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. 2020 Oct 24;8(11):450.
doi: 10.3390/biomedicines8110450.

Modulation of Small RNA Signatures in Schwann-Cell-Derived Extracellular Vesicles by the p75 Neurotrophin Receptor and Sortilin

Nádia P Gonçalves  1 , Yan Yan  2   3 , Maj Ulrichsen  1 , Morten T Venø  2   3 , Ebbe T Poulsen  4 , Jan J Enghild  4 , Jørgen Kjems  2   4 , Christian B Vægter  1
Affiliations

Modulation of Small RNA Signatures in Schwann-Cell-Derived Extracellular Vesicles by the p75 Neurotrophin Receptor and Sortilin

Nádia P Gonçalves et al. Biomedicines. .

Abstract

Schwann cells (SCs) are the main glial cells of the peripheral nervous system (PNS) and are known to be involved in various pathophysiological processes, such as diabetic neuropathy and nerve regeneration, through neurotrophin signaling. Such glial trophic support to axons, as well as neuronal survival/death signaling, has previously been linked to the p75 neurotrophin receptor (p75NTR) and its co-receptor Sortilin. Recently, SC-derived extracellular vesicles (EVs) were shown to be important for axon growth and nerve regeneration, but cargo of these glial cell-derived EVs has not yet been well-characterized. In this study, we aimed to characterize signatures of small RNAs in EVs derived from wild-type (WT) SCs and define differentially expressed small RNAs in EVs derived from SCs with genetic deletions of p75NTR (Ngfr-/-) or Sortilin (Sort1-/-). Using RNA sequencing, we identified a total of 366 miRNAs in EVs derived from WT SCs of which the most highly expressed are linked to the regulation of axonogenesis, axon guidance and axon extension, suggesting an involvement of SC EVs in axonal homeostasis. Signaling of SC EVs to non-neuronal cells was also suggested by the presence of several miRNAs important for regulation of the endothelial cell apoptotic process. Ablated p75NTR or sortilin expression in SCs translated into a set of differentially regulated tRNAs and miRNAs, with impact in autophagy and several cellular signaling pathways such as the phosphatidylinositol signaling system. With this work, we identified the global expression profile of small RNAs present in SC-derived EVs and provided evidence for a regulatory function of these vesicles on the homeostasis of other cell types of the PNS. Differentially identified miRNAs can pave the way to a better understanding of p75NTR and sortilin roles regarding PNS homeostasis and disease.

