Ophthalmology & Visual Sciences, United States.
Biological Chemistry, United States.
Exp Eye Res. 2020 Aug;197:108131. doi: 10.1016/j.exer.2020.108131. Epub 2020 Jul 2.
The retina is one of the most metabolically active tissues, yet the processes that control retinal metabolism remains poorly understood. The mTOR complex (mTORC) that drives protein and lipid biogenesis and autophagy has been studied extensively in regards to retinal development and responses to optic nerve injury but the processes that regulate homeostasis in the adult retina have not been determined. We previously demonstrated that normal adult retina has high rates of protein synthesis compared to skeletal muscle, associated with high levels of mechanistic target of rapamycin (mTOR), a kinase that forms multi-subunit complexes that sense and integrate diverse environmental cues to control cell and tissue physiology. This study was undertaken to: 1) quantify expression of mTOR complex 1 (mTORC1)- and mTORC2-specific partner proteins in normal adult rat retina, brain and liver; and 2) to localize these components in normal human, rat, and mouse retinas. Immunoblotting and immunoprecipitation studies revealed greater expression of raptor (exclusive to mTORC1) and rictor (exclusive for mTORC2) in normal rat retina relative to liver or brain, as well as the activating mTORC components, pSIN1 and pPRAS40. By contrast, liver exhibits greater amounts of the mTORC inhibitor, DEPTOR. Immunolocalization studies for all three species showed that mTOR, raptor, and rictor, as well as most other known components of mTORC1 and mTORC2, were primarily localized in the inner retina with mTORC1 primarily in retinal ganglion cells (RGCs) and mTORC2 primarily in glial cells. In addition, phosphorylated ribosomal protein S6, a direct target of the mTORC1 substrate ribosomal protein S6 kinase beta-1 (S6K1), was readily detectable in RGCs, indicating active mTORC1 signaling, and was preserved in human donor eyes. Collectively, this study demonstrates that the inner retina expresses high levels of mTORC1 and mTORC2 and possesses active mTORC1 signaling that may provide cell- and tissue-specific regulation of homeostatic activity. These findings help to define the physiology of the inner retina, which is key for understanding the pathophysiology of optic neuropathies, glaucoma and diabetic retinopathy.
视网膜是新陈代谢最活跃的组织之一，但控制视网膜代谢的过程仍知之甚少。mTOR 复合物（mTORC）驱动蛋白质和脂质的生物发生和自噬，在视网膜发育和对视神经损伤的反应方面已经得到了广泛的研究，但调节成年视网膜内稳态的过程尚未确定。我们之前的研究表明，与骨骼肌相比，正常成年视网膜具有较高的蛋白质合成率，这与雷帕霉素靶蛋白（mTOR）激酶的水平有关，mTOR 激酶形成多亚基复合物，可感知和整合多种环境线索，以控制细胞和组织生理。本研究旨在：1）定量检测正常成年大鼠视网膜、脑和肝中 mTOR 复合物 1（mTORC1）和 mTORC2 特异性伴侣蛋白的表达；2）定位正常人和大鼠视网膜中这些成分。免疫印迹和免疫沉淀研究显示，与肝脏或大脑相比，正常大鼠视网膜中雷帕霉素复合物相关蛋白（mTORC1 所特有）和rictor（mTORC2 所特有）以及激活 mTORC 的成分 pSIN1 和 pPRAS40 的表达更高。相比之下，肝脏中 mTORC 抑制剂 DEPTOR 的含量更高。三种物种的免疫定位研究表明，mTOR、雷帕霉素复合物相关蛋白和 rictor 以及 mTORC1 和 mTORC2 的大多数其他已知成分主要定位于内视网膜，mTORC1 主要定位于视网膜神经节细胞（RGCs），mTORC2 主要定位于神经胶质细胞。此外，磷酸化核糖体蛋白 S6，mTORC1 底物核糖体蛋白 S6 激酶β-1（S6K1）的直接靶标，在 RGCs 中很容易检测到，表明存在活跃的 mTORC1 信号，并且在人供眼组织中得到了保留。总的来说，这项研究表明，内视网膜表达高水平的 mTORC1 和 mTORC2，并具有活跃的 mTORC1 信号，这可能为维持内稳态活动提供细胞和组织特异性调节。这些发现有助于定义内视网膜的生理学，这对于理解视神经病变、青光眼和糖尿病性视网膜病变的病理生理学至关重要。
