Study on the cellular distribution and molecular function of ALS2, a product of the novel causative gene for famrlial ALS

本研究的目的是描述ALS 2的分子功能，ALS 2是一种隐性运动神经元疾病的致病基因产物，从而阐明ALS 2基因功能缺失突变导致神经元细胞功能障碍和死亡的分子机制。我们已经进行了以下四组分子、生物化学和细胞生物学研究：1）ALS 2亚细胞定位的免疫细胞化学分析，2）鉴定ALS 2的靶向小GTP酶，3）鉴定ALS 2相关/结合因子，和4）探索ALS 2介导的生物化学信号通路。我们在这里证明，ALS 2特异性结合到小GT3 Rab 5和功能作为一个GEF Rab 5。异位表达的ALS 2与Rab 5定位于早期内体区室，并刺激培养的神经元和非神经元细胞内体的扩大。携带MORN/VPS 9结构域的ALS 2的羧基末端不仅通过鸟嘌呤-核苷酸交换反应介导Rab 5的活化，而且介导ALS 2的内体定位，从而通过活化Rab 5-EEAI-SNARE分子途径导致内体融合。我们还表明，ALS 2通过其独特的C-末端区域形成一个嗜同性寡聚体。这种同源寡聚化对于细胞中Rab 5GEF活性和ALS 2介导的内体扩大至关重要。最后，我们已经确定了一种新的ALS 2同源基因，ALS 2 C-末端样（ALS 2CL），这是一种新的因子调节Rab 5介导的细胞内体动力学。两者合计，内体动力学的扰动，这是由ALS 2 Rab 5-GEF活性的丧失引起的，可能是一个关键的分子基础，神经元功能障碍和变性的一些运动神经元疾病引起的ALS 2突变。

The aim of this study was to delineate molecular function of ALS2, a causative gene product underlying a number of recessive motor neuron diseases, thereby clarifying the molecular mechanisms for neuronal cell dysfunction and death resulted from loss of function mutations in the ALS2 gene. We have carried out the following four sets of molecular, biochemical, and cell biological studies; 1) immunocytochemical analysis of the subcellular localization of ALS2, 2) identification of the target small GTPases for ALS2, 3) identification of the ALS2 associating/binding factors, and 4) exploration of the biochemical signal pathways mediated by ALS2. We here demonstrate that ALS2 specifically binds to small GTPase Rab5 and functions as a GEF for Rab5. Ectopically expressed ALS2 localizes with Rab5 onto early endosomal compartments, and stimulates the enlargement of endosomes in the cultured neuronal and non-neuronal cells. The carboxy-terminus of ALS2 carrying the MORN/VPS9 domain mediates not only the activation of Rab5 via a guanine-nucleotide exchanging reaction, but also the endosomal localization for ALS2, thereby leading to endosome fusions by activating the Rab5-EEAI-SNARE molecular pathway. We have also shown that ALS2 forms a homophilic oligomer through its distinct C-terminal regions. This homo-oligomerization is crucial for the Rab5GEF activity and the ALS2-mediated endosome enlargement in the cells. Lastly, we have identified a novel ALS2 homologous gene, ALS2 C-terminal like (ALS2CL), which is a novel factor modulating the Rab5-mediated endosome dynamics in the cells. Taken together, a perturbation of the endosomal dynamics, which is resulted from a loss of ALS2 Rab5-GEF activity, might be a key molecular basis underlying neuronal dysfunction and degeneration in a number of motor neuron diseases caused by the ALS2 mutations.