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

Als2基因的常染色体隐性突变导致一系列临床运动功能障碍，包括青少年起病的肌萎缩侧索硬化症(Als2)、原发性侧索硬化症和遗传性痉挛截瘫。多种体外生化和细胞生物学分析表明，alsin功能障碍通过Rab5介导的机制影响内体转运。在这份报告中，我们扩展了这些早期的研究，揭示了alsin在负向调节内体运输和降解方面的一些新功能。我们发现，Alsin缺乏导致内体融合和降解增加，并降低内体运动性。同时，我们发现内化的AMPA受体的降解在ALS2-/-神经元中显著增强。综上所述，我们的数据表明als2-/-神经元的过度内体降解可能参与了als2及相关运动神经元疾病的发病机制。 Alsin如何影响Rab5介导的内吞作用是了解alsin在运动神经元退行性变中作用的一个焦点。虽然目前的许多文献表明，带有alsin Morn基序的DH/PH和VPS9结构域对于促进Rab5在内吞途径中的活性是必需的，但RLD结构域具有调节作用。最近，在RLD结构域中发现了两个运动神经元疾病相关错义突变(C156Y和G540E)，表明该结构域在alsin的正常功能中也发挥着重要作用。RLD结构域由一个由内部氨基酸重复形成的七个叶片的螺旋桨组成。当ALIN的RLD结构域在细胞系中表达时，其胞浆分布类似于全长ALIN的过度表达。相反，缺乏RLD结构域的alsin的过度表达会导致内体定位。因此，alsin的RLD结构域可能阻止alsin与早期内小体的结合，并对Rab5介导的内体融合起负调控作用。与这一观点一致，我们发现alsin的丢失使更多的Rab5暴露于上游激活剂和下游效应器，并促进了Rab5介导的内体融合，导致扩大的内小体积累增加，正如在alsin缺乏的神经元中观察到的那样。 Rab5的激活也参与了沿微管格子的内体运输。先前的两篇报道表明，在碱性蛋白缺乏的成纤维细胞和神经元中，上皮生长因子(EGF)和脑源性神经生长因子(BDNF)受体的内化速度减慢。为了进一步研究alsin在内体转运中的作用，我们用时间推移成像技术追踪了als2-/-神经元中Rab5阳性小泡的运动。我们发现，在ALS2-/-神经元中，Rab5相关内小体的运动性显著降低。由于基于微管的内小体转运的中断导致Rab5相关的内小体增大，因此在ALS2-/-神经元中观察到的增大的Rab5阳性小泡的聚集可能是由于基于微管的囊泡运输功能障碍所致。然而，由于在转Rab5Q基因的野生型神经元中也观察到了类似的内体运动减少，我们的数据认为内体融合增加可能是导致als2-/-神经元内体运动缺陷的主要原因。 早期以溶酶体依赖的降解途径为目标的内小体在向细胞中心迁移的同时保持大小增长。我们一致地在als2-/-神经元的胞体中发现溶酶体标记LAMP1和Rab5阳性小泡的广泛共存，这表明内体降解增加。由于谷氨酸受体的内化和降解都是由Rab5依赖的内吞途径介导的，因此我们对谷氨酸受体和2-/-神经元的内化和降解进行了量化。我们发现ALS2-/-神经元内化的谷氨酸受体的降解增加。许多研究表明，含有AMPA型谷氨酸受体复合体的钙不透性谷氨酸受体复合体的下调参与了运动神经元的变性。我们先前已经报道，经AMPA处理后，als2-/-神经元中GluR2的细胞/突触表面呈现选择性地减少，这使得als2-/-神经元更容易受到谷氨酸受体介导的毒性应激的影响。ALS2-/-神经元细胞/突触表面GluR2的选择性下调可能是由于内化的GluR2降解增加以及GluR2不能将细胞内的GluR2池靶向质膜所致。

Autosomal recessive mutations in the ALS2 gene lead to a clinical spectrum of motor dysfunction including juvenile onset amyotrophic lateral sclerosis (ALS2), primary lateral sclerosis and hereditary spastic paraplegia. Multiple in vitro biochemical and cell biology assays suggest that alsin dysfunction affects endosomal trafficking through Rab5-mediated mechanism. In this report we extended those early studies and revealed some novel functions of alsin in negatively regulating endosomal trafficking and degradation. We found that alsin-deficiency led to increased endosomal fusion and degradation, and decreased endosomal motility. Concomitantly, we demonstrate that the degradation of internalized AMPA receptors was significantly enhanced in ALS2-/- neurons. Taken together, our data indicate that excessive endosomal degradation in ALS2-/- neurons may contribute to the pathogenesis of ALS2 and related motor neuron diseases. How alsin affects the Rab5-mediated endocytosis is a focal point in understanding the function of alsin in motor neuron degeneration.While much of the current literature suggests that the DH/PH and VPS9 domains with MORN motifs of alsin are necessary to promote Rab5 activity in the endocytic pathway, a regulatory role for the RLD domain has been proposed. Recently, two motor neuron disease-related missense mutations (C156Y and G540E) have been identified within the RLD domain, indicating that this domain also plays an important role in the normal function of alsin. The RLD domain consists of a seven-bladed