ER-stress response in retinal Müller glia occurs significantly earlier than amyloid pathology in in the Alzheimer's mouse brain and retina

Alzheimer's Disease (AD) pathogenesis is thought to begin up to 20 years before cognitive symptoms appear, suggesting the need for more sensitive diagnostic biomarkers of AD. In this report, we demonstrated pathological changes in retinal Müller glia significantly earlier than amyloid pathology in AD mouse models. By utilizing the knock-in NLGF mouse model, we surprisingly discovered an increase in reticulon 3 (RTN3) protein levels in the NLGF retina as early as postnatal day 30 (P30). Despite RTN3 being a canonically neuronal protein, this increase was noted in the retinal Müller glia, confirmed by immunohistochemical characterization. Further unbiased transcriptomic assays of the P30 NLGF retina revealed that retinal Müller glia were the most sensitive responding cells in this mouse retina, compared to other cell types including photoreceptor cells and ganglion neurons. Pathway analyses of differentially expressed genes in glia cells showed activation of ER stress response via the upregulation of unfolded protein response (UPR) proteins such as ATF4 and CHOP. Early elevation of RTN3 in response to challenges by toxic Aß likely facilitated UPR. Altogether, these findings suggest that Müller glia act as a sentinel for AD pathology in the retina and should aid for both intervention and diagnosis. Overall design: Retinas from two wildtype and two nlgf mice were dissected at P30. Cells were isolated into a single-cell suspension and processed using the 10x genomics single-cell RNA sequencing pipeline.

阿尔茨海默病（Alzheimer's Disease, AD）的发病机制被认为在认知症状出现前20年即已启动，这提示亟需开发灵敏度更高的AD诊断生物标志物。本研究在AD小鼠模型中证实，视网膜米勒胶质细胞（retinal Müller glia）的病理变化早于淀粉样蛋白病理改变。借助敲入型NLGF小鼠模型，我们意外发现，在出生后第30天（postnatal day 30, P30）的NLGF小鼠视网膜中，网状蛋白3（reticulon 3, RTN3）的蛋白水平即出现升高。尽管RTN3通常被认为是经典的神经元蛋白，但该蛋白升高现象经免疫组织化学表征证实，确实出现在视网膜米勒胶质细胞中。进一步针对P30龄NLGF小鼠视网膜开展的无偏倚转录组分析显示，与感光细胞、神经节神经元等其他细胞类型相比，视网膜米勒胶质细胞是该模型小鼠视网膜中响应最为敏感的细胞类群。对胶质细胞差异表达基因的通路分析显示，通过上调ATF4、CHOP等未折叠蛋白反应（unfolded protein response, UPR）相关蛋白，内质网应激通路得以激活。早期升高的RTN3在受到毒性Aβ刺激时，可能促进了未折叠蛋白反应的发生。综上，上述研究结果表明米勒胶质细胞可作为视网膜中AD病理的哨兵细胞，有望为AD的临床干预与早期诊断提供新的依据。实验整体设计：于P30龄时剖取2只野生型小鼠与2只nlgf小鼠的视网膜，将细胞解离为单细胞悬液，采用10x Genomics单细胞RNA测序流程完成后续处理。