Costantini L C, Isacson O
Neuroregeneration Laboratory, Program in Neuroscience Harvard Medical School, McLean Hospital, Belmont, MA 02478, USA.
Exp Neurol. 2000 Jul;164(1):60-70. doi: 10.1006/exnr.2000.7417.
Neurotrophic effects of immunophilin ligands have been shown in animal models of peripheral and central nervous system insult. To investigate the specific growth-promoting effects of these compounds, we examined the effects of various immunophilin ligands on primary dopamine (DA) neurons in culture and compared these with a well-known DA trophic factor, glial cell line-derived neurotrophic factor (GDNF). In neuronal cultures from Embryonic Day 14 ventral mesencephalon, enhanced elongation of DA neurites was observed with immunophilin ligands, which inhibited the phosphatase activity of calcineurin (FK506 and cyclosporin A) when compared to vehicle-treated cultures. This elongation was also observed with GDNF, known to exert its trophic effects through phosphorylation-dependent pathways. In contrast, immunophilin ligands that do not inhibit calcineurin (rapamycin and V-10,367) increased branching of DA neurites, suggesting that elongation is dependent upon maintained phosphorylation while branching is not. In addition, both V-10,367 and rapamycin antagonized the elongation effects of FK506 and induced branching. The antagonism of elongation (and reappearance of branching) illustrates the intrinsic abilities of developing DA neurons to either elongate or branch, but not both. We show that the immunophilin FKBP12 (12-kDa FK506-binding protein) is expressed in ventral mesencephalic neuronal cultures and colocalizes with DA neurons. This work elucidates the specific growth-promoting effects by which GDNF and immunophilin ligands modify developmental growth processes of DA neurons, via their interactions with intracellular targets.
免疫亲和素配体的神经营养作用已在周围和中枢神经系统损伤的动物模型中得到证实。为了研究这些化合物的特定促生长作用，我们检测了各种免疫亲和素配体对原代培养的多巴胺（DA）神经元的影响，并将其与一种著名的DA营养因子——胶质细胞系源性神经营养因子（GDNF）进行比较。在胚胎第14天腹侧中脑的神经元培养物中，与溶剂处理的培养物相比，免疫亲和素配体可观察到DA神经突的伸长增强，这些配体抑制了钙调神经磷酸酶（FK506和环孢素A）的磷酸酶活性。GDNF也观察到这种伸长，已知其通过磷酸化依赖性途径发挥营养作用。相比之下，不抑制钙调神经磷酸酶的免疫亲和素配体（雷帕霉素和V-10,367）增加了DA神经突的分支，这表明伸长依赖于持续的磷酸化，而分支则不然。此外，V-10,367和雷帕霉素均拮抗FK506的伸长作用并诱导分支。伸长的拮抗作用（以及分支的再次出现）说明了发育中的DA神经元要么伸长要么分支的内在能力，但不能两者兼具。我们发现免疫亲和素FKBP12（12 kDa FK506结合蛋白）在腹侧中脑神经元培养物中表达，并与DA神经元共定位。这项工作阐明了GDNF和免疫亲和素配体通过与细胞内靶点相互作用来修饰DA神经元发育生长过程的特定促生长作用。
