Exploring exosomes in neurodevelopmental and neuropsychiatric diseases using brain organoids

项目摘要 外泌体是小的细胞外囊泡，其介导脑中的细胞间信号传导，而不需要 细胞之间的直接接触。这些囊泡富含miRNA、蛋白质和脂质。目前的证据 外泌体信号在大脑中的作用指向它们在翻译调节，神经发生，突触 可塑性和神经炎症，所有这些都与几种神经发育疾病有关。 然而，外泌体信号在精神障碍中的变化还有待探索。 外泌体已经从几乎所有的哺乳动物细胞类型中分离出来，包括中枢神经系统中的细胞。 神经系统如神经元、星形胶质细胞、少突胶质细胞和小胶质细胞。神经元和神经胶质细胞释放外泌体， 体外和体内。从神经胶质细胞到神经元的营养支持被认为是由外来体传递的。 外泌体介导的细胞间信号传导与神经退行性疾病如ALS有关， 帕金森氏病、多发性硬化症和阿尔茨海默氏病以及神经发育 紊乱更具体地说，改变的神经元外泌体信号转导与Rett综合征有关， 自闭症谱系障碍（ASD）然而，外泌体信号在ASD和其他精神疾病中的作用， 疾病仍有待充分了解。这个应用的目的是探索外泌体是否 ASD中的内容和数字被改变。我们从健康对照组和患者大脑中分离出外泌体 通过差示超离心法进行类器官培养，并使用透射电镜评价纯化的外泌体 电子显微术、用于生物化学标记物的蛋白质印迹和纳米颗粒表征系统。在 纯化的外泌体组分，我们将首先评估外泌体蛋白质组和核酸谱， 对照组和患者组之间的比较。除了内容，还有证据表明， 外泌体的数量在疾病状态下改变。为了探索神经外泌体在ASD中的作用，我们建议 确定1）从ASD患者类器官分离的外来体的蛋白质组是否不同于外来体 来源于健康对照类器官（例如，通过比较蛋白质谱与串联质量标签[TMT]液体 2）从ASD纯化的外泌体中的核酸含量是否 患者类器官与来自对照的外来体相比发生改变（例如，通过比较RNA/miRNA图谱 用下一代测序，然后是生物信息学），以及3）外泌体的数量是否 从ASD脑细胞释放的蛋白质与对照细胞相比发生了改变（例如，通过评估外泌体的数量 在2D神经元或神经胶质培养基中使用Nanosight跟踪分析）。来自类器官的外来体分析 和2D文化将作为平行的战略，以描绘神经外泌体含量在健康和 疾病我们的研究有可能为ASD的病因学提供新的见解。识别 ASD中脑外泌体的量或含量的改变将揭示未被充分认识的修饰 在精神疾病状态下的细胞通讯。

PROJECT SUMMARY Exosomes are small extracellular vesicles that mediate intercellular signaling in the brain without requiring direct contact between cells. These vesicles are enriched with miRNAs, proteins, and lipids. Current evidence for exosome signaling in the brain points toward their role in translational regulation, neurogenesis, synaptic plasticity, and neuroinflammation, all of which have been implicated in several neurodevelopmental diseases. However, changes in exosome signaling in mental disorders have yet to be explored. Exosomes have been isolated from nearly all mammalian cell types, including cells in the central nervous system such as neurons, astrocytes, oligodendrocytes, and microglia. Neurons and glia release exosomes in vitro and in vivo. Trophic support from glial cells to neurons is thought to be conveyed by exosomes. Exosome-mediated intercellular signaling has been implicated in neurodegenerative diseases such as ALS, Parkinson’s disease, multiple sclerosis, and Alzheimer’s Disease as well as neurodevelopmental disorders. More specifically, altered neuronal exosome signaling has been implicated in Rett syndrome, a form of Autism Spectrum Disorder (ASD). However, the role of exosome signaling in ASD and other mental disorders remains to be fully understood. The goal of this application is to explore whether exosome content and numbers are altered in ASD. We have isolated exosomes from healthy control and patient brain organoid cultures by differential ultracentrifugation and evaluated purified exosomes using transmission electron microscopy, western blotting for biochemical markers, and nanoparticle characterization system. In purified exosome fractions, we will first assess exosome proteomes and nucleic acid profiles that will be compared between control and patient groups. In addition to content, there is also evidence suggesting that exosome quantity is altered in disease states. To explore the role of neural exosomes in ASD, we propose to determine 1) whether the proteome of exosomes isolated from ASD patient organoids differ from exosomes derived from healthy control organoids (e.g., by comparing protein profiles with Tandem mass tag [TMT] liquid chromatography [LC] mass spectrometry), 2) whether nucleic acid content in exosomes purified from ASD patient organoids is altered in comparison to exosomes from control (e.g., by comparing RNA/miRNA profiles with next-generation sequencing followed by bioinformatics), and 3) whether the quantity of exosomes released from ASD brain cells is altered compared to control cells (e.g., by assessing the quantity of exosomes in 2D neuronal or glial culture media using nanosight tracking analysis). Exosome analysis from organoids and 2D cultures will serve as parallel strategies to delineate neural exosome content in health and disease. Our studies have the potential to provide novel insights into the etiology of ASD. Identifying alterations in amount or content of brain exosomes in ASD will reveal underappreciated modifications in cellular communication during mental disease states.