Mourlevat Sophie, Debeir Thomas, Ferrario Juan E, Delbe Jean, Caruelle Daniele, Lejeune Olivier, Depienne Christel, Courty José, Raisman-Vozari Rita, Ruberg Merle
INSERM U679, Hôpital de la Salpêtrière, 47 Boulevard de l'Hôpital, 75013 Paris, France.
Exp Neurol. 2005 Jul;194(1):243-54. doi: 10.1016/j.expneurol.2005.02.015.
To better understand the particular vulnerability of mesencephalic dopaminergic neurons to toxins or gene mutations causing parkinsonism, we have taken advantage of a primary cell culture system in which these neurons die selectively. Antimitotic agents, such as cytosine arabinoside or cAMP, prevent the death of the neurons by arresting astrocyte proliferation. To identify factors implicated in either the death of the dopaminergic neurons or in the neuroprotective effect of cAMP, we constructed cDNA libraries enriched by subtractive hybridization and suppressive PCR in transcripts that are preferentially expressed in either control or cAMP-treated cultures. Differentially expressed transcripts were identified by hybridization of the enriched cDNAs with a commercially available cDNA expression array. The proteoglycan receptors syndecan-3 and the receptor protein tyrosine phosphatase zeta/beta were found among the transcripts preferentially expressed under control conditions, and their ligand, the cytokine pleiotrophin, was highly represented in the cDNA libraries for both conditions. Since pleiotrophin is expressed during embryonic and perinatal neural development and following lesions in the adult brain, we investigated its role in our cell culture model. Pleiotrophin was not responsible for the death of dopaminergic neurons under control conditions, or for their survival in cAMP-treated cultures. It was, however, implicated in the initial and cAMP-dependent enhancement of the differentiation of the dopaminergic neurons in our cultures. In addition, our experiments have provided evidence for a cAMP-dependent regulatory pathway leading to protease activation, and the identification of pleiotrophin as a target of this pathway.
为了更好地理解中脑多巴胺能神经元对导致帕金森症的毒素或基因突变的特殊易损性，我们利用了一种原代细胞培养系统，在该系统中这些神经元会选择性死亡。抗有丝分裂剂，如阿糖胞苷或环磷酸腺苷（cAMP），通过阻止星形胶质细胞增殖来防止神经元死亡。为了确定与多巴胺能神经元死亡或cAMP的神经保护作用相关的因素，我们通过消减杂交和抑制性聚合酶链反应构建了在对照或cAMP处理的培养物中优先表达的转录本富集的cDNA文库。通过将富集的cDNA与市售的cDNA表达阵列杂交来鉴定差异表达的转录本。在对照条件下优先表达的转录本中发现了蛋白聚糖受体syndecan-3和受体蛋白酪氨酸磷酸酶zeta/beta，并且在两种条件下的cDNA文库中它们的配体细胞因子多效生长因子都有高度表达。由于多效生长因子在胚胎期和围生期神经发育期间以及成人大脑损伤后表达，我们在我们的细胞培养模型中研究了它的作用。在对照条件下，多效生长因子对多巴胺能神经元的死亡或在cAMP处理的培养物中的存活没有影响。然而，它与我们培养物中多巴胺能神经元分化的初始和cAMP依赖性增强有关。此外，我们的实验为导致蛋白酶激活的cAMP依赖性调节途径提供了证据，并鉴定出多效生长因子是该途径的一个靶点。
