Solana-Balaguer, JúliaMartín-Flores, NúriaGarcía-Segura, PolCampoy-Campos, GenísPérez-Sisqués, LeticiaChicote-González, AlmudenaFernández Irigoyen, JoaquínSantamaría Martínez, EnriquePérez-Navarro, EstherAlberch, JordiMalagelada, Cristina2024-05-072024-05-072023Solana-Balaguer, J., Martín-Flores, N., Garcia-Segura, P., Campoy-Campos, G., Pérez-Sisqués, L., Chicote-González, A., Fernández-Irigoyen, J., Santamaría, E., Pérez-Navarro, E., Alberch, J., Malagelada, C. (2023) RTP801 mediates transneuronal toxicity in culture via extracellular vesicles. Journal of Extracellular Vesicles, 12(11), 1-26. https://doi.org/10.1002/jev2.12378.2001-307810.1002/jev2.12378https://academica-e.unavarra.es/handle/2454/48057Extracellular vesicles (EVs) play a crucial role in intercellular communication, participating in the paracrine trophic support or in the propagation of toxic molecules, including proteins. RTP801 is a stress-regulated protein, whose levels are elevated during neurodegeneration and induce neuron death. However, whether RTP801 toxicity is transferred trans-neuronally via EVs remains unknown. Hence, we overexpressed or silenced RTP801 protein in cultured cortical neurons, isolated their derived EVs (RTP801-EVs or shRTP801-EVs, respectively), and characterized EVs protein content by mass spectrometry (MS). RTP801-EVs toxicity was assessed by treating cultured neurons with these EVs and quantifying apoptotic neuron death and branching. We also tested shRTP801-EVs functionality in the pathologic in vitro model of 6-Hydroxydopamine (6-OHDA). Expression of RTP801 increased the number of EVs released by neurons. Moreover, RTP801 led to a distinct proteomic signature of neuron-derived EVs, containing more pro-apoptotic markers. Hence, we observed that RTP801-induced toxicity was transferred to neurons via EVs, activating apoptosis and impairing neuron morphology complexity. In contrast, shRTP801-EVs were able to increase the arborization in recipient neurons. The 6-OHDA neurotoxin elevated levels of RTP801 in EVs, and 6-OHDA-derived EVs lost the mTOR/Akt signalling activation via Akt and RPS6 downstream effectors. Interestingly, EVs derived from neurons where RTP801 was silenced prior to exposing them to 6-OHDA maintained Akt and RPS6 transactivation in recipient neurons. Taken together, these results suggest that RTP801-induced toxicity is transferred via EVs, and therefore, it could contribute to the progression of neurodegenerative diseases, in which RTP801 is involved.application/pdfeng© 2023 The Authors. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivsLicense, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.6-OHDAExtracellular vesiclesmTOR signallingNeuronRTP801/REDD1Toxicityinfo:eu-repo/semantics/article2024-05-07info:eu-repo/semantics/openAccess