PATHOGENESIS OF BATTEN DISEASE

我们建议通过关注婴儿型Batten病（CLN 1）和棕榈酰蛋白硫酯酶（PPT 1）缺陷来继续研究Batten病的发病机制。我们将在人神经母细胞瘤LA-N-5细胞、PC 12嗜铬细胞瘤细胞、鸡胚神经元和非神经CHO细胞中过表达PFF 1，使用诱导型启动子、荧光标签和特异性序列将表达引导至离散的亚细胞区室如线粒体。我们将确定PPT 1是如何能够调节抗洗涤剂微结构域（Rafts）中蛋白质棕榈酰化的水平，从而确定其活性水平。 Akt和磷酸酶PTEN，以防止细胞死亡。我们将验证在筏和溶酶体PPT 1之间的关系，筏和PPT 1活性的存在。我们将使用基因阵列技术来确定哪些细胞死亡相关基因在PPT 1过表达时被打开/关闭，并使用原位杂交来跟踪PPT 1在发育中的神经系统中的时空表达。为了模拟PPT 1的缺陷，我们将基于AcG-棕榈酰二氨基丙酸酯-VKIKK（DAP 1）和棕榈酰-半胱氨酸硫酯键的酮酰胺类似物合成PPT 1的特异性、有效的抑制剂。我们将表征这些抑制剂对PPT 1和细胞摄取的最佳抑制。我们将确定它们引起的细胞死亡是否是Akt失活的结果，并使用它们来更好地了解PPT 1在Rafts中的作用。我们广泛的长期目标是了解早期死亡的原因， CLN 1中的皮质神经元以及如何补救。

We propose to continue studies on the pathogenesis of Battens disease by focusing on the Infantile form of Batten Disease (CLN 1) and the deficient enzyme palmitoyl:protein thioesterase (PPT 1). We will overexpress PFF 1 in human neuroblastoma LA-N-5 cells, PC 12 pheochromocytoma cells, embryonic chick neurons, and non-neural CHO cells using inducible promoters, fluorescent tags and specific sequences to direct expression to discrete subcellular compartments such as mitochondria. We will determine how PPT1 is able to regulate the level of protein palmitoylation in detergent-resistant microdomains (Rafts) and hence the level of activity of Akt and the phosphatase PTEN in order to protect against cell death. We will verify the presence of PPT1 activity in Rafts and the relationship between raft and lysosomal PPT1. We will use gene array technology to determine which cell death-associated genes are turned on/off when PPT1 is overexpressed and in situ hybridization to follow the spatio-temporal expression of PPT1 in the developing nervous system. In order to model the deficiency of PPT1 we will synthesize specific, potent inhibitors of PPT1 based on AcG-palmitoyl diamino propionate-VKIKK (DAP1) and ketoamide analogs of the palmitoyl-cysteine thioester linkage. We will characterize these inhibitors for optimum inhibition of PPT1 and cell uptake. We will determine if the cell death they cause results from inactivation of Akt and use them to better understand the role of PPT1 in Rafts. Our broad, long-term objectives are to understand the reason for the early death of cortical neurons in CLN 1 and how this might be remedied.