An AQP4-focused, HTS-compatible, BBB-on-a-chip model

专注于 AQP4、HTS 兼容、BBB 片上模型

• 批准号: 2711834
• 金额: --
• 依托单位: Aston University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2711834
• 关键词: AQP4; focused; HTS; compatible; BBB

"Background: Aquaporin-4 (AQP4) is the main water channel protein in the brain, where it is expressed in astrocytes and enriched at the blood-brain barrier, and allows water to move both between the blood and the brain tissue and through perivascular spaces as part of the recently-described glymphatic system. Following stroke or head injury, water homeostasis is disrupted, which can lead to influx of water into the brain. This excess water causes the brain to swell, increasing intracranial pressure, which can be fatal or lead to long-term disability. AQP4 is therefore established as a drug target for cerebral oedema following stroke and head injury. Despite this, little progress has been made on development of direct AQP4 inhibitors. A recent discovery by my supervisor, Philip Kitchen, has shown that AQP4 can rapidly relocalise from intracellular vesicles to the plasma membrane, and that targeting this relocalisation is a viable therapeutic strategy to prevent or minimise brain and spinal cord oedema in rodent models of CNS injury (Kitchen et al., 2020). However, the drugs used in this study have clear potential for side-effects if used in patients. Further exploitation of this discovery for patient benefit will therefore require a screening campaign to discover novel AQP4 trafficking inhibitors.2-dimensional (2D) astrocyte monocultures have been used to make some progress in understanding the molecular biology of AQP4. However, a key weakness is that monocultured astrocytes are not polarised in the same way as astrocytes in vivo, do not develop endfeet, and AQP4 is not localised to specific vasculature-facing membrane sub-domains. Therefore, an intermediate between simple 2D astrocyte monocultures and physiologically relevant in vivo experiments, using human cells in a high-throughput screening (HTS)-compatible system, would be an ideal platform for AQP4 drug discovery. Such an in vitro system does not currently exist for the study of AQP4. The goal of this project is to develop this system. Research Plan/Methods: We will develop a BBB model using the HTS- compatible microfluidic organ-on-a-chip platform developed by Mimetas BV (Wevers et al, 2018), who are collaborators on my BBSRC Discovery fellowship. Unlike most organ-on-a-chip platforms, the Mimetas system is compatible with existing high-throughput imaging, microplate and robotics technologies, making it ideal for screening projects. In addition, it incorporates a simple, gravity-driven perfusion system, which is crucial for development of full endothelial barrier function. The Mimetas system's "phaseguide" technology means support membranes are not required and that adjacent microfluidic channels allow direct cell-cell contact (here between astrocytes, endothelial cells and pericytes).Cell seeding densities, extracellular matrix composition, and media compositions will be optimised for formation of astrocyte endfeet and endfoot localisation of AQP4. This will be measured in chemically fixed co-cultures using primary astrocytes by immunofluorescence, or by live-cell imaging using iPSC-derived astrocytes which have been edited using CRISPR/Cas9 to have a 3' (C-terminal) eGFP tag on one copy of the AQP4 gene. These cells have already been produced and partially characterised with support from the Joint Research Group Fund. Full characterisation will form part of this project.Expected outcomes: Several publications describing our AQP4-eGFP iPSC astrocytes, and our optimised BBB-on-a-chip model. More importantly, we will have a model to use for screening for novel inhibitors of AQP4 trafficking. My supervisor is a founding shareholder of Estuar Pharmaceuticals which has received significant VC investment to fund several screening projects. There is therefore a clear path to exploitation for any discoveries made during this studentship.ReferencesWevers et al., Fluids Barriers CNS. 2018; 15: 23.Kitchen et al., Cell. 2020; 181(4): 784-799.e19."

“背景：水通道蛋白-4（AQP 4）是脑中的主要水通道蛋白，其中它在星形胶质细胞中表达并在血脑屏障处富集，并且允许水在血液和脑组织之间移动并通过血管周围空间作为最近描述的胶质淋巴系统的一部分。中风或头部受伤后，水的体内平衡被破坏，这可能导致水流入大脑。过量的水分会导致大脑肿胀，增加颅内压，这可能是致命的或导致长期残疾。因此，AQP 4被确定为中风和头部损伤后脑水肿的药物靶点。尽管如此，在开发直接AQP 4抑制剂方面几乎没有取得进展。我的导师Philip Kitchen最近的发现表明，AQP 4可以快速地从细胞内囊泡重新定位到质膜，并且靶向这种重新定位是预防或最小化CNS损伤的啮齿动物模型中的脑和脊髓水肿的可行的治疗策略（Kitchen等人，2020年）。然而，如果用于患者，本研究中使用的药物具有明显的副作用潜力。因此，进一步利用这一发现为患者的利益将需要一个筛选活动，以发现新的AQP 4贩运通道。2维（2D）星形胶质细胞单培养已被用来取得一些进展，了解AQP 4的分子生物学。然而，一个关键的弱点是，单一培养的星形胶质细胞不是以与体内星形胶质细胞相同的方式极化，不发育终足，并且AQP 4不定位于特定的面向血管的膜子域。因此，简单的2D星形胶质细胞单一培养和生理相关的体内实验之间的中间体，使用人类细胞在高通量筛选（HTS）兼容的系统，将是一个理想的AQP 4药物发现的平台。这种体外系统目前不存在用于研究AQP 4。本项目的目标是开发这个系统。研究计划/方法：我们将使用Mimetas BV（Wevers等人，2018）开发的HTS兼容微流体器官芯片平台开发BBB模型，他们是我BBSRC发现奖学金的合作者。与大多数器官芯片平台不同，Mimetas系统与现有的高通量成像，微孔板和机器人技术兼容，使其成为筛选项目的理想选择。此外，它还包括一个简单的重力驱动灌注系统，这对内皮屏障功能的发展至关重要。Mimetas系统的“phaseguide”技术意味着不需要支撑膜，并且相邻的微流体通道允许直接的细胞-细胞接触（在这里是星形胶质细胞、内皮细胞和周细胞之间）。细胞接种密度、细胞外基质成分和培养基成分将被优化以形成星形胶质细胞末端足和AQP 4的末端足定位。这将在使用原代星形胶质细胞的化学固定的共培养物中通过免疫荧光或通过使用iPSC衍生的星形胶质细胞的活细胞成像来测量，所述iPSC衍生的星形胶质细胞已经使用CRISPR/Cas9编辑以在AQP 4基因的一个拷贝上具有3'（C-末端）eGFP标签。在联合研究组基金的支持下，这些细胞已经被生产出来并进行了部分表征。完整的表征将成为该项目的一部分。预期成果：几篇描述我们的AQP 4-eGFP iPSC星形胶质细胞和我们优化的BBB-on-a-chip模型的论文。更重要的是，我们将有一个用于筛选新型AQP 4贩运抑制剂的模型。我的上司是Estuar Pharmaceuticals的创始股东，该公司已获得大量风险投资，为几个筛选项目提供资金。因此，对于在这段学习期间所做的任何发现，都有一条明确的利用途径。液体屏障CNS。2018; 15：23. Kitchen等人，Cell. 2020; 181（4）：784-799.e19。"