Department of Integrative Anatomical Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90089, USA.
Department of Integrative Anatomical Sciences, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90089, USA.
Biochem Biophys Res Commun. 2020 Mar 12;523(3):678-684. doi: 10.1016/j.bbrc.2020.01.009. Epub 2020 Jan 14.
The promotion of axonal regeneration is required for functional recovery from stroke and various neuronal injuries. However, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A. Nogo-66, a C-terminal domain of Nogo-A, binds to the Nogo-A receptor 1 (NgR1) and induces the collapse of growth cones and inhibits neurite outgrowth. NgR1 is also a receptor for additional axonal growth inhibitors, suggesting it is an important target for the prevention of axonal growth inhibition. By using the indirect immunofluorescence method, we show for the first time that a cell-permeable cAMP analog (dibutyryl-cAMP) induced a rapid decrease in the cell surface expression of NgR1 in Neuroscreen-1 (NS-1) cells. The biotinylation method revealed that cAMP indeed induced internalization of NgR1 within minutes. Other intracellular cAMP-elevating agents, such as forskolin, which directly activates adenylyl cyclase, and rolipram, which inhibits cyclic nucleotide phosphodiesterase, also induced this process. This internalization was found to be reversible and influenced by intracellular levels of cAMP. Using selective activators and inhibitors of protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac), we found that NgR1 internalization is independent of PKA, but dependent on Epac. The decrease in cell surface expression of NgR1 desensitized NS-1 cells to Nogo-66-induced growth cone collapse. Therefore, it is likely that besides axonal growth inhibitors affecting neurons, neurons themselves also self-regulate their sensitivity to axonal growth inhibitors, as influenced by intracellular cAMP/Epac. This normal cellular regulatory mechanism may be pharmacologically exploited to overcome axonal growth inhibitors, and enhance functional recovery after stroke and neuronal injuries.
轴突再生的促进对于中风和各种神经元损伤后的功能恢复是必需的。然而，轴突再生受到多种轴突生长抑制剂的抑制，如 Nogo-A。Nogo-66 是 Nogo-A 的 C 端结构域，与 Nogo-A 受体 1（NgR1）结合，诱导生长锥的崩溃，并抑制神经突的生长。NgR1 也是其他轴突生长抑制剂的受体，这表明它是预防轴突生长抑制的重要靶点。通过间接免疫荧光法，我们首次表明，一种细胞通透的 cAMP 类似物（二丁酰环磷腺苷）可迅速降低神经细胞系 NS-1（NS-1）细胞表面 NgR1 的表达。生物素化方法显示 cAMP 确实在数分钟内诱导 NgR1 内化。其他细胞内 cAMP 升高剂，如直接激活腺苷酸环化酶的 forskolin 和抑制环核苷酸磷酸二酯酶的 rolipram，也诱导了这一过程。这种内化是可逆的，并受细胞内 cAMP 水平的影响。使用蛋白激酶 A（PKA）的选择性激活剂和抑制剂以及 cAMP 直接激活的交换蛋白（Epac），我们发现 NgR1 内化不依赖于 PKA，但依赖于 Epac。NgR1 细胞表面表达的减少使 NS-1 细胞对 Nogo-66 诱导的生长锥崩溃脱敏。因此，除了影响神经元的轴突生长抑制剂外，神经元本身可能也通过细胞内 cAMP/Epac 来自我调节对轴突生长抑制剂的敏感性。这种正常的细胞调节机制可能在药理学上被利用来克服轴突生长抑制剂，增强中风和神经元损伤后的功能恢复。
