Analysis of neural cell death using knock-in mouse model and organotypic culture system.

家族性中枢性尿崩症（FDI）是由精氨酸抗利尿素（AVP）基因突变引起的常染色体显性遗传病。AVP的前体形式prepro-AVP由20号染色体上的AVP基因编码，前体由信号肽、AVP、神经磷脂和糖蛋白组成。通过去除信号肽将pre - pro-AVP转化为pro-AVP。在从视上核和室旁核到垂体后叶的轴突运输过程中，原-AVP被包装成囊泡，加工成AVP、NP和糖蛋白。我们使用下丘脑器官型培养和敲入小鼠作为FDI动物模型来研究该疾病的详细发病机制。我们选择了在神经生理素部分预测Cys67停止形成的突变。虽然所有纯合子小鼠在分娩后一周内死亡，但杂合子小鼠在1月龄左右出现多尿和垂体后叶AVP含量低。敲入小鼠3月龄后，AVP神经元树突内正常和异常神经素抗体免疫组化染色密度下降，大细胞核内AVP mRNA表达下降。上述结果提示，AVP表达的显性阴性机制和/或降低可能在早期诱导尿崩症的表型，而视上核和室旁核的神经细胞死亡并不是早期尿崩症的主要原因。另一方面，我们在本研究中通过原位杂交证明了下丘脑器官型培养对研究AVP表达的调节的有效性。我们将使用该动物模型和下丘脑器官型培养来更详细地研究FDI以及神经退行性疾病的发病机制。

Familial central diabetes insipidus(FDI) is an autosomal dominant disease due to the mutation of arginine vasopressin(AVP) gene. The precursor form of AVP, prepro-AVP is encoded by the AVP gene on chromosome 20 and the precursor consists of the signal peptide, AVP, neurophysin and glycoprotein. Prepro-AVP is converted to pro-AVP by the removal of the signal peptide. Pro-AVP is packaged into vesicles and processed into AVP,NP, and glycoprotein during axonal transport from the supraoptic and paraventricular nuclei to the posterior pituitary.We investigated the detailed pathogenesis of the disease using organotypic culture of the hypothalamus and knock-in mouse as a FDI animal model. We selected the mutation which predicts the formation of Cys67 stop in the neurophysin moiety. Although all homozygous mice died within a week after delivery, heterozygous mice indicated polyuria and low content of AVP in the posterior pituitary around one month of age. Immunohistocheimcal staining density examined by antibodies against both normal and abnormal neurophysins in dendrite of AVP neuron decreased in the knock-in mice and the expression of AVP mRNA in the magnocellular nucleus also deteriorated after 3 months of age. These results indicate that dominant negative mechanism and/or the decrease of AVP expression might induce the phenotype of diabetes insipidus at early stage, and neural cell death in supraoptic and paraventricular nuclei is not primary cause at that time.On the other hand, we demonstrated the usefulness of hypothalamic organotypic culture to investigate the regulation of AVP expression by in situ hybridization in this study.We will examine more detailed pathogenesis of not only FDI but also the neuro-degenerative diseases using this animal model and the hypothalamic organotypic culture.