Kimpinski K, Jelinski S, Mearow K
Division of Basic Sciences, Memorial University of Newfoundland, St John's, Canada.
Neuroscience. 1999;93(1):253-63. doi: 10.1016/s0306-4522(99)00156-6.
We have investigated nerve growth factor-dependent neurite growth from adult sensory neurons using the compartmented culture system. The requirement of both TrkA and the p75 neurotrophin receptors in neurite growth was examined using several experimental interventions. Inhibition of TrkA activation using K252a resulted in a total block of distal neurite extension into nerve growth factor-containing compartments. Brain-derived neurotrophic factor and the anti-p75 monoclonal antibody MC192 have been shown to interfere with the binding of nerve growth factor to p75. Brain-derived neurotrophic factor, which binds p75 but not TrkA, competes with nerve growth factorforp75, while the anti-p75 antibody MC192 has been shown to decrease the interaction of nerve growth factor with TrkA. The addition of brain-derived neurotophic factor to nerve growth factor-containing distal compartments inhibited, but did not totally block, distal neurite extension. MC192, on the other hand, totally inhibited nerve growth factor-dependent neurite growth. To test whether MC192 and brain-derived neurotrophic factor might be influencing Trk activation, TrkA phosphorylation was examined biochemically. Both compounds were found to attenuate nerve growth factor-induced Trk phosphorylation, although neither inhibited the activation completely. The possibility that MC192 or brain-derived neurotrophic factor might activate p75 signaling directly (and potentially antagonize TrkA signaling) was also investigated. This was assessed by quantitating the activation and nuclear translocation of the transcription factor NFkB using immunocytochemistry. Only treatment with the anti-p75 antibody MC192 resulted in prolonged and significant increase in the number of neurons displaying nuclear staining for NFkB. Our results demonstrate that both TrkA and p75 play a role in neurite growth response to nerve growth factor, and further suggest that any alteration in optimal TrkA-p75 interactions, or direct activation of p75 at the expense of TrkA, results in an inhibition of nerve growth factor-dependent neurite growth in adult sensory neurons.
我们使用分隔培养系统研究了成年感觉神经元中神经生长因子依赖性的神经突生长。通过多种实验干预措施，研究了TrkA和p75神经营养因子受体在神经突生长中的需求。使用K252a抑制TrkA激活导致远端神经突向含神经生长因子的隔室延伸完全受阻。脑源性神经营养因子和抗p75单克隆抗体MC192已被证明可干扰神经生长因子与p75的结合。脑源性神经营养因子可与p75结合但不与TrkA结合，它与神经生长因子竞争p75，而抗p75抗体MC192已被证明可减少神经生长因子与TrkA的相互作用。向含神经生长因子的远端隔室中添加脑源性神经营养因子可抑制但并未完全阻断远端神经突的延伸。另一方面，MC192完全抑制了神经生长因子依赖性的神经突生长。为了测试MC192和脑源性神经营养因子是否可能影响Trk激活，通过生化方法检测了TrkA磷酸化。发现这两种化合物均会减弱神经生长因子诱导的Trk磷酸化，尽管两者均未完全抑制激活。还研究了MC192或脑源性神经营养因子可能直接激活p75信号传导（并可能拮抗TrkA信号传导）的可能性。通过使用免疫细胞化学定量转录因子NFkB的激活和核转位来评估这一点。仅用抗p75抗体MC192处理会导致显示NFkB核染色的神经元数量长期且显著增加。我们的结果表明，TrkA和p75在对神经生长因子的神经突生长反应中均起作用，并且进一步表明，最佳TrkA - p75相互作用的任何改变，或以TrkA为代价直接激活p75，都会导致成年感觉神经元中神经生长因子依赖性神经突生长受到抑制。
