Spinal Muscular atrophy: is it a motor axon disease?

描述（由申请人提供）：运动神经元疾病是毁灭性的，因为它们剥夺了个体的活动能力，并且由于呼吸系统的神经支配丧失，通常是致命的。脊髓性肌萎缩症（SMA）是一种常染色体隐性遗传病，可引起运动神经元功能障碍，导致瘫痪，严重者死亡，是婴儿/幼儿死亡的主要遗传原因。动物模型分析显示，运动轴突缺陷、未成熟的神经肌肉连接（NMJs）和去神经支配提示运动神经末梢的改变可能引发疾病。存活运动神经元（SMN）基因是SMA的遗传原因，在组装mRNA剪接所需的rna和蛋白质（snRNP组装）中具有明确的作用。然而，我们和其他实验室的数据表明，当SMN水平降低时，SMN可能还有其他功能受到损害。使用斑马鱼作为模型系统，我们已经证明SMN具有snRNP独立的功能，对正常的运动轴突生长很重要。此外，我们已经证明，作为一种肌动蛋白结合蛋白和第一个被发现的人类SMA的修饰物，plastin 3可以修复斑马鱼由低Smn水平引起的运动轴突缺陷。此外，斑马鱼的smn突变体严重降低了活蛋白3的水平。在本研究中，我们将验证一个假设，即plastin 3通过snRNP独立通路与SMN作用，促进正常运动神经元的发育和功能。为了直接验证这一假设，我们将询问其他SMA表型是否由活蛋白3拯救（目的1）。这包括运动神经元和NMJ电生理，NMJ的SV2蛋白和存活。我们将通过进行结构/功能分析（目标2）来确定活素3在SMN方面的功能。在这些实验中，我们将使用活质蛋白3和SMN突变体来定义相关的结构域。我们还将通过检查其他肌动蛋白结合蛋白来验证plastin 3在其修饰SMA表型的能力方面是独特的假设。我们将检验SMN - plastin - 3相互作用独立于SMN的snRNP功能的假设（Aim 3）。最后，我们将使用实时成像来询问SMN和活素3蛋白在运动神经元中的定位位置，以及SMN的减少是否会改变活素3的水平和/或细胞定位（目的4）。从这些目标中获得的数据将直接解决SMN和plastin 3之间的关系，因为它与SMA有关，使用电生理学、分子遗传学、生物化学、细胞生物学和成像的组合。此外，它将建立一个不依赖snRNP的直接影响运动神经元功能的SMN机制，从而极大地促进我们对这种疾病的认识，揭示新的治疗靶点。使用斑马鱼是一个优势，因为我们可以在我们开发的SMA模型中直接分析体内的运动神经元，并且很容易产生新的转基因来提出特定的问题。这是该模型系统的一个独特特征，因此这些研究是高度相关的，并将推进我们对低Smn水平如何导致SMA的理解。

DESCRIPTION (provided by applicant): Motoneuron diseases are devastating in that they rob individuals of the ability to move and are often fatal due to denervation of the respiratory system. Spinal muscular atrophy (SMA) is an autosomal recessive disease that causes motoneuron dysfunction leading to paralysis and in severe cases death making it a leading genetic cause of infant/toddler mortality. Analysis of SMA animal models reveals, motor axon defects, immature neuromuscular junctions (NMJs), and denervation suggesting that changes at the motor nerve terminal may initiate disease. The survival motor neuron (SMN) gene is the genetic cause of SMA and has a clearly defined role in assembling RNAs and proteins needed for mRNA splicing (snRNP assembly). Data from our lab and others, however, suggest that SMN may have other functions that are compromised when SMN levels are decreased. Using zebrafish as a model system, we have shown that SMN has an snRNP independent function important for normal motor axon outgrowth. Moreover, we have shown that plastin 3, an actin binding protein and the first identified modifier of human SMA, can rescue motor axon defects in zebrafish caused by low Smn levels. In addition, zebrafish smn mutants have severely reduced plastin 3 levels. In this proposal we will test the hypothesis that plastin 3 acts with SMN via an snRNP independent pathway to facilitate normal motoneuron development and function. To directly test this hypothesis, we will ask whether other SMA phenotypes are rescued by plastin 3 (Aim 1). This includes motoneuron and NMJ electrophysiology, SV2 protein at the NMJ, and survival. We will determine how plastin 3 is functioning with respect to SMN by performing a structure/function analysis (Aim 2). For these experiments we will use both plastin 3 and SMN mutants to define relevant domains. We will also test the hypothesis that plastin 3 is unique in its ability to modify SMA phenotypes by examining other actin binding proteins. We will test the hypothesis that the SMN plastin 3 interaction is independent of the snRNP function of SMN (Aim 3). Lastly, we will use live imaging to ask where SMN and plastin 3 proteins localize in motoneurons and does decreasing Smn change the levels and/or cellular localization of plastin 3 (Aim 4). Data derived from these Aims will directly address the relationship between SMN and plastin 3 as it relates to SMA using a combination of electrophysiology, molecular genetics, biochemistry, cell biology, and imaging. Moreover, it would establish an snRNP- independent mechanism of SMN that directly affects motoneuron function thus greatly advancing our understanding of this disease and revealing new therapeutic targets. Using zebrafish is a strength in that we can directly analyze motoneurons in vivo in SMA models that we have developed and easily generate novel transgenics to ask specific questions. This is a unique feature of this model system and thus these studies are highly relevant and will advance our understanding of how low Smn levels cause SMA.