MODULATION OF AMYLOID PRECURSOR PROTEIN METABOLISM BY X11ALPHA/MINT-1

神经元适配蛋白X11pha/mint-1与淀粉样前体蛋白(APP)相互作用，调节其在体外的转运和加工。在转基因的非神经细胞中，X11Alpha抑制APP的α-和γ-裂解，但不抑制β-裂解。由于X11Alpha的磷酸酪氨酸结合(PTB)结构域与APP家族特异性地相互作用，X11Alpha似乎特异性地抑制APP的伽马切割，同时阻止Notch和其他调节膜内蛋白降解的底物的伽玛切割。为了确定这些体外数据的体内意义，我们将构建和鉴定新的hX11pha转基因小鼠和X11pha基因敲除小鼠，并检测小鼠APP代谢。将这些小鼠与Tg2576小鼠(转基因HAPP瑞典突变，或hAPPswe)杂交，将探索XI la对hAPPswe代谢和老化脑中部分AD样表型发展的影响。最近的一项人类全基因组分析显示，散发性AD与9号染色体上的单核苷酸多态(SNPs)存在显著关联，可能包括X11pha区域。我们将利用从AD受试者和仔细匹配的对照组受试者样本中提取的基因组DNA来探索X11pha区域的SNPs与散发性AD的遗传联系。其具体目的是：1)建立X11pha基因敲除和hX11pha转基因小鼠并确定其对小鼠脑内和原代神经元培养中APP代谢的影响；2)将X11pha基因敲除和hX11pha转基因小鼠与Tg2576小鼠杂交，以确定a)对脑内和神经元培养中hAPPswe代谢的调节作用，以及b)AD样表型的发展；以及3)阐明AD病例和年龄、性别和ApoE匹配对照的X11pha基因内或附近的SNPs和单倍型，以确定是否存在与散发性AD有统计意义的联系。特殊目标3将研究从病理核心和其他ADRC获得的DNA样本。这些结果将1)通知正常函数 2)在HAPP转基因小鼠模型中利用X11α或其PTB结构域进行AD的病毒载体基因治疗；3)探讨散发性AD的潜在遗传危险因素。

The neuronal adaptor protein X11alpha/mint-1 interacts with amyloid precursor protein (APP) to regulate its trafficking and processing in vitro. In transfected non-neuronal cells, X11alpha inhibits alpha- and gamma- but not beta- cleavage of APP. Because the phosphotyrosine binding (PTB) domain of X11alpha interacts specifically with the APP family, X11alpha appears to inhibit gamma-cleavage of APP specifically while sparing gamma-cleavage of Notch and other substrates of regulated intramembranous proteolysis. To determine the in vivo significance of these in vitro data, we will generate and characterize novel hX11alpha transgenic mice and X11alpha knockout mice and examine murine APP metabolism. Crosses of these mice to Tg2576 mice (transgenic for the Swedish mutation of hAPP, or hAPPswe) will probe the effects of XI la on hAPPswe metabolism and on the development of partial AD-like phenotypes in aging brain. A recent human genome-wide analysis revealed significant linkage of sporadic AD to single nucleotide polymorphisms (SNPs) on chromosome 9, perhaps including the X11alpha region. We will probe genetic linkage of SNPs in the X11alpha region to sporadic AD by using genomic DNA extracted from samples from AD subjects versus carefully-matched control subjects. The specific aims are to: 1) generate X11alpha knockout and hX11alpha transgenic mice and determine effects on murine APP metabolism in brain and in primary neuronal cultures, 2) cross X11alpha knockout and hX11alpha transgenic mice with Tg2576 mice to determine a) modulatory effects on hAPPswe metabolism in brain and in neuronal cultures, and b) development of AD-like phenotypes with aging, and 3) elucidate the SNPs and haplotypes within or adjacent to the X11alpha gene of AD cases and age-, gender-, and ApoE-matched controls to determine if there is a statistically significant link to sporadic AD. Specific aim 3 will study DNA samples obtained from the Pathology Core and from other ADRCs. These results will 1) inform the normal functions of X11alpha as well as APP and its derivatives in CNS neurons, 2) lay the groundwork for viral-vector based gene therapy of AD using either X11alpha or its PTB domain in hAPP transgenic mouse models, and 3) probe a potential genetic risk factor of sporadic AD.