Neuronal RNA processing defects in ALS4 caused by SETX mutations

描述(由研究人员提供)：我们发现了一种少年型常染色体显性遗传性家族性肌萎缩侧索硬化症(ALS)的分子基础--Senataxin基因(SETX)突变。这种形式的肌萎缩侧索硬化症，被称为ALS4，本质上是一种纯运动系统疾病，其特征是四肢无力、严重的肌肉萎缩、锥体体征和缓慢而无情的疾病进展。Senataxin是一个由2677个氨基酸组成的蛋白质，它在C末端含有一个保守的超家族1，DNA/RNA解旋酶(约500个氨基酸)，强烈提示它在RNA加工中具有功能。虽然Senataxin的确切功能尚不清楚，但最令人信服的证据包括10多年来对酵母原基因sen1p的功能研究表明，这些蛋白质在几个RNA加工事件中发挥功能，包括RNAPol II转录终止Short&lt；500-NT转录本。事实证明，在神经退行性变中，RNA处理的改变已经得到了很好的证实，包括在脊髓性肌萎缩症(SMA)中丢失SMN，在脆性X智力低下(Fragile-X)中丢失FMRP。然而，最近发现TDP-43和FUS参与家族性和散发性ALS(FALS和SALS)，再次强调了改变的RNA处理在ALS中的作用。为了进一步研究SETX突变在ALS4中的作用，我们创建了一些重要的试剂，包括转基因(TG)和基因靶向小鼠。我们还将直接从组织中分离神经元RNA，以表征在ALS4和其他FALS/SALS形式中异常的RNA处理途径。为了进一步了解这种形式的FALS，我们最近建立了高水平表达R2136H突变体Senataxin的TG小鼠，该基因来自小鼠的PrP启动子(PrP)。这些R2136H转基因小鼠从6-8个月大的时候开始，通过旋转棒和力量测试显示出运动障碍。此外，我们还通过引入第二个ALS4相关突变L389S产生了SETX基因靶向小鼠。L389S Ki-小鼠表现出更严重但发病较晚的后肢瘫痪(约12-13个月龄)，存活率明显降低。因此，我们建议通过分子遗传分析、行为测试、组织病理学、免疫荧光和电子显微镜来彻底表征这些SETX小鼠模型。接下来，我们的目标是定义神经元表达的RNA转录子集(编码或非编码)，以响应ALS4相关的SETX突变。被确定为受SETX突变影响最大的转录本将通过定量RT-PCR进行验证，并将测试特定转录物类别中SETX在转录终止中的作用。在其他实验中，我们将测试在ALS4中被发现受到影响的不同RNA处理途径是否也可能在其他形式的运动神经元病中受到影响。 与公共卫生相关：已知感觉神经毒素(一种DNA/RNA解旋酶)的叙述性突变会导致两种严重的、衰弱的神经退行性疾病，ALS4和AOA2。这突显了感觉神经毒素功能在各种神经元群体生存中的重要性。我们的研究试图确定导致ALS4运动神经元丢失的分子机制，并进一步表征

DESCRIPTION (provided by investigator): We have discovered mutations in the senataxin gene (SETX) as the molecular basis of a juvenile-onset, autosomal dominant (AD) form of familial amyotrophic lateral sclerosis (ALS). This form of ALS, known as ALS4, is an essentially pure motor systems disorder characterized by limb weakness, severe muscle wasting, pyramidal signs, and slow relentless disease progression. Senataxin is a large 2677 amino acid protein which contains a conserved superfamily 1, DNA/RNA helicase (of ~ 500 amino acids) in the C-terminus, strongly suggesting a functional role in RNA processing. While the precise function of senataxin remains unknown, the most compelling evidence including more than 10 years of functional studies of the yeast ortholog sen1p suggest these proteins function in several RNA processing events including RNA Pol II transcription termination of short < 500-nt transcripts. As it turns out, altered RNA processing in neurodegeneration is well established including loss of SMN in spinal muscular atrophy (SMA) and loss of FMRP in fragile-X mental retardation. However, the recent discovery of the involvement of TDP-43 and FUS in familial and sporadic ALS (FALS and SALS) reemphasizes the role of altered RNA processing in ALS. To further study the role of SETX mutations in ALS4 we have created a number of important reagents including transgenic (Tg) and gene targeted mice. We will also isolate neuronal RNA directly from tissue to characterize RNA processing pathways that are aberrant in ALS4 and other FALS/SALS forms. To further our understanding of this form of FALS we recently established Tg mice expressing R2136H mutant senataxin at high levels from the mouse prion promoter (PrP). These R2136H Tg-mice display a motor deficit by rotarod and strength testing beginning at 6-8 months of age. In addition, we have generated Setx gene-targeted mice by introducing a second ALS4-associated mutation, L389S. The L389S KI-mice show a more severe but later onset hind-limb paralysis (~ 12-13 months age) with apparent reduced survival rates. Therefore, we propose to thoroughly characterize these SETX murine models via molecular genetic analysis, behavioral testing, histopathology, immunofluorescent and electron microscopy. Next, we aim to define the subset of neuron expressed RNA transcripts (coding or non-coding) misregulated in response to ALS4 associated SETX mutation. Transcripts identified as most affected by SETX mutation will be validated by quantitative RT-PCR and the role of SETX in transcription termination in specific transcript classes will be tested. In additional experiments we will test if the different RNA processing pathways found to be affected in ALS4 might also be affected in other forms of motor neuron disease. PUBLIC HEALTH RELEVANCE: Narrative Mutations in senataxin (a DNA/RNA helicase) are known to cause of two severe, debilitating neurodegenerative conditions, ALS4 and AOA2. This underscores the importance of senataxin function in the survival of various neuronal populations. Our studies seek to determine the molecular mechanisms leading to motor neuron loss in ALS4 and further characterize the