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Regulation of endosomal motility and degradation by amyotrophic lateral sclerosis 2/alsin.

Lai C, Xie C, Shim H, Chandran J, Howell BW, Cai H - Mol Brain (2009)

Bottom Line: To directly examine the Rab5-mediated endosomal trafficking in ALS2(-/-) neurons, we introduced green fluorescent protein (GFP)-tagged Rab5 into cultured hippocampal neurons to monitor the morphology and motility of Rab5-associated early endosomes.Excessive accumulation of Rab5-positive vesicles was observed in ALS2(-/-) 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 ALS2(-/-) neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA. laichi@mail.nih.gov

ABSTRACT
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 (ALS2(-/-)) mice have been generated to model this disease. However, it remains elusive whether the Rab5-dependent endocytosis is altered in ALS2(-/-) neurons. To directly examine the Rab5-mediated endosomal trafficking in ALS2(-/-) 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 ALS2(-/-) neurons. Excessive accumulation of Rab5-positive vesicles was observed in ALS2(-/-) 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 ALS2(-/-) 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 ALS2(-/-) neurons may underlie the pathogenesis of motor neuron degeneration in ALS2 and related motor neuron diseases.

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Increased co-localization of LAMP1 with Rab5-associated vesicles in ALS2-/- neurons. (A-D) A lower (left) or higher magnification of Rab5-transfected wild-type (WT, A), or ALS2-/- (KO, B) neurons co-stained with LAMP1 (red) and GFP (green) antibodies. Co-localization of LAMP1 (red) and Rab5 (green) in WT and KO neurons was revealed by z-series stack images. Scale bars = 20 μm (left) and 10 μm (the rest) respectively. C. Quantitative analysis of co-localization of LAMP1 and Rab5 immuno-reactivity in WT (n = 4) and KO (n = 7) neurons. Error bars indicate SEM. * P < 0.05. D-E. In the soma, LAMP1 (red) and Rab5Q (green) were co-localized in either GFP-Rab5Q-transfected wild-type (WT, D) or ALS2-/- (KO, E) neurons. Scale bar = 20 μm.
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Figure 4: Increased co-localization of LAMP1 with Rab5-associated vesicles in ALS2-/- neurons. (A-D) A lower (left) or higher magnification of Rab5-transfected wild-type (WT, A), or ALS2-/- (KO, B) neurons co-stained with LAMP1 (red) and GFP (green) antibodies. Co-localization of LAMP1 (red) and Rab5 (green) in WT and KO neurons was revealed by z-series stack images. Scale bars = 20 μm (left) and 10 μm (the rest) respectively. C. Quantitative analysis of co-localization of LAMP1 and Rab5 immuno-reactivity in WT (n = 4) and KO (n = 7) neurons. Error bars indicate SEM. * P < 0.05. D-E. In the soma, LAMP1 (red) and Rab5Q (green) were co-localized in either GFP-Rab5Q-transfected wild-type (WT, D) or ALS2-/- (KO, E) neurons. Scale bar = 20 μm.

Mentions: Many large Rab5-positive endosomes resulting from fusion of early endosomes are subsequently targeted to lysosomes for degradation and constitutive activation of Rab5 enhances the conversion from endosomes to lysosomes [32]. To examine whether the large Rab5-associated endosomes in ALS2-/- hippocampal neurons are more likely to be targeted to the late endosome and lysosome-dependent degradation pathway, we stained GFP-Rab5 transfected ALS2-/- neurons with an antibody against lysosome membrane protein 1 (LAMP1), a marker for late endosomes/lysosomes [33,34]. We found a substantial increase of co-localization of Rab5 and LAMP1 immunofluorescence signals in the soma of ALS2-/-neurons (Figure 4B–C), which reflects an enhanced conversion of early endosomes to lysosomes for endosomal degradation. In contrast, only very sparse co-localization of Rab5 and LAMP1 staining was observed in wild-type neurons (Figure 4A and 4C, p < 0.05). Moreover, transfection of GFP-Rab5Q induced a similar increase of co-localization of LAMP1 and Rab5 in the soma of both wild-type and ALS2-/- neurons (Figure 4D–E), indicating that increased Rab5-mediated fusion of endosomes promotes the degradation of early endosomes.