Keywords: Schwann cells; exosomes; extracellular vesicles (EVs); miRNAs; p75NTR receptor; sortilin.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Primary Schwann cell (SC) characterization. (a) Schematic representation of the cell isolation strategy used. (b) Depiction of SC primary culture with immunocytochemistry against S100 and p75NTR (both in green) in primary SCs from wild-type (WT) or Ngfr−/− neonatal rats. Images show lack of p75NTR staining in Ngfr−/− derived cells. Nuclei are labeled in blue with Hoechst. Scale bar 50 μm. (c) Double immunocytochemistry between sortilin (red) and S100 (green) for both WT and Sort1−/− derived primary SCs. Hoechst was used to label the cell nuclei (blue). Scale bar 50 μm. (d) Representative immunoblot for sortilin and p75NTR from primary SC lysates, validating sortilin or p75NTR protein absence in Sort1−/− or Ngfr−/− primary SCs, respectively. (e) Live and death assay where viable cells were identified in SC cultures by Calcein green fluorescence, while dead cells were labeled red by ethidium homodimer-1. Scale bar 100 μm (f) Quantification shows that viability of Ngfr−/− or Sort1−/− SCs is similar to that of WT SCs.
Figure 2
Figure 2
Size distribution of vesicles secreted by the primary Schwann cells (SCs). Representative plots with nanoparticle tracking analysis results of extracellular vesicles (EVs) collected from 3 × T175 flasks from (a) WT, (b) Sort1−/− and (c) Ngfr−/− derived rat primary SCs. (d) Immunoblot showing CD81 expression in both the EV pellets and SC lysate, while EEA1 and sortilin were only found present in the SC lysate.
Figure 3
Figure 3
The read mapping distribution for the three types of Schwann cells (SCs) analyzed: (a) WT, (b) Ngfr−/− and (c) Sort1−/− SCs. The tRNA category includes all types of tRNA fragments.
Figure 4
Figure 4
Characterization of WT SC-derived EV cargo in terms of tRNA fragments (tRFs) and halves (tiRNA). (a) Reads compiled on the individual tRNA maps. (b) The length of reads mapping to the various tRNAs.
Figure 5
Figure 5
Pipeline for the miRNA analysis in EVs derived from WT SCs.
Figure 6
Figure 6
Characterization of WT SC-derived EV cargo in terms of miRNA content. Upset plot that shows the instances where more than 2 miRNAs target more than 5 genes. The plot is made using the R package “UpSetR”. The blue bars on the left side of the plot show the number of targets predicted for each miRNA. The right-side bar plot show the number of genes targeted by only a single miRNA (single black dot in the lower right panel) or multiple miRNAs (multiple linked dots in the lower right panel).
Figure 7
Figure 7
WT Schwann cell EV Pathway and Process analysis. (a) Pathway analysis for all genes predicted to be targets of 3 or more miRNAs. Hits from KEGG pathways with p-value < 0.05. Cancer hits were removed. (b) Pathway analysis for all genes predicted to be targets of 3 or more miRNAs. The top 20 hits from Gene Ontology Biological Process. (c) Pathway analysis for predicted targets of each individual miRNA. The 30 most significant hits from KEGG pathways are shown. The heatmap shows the level of significance—red being the most significant, grey meaning not significant. (d) Process analysis for predicted targets of each individual miRNA. The 30 most significant hits from Gene Ontology Biological Process are shown. The heatmap shows the level of significance—red being the most significant and grey representing non significance.
Figure 8
Figure 8
Differentially expressed tRNAs in EVs derived from Ngfr−/− and Sort1−/− compared with EVs from WT Schwann cells. (a) Heatmap covering the most significantly changing tRNAs, with p-values (not multiple-testing corrected) below 0.05 in one or more of the pairwise comparisons. Top annotation shows Sort1−/− in orange, Ngfr−/− in yellow and control WT as blue. Shown are z scores of log2-transformed RPM values. (b) Volcano plot for EV tRNAs from Ngfr−/− versus WT comparison and (c) Volcano plot for EV tRNAs from Sort1−/− versus WT. Plotted are –log10(p-values) vs. −log2(fold change). Red means < 0.05 adjusted p-value. Vertical lines indicate +/− 1 log2(fold change). The names for the 10 most significant tRNA are indicated.
Figure 9
Figure 9
Differentially expressed miRNAs in EVs derived from Ngfr−/− and Sort1−/− compared with EVs from WT Schwann cells (SCs). (a) The 45 most significantly differentially expressed miRNAs (not multiple-testing corrected) are shown as a heatmap covering all samples from WT, Ngfr−/− and Sort1−/− SCs. Top annotation shows Sort1−/− in orange, Ngfr−/− in yellow and control WT as blue. Shown are z scores of log2-transformed RPM values. (b) Volcano plot for EV miRNAs from Ngfr−/− versus WT comparison and (c) Volcano plot for EV miRNAs from Sort1−/− versus WT. Plotted are –log10(p-values) vs. −log2(fold change). Red means < 0.05 adjusted p-value. Vertical lines indicate +/− 1 log2(fold change). The names for the 10 most significant miRNA are indicated.
Figure 10
Figure 10
Transgenic Schwann cell EV cargo characterization (lacking p75NTR or sortilin expression). (a) UpSet plot showing the number of predicted target genes found to be targeted by individual miRNAs or by multiple miRNAs. The plot is made using the R package “UpSetR”. The blue bars on the left side of the plot show the number of targets predicted for each miRNA. The right-side bar plot shows the number of genes targeted by only a single miRNA (single black dot in the lower right panel) or multiple miRNAs (multiple linked dots in the lower right panel). (b) Pathway analysis for all genes predicted to be targets of 3 or more miRNAs. All hits from KEGG pathways with p-value < 0.05. (c) Pathway analysis for all genes predicted to be targets of 3 or more miRNAs. The top 20 hits from Gene Ontology Biological Process.
Figure 11
Figure 11
Pathway analysis for Ngfr−/− and Sort1−/− Schwann cell EV cargo. (a) Pathway analysis for predicted targets of each individual miRNA. The 30 most significant hits from KEGG pathways are shown. The heatmap shows the level of significance—red being most significant, grey means not significant. (b) Pathway analysis for predicted targets of each individual miRNA. The 30 most significant hits from Gene Ontology Biological Process are shown. The heatmap shows the level of significance—red being most significant, grey means not significant.
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