Molecular Mechanisms of Age-related Muscle Loss

描述(由申请人提供)： 年龄相关性骨骼肌萎缩是一种在退伍军人患者中常见的衰弱状况。然而，人们对它知之甚少，也缺乏治疗方法。为了解决这个问题，我们将研究其原因。我们假设，年龄相关性肌肉萎缩发病机制中的一个中心事件是ATF4的表达增加，ATF4是一种由应激诱导的进化古老的转录因子。这一假设是基于我们初步研究中的几条证据。首先，我们和其他人发现，ATF4mRNA是由各种急性萎缩诱导应激(禁食、癌症、糖尿病、尿毒症和肌肉失神经)增加的少数mRNAs之一。其次，我们发现，将编码小鼠ATF4的质粒DNA导入小鼠骨骼肌，足以诱导肌纤维萎缩。第三，我们发现减少骨骼肌ATF4的表达(通过针对ATF4 mRNA的RNA干扰)可以减少禁食诱导的小鼠肌肉萎缩。尽管对年龄相关性肌肉萎缩的发病机制知之甚少，但先前的工作表明，年龄相关性肌肉萎缩至少涉及一些相同的转录调控机制，这些机制介导了更急性形式的肌肉萎缩(如禁食诱导的萎缩)。考虑到这一点，我们假设ATF4可能参与了年龄相关性肌肉萎缩。与这一想法一致，我们对ATF4介导的小鼠骨骼肌基因表达进行了无偏见的全局分析，发现ATF4诱导了五个与萎缩相关的mRNAs(CDKN1A、GADD45A、PEG3、CSRP3和ANKRD1)。有趣的是，先前的工作表明CDKN1A和GADD45A mRNAs在人和小鼠骨骼肌中受到衰老的高度诱导，由这些mRNAs编码的蛋白质被认为是与年龄相关的肌肉萎缩的有吸引力的潜在中介。综上所述，我们的初步数据提出了一种模型，在该模型中，导致萎缩的应激(如衰老和禁食)增加了骨骼肌ATF4，进而诱导CDKN1A、PEG3、GADD45A、CSRP3和ANKRD1mRNAs促进肌肉萎缩。如果这是真的，那么ATF4及其下游介质代表着预防或逆转退伍军人患者年龄相关性肌肉萎缩的治疗的潜在靶点。我们提出了两个目标来测试这个模型。在目标1中，我们将检验ATF4在年龄相关性肌肉萎缩中起重要作用的假设。为此，我们开发了几种方法来特异性地切除小鼠骨骼肌中的ATF4基因。我们将使用这些方法来确定ATF4的结构性丢失是否可以防止与年龄相关的肌肉萎缩，并确定ATF4的急性丢失是否可以逆转老年小鼠的萎缩。在目标2中，我们将检验由ATF4依赖的萎缩相关mRNAs(CDKN1A、GADD45A、Peg3、CSRP3和ANKRD1)编码的蛋白质介导ATF4依赖的萎缩的假设。为了验证这一假设，我们将在小鼠骨骼肌中过表达这些蛋白质，并确定它们是否足以诱导骨骼肌纤维萎缩。我们还将在小鼠骨骼肌中使用RNA干扰来确定这些蛋白质是否是禁食介导的骨骼肌纤维萎缩所必需的。通过这些研究，我们希望阐明年龄相关性肌肉萎缩的中心事件，从而确定影响许多退伍军人患者的致残性疾病的新治疗靶点。

DESCRIPTION (provided by applicant): Age-related skeletal muscle atrophy is a debilitating condition that is common in veteran patients. However, it is poorly understood and lacks a therapy. To begin to address this problem, we will study its causes. We hypothesize that a central event in the pathogenesis of age-related muscle atrophy is increased expression of ATF4, an evolutionarily ancient transcription factor that is induced by stress. This hypothesis is based on several lines of evidence from our preliminary studies. First, we and others found that ATF4 mRNA is one of a small number of mRNAs increased by a variety of acute atrophy-inducing stresses (fasting, cancer, diabetes, uremia and muscle denervation). Second, we found that transfection of mouse skeletal muscle with plasmid DNA encoding mouse ATF4 was sufficient to induce myofiber atrophy. Third, we found that reducing skeletal muscle ATF4 expression (by RNA interference targeting ATF4 mRNA) reduced fasting-induced muscle atrophy in mice. Although little is known about the pathogenesis of age-related muscle atrophy, previous work suggest that age-related muscle atrophy involves at least some of the same transcriptional control mechanisms that mediate more acute forms of muscle atrophy (such as fasting-induced atrophy). This consideration leads us to hypothesize that ATF4 may mediate age-related muscle atrophy. Consistent with this idea, we performed an unbiased, global analysis of ATF4-mediated gene expression in mouse skeletal muscle, and found that ATF4 induced five atrophy-associated mRNAs (CDKN1A, GADD45A, PEG3, CSRP3 and ANKRD1). Interestingly, previous work showed CDKN1A and GADD45A mRNAs are highly induced by aging in human and mouse skeletal muscle, and the proteins encoded by these mRNAs are viewed as attractive potential mediators of age-related muscle atrophy. Taken together, our preliminary data suggest a model in which atrophy-inducing stresses (such as aging and fasting) increase skeletal muscle ATF4, which in turn induces CDKN1A, PEG3, GADD45A, CSRP3 and ANKRD1 mRNAs to promote muscle atrophy. If this is true, then ATF4 and its downstream mediators represent potential targets for therapies to prevent or reverse age-related muscle atrophy in veteran patients. We propose two aims to test this model. In Aim 1, we will test the hypothesis that ATF4 plays an essential role in age-related muscle atrophy. To this end, we have developed several methods to specifically excise the ATF4 gene in mouse skeletal muscle. We will use these methods to determine if constitutive loss of ATF4 prevents age-related muscle atrophy, and to determine if acute loss of ATF4 reverses atrophy in aged mice. In Aim 2, we will test the hypothesis that the proteins encoded by ATF4-dependent atrophy-associated mRNAs (CDKN1A, GADD45A, PEG3, CSRP3 and ANKRD1) mediate ATF4-dependent atrophy. To test this hypothesis, we will overexpress these proteins in mouse skeletal muscle and determine if they are sufficient to induce skeletal myofiber atrophy. We will also use RNA interference in mouse skeletal muscle to determine if these proteins are required for fasting-mediated skeletal myofiber atrophy. Through these studies, we hope to elucidate central events in age-related muscle atrophy, and thus identify novel therapeutic targets for a disabling condition that affects many veteran patients.