Identification of neurotrophic extracellular vesicles

神经生长因子是需要适当布线的交感神经系统， 发展在与其受体TrkA结合后，NGF可以在远端轴突或细胞体中局部地发出信号 来自节后神经元的"信号内体"（SE）。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，并且通过扩展，反映了最终目标的存活。监管不当 这些过程与神经发育障碍如智力迟钝和自闭症有关。 NGF/TrkA内化和逆行运输下轴突在SE中被广泛研究，然而， 我们对SE回到细胞体后的命运的认识存在很大差距。 我们已经发现了一个动态的新的运输途径SE的索马和树突，逆行 转胞吞（RT）。RT包括索马和树突中SE的胞吐和随后的再胞吞 TrkA转化为逃避降解的持久SE。这一建议的前提是我们最近 发现索马中的TrkA-SE数量在6小时后下降50%，但NGF信号传导持续12小时。 或更多小时。这种极长信号持续时间的机制尚不清楚。失踪 来自索马的TrkA-SE的减少可能是由于降解，但我们现在提出一个新的替代假设： RT可能导致TrkA从多泡体（MVB）中的胞吐，不仅导致表面外观， 在索马上激活的TrkA，而且在树突的细胞外囊泡（EV）中分泌NGF-TrkA 它继续发出信号。EV生物学是一个新兴的领域，但是已经描述了EV的一系列功能，主要是 在非神经系统中。EV在组织修复、免疫监视、运输 miRNAs和信号级联的激活。最近有一些EV研究关注于 神经元，然而，EV先前未显示参与营养性神经发育过程。 作为询问TrkA是否可以在EV中分泌以支持持久的信号传导的第一步，我们成功地 从PC12细胞中纯化EV。这些EV含有TrkA，并且可以在SCG神经元中引起功能反应。 由于对TrkA-EV如何产生以及它们在组成方面如何与 从非神经元细胞的EV，我们提出了一套探索性的实验，以严格纯化和分子 定义来自交感神经元的TrkA-EV，并确定TrkA下游的信号传导是否影响它们的 生产我们将使用创新的方法，包括微流控装置，严格的纯化耦合， 质谱法和最先进的流式细胞术，以充分表征TrkA的分子组成。 电动车这些实验是确定这一机制的形式、功能和作用位点的必要的第一步。 潜在的营养信号的新模式。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通信：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.