propeller formed from internal amino acid repeats. When only the RLD domain of alsin is expressed in cell lines, it displays a cytosolic distribution similar to that of over-expression of full-length alsin. By contrast, over-expression of alsin lacking the RLD domain results in endosomal localization. Therefore, the RLD domain of alsin may prevent the association of alsin with early endosomes and act as a negative regulator of Rab5 mediated endosomal fusion. In line with this notion, we found loss of alsin exposed more Rab5 to the upstream activators and downstream effectors, and enhance Rab5-mediated endosomal fusion, resulting in an increased accumulation of enlarged endosomes as observed in alsin-deficient neurons. Activation of Rab5 is also involved in endosomal transport along the microtubule lattice. Two previous reports show that the internalization of epithelium growth factor (EGF) and brain derived neuron growth factor (BDNF) receptors was slowed down in alsin-deficient fibroblasts and neurons. To further investigate the role of alsin in endosomal transport, we traced the movement of Rab5-positive vesicles by time lapse imaging in ALS2-/- neurons. We found that the motility of Rab5-associated endosomes was significantly decreased in ALS2-/- neurons. Since disruption of microtubule-based endosomal transport causes the enlargement of Rab5-associated endosomes, it is possible that the accumulation of enlarged Rab5-positive vesicles observed in ALS2-/- neurons is due to the dysfunction of microtubule-based vesicle transport. However, since a similar reduction of endosomal motility was also observed in Rab5Q-transfected wild-type neurons, our data argue that the increased endosomal fusion may play a main role in causing the endosomal motility defects in ALS2-/- neurons. Early endosomes targeted for the lysosome-dependent degradation pathway keep growing in size while migrating to the cell center. Consistently, we found extensive co-localization of lysosome marker LAMP1 with Rab5-positive vesicles in the soma of ALS2-/- neurons, suggesting an increase of endosomal degradation. Since the internalization and degradation of glutamate receptors are both mediated by the Rab5-dependent endocytic pathway, we quantified the internalization and degradation of glutamate receptors ALS2-/-neurons. We found an increased degradation of internalized glutamate receptors in ALS2-/- neurons. Many studies have suggested that down-regulation of calcium-impermeable GluR2-containing AMPA type glutamate receptor complexes contributes to motor neuron degeneration. We have reported previously that the cell/synaptic surface presentation of GluR2 is selectively decreased in ALS2-/- neurons following AMPA treatment, which renders ALS2-/- neurons more vulnerable to glutamate receptor-mediated toxic stress. The selective down-regulation of GluR2 at cell/synaptic surface of ALS2-/- neurons may likely result from the increased degradation of internalized GluR2 and the deficiency in targeting the intracellular pool of GluR2 to the plasma membrane.