Le Nguyet, Appel Haley, Pannullo Nicole, Hoang Thanh, Blackshaw Seth
Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, United States.
Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, United States.
Front Cell Dev Biol. 2022 Sep 19;10:914386. doi: 10.3389/fcell.2022.914386. eCollection 2022.
Direct reprogramming of retinal Müller glia is a promising avenue for replacing photoreceptors and retinal ganglion cells lost to retinal dystrophies. However, questions have recently been raised about the accuracy of studies claiming efficient glia-to-neuron reprogramming in retina that were conducted using GFAP mini promoter-driven adeno-associated virus (AAV) vectors. In this study, we have addressed these questions using GFAP mini promoter-driven AAV constructs to simultaneously overexpress the mCherry reporter and candidate transcription factors predicted to induce glia-to-neuron conversion, in combination with prospective genetic labeling of retinal Müller glia using inducible Cre-dependent GFP reporters. We find that, while control GFAP-mCherry constructs express faithfully in Müller glia, 5 out of 7 transcription factor overexpression constructs tested are predominantly expressed in amacrine and retinal ganglion cells. These findings demonstrate strong insert-dependent effects on AAV-based GFAP mini promoter specificity that preclude its use in inferring cell lineage relationships when studying glia-to-neuron conversion in retina.
将视网膜穆勒胶质细胞直接重编程是替代因视网膜营养不良而丧失的光感受器和视网膜神经节细胞的一个有前景的途径。然而，最近有人对一些研究的准确性提出了质疑，这些研究声称在视网膜中使用胶质纤维酸性蛋白（GFAP）微型启动子驱动的腺相关病毒（AAV）载体实现了高效的胶质细胞向神经元的重编程。在本研究中，我们使用GFAP微型启动子驱动的AAV构建体来同时过表达mCherry报告基因和预测可诱导胶质细胞向神经元转化的候选转录因子，并结合使用诱导型Cre依赖性GFP报告基因对视网膜穆勒胶质细胞进行前瞻性基因标记，从而解决了这些问题。我们发现，虽然对照GFAP-mCherry构建体在穆勒胶质细胞中忠实表达，但所测试的7种转录因子过表达构建体中有5种主要在无长突细胞和视网膜神经节细胞中表达。这些发现表明，基于AAV的GFAP微型启动子特异性存在强烈的插入依赖性效应，这使得在研究视网膜中胶质细胞向神经元转化时无法用其推断细胞谱系关系。
