Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; Kengun-Sakuragi Eye Clinic, Kumamoto, Japan.
Exp Eye Res. 2018 May;170:160-168. doi: 10.1016/j.exer.2018.02.019. Epub 2018 Feb 24.
Among candidate neuroprotective agents, adenosine is thought to be a possible treatment for central nervous system disorders. Adenosine elicits biological effects through four G protein-coupled receptors (A, A, A, and A). The A and A receptors stimulate adenylyl cyclase (AC) and increase cyclic adenosine monophosphate (cAMP) levels, whereas A and A receptors inhibit AC and decrease cAMP levels. Several studies have investigated the effects of adenosine receptors (AdoRs) in glaucoma, because modulation of A, A, or A receptor regulates intraocular pressure. In addition, AdoR-related phenomena may induce neuroprotective effects in retinal neurons. Notably, A, A, and A receptor agonists reportedly inhibit retinal ganglion cell (RGC) death in in vitro and in vivo glaucoma models. However, there is limited knowledge of the effects of AdoR activation on neurite outgrowth or the regeneration of RGCs. In this report, we described the role of an AdoR subtype in neurite outgrowth and RGC axonal regeneration. The distribution of AdoRs in the retina was evaluated by immunohistochemical analysis. Using primary cultured rat RGCs in vitro and an optic nerve crush model in vivo, neurite elongation was evaluated after stimulation by the following AdoR agonists: CHA, an A receptor agonist; CGS21680, an A receptor agonist; BAY60-6583, an A receptor agonist; and 2-Cl-IB-MECA, an A receptor agonist. To determine the mechanism of neurite promotion, the candidate molecules of signal transduction associated with the neurite elongation of AdoRs were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. All four AdoRs (A, A, A, and A) were present in the inner retinal layers. Among the agonists for AdoR, only 2-Cl-IB-MECA significantly promoted neurite outgrowth in primary cultured RGCs. Signaling pathway analyses showed that 2-Cl-IB-MECA caused upregulated phosphorylation of Akt in cultured RGCs. Additionally, LY294002, an inhibitor of Akt, suppressed the neurite-promoting effects of the A receptor agonist in RGCs. Moreover, 2-Cl-IB-MECA increased the number of regenerating axons in the optic nerve crush model. Taken together, these data indicate that activation of the A receptor, not the A or A receptors, promotes in vitro and in vivo neurite outgrowth during the regeneration of rat RGCs, which is caused by the activation of an Akt-dependent signaling pathway. Therefore, AdoR activation may be a promising candidate for the development of novel regenerative modalities for glaucoma and other optic neuropathies.
在候选神经保护剂中，腺苷被认为是中枢神经系统疾病的一种可能治疗方法。腺苷通过四个 G 蛋白偶联受体（A、A、A 和 A）发挥生物学作用。A 和 A 受体刺激腺苷酸环化酶（AC）并增加环腺苷酸（cAMP）水平，而 A 和 A 受体抑制 AC 并降低 cAMP 水平。几项研究调查了腺苷受体（AdoR）在青光眼中的作用，因为调节 A、A 或 A 受体可调节眼内压。此外，AdoR 相关现象可能在视网膜神经元中诱导神经保护作用。值得注意的是，A、A 和 A 受体激动剂据称可抑制体外和体内青光眼模型中视网膜神经节细胞（RGC）的死亡。然而，对于 AdoR 激活对神经突生长或 RGC 再生的影响知之甚少。在本报告中，我们描述了 AdoR 亚型在神经突生长和 RGC 轴突再生中的作用。通过免疫组织化学分析评估了 AdoR 在视网膜中的分布。使用体外原代培养的大鼠 RGC 和体内视神经挤压模型，在用以下 AdoR 激动剂刺激后评估神经突伸长：CHA，A 受体激动剂；CGS21680，A 受体激动剂；BAY60-6583，A 受体激动剂；和 2-Cl-IB-MECA，A 受体激动剂。为了确定神经突促进的机制，通过酶联免疫吸附测定（ELISA）和 Western blot 分析分别评估了与 AdoR 神经突伸长相关的候选信号转导分子。内视网膜层中存在所有四个 AdoR（A、A、A 和 A）。在 AdoR 的激动剂中，只有 2-Cl-IB-MECA 可显著促进原代培养的 RGC 中的神经突生长。信号通路分析表明，2-Cl-IB-MECA 导致培养的 RGC 中 Akt 的磷酸化上调。此外，Akt 的抑制剂 LY294002 抑制了 RGC 中 A 受体激动剂的神经突促进作用。此外，2-Cl-IB-MECA 增加了视神经挤压模型中再生轴突的数量。总之，这些数据表明，A 受体的激活而不是 A 或 A 受体的激活，通过激活 Akt 依赖性信号通路，促进了大鼠 RGC 再生过程中的体外和体内神经突生长。因此，AdoR 激活可能是开发青光眼和其他视神经病变新型再生方式的有前途的候选物。
