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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.

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Altered transport of GFP-LC3 autophagosomes. Primary hippocampal neurons were transfected with GFP-LC3, treated with PBS or PFFs, and imaged 7 d later. (A) Top, images from movies captured every 1 s for 3 min; scale bar, 10 μm. Kymographs shown below were generated as visual representations of distance traveled over time and used to calculate average velocities as distance traveled over time. Two examples of kymographs generated from independent movies. LC3, number of particles analyzed, 571 for PBS and for 341 PFF (50 axons, PBS; 30 axons, PFF). (B) Percentages of mobile, anterograde and retrograde GFP-LC3 velocities. The Mann–Whitney test revealed significant decreases in the percentage of mobile GFP-LC3 particles in the PFF-exposed neurons. There was no significant difference in the number of GFP-LC3 particles per 50 μm of axonal membrane (C) in PBS- vs. PFF-treated neurons or in number of pauses (D) or number of reversals (E). (F) A Poisson regression on velocities binned with 10 cut points was statistically significant between PBS and PFF groups for anterograde GFP-LC3 velocities (Wald χ2 = 15.98, p < 0.0001). The scatter plot on the right shows the median and interquartile range of the velocities of the anterograde GFP-LC3 particles. The y-axis is broken to help visualize the entire range of velocities. The Mann–Whitney test revealed a statistically significant increase in the velocities of the anterograde GFP-LC3 particles. (G) For retrograde GFP-LC3 velocities, the Poisson regression revealed a statistically significant difference between the PBS- and PFF-treated groups, (Wald χ2 = 18.84, p < 0.0001). The scatter plot on the right shows the median and interquartile range of the velocities of the anterograde GFP-LC3 particles. The y-axis is broken to help visualize the entire range of velocities. The Mann–Whitney test revealed a statistically significant increase in the velocities of the anterograde GFP-LC3 particles.
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Figure 8: Altered transport of GFP-LC3 autophagosomes. Primary hippocampal neurons were transfected with GFP-LC3, treated with PBS or PFFs, and imaged 7 d later. (A) Top, images from movies captured every 1 s for 3 min; scale bar, 10 μm. Kymographs shown below were generated as visual representations of distance traveled over time and used to calculate average velocities as distance traveled over time. Two examples of kymographs generated from independent movies. LC3, number of particles analyzed, 571 for PBS and for 341 PFF (50 axons, PBS; 30 axons, PFF). (B) Percentages of mobile, anterograde and retrograde GFP-LC3 velocities. The Mann–Whitney test revealed significant decreases in the percentage of mobile GFP-LC3 particles in the PFF-exposed neurons. There was no significant difference in the number of GFP-LC3 particles per 50 μm of axonal membrane (C) in PBS- vs. PFF-treated neurons or in number of pauses (D) or number of reversals (E). (F) A Poisson regression on velocities binned with 10 cut points was statistically significant between PBS and PFF groups for anterograde GFP-LC3 velocities (Wald χ2 = 15.98, p < 0.0001). The scatter plot on the right shows the median and interquartile range of the velocities of the anterograde GFP-LC3 particles. The y-axis is broken to help visualize the entire range of velocities. The Mann–Whitney test revealed a statistically significant increase in the velocities of the anterograde GFP-LC3 particles. (G) For retrograde GFP-LC3 velocities, the Poisson regression revealed a statistically significant difference between the PBS- and PFF-treated groups, (Wald χ2 = 18.84, p < 0.0001). The scatter plot on the right shows the median and interquartile range of the velocities of the anterograde GFP-LC3 particles. The y-axis is broken to help visualize the entire range of velocities. The Mann–Whitney test revealed a statistically significant increase in the velocities of the anterograde GFP-LC3 particles.

Mentions: We also analyzed the transport of autophagosomes, since they are retrogradely transported (Lee et al., 2011; Maday et al., 2012), and we recently showed that the autophagy markers LC3II and p62 increase in expression and colocalize with PFF-induced α-syn aggregates, concomitant with impairment of autophagy (Tanik et al., 2013). Thus we asked whether the axonal transport of autophagosomes is altered using GFP-LC3 (Figure 8). There was a significant decrease in the overall mobility of GFP-LC3 in neurons harboring α-syn aggregates in both the anterograde and retrograde directions (Figure 8, A and B). There were no changes in the overall abundance of GFP-LC3 particles within the axons of α-syn aggregate–bearing neurons (Figure 8C) or in the number of pauses (Figure 8D) or reversals (Figure 8E). However, of the GFP-LC3 particles that remained mobile, there was an increase in their velocities in both the anterograde and retrograde directions (Figure 8, F and G). This can be seen in the kymographs (Figure 8A); there was an increase in immobile GFP-LC3 autophagosomes in neurons bearing α-syn aggregates, but the autophagosomes that do move, do so rapidly.


