Altered microRNA profiles in cerebrospinal fluid exosome in Parkinson disease and Alzheimer disease

Abstract

The differential diagnosis of Parkinson's diseases (PD) is challenging, especially in the early stages of the disease. We developed a microRNA profiling strategy for exosomal miRNAs isolated from cerebrospinal fluid (CSF) in PD and AD. Sixteen exosomal miRNAs were up regulated and 11 miRNAs were under regulated significantly in PD CSF when compared with those in healthy controls (relative fold > 2, p < 0.05). MiR-1 and miR-19b-3p were validated and significantly reduced in independent samples. While miR-153, miR-409-3p, miR-10a-5p, and let-7g-3p were significantly over expressed in PD CSF exosome. Bioinformatic analysis by DIANA-mirPath demonstrated that Neurotrophin signaling, mTOR signaling, Ubiquitin mediated proteolysis, Dopaminergic synapse, and Glutamatergic synapse were the most prominent pathways enriched in quantiles with PD miRNA patterns. Messenger RNA (mRNA) transcripts [amyloid precursor protein (APP), α-synuclein (α-syn), Tau, neurofilament light gene (NF-L), DJ-1/PARK7, Fractalkine and Neurosin] and long non-coding RNAs (RP11-462G22.1 and PCA3) were differentially expressed in CSF exosomes in PD and AD patients. These data demonstrated that CSF exosomal RNA molecules are reliable biomarkers with fair robustness in regard to specificity and sensitivity in differentiating PD from healthy and diseased (AD) controls.

Keywords: Alzheimer’s diseases (AD); Parkinson’s diseases (PD); Pathology Section; cerebrospinal fluid (CSF); exosome; microRNA.

Figure 1. Heatmap of CSF exosomal differential miRNA profiles in PD and AD

Heatmap representation of the mean fold change in PD and AD related differential miRNA signature. Two-dimensional grid matrix displaying 27 exosomal miRNAs in CSF was obtained by the functional heat-map in R. Columns refer to time course comparison: 27 healthy controls, 28 AD patients and 47 PD patients. Rows stand for the 27 differential miRNAs. Each entry of the grid refers to relative fold (log2) between the expression level of a given miRNA in exosome relative to RNU6B in healthy controls. The color of each entry is determined by the value of that fold difference, ranging from green (negative values) to red (positive values).

Figure 2. Validation of miRNA array expression using independent samples

TaqMan real-time RT-PCR to validate the expression levels of A. Six miRNAs (miR-1, miR-153, miR-409-3p, miR-19b-3p, miR-10a-5p, and let-7g-3p) in Neurotrophin signaling pathway were selected for further validation using an independent cohort of 78 PD patients, 53AD patients, and 35 healthy controls. B. two miRNAs (miR-136-3p and miR-433) in Dopaminergic synapse pathway using individual CSF exosome samples. Data shown are as mean ± SEM.

Figure 3. Messenger RNA transcripts and long non-coding RNAs were differentially expressed in CSF exosomes in PD and AD patients

A. APP, α-synuclein, Tau, NF-L, DJ-1/PARK7, Fractalkine, and Neurosin, were selected for quantitative RT-PCR experiments using CSF exosomal RNA from 28 AD, 47 PD, and 27 healthy controls. B. Two long non-coding RNAs, RP11-462G22.1 and PCA3, were tested in CSF exosomes using quantitative RT-PCR assay. Data shown are as mean ± SEM.

Figure 4. ROC curves for miRNAs that are significantly different in PD patients as compared to healthy controls

ROC curve with AUC for miR-1, miR-153, miR-409-3p, miR-19b-3p, and miR-10a-5p was performed using SPSS.