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Squalestatin alters the intracellular trafficking of a neurotoxic prion peptide.

Wilson R, Bate C, Boshuizen R, Williams A, Brewer J - BMC Neurosci (2007)

Bottom Line: Treatment with squalestatin reduced neuronal cholesterol levels and caused the redistribution of MoPrP105-132 out of lipid rafts.Squalestatin treatment also reduced the association between MoPrP105-132 and cPLA2/COX-1.As the observed shift in peptide trafficking was accompanied by increased cell survival these studies suggest that the neurotoxicity of this PrP peptide is dependent on trafficking to specific organelles where it activates specific signal transduction pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: 1Division of Immunology, Infection and Inflammation, Western Infirmary, University of Glasgow, G11 6NT, Glasgow. rkw1m@clinmed.gla.ac.uk

ABSTRACT

Background: Neurotoxic peptides derived from the protease-resistant core of the prion protein are used to model the pathogenesis of prion diseases. The current study characterised the ingestion, internalization and intracellular trafficking of a neurotoxic peptide containing amino acids 105-132 of the murine prion protein (MoPrP105-132) in neuroblastoma cells and primary cortical neurons.

Results: Fluorescence microscopy and cell fractionation techniques showed that MoPrP105-132 co-localised with lipid raft markers (cholera toxin and caveolin-1) and trafficked intracellularly within lipid rafts. This trafficking followed a non-classical endosomal pathway delivering peptide to the Golgi and ER, avoiding classical endosomal trafficking via early endosomes to lysosomes. Fluorescence resonance energy transfer analysis demonstrated close interactions of MoPrP105-132 with cytoplasmic phospholipase A2 (cPLA2) and cyclo-oxygenase-1 (COX-1), enzymes implicated in the neurotoxicity of prions. Treatment with squalestatin reduced neuronal cholesterol levels and caused the redistribution of MoPrP105-132 out of lipid rafts. In squalestatin-treated cells, MoPrP105-132 was rerouted away from the Golgi/ER into degradative lysosomes. Squalestatin treatment also reduced the association between MoPrP105-132 and cPLA2/COX-1.

Conclusion: As the observed shift in peptide trafficking was accompanied by increased cell survival these studies suggest that the neurotoxicity of this PrP peptide is dependent on trafficking to specific organelles where it activates specific signal transduction pathways.

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MoPrP105-132 avoids early endosomes and lysosomes. Neuroblastoma incubated with biotinylated MoPrP105-132 at 37°C, then fixed and stained with Texas Red-streptavidin (red) and TfR-FITC (green) or LAMP-1-FITC (green). Nuclei were revealed using Vectashield with DAPI (blue). (A) Co-localisation (yellow) was not observed between MoPrP105-132 and TfR or (B) between MoPrP105-132 and LAMP-1. (C) Co-localisation of biotinylated scrambled MoPrP105-132 (red) with LAMP-1 (green) was evident. Neuroblastoma cells were incubated with biotinylated MoPrP105-132 (red) for 90 minutes, then fixed and stained with GM130 (green) or Grp78 (green). (D) Co-localisation was observed between MoPrP105-132 and GM130 and (E) between MoPrP105-132 and Grp78. Scale bars, 5 μm. (F) Separation of endosomal compartments. Neuroblastoma cells were pulsed with iron dextran beads, before incubation with MoPrP105-132-FITC for 1 hour. Lysosomes (F3) were extracted from whole microsome extracts (MEx) using a magnetic column and early endosomes (F1) and intermediate fraction (F2) isolated using a density gradient. Western blot analysis revealed MoPrP105-132 was present in TfR negative and LAMP-1 negative fractions (F2).
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Figure 2: MoPrP105-132 avoids early endosomes and lysosomes. Neuroblastoma incubated with biotinylated MoPrP105-132 at 37°C, then fixed and stained with Texas Red-streptavidin (red) and TfR-FITC (green) or LAMP-1-FITC (green). Nuclei were revealed using Vectashield with DAPI (blue). (A) Co-localisation (yellow) was not observed between MoPrP105-132 and TfR or (B) between MoPrP105-132 and LAMP-1. (C) Co-localisation of biotinylated scrambled MoPrP105-132 (red) with LAMP-1 (green) was evident. Neuroblastoma cells were incubated with biotinylated MoPrP105-132 (red) for 90 minutes, then fixed and stained with GM130 (green) or Grp78 (green). (D) Co-localisation was observed between MoPrP105-132 and GM130 and (E) between MoPrP105-132 and Grp78. Scale bars, 5 μm. (F) Separation of endosomal compartments. Neuroblastoma cells were pulsed with iron dextran beads, before incubation with MoPrP105-132-FITC for 1 hour. Lysosomes (F3) were extracted from whole microsome extracts (MEx) using a magnetic column and early endosomes (F1) and intermediate fraction (F2) isolated using a density gradient. Western blot analysis revealed MoPrP105-132 was present in TfR negative and LAMP-1 negative fractions (F2).

