Neuronal RNA processing defects in ALS4 caused by SETX mutations

描述（由研究者提供）：我们发现senataxin基因（SETX）突变是家族性肌萎缩侧索硬化症（ALS）青少年发病、常染色体显性（AD）形式的分子基础。这种形式的ALS，称为ALS 4，基本上是一种纯粹的运动系统障碍，其特征是肢体无力，严重的肌肉萎缩，锥体束征和缓慢无情的疾病进展。Senataxin是一个大的2677个氨基酸的蛋白质，其在C-末端包含保守的超家族1，DNA/RNA解旋酶（约500个氨基酸），强烈表明在RNA加工中的功能作用。虽然senataxin的确切功能仍然未知，但最令人信服的证据，包括对酵母直系同源物sen 1 p超过10年的功能研究，表明这些蛋白质在几种RNA加工事件中发挥作用，包括短< 500-nt转录物的RNA Pol II转录终止。事实证明，神经退行性变中RNA加工的改变已经得到了很好的证实，包括脊髓性肌萎缩症（SMA）中SMN的丢失和脆性X智力低下中FMRP的丢失。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加工在ALS中的作用。为了进一步研究SETX突变在ALS 4中的作用，我们已经创建了许多重要的试剂，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ALS 4和其他FALS/SALS形式中异常的RNA加工途径。为了进一步了解这种形式的FALS，我们最近建立了从小鼠朊病毒启动子（PrP）高水平表达R2136 H突变体senataxin的Tg小鼠。这些R2136 H Tg小鼠在6-8月龄开始通过旋转棒和力量测试显示出运动缺陷。此外，我们通过引入第二个ALS 4相关突变L389 S产生了Setx基因靶向小鼠。L389 S KI小鼠表现出更严重但较晚发作的后肢瘫痪（约12-13月龄），存活率明显降低。因此，我们建议通过分子遗传学分析、行为学测试、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转录本（编码或非编码）的子集，在响应ALS 4相关的SETX突变失调。将通过定量RT-PCR验证被鉴定为受SETX突变影响最大的转录物，并将测试SETX在特定转录物类别中的转录终止中的作用。在其他实验中，我们将测试在ALS 4中发现的不同RNA加工途径是否也可能在其他形式的运动神经元疾病中受到影响。 公共卫生相关性：已知senataxin（一种DNA/RNA解旋酶）的突变会导致两种严重的、使人衰弱的神经退行性疾病，ALS 4和AOA 2。这强调了senataxin功能在各种神经元群体存活中的重要性。我们的研究试图确定导致ALS 4运动神经元丢失的分子机制，并进一步表征ALS 4运动神经元的功能。

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