CRMP2 Phosphorylation: A Novel Target for Alzheimer's Disease?

摘要 今天，美国有580万人患有阿尔茨海默病（AD），其中65岁以下的人中有十分之一患有AD。 老年痴呆症协会估计它是该年龄组的第五大死亡原因。尽管 尽管NIH（2019年为24亿美元）和制药公司的投资不断增加，但阿尔茨海默氏症的研究未能提供 治愈，更不用说减缓疾病的治疗了。例如，对淀粉样蛋白假说的教条主义信念已经扼杀了 新思想的出现。我们提出了一种新的方法：阻止CRMP 2磷酸化。脑衰蛋白反应 介体蛋白2（CRMP 2），一种微管相关蛋白，被鉴定为 糖原合成酶激酶3β（GSK 3 β）和细胞周期蛋白依赖性激酶5（Cdk 5）是两种蛋白激酶， 在AD中具有更大的活性。事实上，CRMP 2在GSK 3b靶向的残基上的磷酸化， Cdk 5在从人AD脑分离的皮质中以及在AD动物模型的脑中相对较高， 而磷酸-CRMP 2与淀粉样β（Ab）蛋白一起沿着存在于神经纤维缠结中。在小鼠模型中 在AD中，磷酸化水平的增加发生在病理学之前，这表明CRMP 2的过度磷酸化是 AD发展的早期事件。研究表明， CRMP 2在AD转基因小鼠模型和死后人类AD脑中的表达，但功能性 CRMP 2过度磷酸化对AD发展的影响尚未完全解决。到 迄今为止，只有一项研究研究了CRMP 2磷酸化在AD发病机制中的作用。通过检查 Aβ蛋白核心毒性片段对CRMP 2细胞行为和电生理特性的影响 磷酸化缺陷敲入（crmp 2ki/ki）小鼠，其中残基52 SeràAla，这项研究表明，Ab 25- 35野生型（wt）小鼠中寡聚体诱导的记忆和突触可塑性损伤，但crmp 2ki/ki中没有 小鼠在这里，我们检验了阻止CRMP 2磷酸化（在S522）将阻断CRMP 2受损的假设。 小鼠AD模型中的记忆和突触可塑性。AD的J20小鼠模型将与crmp 2ki/ki 研究CRMP 2磷酸化在AD发病机制中的重要性。在双 转基因J20-crmp 2ki/ki与J20-crmp 2 wt小鼠相比， 识别和Morris水迷宫测试。我们将利用免疫组织化学和生物化学来评估 CRMP 2磷酸化状态、Aβ负荷和突触功能障碍。我们还将评估神经细胞凋亡 使用TUNEL分析。采用电生理学方法，我们将评估海马神经元的长时程增强（LTP）， 切片与CRMP 2磷酸化完整的J20小鼠相比，我们预计J20-crmp 2ki/ki小鼠将具有 降低CRMP 2磷酸化，改善记忆，降低Aβ负荷和突触功能障碍， 细胞死亡减少，LTP无损伤。这些信息将提供初步数据， 更大，更全面的研究，以评估CRMP 2磷酸化的靶向，以减少 AD症状的负担。本行政补充申请是对NOT-AG-20-034的回应。

ABSTRACT Today 5.8 million people in the United States live with Alzheimer’s disease (AD), including 1 in 10 of those 65 and over, estimates the Alzheimer’s Association. It is the fifth leading cause of death in that age group. Despite growing investment by the NIH ($2.4 billion in 2019) and pharma, Alzheimer’s research has failed to deliver a cure, let alone a disease-slowing treatment. Dogmatic belief in the amyloid hypothesis, for example, has stifled emergence of new ideas. We advance a new approach: preventing CRMP2 phosphorylation. Collapsin response mediator protein 2 (CRMP2), a microtubule associated protein, was identified as a physiological substrate for glycogen synthase kinase 3β (GSK3β) and cyclin dependent kinase 5 (Cdk5), two protein kinases reported to have with greater activity in AD. Indeed, phosphorylation of CRMP2, at the residues targeted by GSK3b and Cdk5, is relatively high in cortex isolated from human AD brains, as well as in the brains of animal models of AD, while phospho-CRMP2 is found in neurofibrillary tangles along with amyloid beta (Ab) protein. In mouse models of AD, increased phosphorylation occurs prior to pathology, suggesting that hyperphosphorylation of CRMP2 is an early event in the development of AD. Studies have documented the presence of increased phosphorylation of CRMP2 in transgenic mouse models of AD and in postmortem human AD brains, but the functional consequences of CRMP2 hyperphosphorylation to the development of AD have not been fully addressed. To date, only one study has examined the role of CRMP2 phosphorylation in AD pathogenesis. By examining the effects of a core toxic fragment of Aβ proteins on behavioral and electrophysiological features in CRMP2 phosphorylation-deficient knock-in (crmp2ki/ki) mice, in which residue 52 SeràAla, this study revealed that Ab25– 35 oligomer–induced impairments of memory and synaptic plasticity in wildtype (wt) mice but not in crmp2ki/ki mice. Here, we test the hypothesis that preventing CRMP2 phosphorylation (at S522) will block impairment of memory and synaptic plasticity in a mouse AD model. The J20 mouse model of AD will be bred with the crmp2ki/ki mouse to interrogate the importance of CRMP2 phosphorylation in the pathogenesis of AD. In the double transgenic J20–crmp2ki/ki compared to J20–crmp2wt mice we will evaluate cognition using the novel object recognition and Morris water maze tests. We will utilize immunohistochemistry and biochemistry to evaluate the status of CRMP2 phosphorylation, Aβ burden, and synaptic dysfunction. We will also assess neuronal apoptosis using the TUNEL assay. Using electrophysiology, we will assess long-term potentiation (LTP) in hippocampal slices. Comparing to J20 mice with intact CRMP2 phosphorylation, we expect that J20–crmp2ki/ki mice will have reduced CRMP2 phosphorylation, improved memory, decreased Aβ burden and synaptic dysfunction, decreased cell death, and no impairment in LTP. This information will provide the preliminary data to propose a larger and more comprehensive study to evaluate the targeting of CRMP2 phosphorylation to decrease the burden of AD symptoms. This Administrative Supplement request is in response to NOT-AG-20-034.