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

Reduced transport of TrkB receptor in BDNF-treated neurons. Primary hippocampal neurons were transfected with TrkB-GFP and imaged 7 d after PBS or PFF addition. Images were captured every 1 s for 3 min. Neurons were treated with BDNF for 30 min before imaging, and BDNF was included in the imaging media. BDNF-treated cultures: TrkB, number of particles analyzed, 453 for PBS and 416 for PFF (19 axons, PBS; 17 axons, PFF). (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. (B) Of the mobile particles, the percentages of anterograde and retrograde particles were quantified. There was a significant difference between the percentage of mobile particles between PBS- and PFF-treated groups for particles traveling in both the anterograde and retrograde directions. (C) There was no significant difference in the mean number of TrkB-GFP particles per 50 μm of axonal membrane. Neurons with α-synuclein inclusions showed a significant increase in (D) the number of pauses and (E) the number of reversals. (F) A Poisson regression on velocities binned with 10 cut points was statistically significant between PBS and PFF groups for anterograde TrkB-GFP velocities (Wald χ2 = 61.65, p < 0.0001). The odds ratio of 1.83, indicates that the PBS-treated group is 83% more likely to be in the higher-velocity group. Right, median and interquartile ranges of the anterograde velocities of the mobile TrkB-GFP particles in BDNF-treated neurons (Mann–Whitney test, p = NS). (G) For retrograde TrkB-GFP velocities in BDNF-treated neurons, there was a statistically significant difference between the PBS- and PFF-treated groups (Wald χ2 = 73.3, p < 0.0001). The odds ratio of 2.48 indicates that the PBS-treated group was 148% more likely to be in the higher-velocity group. The scatter plot on the right shows a striking decrease in the velocities of mobile vesicles, and the Mann–Whitney U test revealed a statistically significant decrease (p < 0.0001).
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4263445&req=5

Figure 6: Reduced transport of TrkB receptor in BDNF-treated neurons. Primary hippocampal neurons were transfected with TrkB-GFP and imaged 7 d after PBS or PFF addition. Images were captured every 1 s for 3 min. Neurons were treated with BDNF for 30 min before imaging, and BDNF was included in the imaging media. BDNF-treated cultures: TrkB, number of particles analyzed, 453 for PBS and 416 for PFF (19 axons, PBS; 17 axons, PFF). (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. (B) Of the mobile particles, the percentages of anterograde and retrograde particles were quantified. There was a significant difference between the percentage of mobile particles between PBS- and PFF-treated groups for particles traveling in both the anterograde and retrograde directions. (C) There was no significant difference in the mean number of TrkB-GFP particles per 50 μm of axonal membrane. Neurons with α-synuclein inclusions showed a significant increase in (D) the number of pauses and (E) the number of reversals. (F) A Poisson regression on velocities binned with 10 cut points was statistically significant between PBS and PFF groups for anterograde TrkB-GFP velocities (Wald χ2 = 61.65, p < 0.0001). The odds ratio of 1.83, indicates that the PBS-treated group is 83% more likely to be in the higher-velocity group. Right, median and interquartile ranges of the anterograde velocities of the mobile TrkB-GFP particles in BDNF-treated neurons (Mann–Whitney test, p = NS). (G) For retrograde TrkB-GFP velocities in BDNF-treated neurons, there was a statistically significant difference between the PBS- and PFF-treated groups (Wald χ2 = 73.3, p < 0.0001). The odds ratio of 2.48 indicates that the PBS-treated group was 148% more likely to be in the higher-velocity group. The scatter plot on the right shows a striking decrease in the velocities of mobile vesicles, and the Mann–Whitney U test revealed a statistically significant decrease (p < 0.0001).

Mentions: To determine whether the retrograde transport of other organelles are compromised, we analyzed the movement of TrkB receptors, since these receptors are known to undergo predominantly retrograde axonal transport in endosomes (Deinhardt et al., 2006; Zhou et al., 2012). The most striking change in velocities was found in analysis of retrograde TrkB-GFP transport in brain-derived neurotrophic factor (BDNF)–treated, α-syn inclusion–bearing neurons (Figure 6G and Supplemental Movie S3). Furthermore, the mobility of TrkB-GFP in both the anterograde and retrograde directions also showed significant reductions (Figure 6B), which was reflected by significant increases in the number of pauses (Figure 6D) and reversals (Figure 6E), although the number of particles per 50 μm is unchanged (Figure 6C). In BDNF-treated neurons bearing α-syn inclusions, the velocities of TrkB-GFP showed a significant shift into the lower-velocity bins relative to the control neurons (Figure 6G). In addition, analysis of the median velocities revealed a significant decrease in the neurons exposed to PFFs compared with PBS-treated controls. Images of TrkB-GFP in BDNF-treated neurons with axonal α-syn fibrillar aggregates showed that TrkB-GFP appeared to be localized to more discrete puncta compared with control axons (Figure 6A), possibly reflective of its reduced mobility. The kymographs provide a visual representation of the retrograde movement of TrkB-GFP in BDNF-treated neurons, and it can be seen in both the kymographs and overall quantitation that there were fewer mobile TrkB-GFP particles in neurons exposed to PFFs with α-syn aggregates.


