Extracellular 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.

中文翻译：

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RTP801 通过细胞外囊泡介导培养物中的跨神经元毒性

细胞外囊泡（EV）在细胞间通讯中发挥着至关重要的作用，参与旁分泌营养支持或有毒分子（包括蛋白质）的传播。RTP801 是一种应激调节蛋白，其水平在神经退行性变过程中升高并诱导神经元死亡。然而，RTP801 毒性是否通过 EV 跨神经元转移仍不清楚。因此，我们在培养的皮质神经元中过度表达或沉默 RTP801 蛋白，分离其衍生的 EV（分别为 RTP801-EV 或 shRTP801-EV），并通过质谱（MS）表征 EV 蛋白含量。通过用这些 EV 处理培养的神经元并量化凋亡神经元死亡和分支来评估 RTP801-EV 的毒性。我们还在 6-羟基多巴胺 (6-OHDA) 的病理体外模型中测试了 shRTP801-EV 的功能。RTP801 的表达增加了神经元释放的 EV 数量。此外，RTP801 导致神经元来源的 EV 具有独特的蛋白质组特征，包含更多促凋亡标记。因此，我们观察到 RTP801 诱导的毒性通过 EV 转移到神经元，激活细胞凋亡并损害神经元形态复杂性。相比之下，shRTP801-EVs 能够增加受体神经元的树枝化。6-OHDA 神经毒素升高了 EV 中 RTP801 的水平，并且 6-OHDA 衍生的 EV 通过 Akt 和 RPS6 下游效应器失去了 mTOR/Akt 信号传导激活。有趣的是，来自神经元的 EV 在暴露于 6-OHDA 之前被沉默的 RTP801 保持了受体神经元中的 Akt 和 RPS6 反式激活。总而言之，这些结果表明 RTP801 诱导的毒性是通过 EV 转移的，因此，它可能有助于 RTP801 参与的神经退行性疾病的进展。