Analysis of The Senescence Secretome During Zebrafish Retina Regeneration

Zebrafish possess the innate ability to regenerate any lost or damaged retinal cell type with Müller glia serving as resident stem cells. Recently, we discovered that this process is aided by a population of damage-induced senescent immune cells. As part of the Senescence Associated Secretory Phenotype (SASP), senescent cells secrete numerous factors that can play a role in the modulation of inflammation and remodeling of the retinal microenvironment during regeneration. However, the identity of specific SASP factors that drive initiation and progression of retina regeneration remain unclear. Here, we mined the SASP Atlas and RNAseq datasets to identify differentially expressed SASP factors after retina injury, including two distinct acute damage regimens, as well as a chronic, genetic model of retina degeneration. We discovered a 31 factor “Regeneration-associated Senescence Signature” (RASS) that represents SASP factors and senescence markers that are conserved across all data sets and are upregulated after damage. Among these, we show that depletion of npm1a inhibits retina regeneration. Our data support the model that differential expression of SASP factors promotes regeneration after both acute and chronic retinal damage. Adult AB zebrafish were injected with either NMDA damaging agent alone or in combination with Metformin and ABT-263 senolytic agents. Whole retinas were collected at 3, 12, and 20 days post injury and samples were collected using Trizol based RNA extraction techniques.斑马鱼（Zebrafish）天生具备再生所有丢失或受损视网膜细胞类型的能力，其中穆勒胶质细胞（Müller glia）作为驻留干细胞发挥核心功能。近日，本研究团队发现该再生过程可由一群损伤诱导的衰老免疫细胞辅助推进。作为衰老相关分泌表型（Senescence Associated Secretory Phenotype, SASP）的组成部分，衰老细胞会分泌多种因子，这些因子在再生阶段可参与炎症调控与视网膜微环境重塑。然而，驱动视网膜再生起始与进展的特异性SASP因子的具体身份仍未明确。本研究通过挖掘SASP图谱（SASP Atlas）与RNA测序（RNAseq）数据集，鉴定了视网膜损伤后差异表达的SASP因子，实验涵盖两种不同的急性视网膜损伤模型，以及一种慢性遗传性视网膜退行性变模型。我们最终发现了由31个因子构成的“再生相关衰老特征”（Regeneration-associated Senescence Signature, RASS），该特征包含在所有数据集中均保守存在且在损伤后上调的SASP因子与衰老标志物。其中，我们证实敲降npm1a基因可抑制视网膜再生。本研究数据支持如下模型：SASP因子的差异表达可促进急性与慢性视网膜损伤后的再生过程。成年AB品系斑马鱼被分别注射单一NMDA损伤剂，或联合注射二甲双胍（Metformin）与ABT-263衰老清除剂。分别于损伤后3天、12天和20天收集完整视网膜样本，并采用基于Trizol的RNA提取技术完成样本制备。