Mentions: The intracellular trafficking pathway of MoPrP105-132 was further investigated by incubating biotin-conjugated MoPrP105-132 for 1 hour at 37°C before fixation. Fluorescence microscopy showed that only 6% ± 3 of MoPrP105-132 localised in TfR positive early endosomes (Figure 2A) and only 9% ± 2 of MoPrP105-132 co-localised within LAMP-1 positive lysosomes (Figure 2B). In contrast, 77% ± 6 of scrambled MoPrP105-132 was associated with LAMP-1 positive lysosomes (Figure 2C), confirming the sequence dependence of MoPrP105-132 localisation. Comparable results were obtained in primary cortical neurons (see additional files 2A, 2B). After 90 minutes at 37°C, approximately 41% ± 5 of MoPrP105-132 co-localised with GM130, a marker for cis-Golgi (Fig 2D) and 38% ± 4 with Grp78, which identifies the ER (Fig 2E). These findings are consistent with previous reports that that molecules internalised in lipid rafts traffic to the Golgi/ER [25]. The microscopy studies were complemented by an endosomal fractionation technique, as previously described [26]. In neuroblastoma cells incubated with MoPrP105-132 for 1 hour at 37°C, MoPrP105-132 was detected in the whole microsomal extract and the cell fraction known to be enriched for Golgi and ER compartments [27], but was not found in either TfR positive or LAMP-1 positive fractions (Figure 2F).


Squalestatin alters the intracellular trafficking of a neurotoxic prion peptide.

Wilson R, Bate C, Boshuizen R, Williams A, Brewer J - BMC Neurosci (2007)

