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

ALS 2基因的常染色体隐性突变导致运动功能障碍的临床谱，包括青少年发作的肌萎缩性侧索硬化症（ALS 2）、原发性侧索硬化症和遗传性痉挛性截瘫。多项体外生化和细胞生物学试验表明，alsin功能障碍通过Rab 5介导的机制影响内体运输。在本报告中，我们扩展了这些早期的研究，并揭示了一些新的功能，alsin在负调控内体运输和降解。我们发现，苜蓿蛋白缺乏导致增加的内体融合和降解，并降低内体运动。与此同时，我们证明，降解的内化AMPA受体在ALS 2-/-神经元显着增强。总之，我们的数据表明，在ALS 2-/-神经元内体过度降解可能有助于ALS 2和相关的运动神经元疾病的发病机制。 Alsin如何影响Rab 5介导的内吞作用是了解Alsin在运动神经元变性中功能的焦点。虽然目前的许多文献表明Alsin的DH/PH和VPS 9结构域与MORN基序是促进Rab 5在内吞途径中活性所必需的，但RLD结构域已被提出起调节作用。最近，两个运动神经元疾病相关的错义突变（C156 Y和G540 E）已被确定在RLD结构域内，表明该结构域在alsin的正常功能中也起着重要作用。RLD结构域由内部氨基酸重复序列形成的七叶螺旋桨组成。当仅在细胞系中表达Alsin的RLD结构域时，其显示类似于全长Alsin过表达的胞质分布。相比之下，缺乏RLD结构域的alsin的过表达导致内体定位。因此，Alsin的RLD结构域可能阻止Alsin与早期内体的结合，并作为Rab 5介导的内体融合的负调节剂。根据这一观点，我们发现alsin的缺失使更多的Rab 5暴露于上游激活因子和下游效应因子，并增强Rab 5介导的内体融合，导致在alsin缺陷神经元中观察到的增大的内体的积累增加。 Rab 5的激活也参与了内体沿微管晶格的沿着转运。两个先前的报告表明，上皮细胞生长因子（EGF）和脑源性神经元生长因子（BDNF）受体的内化减慢，在碱性磷酸酶缺陷的成纤维细胞和神经元。为了进一步研究alsin在内体转运中的作用，我们通过延时成像追踪了ALS 2-/-神经元中Rab 5阳性囊泡的运动。我们发现在ALS 2-/-神经元中Rab 5相关内体的运动性显著降低。由于基于微管的内体转运的破坏导致Rab 5相关内体的扩大，因此在ALS 2-/-神经元中观察到的扩大的Rab 5阳性囊泡的积累可能是由于基于微管的囊泡转运的功能障碍。然而，由于在Rab 5 Q转染的野生型神经元中也观察到类似的内体运动性降低，我们的数据认为，增加的内体融合可能在导致ALS 2-/-神经元内体运动缺陷中起主要作用。 靶向溶酶体依赖性降解途径的早期内体在迁移到细胞中心的同时尺寸不断增长。一致地，我们发现溶酶体标记物LAMP 1与Rab 5阳性囊泡在ALS 2-/-神经元的索马胞体中广泛共定位，表明内体降解增加。由于谷氨酸受体的内化和降解都是由Rab 5依赖性内吞途径介导的，因此我们定量了谷氨酸受体ALS 2-/-神经元的内化和降解。我们发现ALS 2-/-神经元中内化谷氨酸受体的降解增加。许多研究表明，钙不可渗透的含GluR 2的AMPA型谷氨酸受体复合物的下调有助于运动神经元变性。我们之前已经报道过，AMPA处理后，ALS 2-/-神经元中GluR 2的细胞/突触表面呈递选择性降低，这使得ALS 2-/-神经元更容易受到谷氨酸受体介导的毒性应激的影响。ALS 2-/-神经元的细胞/突触表面的GluR 2的选择性下调可能是由于内化的GluR 2的降解增加以及GluR 2的细胞内池靶向质膜的缺陷。

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.