Department of Basic Medical Sciences, Neurosciences and Sense Organs, School of Medicine, University of Bari Aldo Moro, 70124 Bari, Italy.
Department of Bioscience, Biotechnology and Biopharmaceutics, University of Bari Aldo Moro, 70125 Bari, Italy.
Int J Mol Sci. 2020 Feb 24;21(4):1531. doi: 10.3390/ijms21041531.
Translational readthrough (TRT) of aquaporin-4 (AQP4) has remarkably expanded the importance of this new post-transcriptional mechanism, as well as the regulation potential of AQP4. The TRT isoform of AQP4, named AQP4ex, is central for both AQP4 polarization and water channel activity in the central nervous system (CNS). Here we evaluate the relevance of the TRT mechanism by analyzing whether AQP4ex is also expressed in peripheral tissues and whether the expression of AQP4ex is necessary for its polarized expression as it occurs in perivascular astrocyte processes. To this purpose, AQP4ex null mice were used, and analysis was performed by immunolocalization and immunoblot. The results demonstrate that AQP4ex is expressed in kidney, stomach, trachea and skeletal muscle with the same localization pattern as the canonical AQP4 isoforms. AQP4ex protein levels vary from 6% to about 13% of the total AQP4 protein levels in peripheral tissues. Immunogold electron microscopy experiments demonstrated the localization of AQP4ex at the astrocytic endfeet, and experiments conducted on AQP4ex null mice CNS confirmed that the expression of AQP4ex is necessary for anchoring of the perivascular AQP4. Without the readthrough isoform, AQP4 assemblies are mis-localized, being uniformly distributed on the astrocyte processes facing the neuropile. No alteration of AQP4 polarization was found in AQP4ex null kidney, stomach, trachea or skeletal muscle, suggesting that AQP4ex does not have a role for proper membrane localization of AQP4 in peripheral tissues. We conclude that a dual role for AQP4ex is limited to the CNS.
水通道蛋白 4（AQP4）的翻译通读（TRT）极大地扩展了这一新的转录后机制以及 AQP4 调节潜力的重要性。AQP4 的 TRT 同工型，命名为 AQP4ex，是中枢神经系统（CNS）中 AQP4 极化和水通道活性的核心。在这里，我们通过分析 AQP4ex 是否也在周围组织中表达以及 AQP4ex 的表达是否对于其在血管周星形胶质细胞过程中发生的极化表达是必要的，来评估 TRT 机制的相关性。为此，使用了 AQP4ex 缺失小鼠，并通过免疫定位和免疫印迹进行了分析。结果表明，AQP4ex 在肾脏、胃、气管和骨骼肌中表达，其定位模式与经典的 AQP4 同工型相同。AQP4ex 蛋白水平在周围组织中的总 AQP4 蛋白水平的 6%至 13%之间变化。免疫金电子显微镜实验证明了 AQP4ex 在星形胶质细胞终足的定位，并且在 AQP4ex 缺失小鼠的 CNS 上进行的实验证实 AQP4ex 的表达对于血管周 AQP4 的锚定是必要的。没有通读同工型，AQP4 组装被错误定位，均匀分布在面对神经丛的星形胶质细胞突起上。在 AQP4ex 缺失的肾脏、胃、气管或骨骼肌中未发现 AQP4 极化的改变，这表明 AQP4ex 在周围组织中对于 AQP4 的正确膜定位没有作用。我们得出结论，AQP4ex 的双重作用仅限于 CNS。
