Prot (a manually annotated and reviewed protein sequence database), Nr (NCBI non-redundant nucleotide sequences) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases.

Quantitative real-time PCR
Quantitative real-time PCR (qRT-PCR) was performed following the MIQE guidelines [85].Total RNA, purified from midbrain tissues of 12-month-old mice and CSF of human, was extracted by a mirVana PARIS Kit (Ambion, PN AM1556) and converted to cDNA using a TaqMan® MicroRNA Reverse Transcription Kit (ABI, USA).A high-capacity cDNA reverse transcription kit (ABI, USA) was used for random primer scheme of miR-615-5p and miR-196a with low concentration [86,87].Selected miRNAs were quantified on an ABI Prism 7500 system (Applied Biosystems, Warrington, UK).The stemloop RT and PCR primers were ordered from TIANGEN Biotech (Supplementary Table 1).U6 in midbrain and miR-34 in CSF were used as an internal control following the 2 −ΔΔCT and ΔCT methods, respectively [33,88].

Statistical analysis
Statistical analyses were performed using SPSS 13.0 (SPSS Inc., Chicago, IL) and Prism 5.0 (GraphPad Software Inc, La Jolla, CA).The normally distributed data were compared by Student's t test, whereas the non-Gaussian data were analyzed using Wilcoxon-Mann-Whitney tests.In the deep sequencing analysis, we calculated the normalized TPM as actual miRNA count/ total count of clean tags×1,000,000 [89].The FC value was calculated following FC = log 2 (TPM of A53T/ TPM of control).The p-value for the analysis of miRNAs was adjusted using the Benjamini-Hochberg approach.The enrichment factor (EF), defined as the ratio of observed to expected for a given enrichment class, was used for geneenrichment analysis, and p-values < 0.05, as determined using Fisher's exact test, was used to assess statistical significance.The ROC analysis and area under ROC curve (AUC) were performed to assess the possibility of miRNA concentration as biomarkers for PD diagnosis.Youden index was used for the selection of cut-off point.According to the study before, the ordinal logistic regression was used for the analysis between miRNAs expression and H&Y scales by STATA (version 13, StataCorp, College Station, Texas) [31].The p values were corrected by the Bonferroni method in multiple testing.All the p values were 2-tailed.

Figure 1 :
Figure 1: A53T mice show increased movement, decreased dopaminenergic neurons and increased α-synuclein aggregation in the midbrain.A. In an open-field test, A53T-α-synuclein mice displayed hyperactive movement at 12 months of age.B. The distances traveled in the total field and inner field in 20 min were compared between A53T-transgenic and wild-type mice (n = 6).C. The ratio of inner field to the total field was increased in A53T mice compared with wild-type mice.D. A53T-α-synucleins in the midbrain (arrows) were labeled with red fluorescence under immunofluorescence double-staining, and the TH-positive neurons were stained with green fluorescence.E. The number of TH positive neurons is accounted in SN.F. Levels of α-synuclein and p-α-synuclein were detected in midbrains by western blot analysis.The three mice in each group were labeled as M1 to M3. Histograms showing the difference in total α-synuclein G. and p-α-synuclein H.. All data are expressed as the mean ± SD, *P < 0.05, the Wilcoxon-Mann-Whitney test was used for the behavior test and the Student t test for the rest comparison.

Figure 2 :
Figure 2: A53T-transgenic mice exhibited a distinct miRNA signature in the midbrain.Distribution of total miRNA sequences A. and specific sequences B. of the midbrain in A53T-transgenic (left, blue) and wild-type mice (right, red).C. Correlation of miRNAs based on a Spearman non-parametric analysis.TPM (transcripts per million) = (Readcount × 1,000,000) / Mapped Reads.D. Volcano plot showing the different miRNAs in A53T-transgenic mice compared to wild-type mice.Red dots indicate a fold-change expression > 2 (|log2 FC| > 1) and P < 0.05 [-log10 (FDR) > 1.4].FC: Fold Change, FDR: False Discovery Rate.E. Star glyph of differentially expressed miRNAs in A53T-transgenic and wild-type mice.The miRNAs contain 40 differentially expressed miRNAs form the results of volcano plot.The axis ratio followed the log 2 (TPM+1) scale.The red and blue curve represented the A53T and wild-type groups.The miRNAs order in Star glyph was same as in hierarchical clustering heatmap.TPM: Transcripts per million clean tags.F. Hierarchical clustering heatmap of miRNAs from star glyph showing comparisons between A53T-transgenic and wild-type mice.The color range gradient from green to red represents the abundance of miRNAs.

Figure 3 :
Figure 3: The expression of candidate miRNAs in CSF from PD. A. The sequencing result of miRNA concentration confirmed by qRT-PCR in the midbrain of A53T-transgenic mice (n = 5).The miRNAs with significant different expression were shown in the histogram, data were expressed as mean ± SE, with corrected P < 0.05 in Wilcoxon-Mann-Whitney test.B. The CT value of miR-24 in CSF was used to evaluate its possibility as control.The relative expression of miR-200a-3p C., miR-144-5p D. and miR-542-3p E. in CSF from PD and health controls by qRT-PCR.Data expressed as mean ± range.***P < 0.001 after Bonferroni correction for 11 tests.

Figure 4 :
Figure 4: The miRNAs in CSF as candidate biomarkers of PD.The feasibility of miR-200a-3p A., miR-144-5p B. and miR-542-3p C. in CSF for PD diagnosis were assessed by receiver operator characteristics (ROC) analysis.The miR-200a-3p D., miR-144-5p E. and miR-542-3p F. were increased in CSF form PD and changed with H&Y scale.The Y axis was the relative expression for each miRNA, while the X axis represented H&Y scale.Data expressed as mean ± SE.AUC: area under ROC curve, H&Y scale: Hoehn and Yahr scale.

Table 1 : Clinical data of PD patients and healthy control
www.impactjournals.com/oncotargetmiRNA signature in A53T-transgenic mice

Table 3 : The CSF miRNAs showed progressive expression trends across increasing H&Y scales by ordinal regression analysis
*SE: the standard error of coefficient.