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, 25 (25), 4010-23

Formation of α-Synuclein Lewy Neurite-Like Aggregates in Axons Impedes the Transport of Distinct Endosomes

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Formation of α-Synuclein Lewy Neurite-Like Aggregates in Axons Impedes the Transport of Distinct Endosomes

Laura A Volpicelli-Daley et al. Mol Biol Cell.

Abstract

Aggregates of α-synuclein (α-syn) accumulate in neurons in Parkinson's disease and other synucleinopathies. These inclusions predominantly localize to axons even in the early stages of the disease, but their affect on axon function has remained unknown. Previously we established a model in which the addition of preformed α-syn fibrils to primary neurons seeds formation of insoluble α-syn inclusions built from endogenously expressed α-syn that closely recapitulate the neuropathological phenotypes of Lewy neurites found in human diseased brains. Here we show, using live-cell imaging, that immobile α-syn inclusions accumulate in axons from the recruitment of α-syn located on mobile α-syn-positive vesicles. 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. However, the α-syn aggregates impair the transport of Rab7 and TrkB receptor-containing endosomes, as well as autophagosomes. In addition, the TrkB receptor-associated signaling molecule pERK5 accumulates in α-syn aggregate-bearing neurons. Thus α-syn pathology impairs axonal transport of signaling and degradative organelles. These early effects of α-syn accumulations may predict points of intervention in the neurodegenerative process.

