Biology and Therapeutic Value of Mammalian Aph-1 Homologues

早老素(PS)与其他几种跨膜蛋白形成高分子量的复合体，称为NCT、APH-1和Pen-2，它们对产生功能性的伽马分泌酶复合体是至关重要的。然而，这些蛋白质的确切作用，特别是在存在两个同源基因的哺乳动物中的APH-1，在调节伽马分泌酶复合体组装方面仍然不确定。尽管最近的数据支持PS、NCT、APH-1和PEN-2由最小的伽马分泌酶复合体组成的概念，但其确切的机制 这四个组分组装成最终的活性复合体仍未确定。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同源基因，即APH-1AL、APH-1AS和APH-1b。根据我们最近发现的APH-1a缺失胚胎的表型与Notch1缺失胚胎或NCT缺失胚胎的表型相似但不完全相同，我们假设APH-1a是胚胎发育所需的早老素依赖的伽马分泌酶复合体中的主要哺乳动物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中通过生成和表征APH-1a空值、APH-1b空值和 APH-1a、APH-1b基因缺失小鼠。根据我们最近的发现，APH-1a的缺失显著降低了成熟和未成熟NCT的水平，再加上APH-1和NCT在物理上相互作用的发现，我们假设APH-1和NCT需要相互调节彼此的稳定性，以形成一个稳定的前复合体来组装PS和Pen-2。在这样的模型中，我们认为三个哺乳动物的APH-1同源物(APH-1AL、APH-1AS和APH-1b)定义了一组不同的功能伽马分泌酶复合体。为了测试这个模型，我们将在目标2中建立和鉴定一系列具有不同APH-1a和APH-1b基因敲除等位基因组合的小鼠以及来自这些小鼠的成纤维细胞。 APH-1b基因缺失的小鼠是存活的，并且APH-1-/-小鼠的脑组织中PS和Pen-2水平降低，我们将测试APH-1b的缺失是否足以改善突变型APP；PS1小鼠大脑中的Abeta沉积。综上所述，这里提出的研究将解决关于哺乳动物APH-1同源物的重要机制问题，并批判性地评估APH-1a和APH-1b作为努力改善AD中Abeta淀粉样变性的治疗靶点。

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.