Beclin 1 regulates neuroprotective TGF-beta signaling

描述(申请人提供)：阿尔茨海默病(AD)仅在美国就有500多万人受到影响，但我们对其发病机制的了解仍然不完整，目前还没有治愈方法。最近，我们报道了自噬相关蛋白Beclin 1在AD脑提取液中的水平降低。小鼠Beclin-1基因杂合性缺失导致突触丢失和神经变性。小胶质细胞中Beclin 1水平的降低损害了吞噬细胞受体CD36的循环，导致淀粉样斑块的清除减少，这是AD的一个病理标志。鉴于Beclin 1在神经元中表达，在哺乳动物中的这种新功能可能会影响对神经元健康至关重要的受体的循环。神经元的存活依赖于营养因子信号通路，在AD中观察到了神经营养信号通路的变化，包括神经保护性的转化生长因子-β通路。我在这里提供的数据显示，在体外，下调Beclin 1，而不是自噬相关蛋白ATG7，会减少转化生长因子-β信号。Beclin 1通过与两种不同的III型磷脂酰肌醇-3-激动素(PI3K)复合体相互作用，在酵母细胞自噬和蛋白质分选中发挥重要作用。PI3K催化亚单位的下调导致转化生长因子-β信号转导减少。此外，酵母中一种专用于蛋白质分选的PI3K复合体成分UVRAG的敲除也降低了转化生长因子-β信号转导，而参与自噬的复合体Atg14的敲除则没有影响。这表明，Beclin 1作为含有UVRAG的PI3K复合体的一部分，在调节转化生长因子-β信号转导中具有特定的作用。这项建议的目的是检验这一假说，即降低的Beclin 1导致神经退行性变，部分是通过损害转化生长因子受体的循环，并通过这一途径剥夺神经元的保护性信号。在目标1中，我确定了Beclin 1和PI3K复合体之间的功能和物理相互作用，PI3K复合体使用一系列突变结构介导转化生长因子-β信号转导。我还通过活细胞成像研究了Beclin 1、转化生长因子β受体和PI3K复合体产物PI3-磷酸之间的关系。在目标2中，我使用流式细胞术、显微镜和免疫沉淀相结合的方法来确定Beclin 1在转化生长因子受体信号通路中的作用，以直接测试Beclin 1 kD对转化生长因子受体内吞和再循环以及下游信号分子募集的影响。在目标3中，我测试了Beclin 1kD是否在体内损害了转化生长因子-β信号，并阻止了转化生长因子-β介导的神经保护作用。这些实验使用生物发光成像和免疫组织化学相结合的方法来确定Beclin 1kD对转化生长因子-β信号转导和神经变性的影响。针对AD中转化生长因子-β途径的治疗方法目前正在研究中。鉴于Beclin 1水平降低对转化生长因子-β信号转导的影响，了解这一过程背后的机制是至关重要的，这样我们才能最大限度地成功开发这种毁灭性疾病的治疗方法。 公共卫生相关性：这项建议的目的是确定在阿尔茨海默病中观察到的一种变化--Beclin 1蛋白水平降低如何影响转化生长因子-β途径中的保护性信号。目前在AD中针对这一途径开发治疗药物的努力旨在识别模拟转化生长因子信号转导的分子。了解Beclin 1缺陷是如何通过转化生长因子受体影响信号传递的，不仅可以加深我们对AD发病机制的理解，还可以为药物开发提供更好的策略。

DESCRIPTION (provided by applicant): Alzheimer Disease (AD) affects over 5 million people in the U.S. alone but our understanding of the mechanisms underlying pathogenesis remains incomplete and there is currently no cure. Recently, we reported that levels of the autophagy-related protein beclin 1 are decreased in AD brain extract. Heterozygous deficiency of beclin 1 in mice results in synaptic loss and neurodegeneration. Reduced beclin 1 levels in microglia impair recycling of the phagocytic receptor CD36, resulting in reduced clearance of amyloid plaques, a pathological hallmark of AD. Given that beclin 1 is expressed in neurons, this novel function of beclin 1 in mammals may affect recycling of receptors important for neuronal health. Neuronal survival depends on trophic factor signaling, and alterations in neurotrophic signaling pathways, including the neuroprotective TGF-¿ pathway, have been observed in AD. I present data here that shows knock-down of beclin 1, but not the autophagy-related protein Atg7, decreases TGF-¿ signaling in vitro. Beclin 1 is known to mediate its roles in autophagy and protein sorting in yeast through interaction with two distinct type III phosphatidylinositol-3-kinae (PI3K) complexes. Knock-down of the catalytic subunit of the PI3K results in decreased TGF-¿ signaling. Furthermore, knock-down of UVRAG, a PI3K complex component specific to protein sorting in yeast, also decreased TGF-¿ signaling, while knock-down of Atg14, specific to the complex involved in autophagy, has no effect. This suggests a specific role for beclin 1, as part of the PI3K complex containing UVRAG, in regulating TGF-¿ signaling. The purpose of this proposal is to test the hypothesis that reduced beclin 1 contributes to neurodegeneration, in part, by impairing TGF-¿ receptor recycling and depriving neurons of protective signaling through this pathway. In Aim 1, I determine the functional and physical interactions between beclin 1, and the PI3K complexes that mediate TGF-¿ signaling using a series of mutant constructs. I also characterize the relationship between beclin 1, the TGF-¿ receptors and PI3-phosphate, the product of the PI3K complex, by live-cell imaging. In Aim 2, I determine where in the TGF-¿ signaling pathway beclin 1 functions using a combination of flow cytometry, microscopy, and immunoprecipitation to directly test the effect of beclin 1 KD on TGF-¿ receptor endocytosis and recycling, and recruitment of downstream signaling molecules. In Aim 3, I test if beclin 1 KD impairs TGF-¿ signaling in vivo and prevents TGF-¿-mediated neuroprotection against excitotoxicity. These experiments use a combination of bioluminescence imaging and immunohistochemistry to determine the effect of beclin 1 KD on TGF-¿ signaling and neurodegeneration. Therapeutic approaches targeting the TGF-¿ pathway in AD are currently under study. Given the effect of reduced beclin 1 levels on TGF-¿ signaling, it is crucial to understand the mechanisms underlying this process so that we maximize our chance of success in developing therapies for this devastating disease. PUBLIC HEALTH RELEVANCE: The purpose of this proposal is to determine how reduced levels of the protein beclin 1, a change observed in Alzheimer Disease, affects protective signaling in the TGF-¿ pathway. Current efforts to develop therapeutics to target this pathway in AD aim to identify molecules that mimic TGF-¿ signaling. Understanding how beclin 1 deficiency affects signaling through TGF-¿ receptors will not only further our understanding of the mechanisms underlying AD, but also lead to better strategies for drug development.