Role of Fibulin-1 in APP Processing

描述(申请人提供)：目前许多抗击阿尔茨海默病(AD)的药理学方法寻求通过抑制淀粉样蛋白生成酶(称为β-和伽马-分泌酶)来阻止Abeta的产生。另一种方法是激活淀粉样前体蛋白(APP)的α-分泌酶处理，这是由金属蛋白去整合素家族的几个成员ADAM9、ADAM10和ADAM17介导的。通过这些α-分泌酶处理APP被认为对AD是有益的，因为它限制了Abeta的产生，并产生了神经保护性的可溶性APPalpha(SAPPalpha)产物。纤维蛋白-1(Fbln1)是一种细胞外基质蛋白，由神经元表达，与APP和sAPPalpha的氨基末端结合。然而，我们发现Fbln1还与其他膜锚定的α-分泌酶底物--肝素结合-表皮生长因子(HB-EGF)和神经调节蛋白-1(NRG1)结合。我们还发现，Fbln1可以抑制可溶性形式的HB-EGF和NRG1的蛋白水解性释放。此外，我们还发现，与野生型小鼠胚胎成纤维细胞相比，Fbln1缺失的小鼠胚胎成纤维细胞的条件培养液中sAPPalpha的水平增加。根据这些发现，推测Fbln1是APPα-分泌酶加工的抑制剂，因此可能是一个治疗靶点，如果被抑制，可能会导致APPα-分泌酶加工的增强和病理性APP切割的减少。为了解决这一假说，有三个具体的目的：1)确定Fbln1基因缺陷小鼠脑内APP蛋白水解酶是否发生改变，2)确定Fbln1转基因过表达是否加速Abeta的产生并加剧AD的发病，以及3)确定Fbln1是否抑制培养的神经细胞中APP的α-分泌酶处理。

DESCRIPTION (provided by applicant): Many current pharmacological approaches to combating Alzheimer's disease (AD) seek to block Abeta production through inhibition of the amyloidogenic enzymes known as beta- and gamma-secretases. An alternative approach is to activate the alpha-secretase processing of amyloid precursor protein (APP), which is mediated by several members of the disintegrin family of metalloproteases, ADAM9, ADAM10 and ADAM17. Processing of APP by these alpha-secretases is thought to be beneficial with respect to AD since it limits production of Abeta and generates the neuroprotective soluble APPalpha (sAPPalpha) product. Fibulin-1 (Fbln1) is an extracellular matrix protein, expressed in the brain by neurons, that binds the amino terminus of APP and sAPPalpha. The significance of this interaction is not yet established however, we have found that Fbln1 also binds to other membrane anchored alpha-secretase substrates, heparin binding-epidermal growth factor (HB-EGF) and neuregulin-1 (NRG1). We also show that Fbln1 acts to inhibit the proteolytic release of soluble forms of HB-EGF and NRG1. Furthermore, we have found increased levels of sAPPalpha in the conditioned culture medium of Fbln1 null mouse embryo fibroblasts (MEFs) as compared to wildtype MEFs. Based on these findings it is hypothesized that Fbln1 serves as an inhibitor of alpha-secretase processing of APP and therefore may represent a therapeutic target that if inhibited might lead to augmented alpha-secretase processing of APP and reduced pathological APP cleavage. To address this hypothesis there are three specific aims: 1) Determine whether brain APP proteolytic cleavage is altered in Fbln1-deficient mice, 2) determine whether transgenic overexpression of Fbln1 accelerates Abeta production and exacerbates AD pathogenesis, and 3) determine whether Fbln1 inhibits alpha-secretase processing of APP in cultured neuronal cells.