ATM (Ataxia Telangiectasia Mutated) signaling through cyclin D1

描述(申请人提供)：共济失调毛细血管扩张症(AT)是一种常染色体隐性遗传病，由共济失调毛细血管扩张症突变(ATM)基因突变引起。ATM是一种蛋白激酶，被DNA双链断裂激活，进而激活DNA修复和细胞周期检查点机制，以维持基因组的稳定。ATM这些功能的丧失解释了AT的许多症状，如癌症易感性、辐射敏感性和免疫缺陷。然而，ATM丢失如何触发神经变性，从而导致AT共济失调(运动功能障碍)尚不清楚。我们最近发现，ATM诱导Thr286残基上细胞周期蛋白D1不稳定的磷酸化，并抑制细胞周期蛋白D1的表达。我们还发现，细胞周期蛋白D1的抑制在诱导DNA损伤后的G1检查点中起着关键作用。我们将探讨ATM和细胞周期蛋白D1在DNA损伤诱导的神经细胞死亡中的作用。神经元中的代谢需求被认为会产生高水平的活性氧物种，从而破坏DNA。ATM感知到这种损伤，并激活细胞周期检查点和DNA修复机制，以应对这种内源性遗传毒性应激。我们假设，当ATM缺陷神经元暴露在遗传毒性应激下时，细胞周期蛋白D1的表达继续，因为它的Thr286残基没有被磷酸化。细胞周期蛋白D1的高表达将超越G1检查点，并有助于在增殖的神经细胞中积累DNA损伤(目标1)。在有丝分裂后神经元中，细胞周期蛋白D1的高表达会引发细胞周期的异常进展和细胞死亡(目标2)。在目标3中，我们将研究在增殖过程中积累的DNA损伤是否会使有丝分裂后神经元对DNA损伤处理敏感。使用ATM-/-和+/+小鼠的原代神经细胞，以及人类神经干细胞，我们检查了ATM依赖的细胞周期蛋白D1抑制是否保护神经元免受氧化应激的影响。基因毒性应激的生物学后果将通过使用特定的抑制剂、shRNA或中和抗体注射来操纵ATM和细胞周期蛋白D1的活性或表达来确定。这些实验正在精心设计，以阐明这一新概念的有效性，即ATM通过其控制细胞周期蛋白D1表达的能力，在遗传毒性应激下的神经细胞中发挥关键作用。 公共卫生相关性：我们发现在DNA损伤条件下，ATM(共济失调毛细血管扩张突变)至少部分地通过其靶向细胞周期蛋白D1的能力来控制细胞周期进展。将ATM-细胞周期蛋白依赖的细胞周期调控这一新概念应用到神经元生物学中，这一建议将解决共济失调毛细血管扩张症(AT)神经元变性的分子机制。这些研究结果可能会促进我们对AT以及其他相关神经退行性疾病，如中风、帕金森病和阿尔茨海默病的发病机制的基本认识。

DESCRIPTION (provided by applicant): Ataxia telangiectasia (AT) is an autosomal recessive disease caused by mutation in ataxia telangiectasia mutated (ATM) gene. ATM, a protein kinase, is activated by DAN double strand break and in turn it activates DNA repair and cell cycle checkpoint mechanisms to maintain genomic stability. The loss of these functions of ATM explains many symptoms of AT such as predisposition to cancer, radiation sensitivity, and immunodeficiency. However, it is not clear how ATM loss triggers neurodegeneration, which causes ataxia (motor dysfunction) in AT. We recently found that ATM induces destabilizing phosphorylation of cyclin D1 on the Thr286 residue, and suppresses cyclin D1 expression. We have also found that the suppression of cyclin D1 plays a critical role in inducing a G1 checkpoint following DNA damage. We will examine the roles of ATM and cyclin D1 in neuronal cell death induced by DNA damage. Metabolic demand in neurons is considered to generate high levels of reactive oxygen species which damage DNA. ATM senses such damage and would activate cell cycle checkpoint and DNA repair mechanism to cope with such endogenous genotoxic stress. We hypothesized that when ATM deficient neurons are exposed to genotoxic stress, cyclin D1 expression continues because its Thr286 residue is not phosphorylated. The high expression of cyclin D1 would override the G1 checkpoint and contribute to accumulation of DNA lesions in proliferating neural cells (Aim 1). In postmitotic neurons, high cyclin D1 expression would trigger aberrant cell cycle progression and cell death (Aim 2). In Aim 3, we will examine, if DNA lesions accumulated during proliferation would sensitize postmitotic neuron to DNA damaging treatment. Using primary neural cells from ATM-/- and +/+ mice, and human neural stem cells, we examine if ATM dependent cyclin D1 suppression protects neurons from oxidative stress. Biological consequence of genotoxic stress will be determined following manipulating the activity or expression of ATM and cyclin D1 using a specific inhibitor, shRNA or neutralizing antibody injection. The experiments are carefully designing to elucidate the validity of the novel concept that through its ability to control cyclin D1 expression, ATM plays a critical role in neuronal cells under genotoxic stress. PUBLIC HEALTH RELEVANCE: We have found that under DNA damaging condition, ATM (ataxia telangiectasia mutated) controls cell cycle progression at least in part by its ability to target cyclin D1. Appling this new concept of ATM-cyclin D1 dependent cell cycle regulation to the biology of the neurons, this proposal will address molecular mechanism of degeneration of neurons in ataxia telangiectasia (AT). The finding obtained from these studies may advance our fundamental understanding on pathogenesis of AT, as well as other related neurodegenerative diseases, such as brain stroke, Parkinson disease, and Alzheimer's disease.