Limits...
Formation of α-synuclein Lewy neurite-like aggregates in axons impedes the transport of distinct endosomes.

Volpicelli-Daley LA, Gamble KL, Schultheiss CE, Riddle DM, West AB, Lee VM - Mol. Biol. Cell (2014)

Bottom Line: Ultrastructural analyses and live imaging demonstrate that α-syn accumulations do not cause a generalized defect in axonal transport; the inclusions do not fill the axonal cytoplasm, disrupt the microtubule cytoskeleton, or affect the transport of synaptophysin or mitochondria.In addition, the TrkB receptor-associated signaling molecule pERK5 accumulates in α-syn aggregate-bearing neurons.These early effects of α-syn accumulations may predict points of intervention in the neurodegenerative process.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Behavioral Neurobiology, University of Alabama, Birmingham, Birmingham, AL 35294 Department of Pathology and Laboratory Medicine, Institute on Aging, and Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 volpicel@uab.edu.

Show MeSH

Related in: MedlinePlus

Abnormal endosome morphology and autophagosome acidification in α-syn aggregate–bearing neurons. Neurons were treated with PBS or PFFs and fixed 7 d later. (A) Neurons were immunostained with antibodies to the late endosome marker Lamp1, p-α-syn, and NeuN, a marker of neuronal nuclei. Laser-scanning confocal microscopy was performed. In control neurons, Lamp1 appears as small puncta, but in neurons with p-α-syn aggregates, Lamp1-enlarged vacuoles also were visible. Scale bar, 10 μm. (B) Transmission EM was performed on neurons 7 d post PBS or PFF treatment to visualize the ultrastructure of late endosomes (arrowhead). In control neurons, late endosomes show a characteristic limiting membrane, with internal vesicles of uniform size. In PFF-treated neurons, the late endosomes appeared enlarged, with abnormal internal vesicles. The boxes highlight filamentous α-syn inclusions. Scale bar, 500 nm. (C) Neurons were transfected with a mCherry-GFP-LC3 construct, treated with PBS or PFFs, and fixed 7 d later. In control neurons, the GFP-fluorescence is dim and diffuse, indicative of GFP quenching in acidified late endosomes/lysosomes. As expected, the mCherry fluorescence is bright, as this fluorophore does not quench in acidic environments. In PFF-treated neurons, the GFP fluorescence is bright and punctate, indicating that the fluorophore was not quenched and thus that the late endosomes/lysosomes are not acidified properly. Scale bar, 10 μm. (D) The relative size of Lamp1 puncta was quantified from the confocal images. The data did not pass a normality test. The scatter plot of the median and interquartile range reveals an increase in the larger Lamp1 endosomes in α-syn aggregate–bearing neurons. Mann–Whitney test, p < 0.0001. (E) Percentage overlap of LC3 with LAMP1 quantified from confocal images. The data fit a normal distribution. There was a statistically significant decrease in the overlap of LC3 autophagosomes with LAMP1 late endosomes/lysosomes (t(23) = 4.275, p = 0.0003).
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4263445&req=5

