Chintala Shravan K, Putris Nahrain, Geno Mason
Laboratory of Ophthalmic Neurobiology, Eye Research Institute of Oakland University, Rochester, Michigan, United States.
Invest Ophthalmol Vis Sci. 2015 Jan 6;56(1):505-14. doi: 10.1167/iovs.14-15539.
To investigate whether activation of Toll-like receptor 3 (TLR3) promotes the degeneration of retinal ganglion cells (RGCs) by upregulating the protein levels of c-jun N-terminal kinase 3 (JNK3).
Toll-like receptor 3-specific activator, Poly(I:C) (polyinosinic-polycytidylic acid), or PBS was injected into the vitreous humor of Thy1-YFP mice. At 24, 48, and 72 hours after treatments, degeneration of RGCs was assessed by using antibodies against brain-specific homeobox/POU domain protein 3a (Brn3a). A TLR3-specific inhibitor was injected into the vitreous humor with or without Poly(I:C). Western blot assays were performed to determine relative levels of TLR3, JNK3, pJNK3, and sterile alpha and HEAT/Armadillo motif-containing 1 (SARM1) proteins in retinal protein extracts, and immunohistochemistry assays were performed to determine their cellular localization in the retina. Mouse eyes were treated with Poly(I:C) or PBS along with MitoTracker Red, and colocalization of MitoTracker Red and JNK3 in the retinas was determined by using antibodies against JNK3.
Poly(I:C) activated TLR3 and upregulated its downstream target protein JNK3 but not SARM1 in the retina. Poly(I:C) activated TLR3 and upregulated JNK3 specifically in RGCs and promoted a significant degeneration of RGCs over a 72-hour time period. Toll-like receptor 3 upregulated the levels of JNK3 protein in the cytoplasm of RGCs, but not in the mitochondria. Toll-like receptor 3-specific inhibitor downregulated Poly(I:C)-mediated upregulation of JNK3 protein, and, in turn, significantly attenuated TLR3-induced degeneration of RGCs.
Results presented in this study show that the activation of TLR3 alone promotes the degeneration of RGCs by upregulating the protein levels of JNK3.
研究Toll样受体3（TLR3）的激活是否通过上调c-jun氨基末端激酶3（JNK3）的蛋白水平促进视网膜神经节细胞（RGCs）的退变。
将TLR3特异性激活剂聚肌苷酸-聚胞苷酸（Poly(I:C)）或磷酸盐缓冲液（PBS）注入Thy1-YFP小鼠的玻璃体内。在处理后24、48和72小时，使用抗脑特异性同源盒/POU结构域蛋白3a（Brn3a）的抗体评估RGCs的退变情况。在有或无Poly(I:C)的情况下，将TLR3特异性抑制剂注入玻璃体内。进行蛋白质免疫印迹分析以确定视网膜蛋白提取物中TLR3、JNK3、磷酸化JNK3（pJNK3）和含无菌α和HEAT/犰狳基序蛋白1（SARM1）的相对水平，并进行免疫组织化学分析以确定它们在视网膜中的细胞定位。用Poly(I:C)或PBS以及线粒体跟踪染料（MitoTracker Red）处理小鼠眼睛，并使用抗JNK3抗体确定视网膜中MitoTracker Red与JNK3的共定位。
Poly(I:C)激活视网膜中的TLR3并上调其下游靶蛋白JNK3，但不影响SARM1。Poly(I:C)激活TLR3并特异性上调RGCs中的JNK3，并在72小时内促进RGCs的显著退变。Toll样受体3上调RGCs细胞质中JNK3蛋白的水平，但不影响线粒体中的水平。TLR3特异性抑制剂下调Poly(I:C)介导的JNK3蛋白上调，进而显著减轻TLR3诱导的RGCs退变。
本研究结果表明，单独激活TLR3通过上调JNK3蛋白水平促进RGCs的退变。
