New Roles for sAPP in Neuroprotection and Neurogenesis

描述(由申请人提供)：淀粉样前体蛋白(APR)及其可溶性分泌片段(Sappa)是一种正常的α-分泌酶切割产物，已在培养系统中被证明对广泛的神经元损伤具有神经保护作用。在某些过度表达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，这些行动背后的机制却鲜有人研究。因此，本应用的目的是研究Sappa功能的分子机制，并评估其在减轻小鼠阿尔茨海默病(AD)模型神经病理方面的有益作用。在初步研究中，我们证明了缺乏APP的小鼠或细胞与应激激酶CDK5活性增加和tau磷酸化增加有关，这可以通过外源Sappa恢复。在NMDA诱导的神经细胞死亡中，SappA具有很强的抗凋亡活性，并通过参与Bcl2的途径与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，这一功能与EGF协同作用。对成年小鼠的慢性输注显示，脑室下区的神经前体细胞含有体内Sappa的主要结合部位。SappA优先与细胞表面的富脂微区结合。综上所述，我们假设Sappa在防止神经元退化和死亡方面发挥着广泛的神经保护作用，而在出生前和出生后阶段的神经发生中，Quinalv起着至关重要的作用。在适当的剂量和时机下，Sappa在中枢神经系统治疗将改善小鼠AD模型的神经病理。因此，我们建议在目标1中评估Sappa在减少Tau TG小鼠模型神经病理方面的作用。我们将使用tau突变(R406W)转基因小鼠测试Sappa治疗是否可以减少tau磷酸化和NFT的形成，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双基因敲除小鼠神经发生中的潜在OLE。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有关，以及慢性注射Sappa是否可以刺激这些小鼠的神经前体/干细胞的增殖并挽救神经发生障碍。目的3了解sAPPA的生物学功能的分子和细胞机制，鉴定sAPPA相关膜蛋白(S)。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，脂筏分离，结合蛋白质组学技术，将被用来分离和鉴定可能的Sappa膜受体(S)。我们的研究将揭示APP/Sappa的新的生理功能以及潜在的分子机制，并将为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 Alzheimer's Disease (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.