Orike N, Middleton G, Borthwick E, Buchman V, Cowen T, Davies A M
Department of Anatomy and Developmental Biology, Royal Free Hospital School of Medicine, London NW3 2PF, United Kingdom.
J Cell Biol. 2001 Sep 3;154(5):995-1005. doi: 10.1083/jcb.200101068. Epub 2001 Aug 27.
By adulthood, sympathetic neurons have lost dependence on NGF and NT-3 and are able to survive in culture without added neurotrophic factors. To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival. Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class IA PI 3-kinase, overexpression of Rukl (a natural inhibitor of Class IA PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase). Phospho-Akt was detectable in adult sympathetic neurons grown without neurotrophic factors and this was lost upon PI 3-kinase inhibition. The neurons died by a caspase-dependent mechanism after inhibition of PI 3-kinase, and were also killed by antisense Bcl-xL and antisense Bcl-2 or by overexpression of Bcl-xS, Bad, and Bax. These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.
到成年期时，交感神经元已不再依赖神经生长因子（NGF）和神经营养因子-3（NT-3），并且在无添加神经营养因子的培养条件下也能够存活。为了了解维持成年神经元存活的分子机制，我们建立了12周龄大鼠颈上神经节神经元的低密度、无胶质细胞培养体系，并对参与调节细胞存活的关键蛋白的功能和/或表达进行了调控。用LY294002或渥曼青霉素对磷脂酰肌醇-3激酶（PI 3-激酶）进行药理学抑制会导致这些神经元死亡，显性负性IA类PI 3-激酶、Rukl（IA类PI 3-激酶的天然抑制剂）的过表达以及显性负性Akt/蛋白激酶B（PKB，PI 3-激酶的下游效应器）也会导致神经元死亡。在无神经生长因子条件下培养的成年交感神经元中可检测到磷酸化的Akt，而在PI 3-激酶受到抑制后这种磷酸化就会消失。在PI 3-激酶受到抑制后，神经元通过一种半胱天冬酶依赖性机制死亡，并且反义Bcl-xL和反义Bcl-2或Bcl-xS、Bad和Bax的过表达也会导致神经元死亡。这些结果表明，PI 3-激酶/Akt信号传导以及Bcl-2家族抗凋亡成员的表达是维持成年交感神经元存活所必需的。
