Astrocyte-Derived Small Extracellular Vesicles Regulate Dendritic Complexity through miR-26a-5p Activity

Abstract

In the last decades, it has been established that astrocytes play key roles in the regulation of neuronal morphology. However, the contribution of astrocyte-derived small extracellular vesicles (sEVs) to morphological differentiation of neurons has only recently been addressed. Here, we showed that cultured astrocytes expressing a GFP tagged version of the stress-regulated astrocytic enzyme Aldolase C (Aldo C-GFP) release small extracellular vesicles (sEVs) which are transferred into cultured hippocampal neurons. Surprisingly, Aldo C-GFP-containing sEVs (Aldo C-GFP sEVs) displayed an exacerbated capacity to reduce the dendritic complexity in developing hippocampal neurons compared to sEVs derived from control (i.e. GFP-expressing) astrocytes. Using bioinformatics and biochemical tools, we found that the total content of overexpressed Aldo C-GFP correlates with an increased content of endogenous miRNA-26a-5p in both total astrocyte homogenates and sEVs. Notably, neurons magnetofected with a nucleotide sequence that mimics endogenous miRNA-26a-5p (mimic 26a-5p) not only decreased the levels of neuronal proteins associated to morphogenesis regulation and also reproduced morphological changes induced by Aldo-C-GFP sEVs. Furthermore, neurons magnetofected with a sequence targeting miRNA-26a-5p (antago 26a-5p) were largely resistant to Aldo C-GFP sEVs. Our results support a novel and complex level of astrocyte-to-neuron communication mediated by astrocyte-derived sEVs and the activity of their miRNA content.Here, we showed that cultured astrocytes that express GFP-tagged Aldo C (Aldo C-GFP) transfer the derived sEVs, carrying the recombinant protein, in order to develop hippocampal neurons, impacting on their dendritic complexity. Using bioinformatics combined with biochemical and molecular approaches, we postulated and then confirmed that the content of miRNA-26a-5 is regulated in Aldo C-GFP-electroporated astrocytes and their sEVs. Finally, we showed that the miRNA-26a-5p-carried by Aldo C-GFP-containing sEVs (Aldo C-GFP sEVs) actively regulate the expression of some relevant neuronal proteins for morphogenesis and dendritic complexity. Our results show that sEVs from astrocytes can regulate dendritic complexity depending on the activity of miRNA-26a-5p.. Preprints (www.preprints.org) | NOT PEER-REVIEWED |

SUMOylation regulates protein cargo in Astrocyte-derived small extracellular vesicles

“…When neurons were incubated with control astrocyte sEVs, a decrease of dendritic arbor complexity was observed. This can be interpreted as increased pruning and thus enhanced network maturation during this time period or decreased growth (Luarte et al, 2020). Intriguingly, sEVs derived from corticosterone treated astrocytes did not affect dendritic length or complexity, but stimulated protein synthesis.…”

“…HeLa cells were maintained in DMEM containing 10% Fetal bovine serum with 100 units/ml of penicillin and 100 µg/ml of streptomycin and 200 µg/ml of G418 (for maintaining stable cell lines) incubated at 37°C, with 5% CO2 and 95% humidity. Astrocytes were obtained from the telencephalon of post-natal rats (at postnatal day 1) as already described (Luarte et al, 2020). Astrocytes were maintained in DMEM containing 10% Fetal bovine serum with 100 units/ml of penicillin and 100 µg/ml of streptomycin incubated at 37°C, with 5% CO2 and 95% humidity.…”

“…Then, the conditioned media of these cells was harvested to isolate sEVs by ultracentrifugation. sEVs were isolated by serial centrifugations as already described (Luarte et al, 2020;Théry, Amigorena, Raposo, & Clayton, 2006): After 30 minutes at 2,000g the supernatant was recovered, the centrifuged for 45 minutes at 10,000g. Then the supernatant was centrifuged for 2 hours at 100,000g, the supernatant was eliminated and the sEV enriched pellet was washed and re-suspended in PBS and stored at -80°C until use.…”

“…in neurons. It was previously shown that astrocyte sEVs can be taken up by neurons (Luarte et al, 2020). Moreover, the relationship between SUMOylation and gene expression or transcription regulation has been described extensively (Liu & Shuai, 2008;Müller, Ledl, & Schmidt, 2004;Rosonina, Akhter, Dou, Babu, & Sri Theivakadadcham, 2017), as well as its relationship with protein translation (Hendriks & Vertegaal, 2016;Nie, Xie, Loo, & Courey, 2009;X.…”

“…The dendritic tree was examined in 3 µm increments. The following parameters were obtained: total dendrite length (i.e., the largest radius at which there is an intersection with a neuronal process) and total number of intersections (i.e., the sum of all intersections with each different radius) (Luarte et al, 2020).…”