A test of the calcium hypothesis of Alzheimers disease

阿尔茨海默病的钙假说检验

• 批准号: 9756294
• 负责人: Katherine R. Sadleir
• 金额: $ 27.65万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9756294
• 关键词: Adult; Age-Months; Aging; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease risk; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Animals; Antibodies; Antibody Therapy; Appearance; Area; Axon; Birth; Brain; Buffers; Ca(2+)-Calmodulin Dependent Protein Kinase; Calcium; Calcium Binding; Calcium Channel Blockers; Calcium Signaling; Cells; Cephalic; Chronic; Cognition; Cognitive; Cognitive deficits; Data; Dendrites; Dependovirus; Deposition; Development; Diet; Distant; Doxycycline; Electrophysiology (science); Environmental Risk Factor; Excision; Fluorescence; Functional disorder; Generations; Genetic; Hippocampus (Brain); Homeostasis; Image; Impaired cognition; Impairment; Implant; Individual; Injections; Interneurons; Intestines; Kidney; Lead; Learning; Life Style; Literature; Measures; Mediating; Memory; Memory impairment; Methods; Modeling; Molecular; Monitor; Mus; Nerve Degeneration; Neurites; Neuronal Dysfunction; Neurons; Organelles; Parvalbumins; Pathology; Pattern; Performance; Population; Pregnant Women; Process; Property; Prosencephalon; Proteins; Proxy; Rest; Risk Factors; Senile Plaques; Signal Transduction; Testing; Tetracyclines; Therapeutic; Time; Trans-Activators; Transgenic Mice; Validation; Vesicle; Viral; Virus; abeta toxicity; aged; amyloid pathology; analog; behavior test; beta amyloid pathology; calbindin; cognitive function; conditioned fear; excitatory neuron; extracellular; functional restoration; healing; hippocampal pyramidal neuron; improved; in vivo; in vivo Model; in vivo evaluation; in vivo monitoring; morris water maze; mouse model; multiphoton imaging; multiphoton microscopy; neuronal cell body; offspring; postnatal; promoter; protein expression; pup; restoration; sensor; therapeutic target; trafficking; transgene expression

The calcium hypothesis of Alzheimer's disease (AD) posits that dysregulation of calcium homeostasis is the point of convergence of the many factors risk factors and molecular mechanisms that lead to development of AD and its associated neurodegeneration. A corollary of the calcium hypothesis of AD is that restoration of calcium homeostasis will ameliorate AD pathophysiology and reverse neuronal dysfunction. Surprisingly, despite being nearly 3 decades old, this hypothesis has not been definitively tested in vivo. We propose to directly test the calcium hypothesis by normalizing resting free intracellular calcium levels through exogenous expression of the simple calcium buffer proteins calbindin-D9k and parvalbumin-α in the brains of 5XFAD mice, a model of amyloid pathology in AD. In Aim 1, we will determine if reduced resting free calcium cures the dystrophic axons and neurites that develop around plaques and likely contribute to neuronal dysfunction and increased amyloid generation In Aim 2, we will determine if increasing calcium buffering in the brains of amyloid plaque-containing 5XFAD mice can restore electrophysiological and cognitive impairments. Two adeno-associated viruses will be co-injected into the ventricles of postnatal P0 mouse pups, resulting in viral transduction throughout the brain and lifelong transgene expression. One virus expresses the calcium sensor GCaMP6f from the calmodulin kinase II (CaMKII) promoter, which drives expression in the excitatory neurons of the forebrain. The second virus co-expresses mCherry and a calcium buffer protein (either calbindin-D9k or parvalbumin-α) under the control of a tetracycline-off (TetO) promoter. In mice that express the tetracycline transactivator protein (tTA) under the control of a CaMKII promoter, the calcium buffer protein and mCherry will be expressed in the same population of cells as the calcium sensor GCaMP6f in the absence of the tetracycline analog doxycycline (dox). The pregnant mothers and offspring will be fed dox-containing chow until 6 months of age while the plaques develop. Mice will then be implanted with a cranial window to allow repeated in vivo multiphoton imaging of calcium levels, as measured by GCaMP6f fluorescence, in the dystrophic neurites around plaques. After baseline calcium levels have been measured, we will discontinue the dox diet to induce calcium buffer protein expression and monitor calcium levels and dystrophies by multiphoton microscopy for several weeks. Four weeks after induction, memory performance will be assessed in the Morris water maze, fear conditioning and the Y-maze, hippocampal memory tasks in which the 5XFAD mouse is impaired. At the end of behavioral testing, electrophysiology will be performed to measure the afterhyperpolarization (AHP), which is increased in aged 5XFAD mice and correlates with impaired memory. We predict that increased calcium buffering will “heal” the dystrophic neurites and improve electrophysiological and cognitive functions, providing strong support for the calcium hypothesis, and validate the use of calcium- reducing therapeutics in AD.

阿尔茨海默氏病（AD）的钙假设认为钙稳态失调是 导致发展的许多因素风险因素和分子机制的收敛点 AD及其相关的神经变性。 钙稳态将占有症状，并反向神经元功能障碍。 尽管已有近3年的历史，但我们建议的假设尚未得到确定的测试 直接通过使静息的游离细胞内钙水平归一化的钙假设直接检验钙假设 在5xFAD小鼠的大脑中，简单钙缓冲蛋白Calbindin-D9K和白蛋白-α的表达， AD 1中的AD淀粉样病理模型。 斑块周围发育，可能导致神经元功能障碍和 在AIM 2中增加淀粉样蛋白的产生，我们将确定在大脑中是否增加了钙缓冲 淀粉样蛋白斑块5xFAD小鼠可以恢复电生理和认知障碍 腺相关病毒将共同注射到产后P0小鼠幼崽的心室中，并在病毒中重塑 整个大脑和终生转基因表达。 来自钙调蛋白激酶II（CAMKII）启动子的GCAMP6F，该启动子在兴奋剂中驱动表达 前脑。 在四环素启动子（TETO）启动子的控制下，白细胞蛋白α）在表示四环素的小鼠中 在CAMKII启动子，钙缓冲蛋白和麦克林遗嘱的控制下，反式激活蛋白（TTA） 在不存在的情况下，在与钙传感器GCAMP6F的细胞中表达 四环素类似物多西环素（DOX）。 直到6个月大，斑块就会发展。 通过gcamp6f荧光测量的钙水平的体内多光子成像在你 测量了基线钙水平后的斑块神经突。 DOX饮食诱导钙缓冲蛋白表达并监测多光子的钙水平和营养不良 在指示后四个星期内，将在莫里斯评估记忆性能 水迷宫，恐惧调节和Y迷宫，5xfad鼠标的海马记忆任务是IS是 在行为测试结束时受损，将进行电生理学 在5xFAD小鼠中增加并与记忆力受损相关的毕竟的辉煌（AHP）。 我们预测，增加的钙缓冲会“治愈”营养不良的神经突并改善电生理学？ 和认知功能，为钙假设提供了强有力的支持，并验证了钙的使用 减少AD中的治疗剂。