Characterization of extracellular vesicles and miRNA released by cerebral organoids

Abstract

Environmental toxicants can contribute to the development of several neurodegenerative diseases. However, the mechanisms behind this pathology are still incompletely understood. Prompt diagnosis of impending neurodegeneration is crucial for early interventions to prevent cognitive decline. Towards this end, accurate biomarkers for early neurodegenerative processes and exposure risk are needed. Extracellular vesicles (EVs) are lipid particles released by cells which contain many bioactive molecules including miRNAs. EVs may serve both as a route of propagating neurotoxic phenotypes and as a source of biomarkers for neurological disease. However, the exact mechanisms though which EVs could spread the deleterious effects of toxicants and the full spectrum of their usage as biomarkers remain unclear. Organoid models have several advantages, including potential for use in high-throughput toxicant testing and applications in personalized medicine and disease models. However, few studies have examined EV release in brain organoids to determine if the EVs could contain useful biomarkers. We employed several technologies to characterize EVs released by human cerebral organoids and their associated miRNAs. We identified that cerebral organoids consistently release EV-associated miRNA in quantities sufficient for robust analysis with NanoString. Further, pathway analyses revealed that terms related to neurodegenerative disease and nervous system signaling are associated with the recovered miRNAs. Together, these data suggest that cerebral organoids have utility as a tool for the discovery of EV-associated miRNAs involved in neurodegenerative disease and neurotoxicity.

Keywords: Cerebral organoids; Extracellular vesicles; Neurodegenerative disease; Neurotoxicity; New approach methodologies; microRNA.

Graphical abstract

Fig. 1

Cerebral organoids release particles with properties of small extracellular vesicles. (A) Representative nanoparticle tracking analysis histogram showing the relative size distribution of particles isolated from mature cerebral organoids. Mean size (B) and total particle count recovered per batch of (approximately 200) organoids each (C) compared in particles extracted from cerebral organoids at three time points post-maturation. Shown are individual data points from separate biological replicates. (D) Tunneling Electron Microscopy (TEM) image of particles isolated from mature cerebral organoids. Scale bar represents 200 nm. (E) Western Blot performed using protein extracted from particles derived from cerebral organoids on growth day 45. Three biological replicates are shown, using antibodies against the EV markers CD63 and TSG101, and the mitochondrial protein VDAC. (F) Droplet-digital PCR showing abundance of RNA transcripts corresponding to GAPDH, mtCO2, or ND1 in EVs isolated from cerebral organoids treated with or without GW 4869. Graph shows average concentrations + S.D. of n = 3 biological replicates. Samples were compared using a one-sample t-test comparing to a hypothetical value of 1.0. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 2

Cerebral-Organoid-Derived EVs contain miRNAs which may target genes associated with neurological disease. (A) Venn diagram illustrating the numbers of miRNAs identified in cerebral organoid tissue versus CO-EVs using NanoString. (B) Top 20 enriched terms identified through a KEGG analysis using the DIANA-miRPath tool.

Fig. 3

CO-EV-associated miRNA targets genes involved in pathways of cellular stress, development, and nervous system signaling. (A) Bubble plot showing the results of IPA core pathway analysis ranked by p-value and number of genes belonging to each pathway within the category. −log(p-value) is shown on the x-axis and pathway category is shown on the y-axis. A −log(p-value) cutoff of 1.3 was used for all categories. Red boxes indicate categories analyzed further in panel B. (B) Bubble plot showing specific pathways contained within the categories of Neurotransmitters and Other Nervous System Signaling and Neuronal System, ranked by p-value and number of genes within the pathway. −log(p-value) is shown on the x-axis and pathway name is shown on the y-axis. A −log(p-value) cutoff of 1.3 was used for all pathways. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

CO-EV-associated miRNA targets genes involved in pathways of cellular stress, development, and nervous system signaling. (A) Bubble plot showing the results of IPA core pathway analysis ranked by p-value and number of genes belonging to each pathway within the category. −log(p-value) is shown on the x-axis and pathway category is shown on the y-axis. A −log(p-value) cutoff of 1.3 was used for all categories. Red boxes indicate categories analyzed further in panel B. (B) Bubble plot showing specific pathways contained within the categories of Neurotransmitters and Other Nervous System Signaling and Neuronal System, ranked by p-value and number of genes within the pathway. −log(p-value) is shown on the x-axis and pathway name is shown on the y-axis. A −log(p-value) cutoff of 1.3 was used for all pathways. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Neurological diseases and functions associated with CO-EV-derived miRNA gene targets. Plot shows the number of genes targeted by CO-EV-associated miRNAs within the categories of neurological disease and nervous system function with a p-value < 0.05 as determined by IPA core analysis of diseases and functions. Number of genes is shown on the x-axis and associated disease or function term on the y-axis.