A Study of MSC Exosomes in axonal growth in stroke and traumatic brain injury

Exosomes derived from mesenchymal stromal cells promote axonal growth of cortical neurons

Abstract
Treatment of brain injury with exosomes derived from mesenchymal stromal cells (MSCs) enhances neurite growth. However, the direct effect of exosomes on axonal growth and molecular mechanisms underlying exosome-enhanced neurite growth are not known. Using primary cortical neurons cultured in a microfluidic device, we found that MSC-exosomes promoted axonal growth, whereas attenuation of argonaut 2 protein, one of the primary microRNA (miRNA) machinery proteins, in MSC-exosomes abolished their effect on axonal growth. Both neuronal cell bodies and axons internalized MSC-exosomes, which was blocked by botulinum neurotoxins (BoNTs) that cleave proteins of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex. Moreover, tailored MSC-exosomes carrying elevated miR-17-92 cluster further enhanced axonal growth compared to native MSC-exosomes. Quantitative RT-PCR and Western blot analysis showed that the tailored MSC-exosomes increased levels of individual members of this cluster and activated the PTEN/mTOR signaling pathway in recipient neurons, respectively. Together, our data demonstrate that native MSC-exosomes promote axonal growth while the tailored MSC-exosomes can further boost this effect and that tailored exosomes can deliver their selective cargo miRNAs into and activate their target signals in recipient neurons. Neuronal internalization of MSC-exosomes is mediated by the SNARE complex. This study reveals molecular mechanisms that contribute to MSC-exosome-promoted axonal growth, which provides a potential therapeutic strategy to enhance axonal growth.

Axonal remodeling is a key repair process, leading to reduction of neurological deficits after stroke and traumatic brain injury (TBI). However, spontaneous axonal regeneration is limited in adult injured brains [1–3]. There are two major conditions that limit neurite regrowth after brain injury, a diminished intrinsic capacity of the neurons to grow and an inhibitory extrinsic environment [4–7]. However, several lines of evidence indicate that axons can sprout after injury in adult central nervous system [8–12]. Recent studies show that distal axons of embryonic cortical neurons contain miRNA machinery proteins, Dicer and argonaut 2 protein (Ago2) and are enriched with miRNAs that can locally regulate axonal growth [13–15]. For example, alteration of the miR-17-92 cluster and miR-29c levels in the cultured neurons promote axonal growth by suppressing their target genes that inhibit axonal growth even under the inhibitory environment with chondroitin sulfate proteoglycans [16,17].
Exosomes are endosome-derived small membrane vesicles (~30–100 nm) and are released by cells in all living systems [18,19]. Exosomes mediate intercellular communication by transferring proteins, lipids, and genomic materials including mRNAs and miRNAs between source and target cells [20]. Emerging data indicate that treatment of stroke and TBI with exosomes derived from MSCs improves neurological function by facilitating interwoven brain repair processes including neurite remodeling [21–23]. In vitro studies indicate that miR-133b in MSC-exosomes mediate neurite growth of cortical neurons [24]. However, mechanisms underlying the effect of MSC-exosomes on axonal growth remain unknown. Using embryonic cortical neurons cultured in a microfluidic device, we investigated whether MSC-exosomes deliver their cargo miRNAs into recipient neurons and promote axonal growth. Our data demonstrated that miRNAs within MSC-exosomes mediated axonal growth and that tailored MSC-exosomes carrying elevated miR-17-92 cluster enhanced axonal growth to a much greater extent than native MSC-exosomes.

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