Spinal muscular atrophy: a novel role of SMN in axonal ribonucleoprotein complexe

描述(由申请人提供)：脊髓性肌萎缩症(SMA)是导致婴儿死亡的最常见的遗传原因。这种常染色体隐性神经肌肉疾病的特征是脊髓前角细胞退化，导致对称性肌肉无力和萎缩。发病机制尚不清楚，目前还没有治愈或治疗方法来阻止其进展。SMA是由编码运动神经元蛋白(SMN)生存的普遍表达基因的突变或缺失引起的。以前的工作主要集中在SMN在所有细胞类型中剪接体核糖核蛋白(RNP)复合体的有效组装和重塑中的重要作用。目前尚不清楚为什么运动神经元对低水平的SMN如此敏感，以及SMN缺乏是如何选择性地导致运动神经元细胞死亡的。在神经元中，SMN定位于胞核和突起中，并以动态颗粒的形式活跃地转运。这提示了SMN的一种新的神经元特异性功能，我们假设轴突SMN相关RNPs的低效可能与SMA有关。为了更好地了解SMN在运动轴突发育和维持中的生物学作用，我们建议研究SMN-RNP复合体的体内定位。我们将产生表达生物功能SMN的转基因小鼠，并将其与荧光蛋白报告基因融合，以研究含有SMN的RNP颗粒在发育过程中的动态定位。此前，我们已经证明，SMN缺陷的初级运动神经元的生长锥体和远端轴突含有降低的肌动蛋白mRNA和蛋白水平。新的数据表明，SMN缺乏会影响额外转录本的运输和/或局部翻译，我们将识别和研究这些受影响的转录本和蛋白质。这项提议将重点放在一种新的方法上，通过使用干细胞来源的运动神经元作为分隔培养的细胞体和轴突生长来克服原代细胞培养的局限性。我们将鉴定野生型和SMN缺陷型运动神经元轴突中转运的RNA，并比较野生型和SMN缺陷型运动神经元轴突中局部翻译蛋白的蛋白质组。我们的结果将阐明SMN在神经元过程中mRNAs的运输、稳定或局部翻译中的潜在作用。由于这些过程与生长锥运动和轴突引导有关，因此了解SMN是如何参与的，以及RNP复合体的传递缺陷如何可能触发或至少调节SMA的疾病过程，是非常有意义的。所提出的研究对于更广泛地理解mRNA定位在神经系统发育过程中的作用也是重要的。脊髓性肌萎缩症(SMA)是一种遗传性儿科疾病，由编码存活运动神经元蛋白(SMN)的基因突变或缺失引起，导致脊髓运动神经元迅速退化，是导致婴儿死亡的主要遗传原因。其发病机制尚不清楚，目前还没有治愈或治疗方法来阻止其进展。我们建议对SMN的轴突功能和SMA的潜在分子病理学进行研究，以揭示运动神经元功能和发育的基本方面，并提出治疗该疾病的策略。

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) represents the most common genetic cause of infant mortality. This autosomal recessive neuromuscular disorder is characterized by degeneration of the anterior horn cells of the spinal cord, leading to symmetrical muscle weakness and atrophy. The pathomechanism is still unclear and currently there is no cure or treatment available to stop its progression. SMA is caused by mutations or deletions in the ubiquitously expressed gene encoding the survival of motor neuron protein (SMN). Previous work has focused mainly on the essential role of SMN in the efficient assembly and remodeling of spliceosomal ribonucleoprotein (RNP) complexes in all cell types. It is still unknown why motor neurons are so specifically vulnerable to low levels of SMN and how SMN deficiency selectively causes motor neuron cell death. In neurons, SMN is found located in both the nucleus and in neurites and it is actively transported in the form of dynamic granules. This suggests a novel neuron-specific function of SMN and we hypothesize that an inefficiency of axonal SMN-associated RNPs may contribute to SMA. To better understand the biological role of SMN in the development and maintenance of motor axons, we propose to investigate the in vivo localization of SMN-RNP complexes. We will generate transgenic mice that express biological functional Smn fused to a fluorescent protein reporter to study the dynamic localization of Smn-containing RNP granules during development. Previously, we have shown that growth cones and distal axons of SMN deficient primary motor neurons contain reduced levels of ¿- actin mRNA and protein. New data suggest that Smn-deficiency affects transport and/or local translation of additional transcripts and we will identify and study these affected transcripts and proteins. This proposal will focus on a novel approach to overcome limitations of primary cell culture by using stem-cell derived motor neurons growing as compartmentalized cultures that separate cell bodies and axons. We will identify RNAs that are transported in axons of wild type and SMN-deficient motor neurons and we will also compare the proteome of locally translated proteins in the axons of wild type and Smn-deficient motor neurons. Our results will clarify a potential role of SMN in the transport, stability or local translation of mRNAs in neuronal processes. As these processes have been linked to growth cone motility and axon guidance, it is of big interest to find out how SMN may be involved and how defects in the delivery of RNP complexes may trigger or at least modulate the disease process in SMA. The proposed research is also important more broadly for understanding the function of mRNA localization during the development of the nervous system. Spinal muscular Atrophy (SMA) is an inherited pediatric disease caused by mutations or deletions in a gene encoding the survival motor neuron protein (SMN) that results in rapid degeneration of spinal cord motor neurons and is the leading genetic cause of infant mortality. Its pathomechanism is still unclear and currently there is no cure or treatment available to stop its progression. We propose studies on the axonal function of SMN and the underlying molecular pathology of SMA that have the potential to reveal essential aspects of motor neuron function and development and also to suggest therapeutic strategies for this disease.