CHOLINERGIC NEURONS FOR TRANSPLANTATION

本项目的总体目标是获得可用于 用于移植以取代阿尔茨海默氏症和其他疾病中丢失的神经元 涉及胆碱能神经元的神经变性疾病。 两个一般 方法将被调查;一个涉及到检测基因的 关键胆碱能蛋白进入细胞能够获得神经元 表型，而另一个涉及使用来自表型的启动子元件。 ChAT基因从胎儿神经细胞中筛选胆碱能神经元 含有胆碱能前体细胞的培养物。 该项目涉及4 明确的目标。 第一种方法是使用细胞转染技术 为了介绍胆碱能细胞的三个主要组成部分， 胆碱转运蛋白、胆碱乙酰转移酶和乙酰胆碱 在一些实施方案中，所述细胞可以是能够获得神经元表型的转运蛋白。 这些 包括NT-2细胞、RN 33 B细胞和胚胎神经元前体细胞。 含有胆碱能特异性基因组序列的构建体将被 用于在胚胎胆碱能神经元中表达耐药基因 前体细胞 在药物存在下的生长将用于 从这个群体中选择胆碱能细胞。 第二 具体目的涉及转染或分离的 细胞的胆碱能功能。 这将侧重于以下能力： 这些细胞合成乙酰胆碱并将其储存在突触囊泡中， 并在去极化时释放乙酰胆碱。 这些能力 细胞与宿主神经元建立功能性相互作用将 最初通过将这些细胞与胎儿细胞共培养 从不同脑区提取脑组织并寻找突触联系。 第三，将测试转染或分离的细胞的生物学特性。 在大鼠脑内植入后存活的能力， 合成、储存和分泌乙酰胆碱。 最后，我们将确定 植入细胞改善特定行为的能力 基底前脑退化动物模型的缺陷。 是 预计这些研究将为进一步的研究提供基础。 可用于基因替换的神经元细胞的发育 阿尔茨海默氏症及相关疾病的治疗。

The overall objective of this project is to obtain cells which can be used for transplantation to replace lost neurons in Alzheimer's and other neurodegenerative diseases involving cholinergic neurons. Two general approaches will be investigated; one involves transfecting genes of the key cholinergic proteins into cells capable of acquiring a neuronal phenotype, while the other involves the use of promoter elements from the ChAT gene to select out cholinergic neurons from fetal neuronal cell cultures containing cholinergic precursor cells. The project involves 4 specific aims. The first involves the use of cell transfection techniques to introduce the three major components of the cholinergic cell, the choline transporter, choline acetyltransferase, and the acetylcholine transporter into cells capable of acquiring a neuronal phenotype. These include NT-2 cells, RN33B cells, and embryonic neuronal precursor cells. Constructs containing the cholinergic specific genomic sequences will be used to express drug resistant genes in embryonic cholinergic neuronal precursor cells. Growth in the presence of the drug will be used to select for the cholinergic cells from this population. The second specific aim involves characterization of the transfected or isolated cells in terms of cholinergic function. This will focus on the ability of these cells to synthesize and store acetylcholine in synaptic vesicles, and to release acetylcholine upon depolarization. The ability of these cells to establish functional interactions with host neurons will initially be examined in vitro by co-culturing these cells with fetal brain tissue from various brain regions and looking for synaptic contacts. Thirdly, the transfected or isolated cells will be tested for their ability to survive intracerebral implantation in rats and to continue to synthesize, store, and secrete acetylcholine. Lastly, we will determine the ability of the implanted cells to ameliorate specific behavioral deficits in animal models of basal forebrain degeneration. It is anticipated that these studies will provide the basis for the further development of neuronal cells which can be used in gene replacement therapy for Alzheimer's and related diseases.