Biology and Therapeutic Value of Mammalian Aph-1 Homologues

早老素 (PS) 与其他几种跨膜蛋白（称为 NCT、Aph-1 和 Pen-2）形成高分子量复合物，这些蛋白对于功能性 γ 分泌酶复合物的生成至关重要。然而，这些蛋白质，特别是存在两个同源基因的哺乳动物中的 Aph-1，在调节 γ-分泌酶复合物组装中的确切作用仍不确定。尽管最近的数据支持 PS、NCT、Aph-1 和 Pen-2 构成最小伽马分泌酶复合物的观点，但其精确机制 这四个组件组装成最终的活性复合体仍然未定义。一个有趣的问题是，为什么存在两个哺乳动物Aph-1基因，即Aph-1a和Aph-1b，编码三个Aph-1同源物，称为Aph-1aL、Aph-1aS和Aph-1b。根据我们最近的发现，Aph-1a无效胚胎的表型与Notch1无效或NCT无效胚胎的表型相似但不相同，我们假设Aph-1a是胚胎发育所需的早老素依赖性γ分泌酶复合物中主要的哺乳动物Aph-1同源物，并且Aph-1同源物受到发育调节。因此，我们计划在目标 1 中通过 Aph-1a null、Aph-1b null 和 Aph-1b null 的生成和表征来解决这些问题。 Aph-1a Aph-1b 无效小鼠。基于我们最近发现 Aph-1a 的缺失显着降低了成熟和未成熟 NCT 的水平，再加上 Aph-1 和 NCT 物理相互作用的发现，我们假设 Aph-1 和 NCT 需要调节彼此的稳定性，以形成用于组装 PS 和 Pen-2 的稳定前复合物。在这样的模型中，我们建议三种哺乳动物 Aph-1 同源物（Aph-1aL、Aph-1aS 和 Aph-1b）定义了一组六种不同的功能性γ分泌酶复合物。为了测试这个模型，我们将在目标 2 中生成并表征一系列具有不同组合的 Aph-1a 和 Aph-1b 敲除等位基因的小鼠以及源自这些小鼠的成纤维细胞。 Aph-1b 无效小鼠是可行的，并且 Aph-1-/- 小鼠大脑中 PS 和 Pen-2 水平降低，我们将测试 Aph-1b 的删除是否足以改善突变 APP;PS1 小鼠大脑中的 Abeta 沉积，目标 3。总而言之，此处提出的研究将解决有关哺乳动物 Aph-1 同源物的生理作用的重要机制问题，并严格评估 Aph-1a 和 Aph-1b改善 AD 中 Abeta 淀粉样变性的治疗目标。

Presenilins (PS) form high molecular weight complexes with several other transmembrane proteins, termed NCT, Aph-1 and Pen-2 that are critical for generation of functional gamma-secretase complexes. However, the exact roles of these proteins, particularly for Aph-1 in mammals where two homologous genes exist, in regulation of gamma-secretase complex assembly remain uncertain. Although recent data support the notion that PS, NCT, Aph-1 and Pen-2 comprise the minimal gamma-secretase complex, the precise mechanism whereby these four components are assembled into the final active complex remain undefined. An interesting question is why there exist two mammalian Aph-1 genes, namely Aph-1a and Aph-1b, encoding three Aph-1 homologues called Aph-1aL, Aph-1aS and Aph-1b. Based on our recent find ings that the phenotype of Aph-1a null embryos resemble but not identical to those of Notch1 null or NCT null embryos, we hypothesize that Aph-1a is the principal mammalian Aph-1 homologue in presenilin-dependent gamma-secretase complexes required for embryonic development and that Aph-1 homologues are developmentally regulated. Thus, we plan in Aim 1 to address these issues by generation and characterization of Aph-1a null, Aph-1b null and Aph-1a+Aph-1b null mice. Based on our recent findings that the deletion of Aph-1a significantly reduces the levels of mature and immature NCT coupled with the finding that Aph-1 and NCT physically interact, we hypothesize that Aph-1 and NCT are required to regulate the stability of each other to form a stable precomplex for assembling PS and Pen-2. In such a model, we suggest that the three mammalian Aph-1 homologues (Aph-1aL, Aph-1aS and Aph-1b) define a set of six distinct functional gamma-secretase complexes. To test this model, we will generate and characterize a series of mice harboring different combination of Aph-1a and Aph-1b knockout allele and fibroblasts derived from these mice in Aim 2. Since we showed that Aph-1b null mice are viable and there is reduction in levels of PS and Pen-2 in brains of Aph-1-/- mice, we will test whether deletion of Aph-1b is sufficient to ameliorate Abeta deposition in brains of mutant APP;PS1 mice in Aim 3. Taken together, studies proposed here will address important mechanistic questions regarding physiological roles of mammalian Aph-1 homologues and critically evaluating Aph-1a and Aph-1b as therapeutic targets in efforts to ameliorate Abeta amyloidosis in AD.