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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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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.
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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.

Mentions: Because the large α-syn–GFP aggregates were relatively immobile, we conducted immuno–electron microscopy (EM) in control PBS- and PFF-treated neurons to determine the spatial relationship between p-α-syn aggregates, membrane organelles, and the cytoskeleton. These experiments were performed in wild-type primary neurons that express α-syn endogenously, since GFP fluorescence was unnecessary. In PBS-treated neurons, microtubules and mitochondria can be seen in axons with (Figure 2A) and without varicosities (Figure 2, B and C). p-α-Syn aggregates in the axons of neurons 14 d after PFF addition (day in vitro [DIV] 19) were visualized using horseradish peroxidase (HRP)–conjugated anti-mouse secondary antibody (Figure 2, D and E) and immunogold (Figure 2, F–I). We found that the α-syn aggregates did not fill the width of the axonal cytoplasm with (Figure 2, D–F) or without (Figure 2, G–I) varicosities, suggesting that transport of organelles along the axon could occur. For example, mitochondria could be seen adjacent to filamentous accumulations with enough distance between the aggregates and axonal membrane that they could bypass them. In addition, microtubules also appeared intact and thus could potentially support axonal transport (Volpicelli-Daley et al., 2011). These representative images demonstrate that LN-like α-syn aggregates do not necessarily fill the axonal cytoplasm and would be unlikely to sterically hinder or block axonal transport.


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: Because the large α-syn–GFP aggregates were relatively immobile, we conducted immuno–electron microscopy (EM) in control PBS- and PFF-treated neurons to determine the spatial relationship between p-α-syn aggregates, membrane organelles, and the cytoskeleton. These experiments were performed in wild-type primary neurons that express α-syn endogenously, since GFP fluorescence was unnecessary. In PBS-treated neurons, microtubules and mitochondria can be seen in axons with (Figure 2A) and without varicosities (Figure 2, B and C). p-α-Syn aggregates in the axons of neurons 14 d after PFF addition (day in vitro [DIV] 19) were visualized using horseradish peroxidase (HRP)–conjugated anti-mouse secondary antibody (Figure 2, D and E) and immunogold (Figure 2, F–I). We found that the α-syn aggregates did not fill the width of the axonal cytoplasm with (Figure 2, D–F) or without (Figure 2, G–I) varicosities, suggesting that transport of organelles along the axon could occur. For example, mitochondria could be seen adjacent to filamentous accumulations with enough distance between the aggregates and axonal membrane that they could bypass them. In addition, microtubules also appeared intact and thus could potentially support axonal transport (Volpicelli-Daley et al., 2011). These representative images demonstrate that LN-like α-syn aggregates do not necessarily fill the axonal cytoplasm and would be unlikely to sterically hinder or block axonal transport.

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