Simultaneous cyclin D1 overexpression and p27kip1 knockdown enable robust Müller glia cell cycle reactivation in uninjured mouse retina. Simultaneous cyclin D1 overexpression and p27kip1 knockdown enable robust Müller glia cell cycle reactivation in uninjured mouse retina

Müller glia (MG) stem cells are dormant retinal stem cells. While teleost fish MG re-enter the cell cycle after retinal damage and regenerate lost retinal neurons through asymmetric cell division of MG-derived progenitors, mammalian MG lack such ability. Here, we show that forced p27 downregulation and Cyclin D1 overexpression in MG using a single adeno-associated virus cell cycle activator (CCA) vector had a strong synergistic effect to induce MG proliferation. We used transcriptome profiling at single-cell level to demonstrate that CCA could induce MG proliferation and reprogram MG into neurogenic and rod-like cells. In addition, retinal injury combined with histone deacetylase inhibitor facilitates CCA to induce more rod-like cells, but not new cell types. Our study suggests new and powerful strategies for awakening the proliferative and neurogenic potential of MG in adult mammalian retinas. Overall design: MG were labelled by Tdtomato in the Glast-CreER; Rosa26-tdTomato mice. MG proliferation was induced by the cell cycle activator (CCA) vector AAV7M8-GFAP-cyclin D1-p27sh. The CCA vector suppressed p27kip1 expression while overexpressed Cyclin D1 specifically in the MG. After receiving the CCA treatment, some mice were further injected with NMDA and TSA (CCA+NMDA+TSA, referred to as CCANT), as NMDA induced retinal injury and histone deacetylase inhibitor have been shown to improve MG reprogramming. The control group was injected with AAV7m8-GFAP-GFP-LacZsh only to control for any non-specific effect caused by virus injection and shRNA expression. Retinas were collected at around 3 weeks post AAV injection, and Tdtomato+ MG were sorted by FACS for scRNA-Seq analysis.

穆勒胶质细胞（Müller glia, MG）是一类静息的视网膜干细胞。硬骨鱼的MG在视网膜损伤后可重新进入细胞周期，并通过MG来源前体细胞的不对称细胞分裂再生丢失的视网膜神经元，而哺乳动物的MG则不具备此种再生能力。本研究表明，利用单腺相关病毒细胞周期激活剂（adeno-associated virus cell cycle activator, CCA）载体在MG中强制下调p27的表达并过表达细胞周期蛋白D1（Cyclin D1），可产生极强的协同效应，诱导MG增殖。我们通过单细胞转录组测序分析证实，CCA可诱导MG增殖，并将MG重编程为神经源性细胞及视杆样细胞。此外，联合视网膜损伤与组蛋白去乙酰化酶抑制剂，可增强CCA诱导产生更多视杆样细胞的能力，但不会催生新的细胞类型。本研究为唤醒成年哺乳动物视网膜中MG的增殖潜能与神经源性潜能提供了全新且高效的策略。

实验设计概述：在Glast-CreER; Rosa26-tdTomato小鼠模型中，通过Tdtomato荧光蛋白标记MG。使用细胞周期激活剂载体AAV7M8-GFAP-cyclin D1-p27sh诱导MG增殖，该CCA载体可特异性在MG中抑制p27kip1的表达并过表达Cyclin D1。部分接受CCA处理的小鼠还会额外注射N-甲基-D-天冬氨酸（NMDA）与曲古抑菌素A（TSA），即CCA+NMDA+TSA组（简称CCANT），因NMDA可诱导视网膜损伤，且已有研究证实组蛋白去乙酰化酶抑制剂可提升MG重编程效率。对照组仅注射AAV7m8-GFAP-GFP-LacZsh，以排除病毒注射与短发夹RNA（short hairpin RNA, shRNA）表达带来的非特异性效应。在AAV注射后约3周收集视网膜组织，通过流式细胞术（Fluorescence Activated Cell Sorting, FACS）分选Tdtomato阳性的MG，用于单细胞RNA测序（single-cell RNA Sequencing, scRNA-Seq）分析。