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. 2021 Apr 2;10(4):791.
doi: 10.3390/cells10040791.

Protein Binding Partners of Dysregulated miRNAs in Parkinson's Disease Serum

Wolfgang P Ruf  1 , Axel Freischmidt  1   2 , Veselin Grozdanov  1 , Valerie Roth  1 , Sarah J Brockmann  1 , Brit Mollenhauer  3 , Dorothea Martin  4 , Bernhard Haslinger  4 , Katrin Fundel-Clemens  5 , Markus Otto  1 , Christine von Arnim  6 , Karlheinz Holzmann  7 , Albert C Ludolph  1   2 , Jochen H Weishaupt  8 , Karin M Danzer  1
Affiliations

Protein Binding Partners of Dysregulated miRNAs in Parkinson's Disease Serum

Wolfgang P Ruf et al. Cells. .

Abstract

Accumulating evidence suggests that microRNAs (miRNAs) are a contributing factor to neurodegenerative diseases. Although altered miRNA profiles in serum or plasma have been reported for several neurodegenerative diseases, little is known about the interaction between dysregulated miRNAs and their protein binding partners. We found significant alterations of the miRNA abundance pattern in serum and in isolated serum-derived extracellular vesicles of Parkinson's disease (PD) patients. The differential expression of miRNA in PD patients was more robust in serum than in isolated extracellular vesicles and could separate PD patients from healthy controls in an unsupervised approach to a high degree. We identified a novel protein interaction partner for the strongly dysregulated hsa-mir-4745-5p. Our study provides further evidence for the involvement of miRNAs and HNF4a in PD. The demonstration that miRNA-protein binding might mediate the pathologic effects of HNF4a both by direct binding to it and by binding to proteins regulated by it suggests a complex role for miRNAs in pathology beyond the dysregulation of transcription.

Keywords: Parkinson’s disease; miRNA; serum.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Differential expression of miRNAs in serum-derived exosomes of Parkinson‘s disease (PD) patients vs. healthy controls (HC). (A) Donut chart showing different non-coding RNA (ncRNA) species detected in serum-derived exosomes with the majority being miRNAs. (B) Functional characterization of detected miRNAs using exRNA forms (miRandola) categorization shows very strong overrepresentation of exosome, microvesicle, and circulation-associated categories. (C) Shows hierarchical clustering of PD patients and HC based on differential miRNA representation of exosomes with many outliers. An adequate separation between PD patients and HC is not possible. (D) Manhattan plot shows differential miRNA expression for every miRNA based on chromosome coordinates. Every plot represents a single miRNA. Blue-filled dots represent miRNAs with an FDR < 0.05. Red line represents a significance level of FDR < 0.05.
Figure 2
Figure 2
Analysis of circulating miRNAs in serum of patients with Parkinson‘s disease (PD) and healthy controls (HC). (A) MiRNA expression measured with miRNA arrays show distinct miRNA expression patterns in PD patients (red) and healthy controls (blue). (B) Unsupervised hierarchical clustering (top) and principal component analysis (PCA, bottom) show robust separation of PD patients from healthy controls (n = 11/10 HC/PD).
Figure 3
Figure 3
Differential expression of miRNAs in serum of Parkinson‘s disease (PD) patients versus healthy controls (HC). (A) Volcano plot showing negative logarithmic p-values versus effect size of differential miRNA expression in PD over healthy controls. Every dot represents a single miRNA (n = 1729), red filled dots represent miRNAs with an FDR < 0.05. Dotted horizontal line represents an FDR < 0.05. (B) Heatmap visualization of the expression of the 14 significantly altered miRNAs with an FDR < 0.05 (row z-score). (C) Validation of altered miRNA expression of top three differentially abundant miRNAs by RT-qPCR in an independent cohort from three different centers (n = 60/60 HC/PD, mean +/− SEM, ** p < 0.01, *** p < 0.001). (D) ROC plot showing diagnostic sensitivity and specificity to predict the disease status (PD) for different cutoffs. Grey represents hsa-mir-3665, light blue represents hsa-mir-1915-3p, and dark blue represents hsa-mir-4745-5p. The red line represents a logistic regression model based on all three miRNAs combined AUC (0.95 CI).
Figure 4
Figure 4
Investigation of miRNA protein binding partners with human protein microarrays. (A) Experimental workflow. (B) miRNA incubation signal on protein microarray. Signal is shown for hsa-mir4747-5p-atto488; panels on the left show the entire microarray spotted with ~9400 recombinant human proteins, the middle panel is an enlarged 484 protein spot sub-array, and the right panel is the enlarged spot for the MTIF3 protein (all proteins spotted in duplicate, sub-array positive controls boxed in red). (C) Shows miRNA/protein binding distribution in relation to background signal.
Figure 5
Figure 5
(A) Venn diagram of numbers of miRNA binding partners of hsa-mir-4745-5p (top regulated miRNA in PD serum), hsa-mir-92a-3p (not regulated in PD), and cel-mir-39-3p (nonhuman controls from C. elegans). (B) Top canonical pathways and upstream regulators of the significant protein binding partners unique for hsa-mir-4745-5p (n = 762) using ingenuity pathway analysis. (C,D) show top PFAM-domains and Gene Ontology (GO) term molecular functions of the significant protein binding partners unique for hsa-mir-4745-5p (n = 762).
Figure 6
Figure 6
Western blot analysis confirms specific binding of hsa-mir-4745-5p with HNF4a protein (A) and MTIF3 protein (B). Non-regulated hsa-mir-92a-3p and nonhuman cel-mir-39-3p from C. elegans do not show protein interaction with HNF4a and MTIF3. (C) Densitometric analysis of western blots. Values are expressed as means +/− SEM, (HNF4a, n = 3).
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