Demographic and clinical data.
Demographic and clinical data of patients are shown in Table 1. Mean age was similar between DLB patients, AD patients and controls, according to the inclusion criteria (75.1 +/- 6.8 years in the DLB group, 73.9 +/- 6.7 years in the AD group; p = 0.236); however, PD patients were significantly younger (66.9 +/- 14.9 years, p = 0.021). The male-female ratio was higher in PD and DLB, than in AD and CTRLs, but no gender-specific expression changes were observed during data analyses. Disease duration was similar between patient groups (p = 0.068).
Platelet characterization and miRNA profile discovery
Analysis of the platelet-rich pellet for possible leukocyte contamination showed no staining for the leukocyte marker CD45 in our samples. Instead, we obtained a high fluorescent signal for the platelet marker CD61, indicating high platelet purity and no leukocyte contamination (Fig. 1a and Sup. Figure 2).
RNA, used for the construction of NGS-libraries, showed an enriched profile of 20–40 nucleotide molecules characteristic for small RNA and miRNA. NGS generated a mean of 1,488,787 ± 921,800 reads per sample in the control group, and 1,210,616 ± 1,868,817 reads per DLB sample. These mapped to 1,279 known different mature miRNAs, and 534 miRNAs fulfilled the criteria of more than 5 reads per sample, corresponding to 430 different miRNA-precursor. Literature search revealed that 304 had been previously associated with platelets (Fig. 1b), and 58.9% had been described in the first platelet-miRNA profiling studies [13, 34]. Our study also confirmed let-7, miR-103 and miR-21  as the most common platelet-miRNA families (Fig. 1c).
The normalized counts from NGS data were analyzed using the Wilcoxon-rank sum test, and 22 miRNAs were differentially expressed between DLB and controls, and were further validated by qPCR (hsa-miR-1343-3p, hsa-miR-191-3p, hsa-miR-6747-3p, hsa-miR-504-5p, hsa-miR-6741-3p, hsa-miR-128-3p, hsa-miR-1468-5p, hsa-miR-139-5p, hsa-let-7d-5p, hsa-let-7d-3p, hsa-miR-142-3p, hsa-miR-132-5p, hsa-miR-150-5p, hsa-miR-23a-5p, hsa-miR-26b-5p, hsa-miR-1301-3p, hsa-miR-625-3p, hsa-miR-146a-5p, hsa-miR-25-3p, hsa-miR-877-3p, hsa-miR-1908-5p, hsa-miR-744-5p).
Validation of miRNA expression
The 22 differentially expressed miRNAs were validated by qPCR in three independent studies.
Study I (2017 ). The first validation study included two cohorts of 21 DLB patients and 21 control individuals. Ten of the 22 miRNAs, hsa-miR-6747-3p, hsa-miR-128-3p, hsa-miR-139-5p, hsa-let-7d-5p, hsa-miR-142-3p, hsa-miR-132-5p, hsa-miR-150-5p, hsa-miR-26b-5p, hsa-miR-146a-5p, hsa-miR-25-3p, were diminished in DLB compared to controls (Table 2).
Study II (2018). Three independent cohorts comprising newly recruited DLB patients (n = 22), AD patients (n = 15) and control subjects (n = 16) were included in the second validation study. Although 9 out of the ten miRNAs were again diminished in DLB when compared to controls, 5 out of these 9 miRNAs failed to produce significant results due to an elevated intra-group variability. Yet, four miRNAs confirmed the results of Study I. miRNAs hsa-miR-128-3p, hsa-miR-139-5p, hsa-miR-150-5p, hsa-miR-25-3p, were significantly down-regulated in DLB compared to controls, with hsa-miR-150-5p showing the most important decrease (Table 3).
The comparison of DLB and AD miRNA expression data revealed that 9 out of the 10 miRNAs were significantly down-regulated in DLB compared with AD (Table 3). Only hsa-miR-150-5p was significantly diminished in AD when compared to controls.
Study III (2019). To the initially recruited patients, four independent cohorts of newly diagnosed DLB (n = 16), AD (n = 13) and PD patients (n = 24), and 14 control subjects were added in the third validation study, analyzing a total of 162 individuals (59 DLB patients, 28 AD patients, 24 PD patients, 51 controls). As a result, two miRNAs (hsa-miR-142-3p, hsa-miR-150-5p) were significantly diminished in DLB compared with controls. Seven miRNAs (hsa-let-7d-5p, hsa-miR-142-3p, hsa-miR-132-5p, hsa-miR-150-5p, hsa-miR-26b-5p, hsa-miR-146a-5p, hsa-miR-25-3p,) were significantly diminished in DLB compared to AD, and two (hsa-miR-150-5p and hsa-miR-26b-5p) were down-regulated in DLB compared to PD (Table 4). When grouping DLB and PD as LBD, only hsa-miR-139-5p was significantly down-regulated, but hsa-miR-128-3p and hsa-miR-139-5p were diminished in PD compared to controls. The expression of four miRNAs (hsa-miR-132-5p, hsa-miR-146a-5p, hsa-miR-25-3p, hsa-miR-6747-3p) was elevated in AD vs CTRLs (Table 4, Fig. 2). Of all miRNAs, only hsa-miR-150-5p was differentially expressed in DLB compared to each of the other cohorts, suggesting disease-specific deregulation (Sup. Figure 3).
