Ophthalmology & Visual Sciences, University of Michigan Medical School, Ann Arbor, Michigan, USA; Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Ophthalmology & Visual Sciences, University of Michigan Medical School, Ann Arbor, Michigan, USA.
J Biol Chem. 2022 Jun;298(6):101944. doi: 10.1016/j.jbc.2022.101944. Epub 2022 Apr 18.
Mechanistic target of rapamycin (mTOR) and mTOR complex 1 (mTORC1), linchpins of the nutrient sensing and protein synthesis pathways, are present at relatively high levels in the ganglion cell layer (GCL) and retinal ganglion cells (RGCs) of rodent and human retinas. However, the role of mTORCs in the control of protein synthesis in RGC is unknown. Here, we applied the SUrface SEnsing of Translation (SUnSET) method of nascent protein labeling to localize and quantify protein synthesis in the retinas of adult mice. We also used intravitreal injection of an adeno-associated virus 2 vector encoding Cre recombinase in the eyes of mtor- or rptor-floxed mice to conditionally knockout either both mTORCs or only mTORC1, respectively, in cells within the GCL. A novel vector encoding an inactive Cre mutant (CreΔC) served as control. We found that retinal protein synthesis was highest in the GCL, particularly in RGC. Negation of both complexes or only mTORC1 significantly reduced protein synthesis in RGC. In addition, loss of mTORC1 function caused a significant reduction in the pan-RGC marker, RNA-binding protein with multiple splicing, with little decrease of the total number of cells in the RGC layer, even at 25 weeks after adeno-associated virus-Cre injection. These findings reveal that mTORC1 signaling is necessary for maintaining the high rate of protein synthesis in RGCs of adult rodents, but it may not be essential to maintain RGC viability. These findings may also be relevant to understanding the pathophysiology of RGC disorders, including glaucoma, diabetic retinopathy, and optic neuropathies.
雷帕霉素靶蛋白（mTOR）及其复合物 1（mTORC1）是营养感应和蛋白质合成途径的关键调节因子，在啮齿动物和人类视网膜的神经节细胞层（GCL）和神经节细胞（RGC）中表达水平较高。然而，mTORC 在控制 RGC 中蛋白质合成的作用尚不清楚。在此，我们应用新生蛋白质标记的表面感应翻译（SUnSET）方法来定位和定量成年小鼠视网膜中的蛋白质合成。我们还使用腺相关病毒 2 载体（在眼睛中编码 Cre 重组酶）在 mtor 或 rptor 条件性敲除小鼠的眼睛中进行眼内注射，分别在 GCL 细胞中条件性敲除两种 mTORC 或仅 mTORC1。一种新型载体编码一种失活的 Cre 突变体（CreΔC）作为对照。我们发现视网膜蛋白质合成在 GCL 中最高，尤其是在 RGC 中。两种复合物或仅 mTORC1 的缺失均显著降低了 RGC 中的蛋白质合成。此外，mTORC1 功能的丧失导致 RNA 结合蛋白多聚体 splicing 的泛 RGC 标志物显著减少，而 RGC 层中的细胞总数减少很少，即使在腺相关病毒-Cre 注射后 25 周也是如此。这些发现表明 mTORC1 信号对于维持成年啮齿动物 RGC 中高蛋白质合成率是必需的，但对于维持 RGC 活力可能不是必需的。这些发现可能与理解 RGC 疾病的病理生理学有关，包括青光眼、糖尿病性视网膜病变和视神经病变。