Regulation of endosomal motility and degradation by amyotrophic lateral sclerosis 2/alsin.

Lai C, Xie C, Shim H, Chandran J, Howell BW, Cai H - Mol Brain (2009)

Increased co-localization of LAMP1 with Rab5-associated vesicles in ALS2-/- neurons. (A-D) A lower (left) or higher magnification of Rab5-transfected wild-type (WT, A), or ALS2-/- (KO, B) neurons co-stained with LAMP1 (red) and GFP (green) antibodies. Co-localization of LAMP1 (red) and Rab5 (green) in WT and KO neurons was revealed by z-series stack images. Scale bars = 20 μm (left) and 10 μm (the rest) respectively. C. Quantitative analysis of co-localization of LAMP1 and Rab5 immuno-reactivity in WT (n = 4) and KO (n = 7) neurons. Error bars indicate SEM. * P < 0.05. D-E. In the soma, LAMP1 (red) and Rab5Q (green) were co-localized in either GFP-Rab5Q-transfected wild-type (WT, D) or ALS2-/- (KO, E) neurons. Scale bar = 20 μm.
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Figure 4: Increased co-localization of LAMP1 with Rab5-associated vesicles in ALS2-/- neurons. (A-D) A lower (left) or higher magnification of Rab5-transfected wild-type (WT, A), or ALS2-/- (KO, B) neurons co-stained with LAMP1 (red) and GFP (green) antibodies. Co-localization of LAMP1 (red) and Rab5 (green) in WT and KO neurons was revealed by z-series stack images. Scale bars = 20 μm (left) and 10 μm (the rest) respectively. C. Quantitative analysis of co-localization of LAMP1 and Rab5 immuno-reactivity in WT (n = 4) and KO (n = 7) neurons. Error bars indicate SEM. * P < 0.05. D-E. In the soma, LAMP1 (red) and Rab5Q (green) were co-localized in either GFP-Rab5Q-transfected wild-type (WT, D) or ALS2-/- (KO, E) neurons. Scale bar = 20 μm.
Mentions: Many large Rab5-positive endosomes resulting from fusion of early endosomes are subsequently targeted to lysosomes for degradation and constitutive activation of Rab5 enhances the conversion from endosomes to lysosomes [32]. To examine whether the large Rab5-associated endosomes in ALS2-/- hippocampal neurons are more likely to be targeted to the late endosome and lysosome-dependent degradation pathway, we stained GFP-Rab5 transfected ALS2-/- neurons with an antibody against lysosome membrane protein 1 (LAMP1), a marker for late endosomes/lysosomes [33,34]. We found a substantial increase of co-localization of Rab5 and LAMP1 immunofluorescence signals in the soma of ALS2-/-neurons (Figure 4B–C), which reflects an enhanced conversion of early endosomes to lysosomes for endosomal degradation. In contrast, only very sparse co-localization of Rab5 and LAMP1 staining was observed in wild-type neurons (Figure 4A and 4C, p < 0.05). Moreover, transfection of GFP-Rab5Q induced a similar increase of co-localization of LAMP1 and Rab5 in the soma of both wild-type and ALS2-/- neurons (Figure 4D–E), indicating that increased Rab5-mediated fusion of endosomes promotes the degradation of early endosomes.

Bottom Line: To directly examine the Rab5-mediated endosomal trafficking in ALS2(-/-) neurons, we introduced green fluorescent protein (GFP)-tagged Rab5 into cultured hippocampal neurons to monitor the morphology and motility of Rab5-associated early endosomes.Excessive accumulation of Rab5-positive vesicles was observed in ALS2(-/-) 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 ALS2(-/-) neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA. laichi@mail.nih.gov

ABSTRACT
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 (ALS2(-/-)) mice have been generated to model this disease. However, it remains elusive whether the Rab5-dependent endocytosis is altered in ALS2(-/-) neurons. To directly examine the Rab5-mediated endosomal trafficking in ALS2(-/-) 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 ALS2(-/-) neurons. Excessive accumulation of Rab5-positive vesicles was observed in ALS2(-/-) 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 ALS2(-/-) 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 ALS2(-/-) neurons may underlie the pathogenesis of motor neuron degeneration in ALS2 and related motor neuron diseases.

Show MeSH
Related in: MedlinePlus