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<p>Abstract</p> <p>Dysfunction of alsin, particularly its putative Rab5 guanine-nucleotide-exchange factor activity, has been linked to one form of juvenile onset recessive familial amyotrophic lateral sclerosis (ALS2). Multiple lines of alsin knockout (<it>ALS2</it>^-/-) mice have been generated to model this disease. However, it remains elusive whether the Rab5-dependent endocytosis is altered in <it>ALS2</it>^-/-neurons. To directly examine the Rab5-mediated endosomal trafficking in <it>ALS2</it>^-/-neurons, we introduced green fluorescent protein (GFP)-tagged Rab5 into cultured hippocampal neurons to monitor the morphology and motility of Rab5-associated early endosomes. Here we report that Rab5-mediated endocytosis was severely altered in <it>ALS2</it>^-/-neurons. Excessive accumulation of Rab5-positive vesicles was observed in <it>ALS2</it>^-/-neurons, which correlated with a significant reduction in endosomal motility and augmentation in endosomal conversion to lysosomes. Consequently, a significant increase in endosome/lysosome-dependent degradation of internalized glutamate receptors was observed in <it>ALS2</it>^-/-neurons. These phenotypes closely resembled the endosomal trafficking abnormalities induced by a constitutively active form of Rab5 in wild-type neurons. Therefore, our findings reveal a negatively regulatory mechanism of alsin in Rab5-mediated endosomal trafficking, suggesting that enhanced endosomal degradation in <it>ALS2</it>^-/-neurons may underlie the pathogenesis of motor neuron degeneration in ALS2 and related motor neuron diseases.</p>