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Extracellular vesicles (EVs) are garnering attention as a safe and efficient biomolecule delivery system. EVs intrinsically play a crucial role in intercellular communication and pathophysiology by transporting functionally active DNA molecules. The internalized DNA pleiotropically affects the recipient cells. Considering these salient features, an intentional incorporation of specific DNA gene cassettes into EVs and their subsequent delivery to the target cells has potential applications in genetic engineering. Moreover, efficient ways to insert the DNA into EVs during their biogenesis is valuable. Our current research is a step in the development of this technology. As such, cancer cells are known to secrete exosomes containing increased amounts of double-stranded DNA than normal cells. The clonal analysis in our previously published data revealed that exosomes released from various cancer cells contained a significantly larger population of NANOGP8 DNA with a 22-base pair insertion in the 3′-untranslated region (UTR) compared to those secreted by normal cells. This finding led us to hypothesize that the 22-base pair insertion may act as a signal to facilitate the incorporation of NANOGP8 DNA into the exosomes. To test this hypothesis, we compared the EV localization of an Enhanced Green Fluorescent Protein (EGFP) gene fused with the NANOGP8 3′-UTR, with and without the 22-base pair insertion. The quantitative PCR analysis showed a significantly higher EGFP DNA accumulation in exosomes released from cells transfected with the gene cassette containing the 3′-UTR with the 22-base pair insertion. The discovery of a DNA localization signal in exosomal DNA's 3'-UTR could pave the way for the development of an EV-based DNA delivery system. This technology will open new possibilities in genetic engineering and innovative therapies using nucleic acid medicine. [ABSTRACT FROM AUTHOR]

We have previously reported the cross-talk between Reelin and Notch-1 signaling pathways, which are 2 major pathways that regulate brain development. We found that Reelin activated Notch-1 signaling, leading to the expression of brain lipid binding protein (BLBP) and the formation of radial glial cells in human neural progenitor cells (hNPCs). In the current study, we investigated the molecular mechanisms by which Reelin activates Notch-1. We show that Reelin- stimulated Notch-1 activation is dependent on Reelin signaling. The induction of Disabled-1 (Dab-1) tyrosine phosphorylation, and the subsequent activation of Src family kinases, were found to be essential steps for the activation of Notch-1 signaling by Reelin. Reelin treatment increased the interaction between Dab-1 and Notch-1 intracellular domain (NICD), and enhanced NICD translocation to the nucleus. This study advances our knowledge of the regulation of Notch-1 activation by Reelin signaling in hNPCs, as an approach to understanding cell fate determination, differentiation, and neurogenesis during brain development. Key words: radial glia, NICD, DAB-1, neural progenitor, reelin, Notch. Nous avons deja rapporte l'existence d'une communication croisee entre les voies de signalisation Reelin et Notch-1, deux sentiers majeurs qui regulent le developpement du cerveau. Nous avons trouve que Reelin activait la voie de signalisation Notch-1 qui conduit a l'expression de la proteine BLBP (Brain Lipid Binding Protein) et a la formation de cellules gliales radiales chez les cellules souches neurales humaines (CSNh). Dans cette etude, nous avons examine les mecanismes moleculaires par lesquels la voie Reelin active Notch-1. Nous montrons que l' activation de Notch-1 stimulee par la voie Reelin depend de la signalisation de Reelin. L'induction de la phosphorylation sur tyrosine de Disabled-1 (Dab-1) et l'activation subsequente des kinases de la famille Src se sont averees essentielles a l'activation de la signalisation de Notch1 par la voie Reelin. Un traitement avec la proteine Reelin augmentait l'interaction entre Dab-1 et le domaine intracellulaire de Notch-1 (NICD) et augmentait la translocation de NICD dans le noyau. Cette etude fait avancer nos connaissances sur la regulation de l' activation de Notch-1 par la voie de signalisation Reelin dans les CSNh, comme approche pour comprendre la determination de la destinee cellulaire, la differentiation et la neurogenese durant le developpement du cerveau. Mots-cles : glie radiale, NICD, DAB-1, cellules souches neurales, reelin, Notch. [Traduit par la Redaction], Introduction Reelin is a large secreted glycoprotein that has been shown to be an important signal for neuronal migration and proper positioning of neurons in the cerebral cortex and cerebellum [...]