Figure 9: Abnormal endosome morphology and autophagosome acidification in α-syn aggregate–bearing neurons. Neurons were treated with PBS or PFFs and fixed 7 d later. (A) Neurons were immunostained with antibodies to the late endosome marker Lamp1, p-α-syn, and NeuN, a marker of neuronal nuclei. Laser-scanning confocal microscopy was performed. In control neurons, Lamp1 appears as small puncta, but in neurons with p-α-syn aggregates, Lamp1-enlarged vacuoles also were visible. Scale bar, 10 μm. (B) Transmission EM was performed on neurons 7 d post PBS or PFF treatment to visualize the ultrastructure of late endosomes (arrowhead). In control neurons, late endosomes show a characteristic limiting membrane, with internal vesicles of uniform size. In PFF-treated neurons, the late endosomes appeared enlarged, with abnormal internal vesicles. The boxes highlight filamentous α-syn inclusions. Scale bar, 500 nm. (C) Neurons were transfected with a mCherry-GFP-LC3 construct, treated with PBS or PFFs, and fixed 7 d later. In control neurons, the GFP-fluorescence is dim and diffuse, indicative of GFP quenching in acidified late endosomes/lysosomes. As expected, the mCherry fluorescence is bright, as this fluorophore does not quench in acidic environments. In PFF-treated neurons, the GFP fluorescence is bright and punctate, indicating that the fluorophore was not quenched and thus that the late endosomes/lysosomes are not acidified properly. Scale bar, 10 μm. (D) The relative size of Lamp1 puncta was quantified from the confocal images. The data did not pass a normality test. The scatter plot of the median and interquartile range reveals an increase in the larger Lamp1 endosomes in α-syn aggregate–bearing neurons. Mann–Whitney test, p < 0.0001. (E) Percentage overlap of LC3 with LAMP1 quantified from confocal images. The data fit a normal distribution. There was a statistically significant decrease in the overlap of LC3 autophagosomes with LAMP1 late endosomes/lysosomes (t(23) = 4.275, p = 0.0003).

Mentions: What is the fate of the autophagosomes once they reach the neuronal soma? We previously demonstrated an increased abundance of autophagy markers in HEK293 cells with α-syn aggregates and that Lamp1-positive late endosomes/lysosomes are dramatically enlarged, suggesting that autophagosome/lysosome fusion is likely impaired (Tanik et al., 2013). Autophagosomes can merge with Lamp1-positive late endosomes/lysosomes to form autophagolysosomes in which autophagy substrates are degraded (Xie and Klionsky, 2007). We confirmed that Lamp1-positive endosomes/lysosomes were also enlarged in primary neurons (Figure 9, A, B, and D). In PBS-treated neurons, Lamp1 appeared as small puncta throughout the cytoplasm. In neurons with axonal α-syn accumulations, small Lamp-1 puncta were still apparent, but a population of enlarged, Lamp1-positive endosomes appeared. The scatter plot presented in Figure 9D demonstrates an increase in the abundance of larger Lamp1-positive endosomes.


Formation of α-synuclein Lewy neurite-like aggregates in axons impedes the transport of distinct endosomes.

Volpicelli-Daley LA, Gamble KL, Schultheiss CE, Riddle DM, West AB, Lee VM - Mol. Biol. Cell (2014)

Abnormal endosome morphology and autophagosome acidification in α-syn aggregate–bearing neurons. Neurons were treated with PBS or PFFs and fixed 7 d later. (A) Neurons were immunostained with antibodies to the late endosome marker Lamp1, p-α-syn, and NeuN, a marker of neuronal nuclei. Laser-scanning confocal microscopy was performed. In control neurons, Lamp1 appears as small puncta, but in neurons with p-α-syn aggregates, Lamp1-enlarged vacuoles also were visible. Scale bar, 10 μm. (B) Transmission EM was performed on neurons 7 d post PBS or PFF treatment to visualize the ultrastructure of late endosomes (arrowhead). In control neurons, late endosomes show a characteristic limiting membrane, with internal vesicles of uniform size. In PFF-treated neurons, the late endosomes appeared enlarged, with abnormal internal vesicles. The boxes highlight filamentous α-syn inclusions. Scale bar, 500 nm. (C) Neurons were transfected with a mCherry-GFP-LC3 construct, treated with PBS or PFFs, and fixed 7 d later. In control neurons, the GFP-fluorescence is dim and diffuse, indicative of GFP quenching in acidified late endosomes/lysosomes. As expected, the mCherry fluorescence is bright, as this fluorophore does not quench in acidic environments. In PFF-treated neurons, the GFP fluorescence is bright and punctate, indicating that the fluorophore was not quenched and thus that the late endosomes/lysosomes are not acidified properly. Scale bar, 10 μm. (D) The relative size of Lamp1 puncta was quantified from the confocal images. The data did not pass a normality test. The scatter plot of the median and interquartile range reveals an increase in the larger Lamp1 endosomes in α-syn aggregate–bearing neurons. Mann–Whitney test, p < 0.0001. (E) Percentage overlap of LC3 with LAMP1 quantified from confocal images. The data fit a normal distribution. There was a statistically significant decrease in the overlap of LC3 autophagosomes with LAMP1 late endosomes/lysosomes (t(23) = 4.275, p = 0.0003).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4263445&req=5