MoPrP105-132 avoids early endosomes and lysosomes. Neuroblastoma incubated with biotinylated MoPrP105-132 at 37°C, then fixed and stained with Texas Red-streptavidin (red) and TfR-FITC (green) or LAMP-1-FITC (green). Nuclei were revealed using Vectashield with DAPI (blue). (A) Co-localisation (yellow) was not observed between MoPrP105-132 and TfR or (B) between MoPrP105-132 and LAMP-1. (C) Co-localisation of biotinylated scrambled MoPrP105-132 (red) with LAMP-1 (green) was evident. Neuroblastoma cells were incubated with biotinylated MoPrP105-132 (red) for 90 minutes, then fixed and stained with GM130 (green) or Grp78 (green). (D) Co-localisation was observed between MoPrP105-132 and GM130 and (E) between MoPrP105-132 and Grp78. Scale bars, 5 μm. (F) Separation of endosomal compartments. Neuroblastoma cells were pulsed with iron dextran beads, before incubation with MoPrP105-132-FITC for 1 hour. Lysosomes (F3) were extracted from whole microsome extracts (MEx) using a magnetic column and early endosomes (F1) and intermediate fraction (F2) isolated using a density gradient. Western blot analysis revealed MoPrP105-132 was present in TfR negative and LAMP-1 negative fractions (F2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: MoPrP105-132 avoids early endosomes and lysosomes. Neuroblastoma incubated with biotinylated MoPrP105-132 at 37°C, then fixed and stained with Texas Red-streptavidin (red) and TfR-FITC (green) or LAMP-1-FITC (green). Nuclei were revealed using Vectashield with DAPI (blue). (A) Co-localisation (yellow) was not observed between MoPrP105-132 and TfR or (B) between MoPrP105-132 and LAMP-1. (C) Co-localisation of biotinylated scrambled MoPrP105-132 (red) with LAMP-1 (green) was evident. Neuroblastoma cells were incubated with biotinylated MoPrP105-132 (red) for 90 minutes, then fixed and stained with GM130 (green) or Grp78 (green). (D) Co-localisation was observed between MoPrP105-132 and GM130 and (E) between MoPrP105-132 and Grp78. Scale bars, 5 μm. (F) Separation of endosomal compartments. Neuroblastoma cells were pulsed with iron dextran beads, before incubation with MoPrP105-132-FITC for 1 hour. Lysosomes (F3) were extracted from whole microsome extracts (MEx) using a magnetic column and early endosomes (F1) and intermediate fraction (F2) isolated using a density gradient. Western blot analysis revealed MoPrP105-132 was present in TfR negative and LAMP-1 negative fractions (F2).
Mentions: The intracellular trafficking pathway of MoPrP105-132 was further investigated by incubating biotin-conjugated MoPrP105-132 for 1 hour at 37°C before fixation. Fluorescence microscopy showed that only 6% ± 3 of MoPrP105-132 localised in TfR positive early endosomes (Figure 2A) and only 9% ± 2 of MoPrP105-132 co-localised within LAMP-1 positive lysosomes (Figure 2B). In contrast, 77% ± 6 of scrambled MoPrP105-132 was associated with LAMP-1 positive lysosomes (Figure 2C), confirming the sequence dependence of MoPrP105-132 localisation. Comparable results were obtained in primary cortical neurons (see additional files 2A, 2B). After 90 minutes at 37°C, approximately 41% ± 5 of MoPrP105-132 co-localised with GM130, a marker for cis-Golgi (Fig 2D) and 38% ± 4 with Grp78, which identifies the ER (Fig 2E). These findings are consistent with previous reports that that molecules internalised in lipid rafts traffic to the Golgi/ER [25]. The microscopy studies were complemented by an endosomal fractionation technique, as previously described [26]. In neuroblastoma cells incubated with MoPrP105-132 for 1 hour at 37°C, MoPrP105-132 was detected in the whole microsomal extract and the cell fraction known to be enriched for Golgi and ER compartments [27], but was not found in either TfR positive or LAMP-1 positive fractions (Figure 2F).

Bottom Line: Treatment with squalestatin reduced neuronal cholesterol levels and caused the redistribution of MoPrP105-132 out of lipid rafts.Squalestatin treatment also reduced the association between MoPrP105-132 and cPLA2/COX-1.As the observed shift in peptide trafficking was accompanied by increased cell survival these studies suggest that the neurotoxicity of this PrP peptide is dependent on trafficking to specific organelles where it activates specific signal transduction pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: 1Division of Immunology, Infection and Inflammation, Western Infirmary, University of Glasgow, G11 6NT, Glasgow. rkw1m@clinmed.gla.ac.uk

ABSTRACT

Background: Neurotoxic peptides derived from the protease-resistant core of the prion protein are used to model the pathogenesis of prion diseases. The current study characterised the ingestion, internalization and intracellular trafficking of a neurotoxic peptide containing amino acids 105-132 of the murine prion protein (MoPrP105-132) in neuroblastoma cells and primary cortical neurons.

Results: Fluorescence microscopy and cell fractionation techniques showed that MoPrP105-132 co-localised with lipid raft markers (cholera toxin and caveolin-1) and trafficked intracellularly within lipid rafts. This trafficking followed a non-classical endosomal pathway delivering peptide to the Golgi and ER, avoiding classical endosomal trafficking via early endosomes to lysosomes. Fluorescence resonance energy transfer analysis demonstrated close interactions of MoPrP105-132 with cytoplasmic phospholipase A2 (cPLA2) and cyclo-oxygenase-1 (COX-1), enzymes implicated in the neurotoxicity of prions. Treatment with squalestatin reduced neuronal cholesterol levels and caused the redistribution of MoPrP105-132 out of lipid rafts. In squalestatin-treated cells, MoPrP105-132 was rerouted away from the Golgi/ER into degradative lysosomes. Squalestatin treatment also reduced the association between MoPrP105-132 and cPLA2/COX-1.

Conclusion: As the observed shift in peptide trafficking was accompanied by increased cell survival these studies suggest that the neurotoxicity of this PrP peptide is dependent on trafficking to specific organelles where it activates specific signal transduction pathways.

Show MeSH
Related in: MedlinePlus