Pyroglutamate Amyloid-beta as an Immunotherapeutic Target for Alzheimer's Disease

描述(申请人提供)：N末端截断和修饰的淀粉样β蛋白(A�)多肽在阿尔茨海默病(AD)患者的大脑淀粉样蛋白沉积中含量丰富。焦谷氨酸A�是在A�的N端截断后，被谷氨酰胺环化酶环化，将3位和11位的谷氨酸转化为焦谷氨酸(A�PE3和A�pE11)。这两种形式都能迅速聚集，抵抗降解，并具有神经毒性。目前尚不清楚在斑块和血管中最初的A�沉积中是否存在这两者之一(即，作为 进一步沉积)或如果它们后来被修改的话。然而，阿尔茨海默病的进展似乎与大脑中A�Pe多肽聚集体的存在有关。我们假设焦谷氨酸盐A�作为A�沉积的种子，加速炎症、神经变性和认知功能下降；因此，通过免疫治疗靶向清除这种有毒物种将减少A�沉积、炎症和神经性营养不良，并在不干扰非致病性A�的情况下保护认知功能。我们提出了四个具体目标。目的1：通过对APP/PS1dE9转基因小鼠海马区或腹腔区注射A-�-Pe脑提取物，观察A-�沉积、炎症、神经退行性变和认知功能减退在体内的变化。目的2：我们将确定早期去除焦谷氨酸A�是否可以防止全身A�斑块沉积和神经元性改变，并通过被动免疫(I.P.)来保护认知能力下降。雄性APP/PS1dE9转基因小鼠，从4-12个月龄开始，在斑块开始之前，每周接种抗A�N3pE单抗(07/1)、抗A�pE11单抗、普通A�单抗(3A1)或PBS。观察指标：行为学、生化和神经病理学分析。目的3：我们将确定在晚期AD中去除焦谷氨酸A�是否能减少总A�和神经退行性变，并通过被动免疫(Ip)改善认知功能障碍。雌性APP/PS1dE9转基因小鼠从12-16个月龄每周一次，在斑块开始之后很好地开始。抗体和结果指标与目标2相同。目标4：我们将在急性体内研究和体外原代培养小胶质细胞中检测小胶质细胞对焦谷氨酸A�单抗的免疫应答。我们在Probiodrug AG(德国)的合作伙伴将友好地为我们提供两种高亲和力、高选择性的焦谷氨酸A�单抗(抗A�PE3和抗A�pE11)、合成的A�Pe多肽，以及从他们的转基因小鼠模型中积累焦谷氨酸-3 A�多肽的脑提取物。我们国家疾病预防控制中心的合作者慷慨地提供了3A1通用A�单抗杂交瘤作为对照。重要的是，我的实验室发起了这项合作，并拥有多年研究焦谷氨酸A�沉积、炎症和A�免疫疗法的经验。我们研究的总体目标是确定焦谷氨酸盐A�蛋白(A�PE3和A�pE11)是否是阿尔茨海默病的治疗靶点，以及针对任一焦谷氨酸A�物种的免疫疗法清除是否将有效地预防和/或治疗阿尔茨海默病。

DESCRIPTION (provided by applicant): N-terminally-truncated and modified amyloid-beta (A�) peptides are abundant in cerebral amyloid deposits in Alzheimer's disease (AD). Pyroglutamate A� is generated upon N-terminal truncation of A� followed by cyclization by glutaminyl cyclase to convert glutamic acid at residues 3 and 11 to pyroglutamate (A�pE3 and A�pE11). Both forms aggregate quickly, resist degradation, and are neurotoxic. It is unclear if either is present in initial A� deposition in plaques and blood vessels (i.e., acting as a seed for further deposition) or if they are modified later. However, Alzheimer's disease progression appears to correlate with the presence of A� pE peptide aggregates in brain. We hypothesize that pyroglutamate A� acts as a seed for A� deposition and accelerates inflammation, neurodegeneration and cognitive decline; therefore, targeted removal of this toxic species by immunotherapy will reduce A� deposition, inflammation and neuritic dystrophy, and protect against cognitive impairment without disturbing non-pathogenic A�. We propose 4 Specific Aims. Aim 1: We will determine if intrahippocampal or intraperitoneal injections of A�pE-containing mouse brain extracts enhance A� deposition, inflammation, neurodegeneration and cognitive decline in APP/PS1dE9 transgenic mice with aging in vivo. Aim 2: We will determine if early removal of pyroGlu A� prevents general A� plaque deposition and neuritic changes, and protects against cognitive decline by passively immunizing (i.p.) male APP/PS1dE9 tg mice with an anti-A�N3pE mAb (07/1), an anti-A�pE11 mAb, a general A� mAb (3A1), or PBS weekly from 4-12 mo of age, starting prior to plaque onset. Outcome measures: behavioral, biochemical, and neuropathological analyses. Aim 3: We will determine if removal of pyroGlu A� in late stage AD reduces total A� and neurodegeneration and improves cognitive deficits by passively immunizing (i.p.) female APP/PS1dE9 tg mice weekly from 12-16 mo of age, starting well after plaque onset. Antibodies and outcome measures are the same as in Aim 2. Aim 4: We will examine the immune response of microglia to pyroGlu A� mAbs in acute in vivo studies and in primary microglial cultures in vitro. Our collaborators at Probiodrug AG (Germany) will kindly provide us with 2 high-affinity, highly selective pyroGlu A� mAbs (anti- A� pE3 and anti-A�pE11), synthetic A�pE peptides, and brain extracts from their transgenic mouse models that accumulate pyroGlu-3 A� peptides. Our collaborators at the CND have generously provided the 3A1 general A� mAb hybridoma as a control. Importantly, my lab initiated this collaboration and has many years of experience investigating pyroGlu A� deposition, inflammation, and A� immunotherapy. The overall goal of our study is to determine whether pyroglutamate A� proteins (A�pE3 and A�pE11) are therapeutic targets for Alzheimer's disease and, whether clearance by immunotherapy specific for either pyroGlu A� species would be efficacious to prevent and/or treat AD.