Figure 9: Abnormal endosome morphology and autophagosome acidification in α-syn aggregate–bearing neurons. Neurons were treated with PBS or PFFs and fixed 7 d later. (A) Neurons were immunostained with antibodies to the late endosome marker Lamp1, p-α-syn, and NeuN, a marker of neuronal nuclei. Laser-scanning confocal microscopy was performed. In control neurons, Lamp1 appears as small puncta, but in neurons with p-α-syn aggregates, Lamp1-enlarged vacuoles also were visible. Scale bar, 10 μm. (B) Transmission EM was performed on neurons 7 d post PBS or PFF treatment to visualize the ultrastructure of late endosomes (arrowhead). In control neurons, late endosomes show a characteristic limiting membrane, with internal vesicles of uniform size. In PFF-treated neurons, the late endosomes appeared enlarged, with abnormal internal vesicles. The boxes highlight filamentous α-syn inclusions. Scale bar, 500 nm. (C) Neurons were transfected with a mCherry-GFP-LC3 construct, treated with PBS or PFFs, and fixed 7 d later. In control neurons, the GFP-fluorescence is dim and diffuse, indicative of GFP quenching in acidified late endosomes/lysosomes. As expected, the mCherry fluorescence is bright, as this fluorophore does not quench in acidic environments. In PFF-treated neurons, the GFP fluorescence is bright and punctate, indicating that the fluorophore was not quenched and thus that the late endosomes/lysosomes are not acidified properly. Scale bar, 10 μm. (D) The relative size of Lamp1 puncta was quantified from the confocal images. The data did not pass a normality test. The scatter plot of the median and interquartile range reveals an increase in the larger Lamp1 endosomes in α-syn aggregate–bearing neurons. Mann–Whitney test, p < 0.0001. (E) Percentage overlap of LC3 with LAMP1 quantified from confocal images. The data fit a normal distribution. There was a statistically significant decrease in the overlap of LC3 autophagosomes with LAMP1 late endosomes/lysosomes (t(23) = 4.275, p = 0.0003).
Mentions: What is the fate of the autophagosomes once they reach the neuronal soma? We previously demonstrated an increased abundance of autophagy markers in HEK293 cells with α-syn aggregates and that Lamp1-positive late endosomes/lysosomes are dramatically enlarged, suggesting that autophagosome/lysosome fusion is likely impaired (Tanik et al., 2013). Autophagosomes can merge with Lamp1-positive late endosomes/lysosomes to form autophagolysosomes in which autophagy substrates are degraded (Xie and Klionsky, 2007). We confirmed that Lamp1-positive endosomes/lysosomes were also enlarged in primary neurons (Figure 9, A, B, and D). In PBS-treated neurons, Lamp1 appeared as small puncta throughout the cytoplasm. In neurons with axonal α-syn accumulations, small Lamp-1 puncta were still apparent, but a population of enlarged, Lamp1-positive endosomes appeared. The scatter plot presented in Figure 9D demonstrates an increase in the abundance of larger Lamp1-positive endosomes.

Bottom Line: Ultrastructural analyses and live imaging demonstrate that α-syn accumulations do not cause a generalized defect in axonal transport; the inclusions do not fill the axonal cytoplasm, disrupt the microtubule cytoskeleton, or affect the transport of synaptophysin or mitochondria.In addition, the TrkB receptor-associated signaling molecule pERK5 accumulates in α-syn aggregate-bearing neurons.These early effects of α-syn accumulations may predict points of intervention in the neurodegenerative process.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Behavioral Neurobiology, University of Alabama, Birmingham, Birmingham, AL 35294 Department of Pathology and Laboratory Medicine, Institute on Aging, and Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 volpicel@uab.edu.

Show MeSH
Related in: MedlinePlus