PATHOGENESIS OF BATTEN DISEASE

我们建议继续研究 Batten 病的发病机制，重点关注婴儿型 Batten 病 (CLN 1) 和缺陷酶棕榈酰：蛋白硫酯酶 (PPT 1)。我们将使用诱导型启动子、荧光标签和特定序列在人神经母细胞瘤 LA-N-5 细胞、PC 12 嗜铬细胞瘤细胞、胚胎鸡神经元和非神经 CHO 细胞中过表达 PFF 1，以将表达引导至离散的亚细胞区室（例如线粒体）。我们将确定 PPT1 如何调节耐洗涤剂微结构域 (Raft) 中的蛋白质棕榈酰化水平，从而调节活性水平 Akt 和磷酸酶 PTEN 的作用，以防止细胞死亡。我们将验证筏中PPT1活性的存在以及筏与溶酶体PPT1之间的关系。我们将利用基因芯片技术来确定当PPT1过表达时哪些细胞死亡相关基因被打开/关闭，并利用原位杂交来追踪PPT1在发育中的神经系统中的时空表达。为了模拟 PPT1 的缺陷，我们将基于 AcG-棕榈酰二氨基丙酸酯-VKIKK (DAP1) 和棕榈酰-半胱氨酸硫酯键的酮酰胺类似物合成特异性、有效的 PPT1 抑制剂。我们将表征这些抑制剂，以实现对 PPT1 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由于 Akt 失活所致，并利用它们更好地了解 PPT1 在 Rafts 中的作用。我们广泛、长期的目标是了解人类早逝的原因 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.