miR-92a-3p and miR-320a are Upregulated in Plasma Neuron-Derived Extracellular Vesicles of Patients with Frontotemporal Dementia

Abstract

Despite the efforts to identify fluid biomarkers to improve diagnosis of Frontotemporal dementia (FTD), only a few candidates have been described in recent years. In a previous study, we identified three circulating miRNAs (miR-92a-3p, miR-320a and miR-320b) differentially expressed in FTD patients with respect to healthy controls and/or Alzheimer's disease (AD) patients. Now, we investigated whether those changes could be due to miRNAs contained in neuron-derived extracellular vesicles (NDEVs). We also evaluated miRNAs content in total plasma EVs and in CSF samples. The analysis of plasma NDEVs carried out on 40 subjects including controls (n = 13), FTD (n = 13) and AD (n = 14) patients, showed that both miR-92a-3p and miR-320a levels were triplicated in the FTD group if compared with CT and AD patients. Increased levels of the same miRNAs were found also in CSF derived from FTD group compared to CTs. No differences were observed in expression levels of miR-320b among the three groups. Worthy of note, all miRNAs analysed were increased in an FTD cell model, MAPT IVS10 + 16 neurons. Our results suggest that miR-92a and miR-320a in NDEVs could be proposed as FTD biomarkers.

Keywords: Alzheimer’s disease; Extracellular vesicles; Frontotemporal dementia; Human iPSCs; MicroRNA.

Fig. 1

Western blot analysis of a representative NDEVs purification from plasma of CT, AD and FTD subjects. (A) An enrichment of neuronal marker (NSE) is observed in the NDEVs fraction concerning T-N EVs, (B) which is undetectable in the TEVs fractions. CD9 is a common exosome marker. 4 µg of mouse brain cortex (CTX, positive control) and 100 µg of EVs have been loaded in each lane. (C, D, E) Densitometric analysis of NSE in NDEVs (C), CD9 in NDEVs (D), and CD9 in TEVs (E). NSE and CD9 expression levels have been analysed using One-way ANOVA with a post hoc Tukey test. No differences are observed for the number of EVs extracted (both NDEVs and TEVs) in the CT group concerning AD and FTD ones. Values are expressed as % concerning the CT group. Each point in the frame depicts the value for a single subject, while bars represent the median value ± standard deviation. Each represents the mean of 2–3 replicates.

Fig. 2

Nanoparticle tracking analysis of both size distribution and relative concentration of microvesicles. TEVs were isolated from plasma samples (1 mL) collected from two healthy volunteers. As expected for the exosomal fraction, both plots show that the majority of the EV population is distributed between 50 and 150 nm

Fig. 3

Scatter plots of the miRNA levels in plasmatic NDEVs from CT, AD and FTD groups. Relative quantification of miRNAs in FTD and AD patients compared to CTs in NDEVs. The bold bars represent the average value ± standard error. *p < 0.05; ***p ≤ 0.001

Fig. 4

Scatter plots of the miRNA levels in plasmatic TEVs from CT, AD and FTD groups. Relative quantification of miRNAs in FTD and AD patients compared to CTs in TEVs. The bold bars represent the average value ± standard error. *p < 0.05; **p ≤ 0.01

Fig. 5

Scatter plots of the miRNA levels in CSF from CT, AD and FTD groups. Relative quantification of miRNAs in FTD and AD patients compared to CT in CSF samples. The bold bars represent the average value ± standard error. *p < 0.05

Fig. 6

miRNA levels in FTD cellular model. Quantification of the miRNA levels (expressed as 2^-ΔCt) in (A) neurons and (B) culture medium derived from hiPSCs. White columns are referred to wild-type hiPSC, while the black ones to the MAPT IVS10 + 16 mutated hiPSCs, after 120 days from differentiation inputs (n = 4 for each group). *p < 0.05; **p < 0.01; ***p < 0.001