Exosomal miRNA in neuron to astroglial communication in the CNS

摘要 神经元与（星形）胶质细胞之间的通讯对于神经元的功能性突触传递和生理学是必不可少的。 CNS。尽管星形胶质细胞在突触功能中具有重要的调节作用， 调节神经元-星形胶质细胞功能单位在很大程度上是不确定的。外泌体（直径50-150 nm）是一种主要的 一种分泌型细胞外囊泡（EV），来源于早期内体细胞中的管腔内囊泡（ILV）， 在核内体成熟过程中从细胞中释放。EV和外泌体分泌自 各种CNS细胞类型已经成为CNS中新的和重要的细胞间通讯途径。 特别是，miRNAs（miRs）通常存在于外泌体中，在细胞之间穿梭以进行细胞间信号传导。 已经在CNS细胞中观察到miR的细胞间转移以调节谷氨酸转运蛋白功能， 促进髓鞘形成，维持脑血管的完整性。外泌体信号传导也与 CNS的病理状况，包括神经损伤、神经变性疾病和胶质母细胞瘤。 尽管先前的研究表明外泌体途径在CNS细胞中的重要性， 这些研究主要基于培养模型或人CSF样品，外泌体信号传导， 在中枢神经系统中的原位仍然基本上未被探索。此外，细胞生物学的基本重要方面， 这一途径，如神经元活动的影响，外泌体内化机制，和下游 受体CNS细胞中的调节也仍然未知。这对于CNS细胞来说尤其重要 类型与癌症/免疫细胞高度不同，其中目前获得了大多数外泌体知识， 外泌体信号传导机制可以是非常细胞类型异质的。 基于我们已发表的研究和其他初步结果，我们提出了以下目标 项目：目的1：确定神经元活性对ILV和神经元亚细胞定位的影响 外泌体分泌;目的2：解剖参与星形胶质细胞的识别途径和进入机制 目的3：研究神经元外泌体miR-124的遗传调控， 我们已经生成了大量的初步数据来支持我们的理论，并证明 提出的目标的可行性。我们将使用老鼠遗传学，分子生物学，病毒注射，各种成像， 和生物化学的方法来完成这些目标。该项目的结果将提供体内证据 以支持一种以前未被认识到的中枢神经系统中神经元与神经胶质之间的通讯模式。它还将提供 急需的细胞生物学知识和见解，以了解神经元到神经胶质细胞中的外泌体信号传导 特别是关于miR-124- 3 p在星形胶质细胞中的非细胞自主遗传调控， 内化由于神经元与（星形）胶质细胞的通讯改变， 疾病，这些知识和见解可以显着帮助理解这一途径是如何参与中枢神经系统 疾病

Abstract Neuron to (astro)glia communication is essential for functional synaptic transmission and physiology in the CNS. Despite the important modulatory roles of astroglia in synapse function, molecular pathways that regulate the neuron-astroglia functional unit are largely undefined. Exosomes (50-150 nm in diameter), a major type of secreted extracellular vesicles (EVs), are derived from intraluminal vesicles (ILVs) in the early endosomal compartment and are released from cells during endosome maturation. EVs and exosomes secreted from various CNS cell types have emerged as a novel and important intercellular communication pathway in the CNS. In particular, miRNAs (miRs) are often found in exosomes to shuttle between cells for intercellular signaling. Intercellular transfers of miRs have been observed in CNS cells to regulate glutamate transporter function, promote myelination, and maintain brain vascular integrity. Exosomal signaling has also been implicated in pathological conditions of the CNS, including neurological injury, neurodegenerative diseases, and glioblastoma. Despite the strong rigor in prior studies to suggest the importance of the exosomal pathway in CNS cell communication, these studies are largely based on culture models or human CSF samples, exosome signaling in situ in the CNS remains essentially unexplored. In addition, fundamentally important cell biology aspects of this pathway, such as neuronal activity's influence, exosome internalization mechanisms, and downstream regulation in recipient CNS cells also remain unknown. This is particularly important to address as CNS cell types are highly distinct from cancer/immune cells where most of exosome knowledge is currently gained and exosome signaling mechanisms can be very cell-type heterogeneous. Based on our published study and additional preliminary results, we propose the following aims in this project: Aim 1: Determine the effect of neuronal activity on the subcellular localization of ILVs and neuronal exosome secretion; Aim 2: Dissect recognition pathways and entry mechanisms involved in astroglial internalization of neuronal exosomes; Aim 3: Investigate genetic regulation of neuronal exosomal miR-124 in astroglia; We have generated a large amount of preliminary data to support our rationales and to demonstrate feasibility for proposed aims. We will employ mouse genetics, molecular biology, virus injections, various imaging, and biochemical approaches to complete these aims. Outcomes from this project will present in vivo evidence to support a previously unrecognized mode of communication from neurons to glia in the CNS. It will also provide much-needed cell biological knowledge and insights for understanding exosome signaling in neuron to glia communication, especially about miR-124-3p's non-cell autonomous genetic regulation in astroglia following its internalization. As altered neuron to (astro)glia communication is clearly implicated in many neurological diseases, this knowledge and insights can significantly help understand how this pathway is involved in CNS diseases.