Abstract
In the last few 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) that 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, but 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.
Highlights
Astrocyte clues such as secreted factors and cell–cell contact signals are essential for proper development, maintenance, and functioning of individual neurons, as well as for the wiring of the central nervous system (CNS) by controlling axonal guidance and dendritic complexity [1,2,3].Several studies have addressed the molecular mechanisms mediating the role of the astrocyte-derived secretome on dendritic morphology
In order to unequivocally identify small extracellular vesicles (sEVs) derived from astrocytes, we electroporated in utero rat cerebral cortices with plasmids to overexpress Aldolase C-GFP (Aldo C-GFP) or GFP (GFP) as control, using the cytomegalovirus early enhancer/chicken β actin promoter (CAG)
We observed abundant GFP-positive puncta (GFP+) in neurites and cell bodies of neurons co-cultured with Aldo C-GFP-expressing astrocytes, while virtually no GFP signal was found in neurons co-cultured with GFP-expressing astrocytes (Figure 1D)
Summary
Astrocyte clues such as secreted factors (secretome) and cell–cell contact signals are essential for proper development, maintenance, and functioning of individual neurons, as well as for the wiring of the central nervous system (CNS) by controlling axonal guidance and dendritic complexity [1,2,3].Several studies have addressed the molecular mechanisms mediating the role of the astrocyte-derived secretome on dendritic morphology. Astrocyte clues such as secreted factors (secretome) and cell–cell contact signals are essential for proper development, maintenance, and functioning of individual neurons, as well as for the wiring of the central nervous system (CNS) by controlling axonal guidance and dendritic complexity [1,2,3]. It has been shown that astrocyte-derived small extracellular vesicles (sEVs) act as regulators of cell functions and signaling in CNS cells, especially between astrocytes and neurons [7,8,9]. The most studied types of sEVs, known as exosomes, are vesicles of 30–120 nm in diameter with an endocytic origin. These nanosized sEVs are released to the extracellular space from multivesicular bodies (MVBs) after their fusion with the plasma membrane [10]. Mature miRNAs, i.e., small non-coding RNAs, 20–22 nucleotides long, recognize specific sequences located mainly at the
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