Regulation of SMN and Identification of its Downstream Target

描述（由申请人提供）：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，是婴儿死亡的最常见遗传原因。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主要编码快速退化的截短形式（smn7）和全长形式。在最严重的1型SMA中，SMN2基因有1或2个拷贝，患者在2岁内因呼吸衰竭而死亡。然而，SMN2基因拷贝数较多的患者表现为不那么严重的SMA （III型SMA）。脊髓萎缩症的发生是由于脊髓运动神经元中FL-SMN蛋白的减少。因此，SMA治疗干预的大部分努力都集中在增加FL-SMN蛋白产物的水平上。我们的初步结果表明，SMN存在于一个包含E3泛素连接酶、后期促进复合物（APC）和RNA结合蛋白HuD的复合物中。APC将蛋白质靶向蛋白酶体降解，而HuD则稳定mrna。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。我们也从以前的研究中知道，SMN可能是神经突中某些mrna的运输、稳定和/或翻译所必需的。这些数据共同促使我们制定了一个工作模型：1)APC的泛素化调节SMN或SMN复合物其他成员的稳定性和/或功能；2)轴突中假定的hud -SMN相关mrna对运动神经元的生长和存活很重要。在本应用程序的第一部分，我们将研究APC在SMN稳定性和功能调节中的作用。我们将首先描述神经元中APC和SMN之间的相互作用。然后，我们将研究抑制APC-SMN相互作用对SMN蛋白功能、稳定性和定位的影响。在第二部分中，我们将重点关注一个可能在轴突生长中发挥作用的轴突SMN靶mRNA。由于FL-SMN的数量与疾病的严重程度密切相关，因此了解APC和SMN之间的相互作用以及阐明SMN的下游靶点将为SMA的生物学提供重要见解，并有可能为该疾病提供新的治疗方案。

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.