New Roles for sAPP in Neuroprotection and Neurogenesis

描述（由申请人提供）：淀粉样蛋白前体蛋白（APR）及其可溶性分泌片段（sAPPa）的神经保护作用，是一种正常的a-分泌酶裂解产物，已经在培养系统中证明了抗广谱神经元损伤的作用。在某些过表达APP的转基因（Tg）小鼠模型中观察到的几种保护作用也被推断为sAPPa。然而，这些行为背后的机制却很少被研究。因此，本应用的目的是研究sAPPa功能的分子机制，并评估其对减轻小鼠AD模型神经病变的有益作用。在初步研究中，我们证明缺乏APP的小鼠或细胞与应激激酶CDK5活性增加和tau磷酸化升高有关，这可以通过外源性sAPPa恢复。sAPPa在nmda诱导的神经元死亡中具有较强的抗凋亡活性，并通过Bcl-2通路与IGF协同作用。重要的是，我们在年轻的成年APP小鼠中发现了神经源性缺陷，并在各种细胞/组织培养中发现了sAPPa的神经营养功能，该功能与EGF协同作用。长期输注于成年小鼠体内，发现脑室下区神经元祖细胞含有sAPPa的主要结合位点。sAPPa优先结合到细胞表面富含脂质的微结构域。综上所述，我们推测sAPPa在预防神经元变性和死亡方面发挥着广泛的神经保护作用，而在产前和产后阶段，sAPPa在神经发生中发挥着更重要的作用。适当剂量和时间的中枢神经系统sAPPa治疗可改善小鼠AD模型的神经病变。因此，我们在Aim 1中提出评估sAPPa在Tau Tg小鼠模型中减少神经病理的作用。我们将使用tau突变体（R406W）转基因小鼠测试sAPPa处理是否可以减少tau磷酸化和NFT形成，提供兴奋保护，并防止记忆丧失。目的2将评估sAPPa在APP ' '*和APP/APLP2双敲除小鼠神经发生中的潜在作用。我们将研究这些小鼠的神经发生水平是否与它们的死亡率相关，以及慢性输注sAPPa是否可以刺激这些小鼠神经祖细胞/干细胞的增殖并挽救神经发生损伤。目的3是了解sAPPa生物学功能的分子和细胞机制，并鉴定sAPPa相关的膜蛋白。分子和细胞生物学方法，包括膜蛋白与结合的生物素化sAPPa的交联，脂质筏分离，结合蛋白质组学技术，将用于分离和鉴定sAPPa的假定膜受体。我们的研究将揭示APP/sAPPa的新的生理功能及其潜在的分子机制，并为sAPPa未来的潜在治疗窗口提供线索。

DESCRIPTION (provided by applicant): The neuroprotective roles for amyloid precursor protein (APR) and its soluble secreted fragment (sAPPa), a normal a-secretase cleavage product, have been demonstrated in culture systems against a broad spectrum of neuronal insults. Several protective roles observed in certain transgenic (Tg) mouse models overexpressing APP have also been inferred to sAPPa. The mechanisms underlying these actions, are however, scarcely studied. The objectives of this application are thus to investigate the molecular mechanisms of sAPPa's functions and to evaluate its beneficial effects on alleviating neuropathologies in mouse AD models. In preliminary studies, we demonstrated that mice or cells lacking APP are associated with increased stress kinase CDK5 activity and elevated tau phosphorylation, which can be restored by exogenous sAPPa. sAPPa possesses strong anti-apoptotic activity in NMDA-induced neuronal death which synergizes with IGF via a pathway involving Bcl-2. Importantly, we found neurogenic defect in young adult APP"'" mice and identified a neurotrophic function of sAPPa in various cell/tissue cultures, which is synergized by EGF. Chronic infusion of into adult mice reveals that the neuronal progenitor cells in the subventricular zone contain major binding sites for sAPPa in vivo. sAPPa preferentially binds to the lipid-enriched microdomains on the cell surface. Taken together, we hypothesize that sAPPa plays broad neuroprotective roles in preventing neuronal degeneration and death, and more intriQuinalv, a crucial role in neurogenesis at both the prenatal and postnatal stages. sAPPa treatment in CNS at a proper dosage and timing will improve neuropathologies developed in mouse AD models. We therefore propose in Aim 1 to assess the roles of sAPPa in reducing neuropathologies in Tau Tg mouse model. We will test whether sAPPa treatment can reduce tau-phosphorylation and NFT formation, provide excitoprotection, and prevent memory loss using tau mutant (R406W) transgenic mice. Aim 2 will assess the potential oles of sAPPa in neurogenesis in APP"'* and APP/APLP2 double knockout mice. We will examine whether neurogenesis levels in these mice associate with their mortality, and whether chronic i9nfusion of sAPPa can stimulate proliferation of neuronal progenitor/stem cells in these mice and rescue neurogenesis impairment. Aim 3 is to understand the molecular and cellular mechanism underlying the biological functions of sAPPa and to identify sAPPa-associated membrane protein(s). Molecular and cell biological approaches including cross linking of membrane proteins with bound biotinylated sAPPa, lipid raft isolation, in combination with proteomic technologies, will be used to isolate and identify putative membrane-receptor(s) for sAPPa. Our study will reveal novel physiological functions of APP/sAPPa as well as the underlying molecular mechanisms, and should shed light on potential therapeutic windows for sAPPa in the future.