Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, Bari, Italy.
Fondazione IRCCS Casa Sollievo della Sofferenza, Cancer Stem Cells Unit, San Giovanni Rotondo (FG), Italy.
Glia. 2021 Sep;69(9):2235-2251. doi: 10.1002/glia.24032. Epub 2021 May 26.
In astrocytes, unknown mechanisms regulate the expression of M1 and M23 isoforms of water channel aquaporin-4 (M1-AQP4 and M23-AQP4). The ratio between these two isoforms controls the AQP4 assembly state in the plasma membrane known as orthogonal arrays of particles (OAPs). To give new insights into these mechanisms, here, we explore the regulation of AQP4 expression in the spinal cord of a CRISPR/Cas9 M23-null mouse model (M23-null). In the M23-null spinal cord OAP assembly, the perivascular localization of AQP4 and M1-AQP4 protein were drastically reduced. In heterozygous, M1-AQP4 was proportionally reduced with M23-AQP4, maintaining the isoform ratio unaffected. We hypothesize a role of the M23-AQP4 in the regulation of M1-AQP4 expression. M1-AQP4 transcription, splicing and M1-AQP4 protein degradation were found to be unaffected in M23-null spinal cord and in M23-null astrocyte primary culture. The translational control was investigated by mRNA-protein pull down and quantitative mass spectrometry, to isolate and quantify AQP4 mRNA binding proteins (AQP4-RBPs). Compared to WT, in M23-null spinal cord, the interaction between AQP4 mRNA and polypyrimidine tract binding protein 1, a positive regulator of AQP4 translation, was higher, while interaction with the RNA helicase DDX17 was lower. In astrocyte primary cultures, DDX17 knockdown upregulated AQP4 protein expression and increased cell swelling, leaving AQP4 mRNA levels unchanged. Here, we identify AQP4-RBPs and provide evidence that in mouse spinal cord M23-AQP4 deletion changes the interaction between AQP4 mRNA and some RBPs involved in AQP4 translation. We describe for the first time the RNA helicase DDX17 as a regulator of AQP4 expression in astrocytes.
在星形胶质细胞中，未知的机制调节水通道 Aquaporin-4（M1-AQP4 和 M23-AQP4）的 M1 和 M23 异构体的表达。这两种异构体之间的比例控制着质膜中称为正交排列的粒子（OAP）的 AQP4 组装状态。为了深入了解这些机制，我们在此探索了 CRISPR/Cas9 M23 缺失型小鼠模型（M23-null）脊髓中 AQP4 表达的调节。在 M23-null 脊髓中，OAP 组装中 AQP4 和 M1-AQP4 蛋白的血管周定位明显减少。在杂合子中，M1-AQP4 与 M23-AQP4 成比例减少，保持异构体比例不受影响。我们假设 M23-AQP4 在 M1-AQP4 表达的调节中起作用。在 M23-null 脊髓和 M23-null 星形胶质细胞原代培养物中，未发现 M1-AQP4 的转录、剪接和 M1-AQP4 蛋白降解受到影响。通过 mRNA-蛋白下拉和定量质谱法研究了翻译控制，以分离和定量 AQP4 mRNA 结合蛋白（AQP4-RBPs）。与 WT 相比，在 M23-null 脊髓中，AQP4 mRNA 与多嘧啶 tract 结合蛋白 1（一种 AQP4 翻译的正调节剂）之间的相互作用更高，而与 RNA 解旋酶 DDX17 的相互作用更低。在星形胶质细胞原代培养物中，DDX17 敲低上调了 AQP4 蛋白表达并增加了细胞肿胀，而 AQP4 mRNA 水平保持不变。在这里，我们确定了 AQP4-RBPs，并提供了证据表明，在小鼠脊髓中，M23-AQP4 缺失改变了 AQP4 mRNA 与一些参与 AQP4 翻译的 RBPs 之间的相互作用。我们首次描述 RNA 解旋酶 DDX17 是星形胶质细胞中 AQP4 表达的调节剂。
