Brain-Derived Extracellular Vesicle microRNA Signatures Associated with In Utero and Postnatal Oxycodone Exposure

Abstract

Oxycodone (oxy) is a semi-synthetic opioid commonly used as a pain medication that is also a widely abused prescription drug. While very limited studies have examined the effect of in utero oxy (IUO) exposure on neurodevelopment, a significant gap in knowledge is the effect of IUO compared with postnatal oxy (PNO) exposure on synaptogenesis-a key process in the formation of synapses during brain development-in the exposed offspring. One relatively unexplored form of cell-cell communication associated with brain development in response to IUO and PNO exposure are extracellular vesicles (EVs). EVs are membrane-bound vesicles that serve as carriers of cargo, such as microRNAs (miRNAs). Using RNA-Seq analysis, we identified distinct brain-derived extracellular vesicle (BDEs) miRNA signatures associated with IUO and PNO exposure, including their gene targets, regulating key functional pathways associated with brain development to be more impacted in the IUO offspring. Further treatment of primary 14-day in vitro (DIV) neurons with IUO BDEs caused a significant reduction in spine density compared to treatment with BDEs from PNO and saline groups. In summary, our studies identified for the first time, key BDE miRNA signatures in IUO- and PNO-exposed offspring, which could impact their brain development as well as synaptic function.

Keywords: RNA-Seq; brain derived EVs; in utero; oxycodone; postnatal.

Figure 1

Characterization of BDEs. (A) Western blot analysis on BDEs from three P14 saline animals (#1, #2, and #3) show the expression of the positive markers CD81, flotillin-1, and Hsp70. Note the negative marker was enriched in the whole tissue lysate (WTL) but absent in the EVs fraction (BDE). (B) BDEs isolated using a sucrose density gradient from the three groups were characterized using TEM. (C) Nano tracking analysis (NTA) showing the different size distribution; no significant changes in concentrations of isolated BDEs from the three groups was observed.

Figure 2

BDEs miRNAs signatures for IUO and PNO offspring. 2D hierarchical clustering heatmaps for the significant miRNAs and samples in the three comparisons, IUO vs. PNO, IUO vs. saline, and PNO vs. saline. The horizontal axis shows the clustering of samples within the two groups of each comparison, and the vertical axis represents the clustering of the significant miRNAs (p ≤ 0.05) for each comparison.

Figure 3

Mapping of biological processes. Clue-Go analysis of differentially expressed miRNAs targeted gene functions in the PNO and IUO groups. Pie diagrams show enriched biological processes involved in developmental, neurological, and psychological disorders (boxed), which are more impacted in the IUO offspring. The asterisks represent the group term p-value representing each category. ** p < 0.001.

Figure 4

Confocal imaging of dendritic spines. Representative images of dendritic spines (50 µm) from saline, PNO, and IUO BDEs treated (for 24 h) DIV14 primary rat cortical neurons. Neurons were immunostained for MAP2 (neuronal marker, green) and Drebrin (dendritic spine marker, red). Histograms show the overall spine counts for the three groups. Confocal images were processed by ImageJ and the number of dendritic spines on 50-µm-long dendrites were manually counted. All the experiments were performed in three replicates (n = 3) and a total of 10 neurons from each replicate were used for analysis. For the final compilation of the data, 50 μm dendrite segments and 45 dendrites per condition were used for the final analysis. Error bars = SEM. One-way ANOVA (hatched line) with Tukey’s multiple comparison test was used to determine significance. p-values are represented above for each comparison.

Figure 5

Validation of DRD1 expression, Western blot analysis of crude synaptosomal extracts isolated from saline (SAL), PNO, and IUO groups for dopamine receptors DRD1. GAPDH was used as an internal control. One-way ANOVA (hatched line) with Tukey’s multiple comparison test was used to determine significance. p-values are represented above for each comparison.