Zhou Shuyan, Hum Jacob, Taskintuna Kaan, Olaya Stephanie, Steinman Jeremy, Ma Junfeng, Golestaneh Nady
Department of Ophthalmology, Georgetown University Medical Center, Washington, DC 20057, USA.
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.
Antioxidants (Basel). 2023 Feb 5;12(2):385. doi: 10.3390/antiox12020385.
Initially discovered by Makuto Kuro-o in 1997, Klotho is a putative aging-suppressor gene when overexpressed and accelerates aging when deleted in mice. Previously, we showed that α-Klotho regulates retinal pigment epithelium (RPE) functions and protects against oxidative stress. However, the mechanisms by which Klotho influences RPE and retinal homeostasis remain elusive. Here, by performing a series of in vitro and in vivo experiments, we demonstrate that Klotho regulates cell viability under oxidative stress, mitochondrial gene expression and activity by inducing the phosphorylation of AMPK and p38MAPK, which in turn phosphorylate and activate CREB and ATF2, respectively, triggering PGC-1α transcription. The inhibition of Klotho in human RPE cells using CRISPR-Cas9 gene editing confirmed that a lack of Klotho negatively affects RPE functions, including mitochondrial activity and cell viability. Proteomic analyses showed that myelin sheath and mitochondrial-related proteins are downregulated in the RPE/retina of compared to WT mice, further supporting our biochemical observations. We conclude that Klotho acts upstream of the AMPK/PGC-1α pathway and regulates RPE/retinal resistance to oxidative stress, mitochondrial function, and gene and protein expressions. Thus, KL decline during aging could negatively impact retinal health, inducing age-related retinal degeneration.
1997年，黑尾真琴首次发现了Klotho基因。在小鼠中，该基因过度表达时被认为是一种衰老抑制基因，而缺失时则会加速衰老。此前，我们发现α-Klotho可调节视网膜色素上皮（RPE）的功能，并抵御氧化应激。然而，Klotho影响RPE和视网膜内环境稳态的机制仍不清楚。在此，通过进行一系列体外和体内实验，我们证明Klotho通过诱导AMPK和p38MAPK的磷酸化来调节氧化应激下的细胞活力、线粒体基因表达和活性，并依次使CREB和ATF2磷酸化并激活，从而触发PGC-1α转录。使用CRISPR-Cas9基因编辑技术抑制人RPE细胞中的Klotho，证实了Klotho的缺失会对RPE功能产生负面影响，包括线粒体活性和细胞活力。蛋白质组学分析表明，与野生型小鼠相比，Klotho基因敲除小鼠的RPE/视网膜中髓鞘和线粒体相关蛋白表达下调，进一步支持了我们的生化观察结果。我们得出结论，Klotho在AMPK/PGC-1α信号通路的上游发挥作用，调节RPE/视网膜对氧化应激的抗性、线粒体功能以及基因和蛋白质表达。因此，衰老过程中Klotho水平的下降可能会对视网膜健康产生负面影响，引发与年龄相关的视网膜变性。