Figures

FIGURE 1:
FIGURE 1:
PFFs induce formation of α-syn-GFP aggregates that are immobile and grow by recruitment of mobile α-syn vesicular carriers. Primary hippocampal neurons from α-syn KO mice were transfected with α-syn-GFP and treated with PBS or PFFs and imaged 7 d later. Syn-GFP, number of particles analyzed, 484 for PBS and 387 for PFF (18 axons, PBS; 22 axons, PFF). (A) In PBS-treated neurons (top), α-syn-GFP localized to puncta corresponding to presynaptic terminals. α-Syn-GFP was soluble and thus extractable when fixed with paraformaldehyde containing 1% Triton X-100. PBS-treated neurons showed minimal p-α-syn immunoreactivity. Seven days after PFF treatment, α-syn-GFP localized to longer, more serpentine aggregates and small puncta. These aggregates were not extractable with 1% Triton X-100. The insoluble aggregates were extensively phosphorylated, as revealed by immunofluorescence with an antibody specific for p-Ser-129. Scale bar, 50 μm (low magnification), 20 μm (high magnification). (B) Neurons were cotransfected with α-syn-GFP and mRFP-ubiquitin and imaged 7 d post-PFF. Neurons were imaged with the spinning disk confocal, and mRFP-ubiquitin could be seen to coaccumulate with α-syn-GFP aggregates. (C) Live movies of α-syn-GFP were captured every 1 s for 3 min. Top two snapshots show α-syn-GFP in PBS- and PFF-treated neurons. Small axonal puncta were visible in the PBS-treated neurons, whereas longer α-syn-GFP serpentine aggregates were visible in axons from PFF treated neurons. Bottom two kymographs demonstrate that in PBS-treated neurons, there were some mobile and some immobile α-syn-GFP particles. In the PFF-treated neurons, there appeared to be more immobile particles that were larger in size. The mobile α-syn-GFP particles seem to approach an immobile α-syn-GFP aggregate but not bypass it. Scale bar, 10 μm. (D) Quantified percentage of mobile anterograde and retrograde particles. There was a significant decrease in the percentage of anterograde-moving mobile α-syn-GFP particles in neurons 7 d after PFF treatment. (E) Scatter plot of median velocities of mobile particles with interquartile range. The Mann–Whitney test did not reveal a significant difference between velocities. (F) Neurons expressing α-syn-GFP were treated with PFFs and imaged 7 d later. Images were captured every 3 min over 5 h. The larger aggregates were immobile (arrowheads). Smaller, mobile puncta can be seen to merge with the larger aggregates (arrows). At 240 min, a syn-GFP aggregate can be seen to break away from the larger aggregate, but it remerged at 290 min. Scale bar, 10 μm.
FIGURE 2:
FIGURE 2:
Ultrastructure of endosomes in α-syn inclusion–bearing axons. (A, D, E) Immuno-EM of HRP-labeled p-α-syn inclusions in axons. (B, C, F–I) Immunogold-labeled p-α-syn inclusions in axons. (A–C) Three examples of PBS-treated neurons. No HRP immunoreactivity was found in PBS-treated controls. (D–I) Six examples of neurons 14 d after PFF exposure. p-α-Syn aggregates were visible with HRP (asterisks). Immunogold labeling allowed visualization of the 10- to 15-nm filamentous α-syn inclusions. Note that the inclusions did not fill the entire axonal cytoplasm. Arrows point to examples of membrane organelles juxtaposed to the aggregates. Scale bar, 500 nm.
FIGURE 3:
FIGURE 3:
Normal transport of synaptophysin-GFP in neurons with α-syn aggregates. Primary hippocampal neurons were transfected with synaptophysin-GFP, treated with PBS or PFFs, and imaged 7 d later. Synaptophysin-GFP, number of particles analyzed, 2044 for PBS and 1944 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 the images were generated as visual representations of distance traveled over time. (B) Of the mobile particles, the percentages of anterograde and retrograde particles were also quantified. There were no significant differences between the PBS- and PFF-treated groups. (C) There were no significant differences in the mean number of synaptophysin-GFP particles per 50 μm of axonal membrane. In addition, there were no significant differences in the number of pauses (D) or reversals (E) between the two groups. A Poisson regression on velocities binned with 10 cut points was not statistically significant between PBS and PFF groups for anterograde synaptophysin-GFP velocities (Wald χ2 = 1.420, p = NS; F) or for retrograde synaptophysin-GFP velocities (Wald χ2 = 3.246, p = NS; G). (F, G) Right, median and interquartile ranges of the velocities of the mobile synaptophysin-GFP particles. The Mann–Whitney test did not produce significant differences for anterograde or retrograde velocities.
FIGURE 4:
FIGURE 4:
Normal transport of YFP-Mito in neurons with α-syn aggregates. Primary hippocampal neurons were transfected with YFP-Mito, treated with PBS or PFFs, and imaged 7 d later. YFP-Mito, number of particles analyzed, 162 for PBS and 244 for PFF (10 axons, PBS; 9 axons, PFF). (A) Top, images from movies captured every 1 s for 3 min; scale bar, 10 μm. Kymographs shown below the images 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 PBS- and PFF-treated groups. There were no significant differences in the mean number of YFP-Mito particles per 50 μm of axonal membrane (C), number of pauses (D), or number of reversals (E). A Poisson regression on velocities binned with 10 cut points was not statistically significant between PBS and PFF groups for anterograde YFP-Mito velocities (Wald χ2 = 0.713, p = NS; F) or for retrograde synaptophysin-GFP velocities (Wald χ2 = 2.886, p = NS; G). (F, G) Right, median and interquartile ranges of the velocities of the mobile synaptophysin-GFP particles. The Mann–Whitney test did not produce significant differences for anterograde or retrograde velocities.
FIGURE 5:
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.
FIGURE 6:
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).
FIGURE 7:
FIGURE 7:
Accumulation of endosomes and endosomal-associated signaling molecules in neurons with α-syn aggregates. (A) Neurons were cotransfected with TrkB-GFP and mRFP-Rab7 and imaged by confocal microscopy. Neurons were treated with BDNF for 30 min before imaging. TrkB-GFP appeared to localize at or near plasma membrane and intracellular puncta in neuronal soma of control neurons. In α-syn aggregate–bearing neurons, TrkB-GFP did not appear to localize to the plasma membrane but showed enlarged intracellular accumulations. Scale bar, 10 μm. (B) The percentage of colocalization of TrkB-GFP with mRFP-Rab7 late endosomes was significantly increased in PFF-treated neurons (t = 3.3, p = 0.004). Scale bar, 10 μm. (C) Neurons (in this case, not transfected with TrkB-GFP or other plasmid) were treated with PBS or PFFs and fixed 7 d later. Immunostaining was performed with antibodies to p-ERK5, p-α-syn, and NeuN as a marker for neuronal soma. Neurons were imaged by confocal microscopy. In control neurons, p-ERK5 showed minimal immunofluorescence. In α-syn aggregate–bearing neurons, p-ERK5 showed increased immunofluorescence and localized to perinuclear puncta juxtaposed to p-α-syn aggregates. Right, higher-magnification image shows that pERK5 puncta can be found juxtaposed to the α-syn aggregates. Scale bar, 10 μm.
FIGURE 8:
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.
FIGURE 9:
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).

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