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)

Reduced transport of TrkB receptor in BDNF-treated neurons. Primary hippocampal neurons were transfected with TrkB-GFP and imaged 7 d after PBS or PFF addition. Images were captured every 1 s for 3 min. Neurons were treated with BDNF for 30 min before imaging, and BDNF was included in the imaging media. BDNF-treated cultures: TrkB, number of particles analyzed, 453 for PBS and 416 for PFF (19 axons, PBS; 17 axons, PFF). (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. (B) Of the mobile particles, the percentages of anterograde and retrograde particles were quantified. There was a significant difference between the percentage of mobile particles between PBS- and PFF-treated groups for particles traveling in both the anterograde and retrograde directions. (C) There was no significant difference in the mean number of TrkB-GFP particles per 50 μm of axonal membrane. Neurons with α-synuclein inclusions showed a significant increase in (D) the number of pauses and (E) the number of reversals. (F) A Poisson regression on velocities binned with 10 cut points was statistically significant between PBS and PFF groups for anterograde TrkB-GFP velocities (Wald χ2 = 61.65, p < 0.0001). The odds ratio of 1.83, indicates that the PBS-treated group is 83% more likely to be in the higher-velocity group. Right, median and interquartile ranges of the anterograde velocities of the mobile TrkB-GFP particles in BDNF-treated neurons (Mann–Whitney test, p = NS). (G) For retrograde TrkB-GFP velocities in BDNF-treated neurons, there was a statistically significant difference between the PBS- and PFF-treated groups (Wald χ2 = 73.3, p < 0.0001). The odds ratio of 2.48 indicates that the PBS-treated group was 148% more likely to be in the higher-velocity group. The scatter plot on the right shows a striking decrease in the velocities of mobile vesicles, and the Mann–Whitney U test revealed a statistically significant decrease (p < 0.0001).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Reduced transport of TrkB receptor in BDNF-treated neurons. Primary hippocampal neurons were transfected with TrkB-GFP and imaged 7 d after PBS or PFF addition. Images were captured every 1 s for 3 min. Neurons were treated with BDNF for 30 min before imaging, and BDNF was included in the imaging media. BDNF-treated cultures: TrkB, number of particles analyzed, 453 for PBS and 416 for PFF (19 axons, PBS; 17 axons, PFF). (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. (B) Of the mobile particles, the percentages of anterograde and retrograde particles were quantified. There was a significant difference between the percentage of mobile particles between PBS- and PFF-treated groups for particles traveling in both the anterograde and retrograde directions. (C) There was no significant difference in the mean number of TrkB-GFP particles per 50 μm of axonal membrane. Neurons with α-synuclein inclusions showed a significant increase in (D) the number of pauses and (E) the number of reversals. (F) A Poisson regression on velocities binned with 10 cut points was statistically significant between PBS and PFF groups for anterograde TrkB-GFP velocities (Wald χ2 = 61.65, p < 0.0001). The odds ratio of 1.83, indicates that the PBS-treated group is 83% more likely to be in the higher-velocity group. Right, median and interquartile ranges of the anterograde velocities of the mobile TrkB-GFP particles in BDNF-treated neurons (Mann–Whitney test, p = NS). (G) For retrograde TrkB-GFP velocities in BDNF-treated neurons, there was a statistically significant difference between the PBS- and PFF-treated groups (Wald χ2 = 73.3, p < 0.0001). The odds ratio of 2.48 indicates that the PBS-treated group was 148% more likely to be in the higher-velocity group. The scatter plot on the right shows a striking decrease in the velocities of mobile vesicles, and the Mann–Whitney U test revealed a statistically significant decrease (p < 0.0001).
Mentions: To determine whether the retrograde transport of other organelles are compromised, we analyzed the movement of TrkB receptors, since these receptors are known to undergo predominantly retrograde axonal transport in endosomes (Deinhardt et al., 2006; Zhou et al., 2012). The most striking change in velocities was found in analysis of retrograde TrkB-GFP transport in brain-derived neurotrophic factor (BDNF)–treated, α-syn inclusion–bearing neurons (Figure 6G and Supplemental Movie S3). Furthermore, the mobility of TrkB-GFP in both the anterograde and retrograde directions also showed significant reductions (Figure 6B), which was reflected by significant increases in the number of pauses (Figure 6D) and reversals (Figure 6E), although the number of particles per 50 μm is unchanged (Figure 6C). In BDNF-treated neurons bearing α-syn inclusions, the velocities of TrkB-GFP showed a significant shift into the lower-velocity bins relative to the control neurons (Figure 6G). In addition, analysis of the median velocities revealed a significant decrease in the neurons exposed to PFFs compared with PBS-treated controls. Images of TrkB-GFP in BDNF-treated neurons with axonal α-syn fibrillar aggregates showed that TrkB-GFP appeared to be localized to more discrete puncta compared with control axons (Figure 6A), possibly reflective of its reduced mobility. The kymographs provide a visual representation of the retrograde movement of TrkB-GFP in BDNF-treated neurons, and it can be seen in both the kymographs and overall quantitation that there were fewer mobile TrkB-GFP particles in neurons exposed to PFFs with α-syn aggregates.

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