The 5 miRNA sets were further studied for their usefulness as biomarkers (Fig. 3a).
ROC curve analysis
ROC curves were calculated for all five miRNA sets to assess their discrimination potential between groups. The combination of the seven differentially expressed miRNAs between DLB and AD (miRNAs hsa-let-7d-5p, hsa-miR-132-5p, hsa-miR-142-3p, hsa-miR-146a-5p, hsa-miR-150-5p, hsa-miR-25-3p and hsa-miR-26b-5p) presented the highest specificity (100%) and sensitivity (100%) to distinguish DLB patients from AD patients, with an AUC of 1 (Fig. 3b).
The ROC curve for hsa-miR-142-3p and hsa-miR-150-5p, differentially expressed between DLB and CTRLs, yielded an AUC = 0.85 (95% C.I. 0.74–0.95; 82% sensitivity, 70% specificity). Comparison of AD and CTRLs, miRNAs hsa-miR-132-5p, hsa-miR-146a-5p, hsa-miR-25-3p, and hsa-miR-6747-3p resulted in AUC = 0.94 (95% C.I. 0.86-1.00; 89% sensitivity, 80% specificity); and AUC = 0.81 (95% C.I. 067-0.94; 84% sensitivity, 76% specificity) was obtained for hsa-miR-128-3p and hsa-miR-139-5p comparing PD and CTRLs. AUC = 0.83 (95% C.I. 0.73–0.98; 90% sensitivity, 73.7% specificity), was obtained for hsa-miR-150-5p and hsa-miR-26b-5p when comparing DLB and PD (Fig. 3b).
miRNA expression in whole blood
To assess whether the results were platelet-specific, we analyzed the 10 differentially expressed miRNAs in whole blood of DLB, PD and AD patients, and controls (n = 16, each). Only the expression of hsa-let-7d-5p and hsa-miR-132-5p was diminished in blood of PD patients in comparison with controls (Sup. Table 1, Additional file 1). Specifically, these miRNAs did not show expression changes in platelets of PD patients. No additional differences were found.
miRNA target prediction
Target-gene lists were obtained for the five miRNA sets (Fig. 3a).
When comparing DLB and controls, the target screening of miRNAs hsa-miR-150-5p and hsa-miR-142-3p identified 81 different genes. Genes involved in transcriptional regulation (p = 0.001), cellular response to stress (p = 8.36·10− 5) and immune response (p = 0.021), were overrepresented (Fig. 4a).
The seven miRNAs down-regulated in DLB compared to AD were predicted to target genes involved in integrin cell surface interactions (p = 1.5·10− 4), cell death pathways (p = 0.002), and transcriptional regulation (p = 0.004) (Fig. 4a). When analyzing miRNAs diminished in DLB compared with PD, formation of senescence-associated heterochromatin foci (SAHF) was the most representative pathway (p = 2.4·10− 4).
The analysis of the miRNA-set that distinguishes AD from controls (Fig. 4a) rendered 393 possible target genes. Of these, 27.7% were involved in transcriptional regulation, 43.3% in signal transduction and 54.2% were phosphoprotein-coding. Gene clusters related to stress (p = 8.3·10− 5) and immune response (p = 1.58·10− 5) were identified, including TDRD7 (tudor-domain-containing protein 7) and TIA1 (TIA1 cytotoxic granule associated RNA binding protein), both involved in stress granule formation.
MiRNAs hsa-miR-128-3p and hsa-miR-139-5p, down-regulated in PD compared to controls, were predicted to target 78 different genes. Signal transduction (p = 1.38·10− 10) and PIP-AKT signaling (p = 4.9·10− 10) were the most enriched pathways (Fig. 4a). A protein-phosphorylation (p = 0.0376) and a MAPK-pathway (p = 0.015) cluster were identified including FOS, MTOR and RICTOR.
Comparison of the five pathway lists revealed that 31 pathways were altered specifically in DLB (Fig. 4b). RNA and small RNA metabolism (mitochondrial tRNA processing), and RNA silencing by small RNA were found. In AD, specific pathway enrichment comprised cell death-related pathways (p = 5.04·10− 8). In PD, 78 pathways were specifically enriched (Fig. 4b), including MAP kinase, protein phosphorylation pathways (p = 8.3·10− 8), negative regulation of cell death (p = 0.009), and serotonin and dopamine receptor-related pathways (p = 0.05).