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

Abstract

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.

Keywords: A53T mutation; Gerotarget; Parkinson’s disease; deep sequencing; microRNAs.

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. 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 log₂ (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. 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. 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.