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

描述（由申请人提供）：N-末端截短和修饰的淀粉样蛋白-β（A？）肽在阿尔茨海默病（AD）的大脑淀粉样蛋白沉积物中丰富。焦谷氨酸A在A <$的N-末端截短后，通过N-氨基环化酶环化，将残基3和11处的谷氨酸转化为焦谷氨酸（A <$pE3和A <$pE11）。这两种形式都能迅速聚集，抵抗降解，并具有神经毒性。目前还不清楚是否存在于斑块和血管中的初始A？沉积中（即，作为种子， 进一步沉积）或者如果它们稍后被修改。然而，阿尔茨海默病的进展似乎与脑中A pE肽聚集体的存在相关。我们假设焦谷氨酸A <$充当A <$沉积的种子并加速炎症，神经变性和认知衰退;因此，通过免疫疗法有针对性地去除这种有毒物质将减少A <$沉积，炎症和神经炎性营养不良，并在不干扰非致病性A <$的情况下防止认知障碍。我们提出了四个具体目标。目标1：我们将确定是否海马内或腹腔内注射含有A？pE的小鼠脑提取物增强A？沉积，炎症，神经退行性变和认知能力下降的APP/PS1 dE 9转基因小鼠体内老化。目标二：我们将确定早期去除pyroGlu A <$是否可以防止一般A <$斑块沉积和神经炎变化，并通过被动免疫（i. p.）从4-12月龄开始，在斑块开始前，每周给予雄性APP/PS1 dE 9 tg小鼠抗A <$N3 pE mAb（07/1）、抗A <$pE11 mAb、一般A <$mAb（3A 1）或PBS。结果测量：行为，生化和神经病理学分析。目标3：我们将确定在晚期AD中去除pyroGlu A <$是否会减少总A <$和神经变性，并通过被动免疫（i. p.）雌性APP/PS1 dE 9 tg小鼠，从12-16月龄开始，每周一次，在斑块发作后开始。抗体和结局指标与目标2相同。目的4：我们将在急性体内研究和体外原代小胶质细胞培养中检查小胶质细胞对pyroGlu A？mAb的免疫应答。我们在Probiodrug AG（德国）的合作者将友好地为我们提供2种高亲和力、高选择性的pyroGlu A <$mAb（抗A <$pE3和抗A <$pE11）、合成A <$pE肽和来自其积累pyroGlu-3 A <$肽的转基因小鼠模型的脑提取物。我们在CND的合作者慷慨地提供了3A 1通用A？mAb杂交瘤作为对照。重要的是，我的实验室发起了这项合作，并有多年的研究pyroGlu A？沉积，炎症和A？免疫疗法的经验。我们研究的总体目标是确定焦谷氨酸A <$蛋白（A <$pE3和A <$pE11）是否是阿尔茨海默病的治疗靶点，以及通过对任一种pyroGlu A <$物种特异性的免疫疗法清除是否有效预防和/或治疗AD。

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.