PDF(Pubmed)
Abstract：
Aquaporin-4 (AQP4) is a water channel protein that links the astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, which preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel AQP4 mouse line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wild-type (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to WT and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. An increased dose of AQP4x enhanced perivascular localization of α-syntrophin and AQP4, while total protein expression of the two was unchanged. However, at 100% readthrough, AQP4x localization and the formation of higher order complexes were disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for an increased proportion of endothelial cells with budding vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels.
摘要：
水通道蛋白4（AQP4）是一种水通道蛋白，可将星形胶质细胞末端足与血脑屏障（BBB）连接，并调节大脑中的水和钾稳态，以及废物的淋巴清除，否则会加剧神经系统疾病。最近,翻译连读显示产生AQP4的C末端延伸变体，称为AQP4x，通过与支架蛋白α-syntrophin相互作用，优先定位于BBB周围，AQP4x的丢失会破坏大脑中的废物清除。为了研究AQP4x的功能，我们产生了新的AQP4小鼠系(AllX),以增加野生型(WT)小鼠脑中AQP4的约15%以上的连读变体的相对水平。我们验证了这条线，并评估了受AQP4x存在影响的特征，包括AQP4和α-syntrophin定位，BBB的完整性，和神经血管耦合。我们将AllXHom和AllXHet小鼠与WT以及先前表征的AQP4NoXHet和NoXHom小鼠进行了比较，不能生产AQP4x。AQP4x剂量的增加增强了α-syntrophin和AQP4的血管周定位，而两者的总蛋白表达没有变化。然而,在100%通读时，AQP4x的定位和高级复合物的形成被破坏。电子显微镜显示，除了NoXHom小鼠中具有出芽囊泡的内皮细胞比例增加外，整体血管形态没有变化，这可能对应于使用MRI在NoX小鼠中鉴定的渗漏BBB或改变的外排。这些数据表明，AQP4x在维持BBB完整性以及向血管募集结构和功能支持蛋白方面起着很小但可测量的作用。这也建立了一套新的遗传工具来定量调节AQP4x水平。
