Identification of neurotrophic extracellular vesicles

神经营养性细胞外囊泡的鉴定

基本信息

批准号：
9765756
负责人：
Bettina R Winckler
金额：
$ 44.41万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-03-15 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9765756
关键词：
Affect Alzheimer&apos s Disease Appearance Autocrine Communication Axon Axonal Transport Back Binding Biogenesis Biology Calcium Cell Survival Cell membrane Cells Communication Coupled Data Dendrites Development Developmental Process Distal Endocytosis Endosomes Etiology Event Exocytosis Exploratory/Developmental Grant Flow Cytometry Fluorescence Foundations Funding Mechanisms Future Goals Growth Factor Hour Humulus Immunologic Surveillance Investigation Knowledge Label Lead Link Logic MAP Kinase Gene MAPK1 gene Mass Spectrum Analysis Mediating Mental Retardation MicroRNAs Microfluidic Microchips Molecular Multivesicular Body Nerve Growth Factors Nervous system structure Neurodevelopmental Disorder Neuroglia Neurons Neurotrophic Tyrosine Kinase Receptor Type 1 PC12 Cells Pathology Pathway interactions Pattern Peripheral Nervous System Pharmacology Presynaptic Terminals Process Production Proteome Proto-Oncogene Proteins c-akt Publishing Regulation Research Rest Role Signal Pathway Signal Transduction Sorting - Cell Movement Spinal Cord Surface Sympathetic Ganglia Sympathetic Nervous System Synapses System Techniques Testing Transportation Ultracentrifugation Vesicle Work autism spectrum disorder autocrine axon growth base experimental study extracellular vesicles in vivo innovation insight life history neuronal cell body neuronal survival neurotransmission neurotrophic factor novel phospholipase C gamma postsynaptic presynaptic neurons response retrograde transport synaptogenesis tissue repair trafficking transcytosis translational approach

项目摘要

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.

NGF是需要在同情神经系统上正确接线的发展。与其接收器TRKA结合后，NGF可以在远端轴突或细胞体中局部信号源自后神经神经元的“信号内体”（SES）。将TRKA-SE贩运到细胞体是对于许多发育过程至关重要，包括生存和突触形成。另外，突触前神经元的生存反映了同情神经节的生存，并扩大了最终目标。不当调节这些过程与神经发育障碍（例如智力障碍和自闭症）有关。但是，NGF/TRKA内在化和逆行运输在SES中的轴突上被广泛研究，但是，一旦回到细胞体，就可以涉及到SE的命运时，这是我们所知的巨大差距。我们已经发现了SES和树突中SES的动态小说贩运途径，逆行转胞病（RT）。 RT由SOSA和树突中的SES胞吐作用以及随后的再胞吞作用 trka的长期SES逃避降解。该提案的前提取决于我们的最新发现SOMA中的TRKA-SE数量在6小时后下降了50％，但NGF信号持续12 或更多小时。该非常长的信号持续时间的基础机制尚不清楚。失踪 soma的trka-ses可能是由于退化而引起的，但我们现在提出了一个新颖的替代假设： RT可能会导致多种体（MVB）的TRKA胞吐作用，不仅导致表面外观在soma上活化的trka，但也在树突外蔬菜中的NGF-trka分泌 EV生物学是一个新生的领域，但主要描述了一系列EV功能在非神经系统中。电动汽车在组织修复，免疫监视，运输，运输中表现出作用 miRNA和信号级联激活。最近有少数电动汽车研究的重点但是，神经元以前尚未证明EV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，我们成功地了从PC12细胞中纯化电动汽车。这些电动汽车包含TRKA，可以在SCG神经元中引起功能反应。由于对TRKA-EV的产生以及它们如何在组成方面进行比较，因为来自非神经元细胞的电动汽车，我们提出了一组探索性实验集，以严格纯化和分子从交感神经元中定义trka-evs，并确定TRKA下游的信号是否影响其生产。我们将使用包括微流体设备在内的创新方法，严格的纯化耦合到质谱法和最先进的流式细胞仪，以完全表征TRKA-的分子构成电动汽车。这些实验是确定此的形式，功能和作用源的重要第一步潜在的营养信号传导模式。我们的长期目标是探索一种新型的神经元 - 神经元可能对发展功能电路至关重要的交流：神经营养性TRKA-EV的分泌。