Cell-specific Studies Through Novel Mouse Model Reveal the Transcriptomic Response of Retinal Müller Glia to Acute and Chronic Stress [ONC]

Epigenetic regulation of the genome through DNA modifications, mainly methylcytosine (mC) and hydroxymethylcytosine (hmC), alters DNA accessibility, genomic organization and gene expression and is thought to be altered during neurodegenerative diseases, such as  age-related macular degeneration (AMD). Analysis of retina epigenetic and transcriptomic signatures at the cell-type specific level is crucial to understanding the pathophysiology of retinal degeneration. We have discovered that Aldh1l1 is specifically expressed in the major macroglia of the retina, the Müller glia, and, unlike the brain, is not expressed in retinal astrocytes. This allows a novel model to study paired epigenetic and transcriptomic signatures in Müller glia using Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) for temporally controlled labeling and isolation of Müller glial DNA and RNA. As validated through a variety of approaches, the Aldh1l1cre/ERT2-NuTRAP model provides Müller glia specific translatome and epigenome profiles. Application of this approach to models of acute injury (optic nerve crush) and chronic stress (aging) uncovered common Müller glia-specific transcriptome changes in inflammatory pathways, as well as differential signatures for each stimulus. The expression of components of the IL1b signalling axis, complement system, and markers of gliosis was enhanced in Müller glia in response to optic nerve crush but not in response to aging. The expression of components of the purinergic receptor and focal adhesion signalling pathways changed uniquely in response to aging but not with optic nerve crush. The Aldh1l1cre/ERT2-NuTRAP model allows focusing molecular analyses to a single, minority cell type within the retina, providing more substantial effect sizes than whole tissue analyses. The NuTRAP model, nucleic acid isolation, and ­validation approaches presented here can be applied to any retina cell type for which a cell type-specific cre is available. Mouse retinal RNA from Muller glia in treated (ONC) and untreated (control) samples (n = 5)

通过DNA修饰（主要为胞嘧啶甲基化（methylcytosine, mC）与羟甲基胞嘧啶（hydroxymethylcytosine, hmC））实现的基因组表观遗传调控（epigenetic regulation），可改变DNA可及性（DNA accessibility）、基因组组织（genomic organization）与基因表达（gene expression），且在神经退行性疾病（neurodegenerative diseases）进程中被发现存在异常，例如年龄相关性黄斑变性（age-related macular degeneration, AMD）。在细胞类型特异性水平（cell-type specific level）上解析视网膜表观遗传与转录组特征，对于阐明视网膜变性（retinal degeneration）的病理生理学（pathophysiology）机制至关重要。
本研究发现，乙醛脱氢酶1L1（Aldh1l1）特异性表达于视网膜的主要大胶质细胞（macroglia）——米勒胶质细胞（Müller glia），且与脑部情况不同，其在视网膜星形胶质细胞（retinal astrocytes）中无表达。这一特性为利用核标记与翻译核糖体亲和纯化（Nuclear Tagging and Translating Ribosome Affinity Purification, NuTRAP）技术，实现时空可控标记（temporally controlled labeling）并分离米勒胶质细胞的DNA与RNA，进而研究米勒胶质细胞中成对的表观遗传与转录组特征提供了全新的研究模型。经多种实验方法验证，Aldh1l1^{cre/ERT2-NuTRAP}模型可获取米勒胶质细胞特异性的翻译组（translatome）与表观基因组（epigenome）图谱。
将该模型应用于急性损伤（acute injury）模型（视神经钳夹术，optic nerve crush）与慢性应激（chronic stress）模型（衰老）后，研究人员发现米勒胶质细胞中存在炎症通路（inflammatory pathways）共有的转录组变化，以及两种刺激各自对应的特异性差异特征（differential signatures）。响应视神经钳夹术时，米勒胶质细胞中IL1β信号轴（IL1b signalling axis）组分、补体系统（complement system）及胶质增生（gliosis）标志物的表达水平显著上调，而衰老应激未引发此类变化；反之，嘌呤能受体（purinergic receptor）与黏着斑信号通路（focal adhesion signalling pathways）组分的表达变化仅见于衰老应激，而非视神经钳夹术。
Aldh1l1^{cre/ERT2-NuTRAP}模型可将分子分析精准聚焦于视网膜内单一的少数细胞类型（minority cell type），其获得的效应量（effect sizes）远高于全组织分析（whole tissue analyses）结果。本研究中所介绍的NuTRAP模型、核酸分离与验证方法，可推广至所有拥有细胞类型特异性cre重组酶（cell type-specific cre）的视网膜细胞类型。本数据集包含经处理（视神经钳夹术，ONC）与未处理（对照）样本中米勒胶质细胞的小鼠视网膜RNA（每组n=5）。