Beclin 1 regulates neuroprotective TGF-beta signaling

描述（由申请人提供）：阿尔茨海默病（AD）仅在美国就影响了超过500万人，但我们对其潜在发病机制的了解仍然不完整，目前尚无治愈方法。最近，我们报道了AD脑提取物中自噬相关蛋白beclin 1的水平降低。小鼠beclin - 1杂合子缺乏可导致突触丧失和神经变性。小胶质细胞中beclin 1水平的降低损害了吞噬受体CD36的再循环，导致淀粉样斑块的清除减少，这是AD的病理标志。鉴于beclin 1在神经元中表达，哺乳动物中beclin 1的这种新功能可能会影响对神经元健康重要的受体的再循环。神经元的存活依赖于营养因子信号传导，在AD中已经观察到神经营养信号通路的改变，包括神经保护TGF-¿通路。我在这里提供的数据显示，在体外，敲除beclin 1，而不是自噬相关蛋白Atg7，会降低TGF-¿信号。已知Beclin 1通过与两种不同的III型磷脂酰肌醇-3-激酶（PI3K）复合物相互作用介导酵母自噬和蛋白质分选。敲除PI3K的催化亚基导致TGF-¿信号传导减少。此外，敲除酵母中PI3K复合物特异性蛋白分选组分UVRAG也会降低TGF-¿信号，而敲除参与自噬的复合物特异性Atg14则没有影响。这表明beclin 1作为包含UVRAG的PI3K复合体的一部分，在调节TGF-信号传导中具有特殊作用。本研究的目的是验证这样一种假设，即减少beclin 1有助于神经退行性变，部分原因是通过破坏TGF-受体的再循环和剥夺神经元通过该途径获得的保护性信号。在Aim 1中，我使用一系列突变体结构确定了beclin 1和介导TGF-¿信号的PI3K复合物之间的功能和物理相互作用。我还通过活细胞成像描述了TGF-受体beclin 1和PI3K复合物产物pi3 -磷酸之间的关系。在Aim 2中，我使用流式细胞术、显微镜和免疫沉淀相结合的方法确定了beclin 1在TGF-¿信号通路中的作用，直接测试了beclin 1 KD对TGF-¿受体内吞和再循环以及下游信号分子募集的影响。在Aim 3中，我测试了beclin 1 KD是否在体内损害TGF-¿信号并阻止TGF-¿介导的神经保护对兴奋性毒性的作用。这些实验采用生物发光成像和免疫组织化学相结合的方法来确定beclin 1 KD对TGF-¿信号传导和神经退行性变的影响。针对TGF-¿通路的AD治疗方法目前正在研究中。考虑到beclin 1水平降低对TGF-信号传导的影响，了解这一过程的机制是至关重要的，这样我们才能最大限度地成功开发出治疗这种毁灭性疾病的方法。

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.