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，它在星形胶质细胞中表达，并在血脑屏障富集，作为最近描述的淋巴系统的一部分，它允许水在血液和脑组织之间以及通过血管周围空间移动。中风或头部受伤后，水平衡被破坏，这可能导致水涌入大脑。过量的水分会导致大脑膨胀，增加颅内压，这可能是致命的或导致长期残疾。因此，AQP4被确定为脑卒中和头部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。我的导师Philip Kitchen最近的一项发现表明，AQP4可以迅速从细胞内囊泡重新定位到质膜，并且针对这种重新定位是一种可行的治疗策略，可以预防或减少CNS损伤啮齿动物模型中的脑和脊髓水肿（Kitchen等人，2020）。然而，这项研究中使用的药物如果用于患者，显然有潜在的副作用。因此，要进一步利用这一发现造福患者，需要开展筛查活动，发现新的AQP4贩运抑制剂。二维星形胶质细胞单培养在了解AQP4的分子生物学方面取得了一些进展。然而，一个关键的弱点是，单培养的星形胶质细胞不像体内的星形胶质细胞那样极化，不发育终足，AQP4不定位于特定的面向血管的膜亚域。因此，在简单的二维星形胶质细胞单培养和生理相关的体内实验之间，在高通量筛选（HTS）兼容系统中使用人类细胞，将是AQP4药物发现的理想平台。目前尚不存在这样的体外系统来研究AQP4。这个项目的目标就是开发这个系统。研究计划/方法：我们将使用Mimetas BV （Wevers et al ., 2018）开发的HTS兼容微流控器官芯片平台开发BBB模型，他们是我BBSRC Discovery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，它还包含了一个简单的、重力驱动的灌注系统，这对于内皮屏障功能的全面发展至关重要。Mimetas系统的“相位引导”技术意味着不需要支持膜，相邻的微流体通道允许直接的细胞-细胞接触（这里是星形胶质细胞、内皮细胞和周细胞之间）。细胞播种密度、细胞外基质组成和培养基组成将优化星形细胞终足的形成和AQP4终足的定位。这将在化学固定共培养中测量，使用原代星形胶质细胞，通过免疫荧光，或使用ipsc衍生的星形胶质细胞，使用CRISPR/Cas9编辑，在AQP4基因的一个拷贝上具有3' （c端）eGFP标签。在联合研究小组基金的支持下，这些细胞已经生产出来，并进行了部分表征。完整的特征描述将是这个项目的一部分。预期结果：一些出版物描述了我们的AQP4-eGFP iPSC星形细胞，以及我们优化的BBB-on-a-chip模型。更重要的是，我们将有一个模型用于筛选AQP4转运的新型抑制剂。我的导师是Estuar Pharmaceuticals的创始股东，该公司获得了大量的VC投资，为几个筛选项目提供资金。因此，在此期间，任何发现都有明确的开发途径。参考文献wevers等，《流体屏障中枢神经系统》。2018年;15: 23。Kitchen et al., 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."