Spinal Muscular atrophy: is it a motor axon disease?

描述(申请人提供)：运动神经元疾病是毁灭性的，因为它们剥夺了个人的行动能力，往往是致命的，因为呼吸系统的失神经。脊髓性肌萎缩症(SMA)是一种常染色体隐性遗传病，可导致运动神经元功能障碍，严重时可导致瘫痪和死亡，是导致婴幼儿死亡的主要遗传原因。对SMA动物模型的分析显示，运动轴突缺陷、未成熟的神经肌肉接头(NMJ)和失神经提示运动神经末梢的变化可能引发疾病。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，来自我们实验室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影响。以斑马鱼为模型系统，我们证明了SMN具有非依赖于SNRNP的功能，对正常运动轴突突的生长具有重要作用。此外，我们已经证明，第一个被发现的人SMA的修饰物，肌动蛋白结合蛋白3，可以修复由于低水平的SMN导致的斑马鱼的运动轴突缺陷。此外，斑马鱼SMN突变体严重降低了纤溶酶原3的水平。在这个方案中，我们将检验这一假说，即纤溶酶3通过非依赖于SnRNP的途径与SMN作用，以促进正常运动神经元的发育和功能。为了直接验证这一假说，我们将询问其他SMA表型是否被纤溶酶3拯救(目标1)。这包括运动神经元和NMJ的电生理学、NMJ的SV2蛋白和存活率。我们将通过进行结构/功能分析来确定与SMN相关的纤溶酶原3的功能(目标2)。对于这些实验，我们将使用纤溶酶原3和SMN突变体来定义相关的结构域。我们还将通过检测其他肌动蛋白结合蛋白来检验这一假设，即纤溶酶3在其修改SMA表型的能力方面是独一无二的。我们将检验这样的假设，即SMN与纤溶酶原3的相互作用独立于SMN的SnRNP功能(目标3)。最后，我们将使用实时成像来询问SMN和纤溶酶3蛋白在运动神经元中的定位，以及减少SMN是否会改变纤溶酶3的水平和/或细胞定位(目标4)。从这些目标获得的数据将直接解决SMN和PLATIN3之间的关系，因为它涉及SMA，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，它还将建立一种直接影响运动神经元功能的不依赖于SNRNP的SMN机制，从而极大地促进我们对该疾病的认识，并揭示新的治疗靶点。使用斑马鱼是一个优势，因为我们可以直接在体内分析我们开发的SMA模型中的运动神经元，并很容易产生新的转基因物质来提出特定的问题。这是这个模型系统的一个独特特征，因此这些研究具有很高的相关性，并将促进我们对SMN水平低如何导致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.