The Role of ALS2/Alsin in ALS and Motor Neuron Diseases

背景和具体目的：最近，在几个家系中，一个名为als2的基因被与青少年ALS的隐性形式联系在一起。ALS2编码一种名为alsin的蛋白质，它与GTP酶调节蛋白(鸟嘌呤核苷酸交换因子)具有同源性，后者参与关键的细胞功能，包括信号转导、细胞骨架调节和细胞内转运。Als2基因突变导致编码蛋白过早截断，表明该病与als2功能丧失有关。因此，我们选择使用遗传方法来去除编码als2的基因，以模拟这种罕见的ALS形式。我们对项目1的具体目标是： 目的1：建立并鉴定als2基因敲除小鼠； 目的2：确定als2/alsin参与的生化途径和细胞内过程 进度报告： 目标1：我们通过破坏als2等位基因的第二个编码外显子，产生了als2基因敲除(als2-/-)小鼠。尽管2-/-小鼠缺乏明显的发育异常，但它们在运动协调和运动学习方面表现出年龄依赖性的缺陷。此外，在旷野和高架迷宫任务中，2-/-小鼠表现出更高的焦虑反应。尽管他们未能总结出与20个月龄以下运动神经元疾病一致的临床或神经病理表型，但与野生型对照相比，2/-小鼠和来自这些小鼠的原代培养的神经元更容易受到氧化应激的影响。这些观察表明，als2功能的丧失不足以导致小鼠模型中的主要运动缺陷或运动神经元退化，但会使神经元容易受到氧化应激的影响。其中一些工作已经发表在《神经科学杂志》(蔡等人，2005年)上。 对于AIM2：为了确定als2/alsin参与的细胞内分子通路，我们筛选了与als2/alsin相互作用的蛋白质。我们发现als2与谷氨酸受体相互作用蛋白(GRIP1)有关，在异源表达系统和小鼠脑中的提取物中als2和GRIP1的免疫共沉淀证明了这一点。在原代培养的神经元中，ALS2也与GRIP1共定位。GRIP1参与调节突触中AMPA型谷氨酸受体2型的运输和稳定(Song和Huganir，2002)。ALS2在脑裂解液突触体膜部分的丰富，以及GRIP1在ALS2脊髓运动神经元中分布的改变，表明ALS2参与了AMPA受体运输的调节。为了支持这一假说，我们发现，与野生型对照相比，als2-/-神经元中AMPA受体介导的突触反应的反向整流增加(图3)，表明突触中缺乏钙不通透的AMPA受体。此外，我们还发现，与野生型神经元相比，经AMPA处理后，ALS2-/-神经元细胞表面不透钙的AMPA受体的呈现性显著降低。这些观察结果与我们发现在器官型脊髓切片培养和动物中，als2/-脊髓运动神经元对AMPA或海人酸致神经毒性的敏感性增加有关。因此，我们认为als2的缺失增加了运动神经元对谷氨酸受体介导的兴奋性毒性的易感性，这是由于细胞/突触表面缺乏钙不通透的AMPA受体：突变的als2导致的ALS的一个潜在的致病机制。这项研究发表在《神经科学杂志》(Lai等人，2006)上。 未来发展方向： 此外，全球7个不同的实验室已经培育出2只KO小鼠。它们都没有表现出任何明显的脊髓运动神经元变性。然而，最近的一项研究表明，在als2的第4个编码外显子上存在一个替代的翻译起始点，该外显子在许多als2 KO鼠系中保持完整。为了解决alsin的替代翻译变体是否补偿全长蛋白质的损失，我们将产生一个新的als2KO小鼠品系，该品系通过基因捕获技术扰乱外显子4和其他下游外显子的转录。我们将仔细研究这些新发育的ALS2 KO小鼠的神经病理表型的运动行为，并进一步研究alsin的丢失是否会导致运动神经元疾病。

Background and Specific Aims: Recently, a gene termed ALS2 was linked to a recessive form of juvenile ALS in several families. ALS2 encodes a protein termed alsin, which shares homology to GTPase regulatory proteins (guanine-nucleotide exchange factors) that participate in critical cellular functions including signal transduction, regulation of the cytoskeleton and intracellular trafficking. Mutations in ALS2 lead to a premature truncation of encoded protein, suggesting that the disease is associated with a loss of ALS2 function. Thus, we opted to use a genetic approach to ablate the gene encoding ALS2 to model this rare form of ALS. Our specific aims for project 1 are: Aim 1: To generate and characterize ALS2 knockout mice; Aim 2: To define the biochemical pathways and intracellular processes in which ALS2/alsin participates Progress Report: For Aim 1: we have generated ALS2 knockout (ALS2-/-) mice by disrupting the second coding exon of the ALS2 allele. While ALS2-/- mice lacked obvious developmental abnormalities, they exhibited age-dependent deficits in motor coordination and motor learning. Moreover, ALS2-/-mice showed a higher anxiety response in the open field and elevated plus maze tasks. Although they failed to recapitulate clinical or neuropathological phenotypes consistent with motor neuron disease up to 20 months of age, ALS2-/- mice and primary cultured neurons derived from these mice were more susceptible to oxidative stress compared to wild