Regulation of SMN and Identification of its Downstream Target

描述(申请人提供)：脊髓性肌萎缩症(SMA)是一种由存活运动神经元1(SMN1)基因突变引起的疾病，是导致婴儿死亡的最常见的遗传原因。人类有两个SMN基因拷贝，端粒SMN1编码全长形式(FL-SMN)，着丝粒SMN2主要编码快速降解的截短形式(SMN 7)以及全长形式。在最严重的类型-1型SMA中，有1到2个SMN2基因拷贝，患者在2岁内死于呼吸衰竭。然而，SMN2基因拷贝较多的患者表现出一种不太严重的SMA(III型SMA)。SMA的发生是由于脊髓运动神经元FL-SMN蛋白含量减少所致。因此，SMA治疗干预的大部分努力都集中在提高FL-SMN蛋白产品的水平上。我们的初步结果表明，SMN存在于包含E3泛素连接酶后期促进复合体(APC)和RNA结合蛋白HUD的复合体中。APC将蛋白质定位于蛋白酶体进行降解，而HUD则稳定mRNAs。此外，有证据表明APC在神经元存活、轴突生长和突触功能中发挥作用，而HUD参与神经元的成熟和维持。我们还从以往的研究中了解到，SMN可能是某些mRNAs在神经突起中运输、稳定和/或翻译所必需的。这些数据促使我们建立了一个工作模型，其中：1)APC的泛素化调节SMN或SMN复合体的其他成员的稳定性和/或功能；2)轴突中可能与HUD-SMN相关的mRNAs对运动神经元的生长和存活至关重要。在本应用的第一部分，我们将研究APC在调节SMN稳定性和功能中的作用。我们将首先描述APC和SMN在神经元中的相互作用。然后，我们将研究抑制APC-SMN相互作用对SMN蛋白的功能、稳定性和定位的影响。在第二部分中，我们将重点介绍一个可能在轴突生长中发挥作用的轴突SMN靶基因。由于FL-SMN的数量与疾病的严重程度密切相关，了解APC和SMN之间的相互作用以及阐明SMN的下游靶点将为SMA的生物学提供重要的见解，并有可能为SMA提供新的治疗方案。 公共卫生相关性：脊髓性肌萎缩症(SMA)是美国婴儿死亡的主要遗传原因。我们建议使用最先进的蛋白质组学和RNA分析来研究这种疾病的细胞机制。了解这些细胞机制可能最终对设计SMA的治疗方法很重要。

DESCRIPTION (provided by applicant): Spinal Muscular Atrophy (SMA), a disease caused by the mutations of Survival Motor Neuron 1 (SMN1) gene, is the most common genetic cause of infant mortality. Humans have two copies of the SMN gene, the telomeric SMN1, which encodes for a full-length form (FL-SMN), and the centromeric SMN2, which encodes primarily for a rapidly-degraded truncated form (SMN 7) as well as the full-length form. In the most severe form, Type 1 SMA, there are 1 or 2 copies of the SMN2 gene, and patients die within 2 years of age due to respiratory failure. Patients with more copies of the SMN2 gene, however, manifest a less severe form of SMA (Type III SMA). SMA occurs due to decreased amount of FL-SMN protein in spinal motor neurons. Therefore, much of the effort for therapeutic interventions in SMA has focused on increasing the level of FL-SMN protein products. Our preliminary results show that SMN is found in a complex containing an E3 ubiquitin ligase the Anaphase- Promoting Complex (APC) and HuD, a RNA binding protein. The APC targets proteins to the proteasome for degradation whereas HuD stabilizes mRNAs. Furthermore, evidence indicates a role for APC in neuronal survival, axonal growth, and synaptic function, and HuD is involved in the maturation and maintenance of neurons. We also know from previous studies that SMN may be necessary for the transport, stability and/or translation of certain mRNAs in neurites. These data together prompted us to formulate a working model in which: 1) ubiquitination by APC regulates the stability and/or function of SMN or other members of the SMN complex and 2) the putative HuD-SMN-associated mRNAs in axons are important for the growth and survival of motor neurons. In the first part of this application, we will investigate the role of APC in regulation of SMN stability and function. We will first characterize the interaction between APC and SMN in neurons. Then, we will investigate the effect of inhibiting the APC-SMN interaction on the function, stability, localization, of SMN protein. In the second part, we will focus on one axonal SMN target mRNA which is likely to play a role in axon outgrowth. Since there is a tight correlation between the amount of FL-SMN and the severity of disease, understanding the interaction between APC and SMN as well as elucidating the downstream targets of SMN will provide important insights into the biology of SMA and has the potential to generate new treatment options for this disease. PUBLIC HEALTH RELEVANCE: Spinal muscular atrophy (SMA) is the leading genetic cause of infant deaths in the United States. We propose to investigate the cellular mechanisms of this disease using state of the art proteomics and RNA analysis. Understanding these cellular mechanisms may ultimately be important for designing therapies for SMA.