Ferroportin and APP: Regulation of Iron Trafficking at the Blood-Brain Barrier

氧化还原活性的第一排过渡金属，如铁和铜，与病理生理有关。 淀粉样变性疾病；这些相关性在机制水平上知之甚少。我们还有 对淀粉样前体蛋白(APP)的生理作用认识有限。 然而，APP转录本包含铁反应结合蛋白(IRBP)茎环结构的事实 与铁蛋白一样，APP蛋白质合成与细胞铁含量直接相关，这表明APP是 “铁规。”APP对铁的调节仍然是一个未知的关于铁代谢和 淀粉样变；在这里，我们建议测试一个关于APP和APP的生理功能的特定前提。 它的分泌酶依赖产物。首先，我们实验室发表的工作提供了关于这种联系的线索 APP和铁代谢之间的关系。我们已经确定了一个由22个氨基酸残基所展示的独特功能 APP中E2结构域的BC螺旋束N-末端的多肽。vbl.使用 重组BC或合成肽(BC内的第327-348残基)，我们证明该序列系链 APP结合并稳定质膜中铁转运蛋白(FPN)。FPN是唯一的哺乳动物铁出口商； SAPP稳定PM中的FPN，刺激细胞铁外流。我们已将此物种指定为 以多肽为靶标的铁蛋白。在与FPN的结合中，SAPP/FTP是FPN关键调节因子的拮抗剂 功能，表明Sapp是任何哺乳动物细胞铁外流的关键调节因子， 表达这种淀粉样蛋白或共享包括另一种细胞类型的间质空间。 虽然SAPP在FPN质膜显示中的作用已被证实，但其分子基础 活动及其生理功能仍然是重要的未知数，重要的原因是：1)关键作用APP 及其产品在阿尔茨海默氏症和脑淀粉样血管病中发挥作用；2)隐含的作用 铁在这种神经退行性疾病中发挥作用；以及3)FTP或其合成同系物可能 作为(A)铁代谢的药理调节剂。这个应用程序的关键要素是我们的血脑 由脑毛细血管内皮细胞、星形胶质细胞和神经元组成的屏障Transwell模型系统，即 神经血管单位的细胞。我们的前提是：1)铁调节APP的表达，从而调节 大脑开放空间中的铁水平；2)铁也调节APP的处理到A？；3)A？ 反过来起到铁清除剂的作用，通过这种机制具有细胞保护作用。我们将通过以下方式测试这一前提 生物物理结构-功能、细胞生物学和遗传学方法的结合，包括使用初级 从精心挑选的小鼠KO系培养的细胞。这项研究计划将：1)提供对 FPN/APP相互作用机制及其在细胞铁转运中的作用；2)检验我们的假设 对这种铁的管理不善会导致我们的血脑屏障模型的渗透率增加；3)测试我们的假设：A？ 通过抑制铁作为一种有效的促氧化剂的活性，在防止这种毛细血管渗漏方面发挥了关键作用。

Redox active first row transition metals such as iron and copper have been implicated in the pathophysiology of amyliodogenic diseases; these correlations are poorly understood at the level of mechanism. We also have limited understanding of the physiologic role for which the amyloid precursor protein (APP) has been selected. However, the fact that the APP transcript contains an iron response binding protein (IRBP) stem-loop structure and like ferritin, APP protein synthesis directly correlates with cell iron content indicating that APP is part of the “iron regulon.” This iron regulation of APP remains an unknown with respect to both iron metabolism and amyloidogenesis; here we propose to test a specific premise with respect to a physiologic function of APP and its secretase-dependent products. First, published work from our lab provides a clue as to the connection between APP and iron metabolism. We have identified a unique function exhibited by a 22-amino acid residue peptide element at the N-terminus of the BC helical bundle contained in the E2 domain in APP. Using recombinant BC or a synthetic peptide (residues 327-348 within BC), we show that this sequence tethers APP to and stabilizes ferroportin (Fpn) in the plasma membrane. Fpn is the sole mammalian iron exporter; by stabilizing Fpn in the PM, sAPP stimulates cell iron-efflux. We have designated this species FTP for Ferroportin Targeting Peptide. In binding to Fpn, sAPP/FTP acts as antagonist of the key regulator of Fpn function, hepcidin (Hepc) indicating that sAPP is a key regulator of iron efflux from any mammalian cell that expresses this amyliodogenic protein or shares an interstitial space that includes another cell type that does. Although the effect of sAPP on Fpn plasma membrane display is now established, the molecular basis of this activity and its physiologic function remain significant unknowns, significant because of: 1) the key role APP and its products play in Alzheimer's disease and in cerebral amyloid angiopathy, CAA; 2) the implied role that iron plays in this neurodegenerative disorder; and 3) the potential that FTP or its synthetic congeners may have as (a) pharmacologic regulator of iron metabolism. The key element of this Application is our blood-brain barrier transwell model system composed of brain capillary endothelial cells, astrocytes and neurons, i.e, the cells of the neurovascular unit. Our premise is that: 1) iron regulates the expression of APP so as to modulate the level of iron in the brain's abluminal space; 2) iron also modulates the processing of APP to Aß; and 3) Aß in turn serves as an iron scavenger and by this mechanism is cytoprotective. We will test this premise by a combination of biophysical structure-function, cell biologic and genetic approaches including the use of primary cells cultured from strategically chosen mice KO lines. This research plan will: 1) provide novel insight into the mechanism of the Fpn/APP interaction and its role in cellular trafficking of iron; 2) test our premise that mismanagement of this iron leads to increased permeability in our BBB model; and 3) test our premise that Aß is a key player in preventing this capillary leakage by suppressing iron's activity as a potent pro-oxidant.