Recombinant DDX58 associates with SOD1 protein and selective miRNA changes in secreted EVs
Creators: name: Fabbiano, Fabrizio
Publisher: name: Università degli studi di Trento
Year: 2023

description: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease which results in the focal degeneration of motor neurons. The contribution of different cell types in the disease progression leads to hypothesize non-cell-autonomous defects, and possibly intercellular miscommunication. Extracellular Vesicles (EVs) are cell-secreted nanosized heterogenous particles proposed as vehicles of neurotoxic cargoes contributing to ALS progression. EVs are ubiquitously released by cells and can be detected in biofluids. Their biological content, reflecting the cell of origin, includes proteins, lipids, and nucleic acids, and could represent a precious source of disease biomarkers. The vesicular RNA content can be intended as a result of the post-transcriptional control exerted by RNA-binding proteins at intracellular level. Here, we studied a novel EV-associated function for DExD/H-Box Helicase 58 (DDX58) RNA-binding protein, also known as RIG-I (retinoic acid inducible gene I). DDX58 is a cytosolic RNA sensor involved in innate immunity against viruses. Upon viral RNA (as well as host ncRNA) binding, it associates to mitochondria and activates the interferon response. Interestingly, DDX58 recently emerged as a novel RBP associated to ALS, as it was found upregulated in SOD1 and TDP-43 ALS models. Therefore, we decided to investigate DDX58 in connection to EVs and their paracrine effects. In NSC34 cells, we observed that recombinant DDX58-tGFP displays an intracellular distribution that partially reaches the organelle-enriched fraction of the endogenous Ddx58. We showed DDX58 local interactions with wt and G93A mutant SOD1 proteins, but not with TDP-43. By processing the cell secretome with orthogonal approaches for EV isolation, we found that DDX58 is included as EV cargo and influences SOD1 protein EV-enrichment. Interestingly, we observed that an acute overexpression of recombinant DDX58 resulted in significant EV-RNA release, further enhanced upon DDX58-tGFP and SOD1 G93A co-expression. This increase was also confirmed in the secretome of SOD1 G93A mouse primary astrocytes. RNA from NSC34 and astrocyte cellular models, along with corresponding EVs samples, was therefore analysed by RNA-Seq. Exploiting FACS, we included tGFP-positive and negative NSC34 cells and corresponding EVs samples. From tGFP-sorted cells, we identified clusters of differentially expressed miRNAs at intracellular level but with surprisingly no effect on EV release, except for a couple of miRNA targets. In astrocytes EVs, we identified 8 miRNAs modulated upon G93A mutation. Interestingly, in NSC34, DDX58 and SOD1 G93A co-expression resulted in a significant enrichment of miRNA transcripts in EVs compared to DDX58 overexpression, confirming an indirect role of SOD1 in EV-miRNA trafficking. We started a preliminary validation of DDX58-associated miRNAs in HEK293T cells and iPS-derived motor neuron progenitors. In mature motor neurons, we also observed increased DDX58 mRNA upon TDP-43 silencing. In conclusion, we explored the involvement of DDX58 in EV biology, describing some molecular clues of selective EV-RNA and protein cargo in ALS cellular models.