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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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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.
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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.

Mentions: Previously we showed that high-density neuronal cultures develop α-syn aggregates resembling LNs in the majority of axons treated with exogenously applied α-syn PFFs. To visualize and monitor in real time the templated recruitment of endogenous α-syn into LN-like aggregates in axons, we expressed C-terminally tagged α-syn–green fluorescent protein (GFP) in primary neurons by transfection. For these experiments, we used hippocampal neurons generated from α-syn–knockout (KO) mice, so that all α-syn evaluated represented α-syn–GFP and not endogenous α-syn, which may coaggregate with α-syn–GFP. The expression of α-syn–GFP was approximately equivalent to expression of endogenous α-syn (Supplemental Figure S2A). In addition, phosphate-buffered saline (PBS)–treated and PFF-treated α-syn KO showed no abnormalities in overall neuritic morphology (Supplemental Figure S2B), and neurons lacking α-syn do not have significant changes in synaptic ultrastructure or physiology (Chandra et al., 2004). In control, PBS-treated neurons (i.e., no α-syn PFF exposure), α-syn–GFP (Figure 1, A and C, top left) localized to small puncta in neurites and showed diffuse localization in the neuronal soma. α-Syn–GFP was completely extracted by 1% Triton X-100, as shown previously for endogenous, soluble α-syn (Volpicelli-Daley et al., 2011). In PFF-treated neurons, α-syn–GFP aggregates were found in neurites and soma (Figure 1, A and B) that were morphologically similar to the inclusions formed by PFF-induced recruitment of endogenous (i.e., untagged) α-syn (Volpicelli-Daley et al., 2011). The PFF-induced α-syn–GFP aggregates remained after extraction with 1% Triton X-100 and thus were insoluble, a defining feature of pathological 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)

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.
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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.
Mentions: Previously we showed that high-density neuronal cultures develop α-syn aggregates resembling LNs in the majority of axons treated with exogenously applied α-syn PFFs. To visualize and monitor in real time the templated recruitment of endogenous α-syn into LN-like aggregates in axons, we expressed C-terminally tagged α-syn–green fluorescent protein (GFP) in primary neurons by transfection. For these experiments, we used hippocampal neurons generated from α-syn–knockout (KO) mice, so that all α-syn evaluated represented α-syn–GFP and not endogenous α-syn, which may coaggregate with α-syn–GFP. The expression of α-syn–GFP was approximately equivalent to expression of endogenous α-syn (Supplemental Figure S2A). In addition, phosphate-buffered saline (PBS)–treated and PFF-treated α-syn KO showed no abnormalities in overall neuritic morphology (Supplemental Figure S2B), and neurons lacking α-syn do not have significant changes in synaptic ultrastructure or physiology (Chandra et al., 2004). In control, PBS-treated neurons (i.e., no α-syn PFF exposure), α-syn–GFP (Figure 1, A and C, top left) localized to small puncta in neurites and showed diffuse localization in the neuronal soma. α-Syn–GFP was completely extracted by 1% Triton X-100, as shown previously for endogenous, soluble α-syn (Volpicelli-Daley et al., 2011). In PFF-treated neurons, α-syn–GFP aggregates were found in neurites and soma (Figure 1, A and B) that were morphologically similar to the inclusions formed by PFF-induced recruitment of endogenous (i.e., untagged) α-syn (Volpicelli-Daley et al., 2011). The PFF-induced α-syn–GFP aggregates remained after extraction with 1% Triton X-100 and thus were insoluble, a defining feature of pathological 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.

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Related in: MedlinePlus