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

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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.