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

背景：水通道蛋白-4(AQP4)是大脑中主要的水通道蛋白，在星形胶质细胞中表达，并在血脑屏障中丰富，允许水在血液和脑组织之间流动，并作为最近描述的淋巴系统的一部分通过血管周围空间。中风或头部受伤后，水的动态平衡被破坏，这可能导致水流入大脑。这种过量的水分会导致大脑肿胀，增加颅内压，这可能是致命的，也可能导致长期残疾。因此，AQP4被确定为治疗中风和脑损伤后脑水肿的药物靶点。尽管如此，直接AQP4抑制剂的开发进展甚微。我的主管Philip Kitchen最近的一项发现表明，AQP4可以迅速从细胞内的小泡重新定位到质膜，并且针对这种重新定位是一种可行的治疗策略，可以在中枢神经系统损伤的啮齿动物模型中预防或减少脑和脊髓水肿(Kitchen等人，2020)。然而，这项研究中使用的药物如果用于患者，显然有可能产生副作用。因此，进一步利用这一发现造福患者将需要一场筛选活动，以发现新的AQP4运输抑制剂。二维(2D)星形胶质细胞单培养已被用于在理解AQP4的分子生物学方面取得一些进展。然而，一个关键的弱点是，单一培养的星形胶质细胞在体内的极化方式与星形胶质细胞不同，不发育终足，AQP4不定位于特定的面向血管的膜亚结构域。因此，介于简单的2D星形胶质细胞单一培养和生理相关的活体实验之间，在高通量筛选(HTS)兼容系统中使用人类细胞，将是发现AQP4药物的理想平台。目前还不存在这样的体外系统来研究AQP4。本项目的目标是开发该系统。研究计划/方法：我们将使用Mimetas BV(Wevers等人，2018)开发的HTS兼容微流控芯片上器官平台开发BBB模型，Mimetas BV等人是我的BBSRC发现奖学金的合作者。与大多数芯片上器官平台不同，Mimetas系统与现有的高通量成像、微型板和机器人技术兼容，使其成为筛选项目的理想选择。此外，它还结合了一个简单的重力驱动的灌流系统，这对于发展完整的内皮屏障功能至关重要。Mimetas系统的“PhaseGuide”技术意味着不需要支撑膜，相邻的微流体通道允许细胞与细胞直接接触(这里是星形胶质细胞、内皮细胞和周细胞之间)。细胞种植密度、细胞外基质组成和介质组成将为星形胶质细胞端脚的形成和AQP4的内足定位进行优化。这将在使用原代星形胶质细胞的化学固定共培养中通过免疫荧光进行测量，或者通过使用IPSC来源的星形胶质细胞进行活细胞成像来测量，这些星形胶质细胞已经使用CRISPR/Cas9编辑，在AQP4基因的一个副本上具有3‘(C-末端)EGFP标签。这些小组已经产生，并在联合研究小组基金的支持下部分确定了其特点。预期结果：多份出版物描述了我们的AQP4-EGFP iPSC星形胶质细胞，以及我们优化的BBB芯片模型。更重要的是，我们将有一个模型用于筛选AQP4贩运的新抑制剂。我的主管是Estuar PharmPharmticals的创始股东之一，该公司获得了大量风险投资，为几个筛查项目提供了资金。因此，有一条明确的途径可以利用在本学习期间所取得的任何发现。参考文献Wevers等人，流体屏障CNS。2018；15：23.Kitchen等人，Cell.2020；181(4)：784-799.e19。

"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."