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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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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.
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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.

Mentions: In the soma of control neurons treated with BDNF, TrkB-GFP appeared to localize both to the plasma membrane and intracellular puncta that showed partial overlap with mRFP-Rab7 (Figure 7A). In α-syn aggregate–bearing neurons, TrkB-GFP showed accumulations in enlarged Rab7-positive puncta. Colocalization of TrkB-GFP with mRFP-Rab7 was increased in neurons with abnormal α-syn aggregates (Figure 7B), suggestive of trapping of TrkB receptors in endosomes such that they cannot be targeted to lysosome for degradation. Alternatively, the inability to recycle back to the plasma membrane could also account for this intracellular accumulation.


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: In the soma of control neurons treated with BDNF, TrkB-GFP appeared to localize both to the plasma membrane and intracellular puncta that showed partial overlap with mRFP-Rab7 (Figure 7A). In α-syn aggregate–bearing neurons, TrkB-GFP showed accumulations in enlarged Rab7-positive puncta. Colocalization of TrkB-GFP with mRFP-Rab7 was increased in neurons with abnormal α-syn aggregates (Figure 7B), suggestive of trapping of TrkB receptors in endosomes such that they cannot be targeted to lysosome for degradation. Alternatively, the inability to recycle back to the plasma membrane could also account for this intracellular accumulation.

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