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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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Reduced retrograde transport of GFP-Rab7–positive late endosomes in neurons with α-syn aggregates. Primary hippocampal neurons were transfected with GFP-Rab7, treated with PBS or PFFs, and imaged 7 d later. Rab7, number of particles analyzed, 260 for PBS and 179 for PFF (19 axons, PBS; 24 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 no significant difference between the percentages of mobile particles between PBS- and PFF-treated groups. There was no significant difference in the mean number of GFP-Rab7 particles per 50 μm of axonal membrane (C) or number of pauses (D). There was, however, a significant increase in the number of reversals (E). (F) A Poisson regression on velocities binned with 10 cut points was not statistically significant between PBS and PFF groups for anterograde GFP-Rab7 velocities (Wald χ2 = 2.316, p = NS). Right, median and interquartile ranges of the velocities of the mobile GFP-Rab7 particles. The Mann–Whitney test did not produce significant differences for anterograde velocities. (G) For retrograde GFP-Rab7 velocities, there was a statistically significant difference between the PBS- and PFF-treated groups (Wald χ2 = 13.1, p < 0.001). The odds ratio of 1.30 indicates that the PBS-treated group is 30% more likely to be in the higher-velocity group. Right, median and interquartile ranges of the velocities of the mobile GFP-Rab7 particles. The Mann–Whitney test was significantly different for retrograde velocities.
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Figure 5: Reduced retrograde transport of GFP-Rab7–positive late endosomes in neurons with α-syn aggregates. Primary hippocampal neurons were transfected with GFP-Rab7, treated with PBS or PFFs, and imaged 7 d later. Rab7, number of particles analyzed, 260 for PBS and 179 for PFF (19 axons, PBS; 24 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 no significant difference between the percentages of mobile particles between PBS- and PFF-treated groups. There was no significant difference in the mean number of GFP-Rab7 particles per 50 μm of axonal membrane (C) or number of pauses (D). There was, however, a significant increase in the number of reversals (E). (F) A Poisson regression on velocities binned with 10 cut points was not statistically significant between PBS and PFF groups for anterograde GFP-Rab7 velocities (Wald χ2 = 2.316, p = NS). Right, median and interquartile ranges of the velocities of the mobile GFP-Rab7 particles. The Mann–Whitney test did not produce significant differences for anterograde velocities. (G) For retrograde GFP-Rab7 velocities, there was a statistically significant difference between the PBS- and PFF-treated groups (Wald χ2 = 13.1, p < 0.001). The odds ratio of 1.30 indicates that the PBS-treated group is 30% more likely to be in the higher-velocity group. Right, median and interquartile ranges of the velocities of the mobile GFP-Rab7 particles. The Mann–Whitney test was significantly different for retrograde velocities.

Mentions: Our ultrastructural images showed accumulations of membranous organelles approximately the size and morphology of endosomes near α-syn pathology in axons (see arrows in Figure 2, D and E). Rab7 localizes to endosomes and plays a major role in transport of these organelles along the axon (Deinhardt et al., 2006). The presence of abnormal α-syn fibrils in axons did not affect the overall mobility of GFP-Rab7 in axons (Figure 5B), the abundance of GFP-Rab7 particles along the membrane (Figure 5C), or the number of pauses (Figure 5D). In addition, a Poisson regression on binned velocities of anterograde traveling particles (Figure 5F) was not statistically significant between PBS and PFF groups However, for retrograde GFP-Rab7 velocities, there was a significant difference between the PBS- and PFF-treated groups (Figure 5G), with a significant reduction in the median velocities in the PFF-treated group. This reduction in velocities could be accounted for by the increase in number of reversals (Figure 5E). Thus the presence of α-syn pathology disrupts the axonal transport of Rab7-positive late endosomes in the retrograde direction.


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 retrograde transport of GFP-Rab7–positive late endosomes in neurons with α-syn aggregates. Primary hippocampal neurons were transfected with GFP-Rab7, treated with PBS or PFFs, and imaged 7 d later. Rab7, number of particles analyzed, 260 for PBS and 179 for PFF (19 axons, PBS; 24 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 no significant difference between the percentages of mobile particles between PBS- and PFF-treated groups. There was no significant difference in the mean number of GFP-Rab7 particles per 50 μm of axonal membrane (C) or number of pauses (D). There was, however, a significant increase in the number of reversals (E). (F) A Poisson regression on velocities binned with 10 cut points was not statistically significant between PBS and PFF groups for anterograde GFP-Rab7 velocities (Wald χ2 = 2.316, p = NS). Right, median and interquartile ranges of the velocities of the mobile GFP-Rab7 particles. The Mann–Whitney test did not produce significant differences for anterograde velocities. (G) For retrograde GFP-Rab7 velocities, there was a statistically significant difference between the PBS- and PFF-treated groups (Wald χ2 = 13.1, p < 0.001). The odds ratio of 1.30 indicates that the PBS-treated group is 30% more likely to be in the higher-velocity group. Right, median and interquartile ranges of the velocities of the mobile GFP-Rab7 particles. The Mann–Whitney test was significantly different for retrograde velocities.
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Figure 5: Reduced retrograde transport of GFP-Rab7–positive late endosomes in neurons with α-syn aggregates. Primary hippocampal neurons were transfected with GFP-Rab7, treated with PBS or PFFs, and imaged 7 d later. Rab7, number of particles analyzed, 260 for PBS and 179 for PFF (19 axons, PBS; 24 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 no significant difference between the percentages of mobile particles between PBS- and PFF-treated groups. There was no significant difference in the mean number of GFP-Rab7 particles per 50 μm of axonal membrane (C) or number of pauses (D). There was, however, a significant increase in the number of reversals (E). (F) A Poisson regression on velocities binned with 10 cut points was not statistically significant between PBS and PFF groups for anterograde GFP-Rab7 velocities (Wald χ2 = 2.316, p = NS). Right, median and interquartile ranges of the velocities of the mobile GFP-Rab7 particles. The Mann–Whitney test did not produce significant differences for anterograde velocities. (G) For retrograde GFP-Rab7 velocities, there was a statistically significant difference between the PBS- and PFF-treated groups (Wald χ2 = 13.1, p < 0.001). The odds ratio of 1.30 indicates that the PBS-treated group is 30% more likely to be in the higher-velocity group. Right, median and interquartile ranges of the velocities of the mobile GFP-Rab7 particles. The Mann–Whitney test was significantly different for retrograde velocities.
Mentions: Our ultrastructural images showed accumulations of membranous organelles approximately the size and morphology of endosomes near α-syn pathology in axons (see arrows in Figure 2, D and E). Rab7 localizes to endosomes and plays a major role in transport of these organelles along the axon (Deinhardt et al., 2006). The presence of abnormal α-syn fibrils in axons did not affect the overall mobility of GFP-Rab7 in axons (Figure 5B), the abundance of GFP-Rab7 particles along the membrane (Figure 5C), or the number of pauses (Figure 5D). In addition, a Poisson regression on binned velocities of anterograde traveling particles (Figure 5F) was not statistically significant between PBS and PFF groups However, for retrograde GFP-Rab7 velocities, there was a significant difference between the PBS- and PFF-treated groups (Figure 5G), with a significant reduction in the median velocities in the PFF-treated group. This reduction in velocities could be accounted for by the increase in number of reversals (Figure 5E). Thus the presence of α-syn pathology disrupts the axonal transport of Rab7-positive late endosomes in the retrograde direction.

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