type controls. These observations suggest that loss of ALS2 function is insufficient to cause major motor deficits or motor neuron degeneration in a mouse model, but predisposes neurons to oxidative stress. Some of this work has been published in the Journal of Neuroscience (Cai et al., 2005). For Aim2: to identify the intracellular molecular pathways in which ALS2/alsin is involved, we screened proteins interacting with ALS2/alsin. We found that ALS2 is associated with the glutamate receptor interacting protein (GRIP1) as evidenced by co-immunoprecipitation of ALS2 and GRIP1 in extracts from heterologous expression systems and from mouse brain. ALS2 also co-localizes with GRIP1 in primary cultured neurons. GRIP1 is involved in the regulation of transportation and stabilization of AMPA type glutamate receptor type 2 in the synapses (Song and Huganir, 2002). The enrichment of ALS2 in the synaptic membrane fraction of the brain lysate in conjunction with altered distribution of GRIP1 in ALS2-/- spinal motor neurons implies that ALS2 is involved in the regulation of AMPA receptor trafficking. In support of this hypothesis, we found that loss of ALS2 leads to increased reverse rectification of AMPA receptor-mediated synaptic response in ALS2-/- neurons as compared to wild type controls (Fig. 3), indicating a lack of calcium-impermeable AMPA receptors in the synapses. Furthermore, we found that cell surface presentation of calcium-impermeable AMPA receptors was significantly decreased in ALS2-/- neurons as compared to wild type neurons following AMPA treatment. These observations are correlated with our finding of increased susceptibility of ALS2-/- spinal motor neurons to AMPA or kainic acid-induced neurotoxicity in organotypic spinal cord slice culture and in animals. Therefore, we propose that loss of ALS2 increases the vulnerability of motor neurons to glutamate receptor-mediated excitotoxicity due to a lack of calcium-impermeable AMPA receptors at the cell/synaptic surface: a potential pathogenic mechanism of ALS resulting from mutant ALS2. This work has been published in the Journal of Neuroscience (Lai et al., 2006). Future Directions: ALS2 KO mice have been generated by 7 different laboratories around the globe. None of them display any obvious spinal motor neuron degeneration. However, a recent work indicated the existence of an alternative translational initiation site at the 4th coding exon of ALS2, which remains intact in many of these ALS2 KO mouse lines. To address whether the alternative translational variants of alsin compensate for the loss of full-length protein, we will generate a new line of ALS2 KO mice that disrupts the transcription of exon 4 and other down-stream exons by the gene-trapping technique. We will closely examine the motor behavioral of neuropathological phenotypes of these newly developed ALS2 KO mice and to further investigate whether the loss of alsin causes motor neuron diseases.