Identification of neurotrophic extracellular vesicles

NGF是交感神经系统正常连接所必需的。 发展。当NGF与其受体TrkA结合后，可以在远端轴突或细胞体中局部发出信号 来自节后神经元的“信号内小体”(Ses)。TrkA-SE向细胞体的运输是 对许多发育过程至关重要，包括生存和突触形成。此外，突触前 神经元的存活反映了交感神经节的存活，进而也反映了最终的目标。不适当的监管 这些过程与神经发育障碍有关，如智力低下和自闭症。 NGF/TrkA在自发性硬化症中的内化和轴突的逆行运输得到了广泛的研究，然而，有 当SE回到细胞体后，我们对SE的命运的了解存在很大差距。 我们发现了Se在胞体和树突中的一种动态的新的逆行运输途径 细胞穿透(RT)。RT由胞体和树突中Se的胞吐作用和随后的再内吞作用组成 将TrkA转化为持久的SES，从而避免了退化。这项提议的前提是我们最近 发现胞体内TrkA-SE数量在6小时后下降50%，但NGF信号持续12小时 或者更长的时间。这种极长的信号持续时间背后的机制尚不清楚。失踪案 来自胞体的TrkA-Ses可能是由于退化，但我们现在提出了一个新的替代假设： RT可导致TrkA从多囊泡小体(MVB)中胞吐，不仅导致表面外观 激活的TrkA在胞体上的表达，以及树突细胞外小泡中NGF-TrkA的分泌 这继续发出信号。EV生物学是一个新的领域，但主要描述了EV的一系列功能 在非神经系统中。电动汽车在组织修复、免疫监测、运输等方面发挥着重要作用。 MiRNAs，并激活信号级联。最近有几项电动汽车研究集中在 然而，EVS以前并没有被证明参与营养神经发育过程。 作为询问TrkA是否可以在电动汽车中分泌以支持持久信号的第一步，我们成功地 从PC12细胞中提纯EV。这些EV含有TrkA，可以在SCG神经元中引发功能性反应。 由于对TrkA-EV是如何产生的以及它们在组成方面如何与 来自非神经细胞的EVS，我们提出了一套探索性的实验来严格纯化和分子 定义来自交感神经元的TrkA-EV，并确定TrkA下游的信号是否影响它们的 制作。我们将使用创新的方法，包括微流控设备，严格的净化耦合到 质谱仪和最先进的流式细胞术，以全面表征TrkA- 电动汽车。这些实验是确定这一现象的形式、功能和作用轨迹的重要第一步 潜在的营养信号新模式。我们的长期目标是探索一种新型的神经元--神经元 可能对功能回路的发展至关重要的交流：神经营养性TrkA-EV的分泌。

NGF is required for proper wiring of the sympathetic nervous system during development. Upon binding to its receptor TrkA, NGF can either signal locally in distal axons or in the cell body from “signaling endosomes” (SEs) of the postganglionic neuron. Trafficking of the TrkA-SE to the cell body is critical for many developmental processes, including survival and synapse formation. Additionally, presynaptic neuron survival mirrors that of sympathetic ganglia and, by extension, the final target. Improper regulation of these processes has been linked to neurodevelopmental disorders such as mental retardation, and autism. NGF/TrkA internalization and retrograde transport down the axon in SEs is widely studied, however, there is a substantial gap in our knowledge when it comes to the fate of the SE once it gets back to the cell body. We have discovered a dynamic novel trafficking pathway of SEs in the soma and dendrites, retrograde transcytosis (RT). RT consists of exocytosis of SEs in the soma and dendrites and subsequent re-endocytosis of TrkA into long-lasting SEs which evade degradation. The premise for this proposal rests on our recent discovery that TrkA-SE number in the soma declines by 50% after 6 hours, but NGF signaling continues for 12 or more hours. The mechanism underlying this extremely long signal duration is unknown. The disappearance of TrkA-SEs from the soma could be due to degradation, but we now propose a novel alternative hypothesis: RT might lead to exocytosis of TrkA from multivesicular bodies (MVBs), resulting not only in surface appearance of activated TrkA on the soma, but also in secretion of NGF-TrkA in extracellular vesicles (EVs) in dendrites which continue to signal. EV biology is a nascent field, but a range of EV functions have been described mainly in non-neuronal systems. EVs have a demonstrated role in tissue repair, immune surveillance, transportation of miRNAs, and activation of signaling cascades. There have been a handful of recent EV studies focusing on neurons, however EVs have not previously been shown to participate in trophic neurodevelopmental processes. As a first step to ask if TrkA can be secreted in EVs to support long-lasting signaling, we succeeded in purifying EVs from PC12 cells. These EVs contain TrkA and can elicit functional responses in SCG neurons. Since nothing is known about how TrkA-EVs are generated and how they compare in terms of composition to EVs from non-neuronal cells, we propose an exploratory set of experiments to rigorously purify and molecularly define TrkA-EVs from sympathetic neurons, and to determine if signaling downstream of TrkA affects their production. We will use innovative approaches including microfluidic devices, rigorous purification coupled to mass spectrometry, and state-of-the art flow cytometry to fully characterize the molecular constituents of TrkA- EVs. These experiments are an essential first step in determining the form, function and locus of action of this potentially novel mode of trophic signaling. Our long-term goal is to explore a new type of neuron-neuron communication that may be critical for development of a functional circuit: secretion of neurotrophic TrkA-EVs.