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)

Altered transport of GFP-LC3 autophagosomes. Primary hippocampal neurons were transfected with GFP-LC3, treated with PBS or PFFs, and imaged 7 d later. (A) Top, images from movies captured every 1 s for 3 min; scale bar, 10 μm. Kymographs shown below were generated as visual representations of distance traveled over time and used to calculate average velocities as distance traveled over time. Two examples of kymographs generated from independent movies. LC3, number of particles analyzed, 571 for PBS and for 341 PFF (50 axons, PBS; 30 axons, PFF). (B) Percentages of mobile, anterograde and retrograde GFP-LC3 velocities. The Mann–Whitney test revealed significant decreases in the percentage of mobile GFP-LC3 particles in the PFF-exposed neurons. There was no significant difference in the number of GFP-LC3 particles per 50 μm of axonal membrane (C) in PBS- vs. PFF-treated neurons or in number of pauses (D) or number of reversals (E). (F) A Poisson regression on velocities binned with 10 cut points was statistically significant between PBS and PFF groups for anterograde GFP-LC3 velocities (Wald χ2 = 15.98, p < 0.0001). The scatter plot on the right shows the median and interquartile range of the velocities of the anterograde GFP-LC3 particles. The y-axis is broken to help visualize the entire range of velocities. The Mann–Whitney test revealed a statistically significant increase in the velocities of the anterograde GFP-LC3 particles. (G) For retrograde GFP-LC3 velocities, the Poisson regression revealed a statistically significant difference between the PBS- and PFF-treated groups, (Wald χ2 = 18.84, p < 0.0001). The scatter plot on the right shows the median and interquartile range of the velocities of the anterograde GFP-LC3 particles. The y-axis is broken to help visualize the entire range of velocities. The Mann–Whitney test revealed a statistically significant increase in the velocities of the anterograde GFP-LC3 particles.
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Related In: Results  -  Collection

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Figure 8: Altered transport of GFP-LC3 autophagosomes. Primary hippocampal neurons were transfected with GFP-LC3, treated with PBS or PFFs, and imaged 7 d later. (A) Top, images from movies captured every 1 s for 3 min; scale bar, 10 μm. Kymographs shown below were generated as visual representations of distance traveled over time and used to calculate average velocities as distance traveled over time. Two examples of kymographs generated from independent movies. LC3, number of particles analyzed, 571 for PBS and for 341 PFF (50 axons, PBS; 30 axons, PFF). (B) Percentages of mobile, anterograde and retrograde GFP-LC3 velocities. The Mann–Whitney test revealed significant decreases in the percentage of mobile GFP-LC3 particles in the PFF-exposed neurons. There was no significant difference in the number of GFP-LC3 particles per 50 μm of axonal membrane (C) in PBS- vs. PFF-treated neurons or in number of pauses (D) or number of reversals (E). (F) A Poisson regression on velocities binned with 10 cut points was statistically significant between PBS and PFF groups for anterograde GFP-LC3 velocities (Wald χ2 = 15.98, p < 0.0001). The scatter plot on the right shows the median and interquartile range of the velocities of the anterograde GFP-LC3 particles. The y-axis is broken to help visualize the entire range of velocities. The Mann–Whitney test revealed a statistically significant increase in the velocities of the anterograde GFP-LC3 particles. (G) For retrograde GFP-LC3 velocities, the Poisson regression revealed a statistically significant difference between the PBS- and PFF-treated groups, (Wald χ2 = 18.84, p < 0.0001). The scatter plot on the right shows the median and interquartile range of the velocities of the anterograde GFP-LC3 particles. The y-axis is broken to help visualize the entire range of velocities. The Mann–Whitney test revealed a statistically significant increase in the velocities of the anterograde GFP-LC3 particles.
Mentions: We also analyzed the transport of autophagosomes, since they are retrogradely transported (Lee et al., 2011; Maday et al., 2012), and we recently showed that the autophagy markers LC3II and p62 increase in expression and colocalize with PFF-induced α-syn aggregates, concomitant with impairment of autophagy (Tanik et al., 2013). Thus we asked whether the axonal transport of autophagosomes is altered using GFP-LC3 (Figure 8). There was a significant decrease in the overall mobility of GFP-LC3 in neurons harboring α-syn aggregates in both the anterograde and retrograde directions (Figure 8, A and B). There were no changes in the overall abundance of GFP-LC3 particles within the axons of α-syn aggregate–bearing neurons (Figure 8C) or in the number of pauses (Figure 8D) or reversals (Figure 8E). However, of the GFP-LC3 particles that remained mobile, there was an increase in their velocities in both the anterograde and retrograde directions (Figure 8, F and G). This can be seen in the kymographs (Figure 8A); there was an increase in immobile GFP-LC3 autophagosomes in neurons bearing α-syn aggregates, but the autophagosomes that do move, do so rapidly.

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.

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Related in: MedlinePlus