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

背景和具体目的：最近，一个名为ALS 2的基因与几个家族中的隐性形式的青少年ALS有关。ALS 2编码称为alsin的蛋白质，其与参与关键细胞功能（包括信号转导、细胞骨架调节和细胞内运输）的GT3调节蛋白（鸟嘌呤-核苷酸交换因子）具有同源性。ALS 2的突变导致编码蛋白的过早截短，这表明该疾病与ALS 2功能的丧失有关。因此，我们选择使用遗传方法来消除编码ALS 2的基因，以模拟这种罕见的ALS。项目1的具体目标是： 目的1：建立和鉴定ALS 2基因敲除小鼠; 目的2：明确ALS 2/alsin参与的生化途径和细胞内过程 进度报告： 对于目标1：我们通过破坏ALS 2等位基因的第二编码外显子来产生ALS 2敲除（ALS 2-/-）小鼠。虽然ALS 2-/-小鼠没有明显的发育异常，但它们在运动协调和运动学习方面表现出年龄依赖性缺陷。此外，ALS 2-/-小鼠在旷场和高架十字迷宫任务中表现出更高的焦虑反应。尽管他们未能概括与长达20个月的运动神经元疾病一致的临床或神经病理学表型，但与野生型对照相比，ALS 2-/-小鼠和源自这些小鼠的原代培养神经元对氧化应激更敏感。这些观察结果表明，ALS 2功能的丧失不足以在小鼠模型中引起主要的运动缺陷或运动神经元变性，但使神经元易于氧化应激。其中一些工作已经发表在Journal of Neuroscience（Cai et al.，2005年）。 对于Aim 2：为了鉴定ALS 2/alsin参与的细胞内分子途径，我们筛选了与ALS 2/alsin相互作用的蛋白质。我们发现，ALS 2与谷氨酸受体相互作用蛋白（GRIP 1），证明了从异源表达系统和小鼠大脑的提取物中的ALS 2和GRIP 1的免疫共沉淀。ALS 2还与GRIP 1共定位于原代培养的神经元中。GRIP 1参与突触中AMPA型谷氨酸受体2型的运输和稳定的调节（Song和Huganir，2002）。ALS 2在脑裂解物的突触膜部分中的富集连同GRIP 1在ALS 2-/-脊髓运动神经元中的改变的分布暗示ALS 2参与AMPA受体运输的调节。为了支持这一假设，我们发现，与野生型对照相比，ALS 2的缺失导致ALS 2-/-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可渗透的AMPA受体。此外，我们发现，与AMPA处理后的野生型神经元相比，在ALS 2-/-神经元中钙不可渗透的AMPA受体的细胞表面呈递显著降低。这些观察结果与我们在器官型脊髓切片培养和动物中发现的ALS 2-/-脊髓运动神经元对AMPA或红藻氨酸诱导的神经毒性的易感性增加相关。因此，我们提出，ALS 2的丢失增加了运动神经元对谷氨酸受体介导的兴奋性毒性的脆弱性，这是由于细胞/突触表面缺乏钙不可渗透的AMPA受体：突变ALS 2导致ALS的潜在致病机制。这项工作已经发表在Journal of Neuroscience（Lai et al.，2006年）。 未来发展方向： ALS 2 KO小鼠由地球仪的7个不同实验室生产。无一例出现明显的脊髓运动神经元变性。然而，最近的研究表明，在ALS 2的第4个编码外显子处存在替代翻译起始位点，该位点在许多这些ALS 2 KO小鼠系中保持完整。为了解决alsin的替代翻译变体是否补偿全长蛋白的损失，我们将产生一个新的ALS 2 KO小鼠系，通过基因捕获技术破坏外显子4和其他下游外显子的转录。我们将仔细检查这些新开发的ALS 2基因敲除小鼠的神经病理表型的运动行为，并进一步研究是否alsin的丢失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.