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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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Squalestatin reroutes MoPrP105-132 into lysosomes. Neuroblastoma cells were incubated with 1 μM squalestatin for 24 hours before incubation with 30 μM MoPrP105-132-biotin for 90 minutes, then fixed and stained with Texas Red-streptavidin (red) and with anti-GM130, anti-Grp78, anti-TfR-FITC or anti-LAMP-1-FITC (all green) (A) Co-localisation between MoPrP105-132 and GM130, or (B) between MoPrP105-132 and Grp78. (C) In squalestatin treated cells co-localisation was observed between MoPrP105-132 and TfR and (D) between MoPrP105-132 and LAMP-1. Scale bars, 5 μm. (E) In squalestatin-treated cells, following fractionation of whole microsomal extracts (MEx) using iron dextran and density gradient centrifugation, MoPrP105-132 was detected in the lysosome fraction (F3).
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Figure 4: Squalestatin reroutes MoPrP105-132 into lysosomes. Neuroblastoma cells were incubated with 1 μM squalestatin for 24 hours before incubation with 30 μM MoPrP105-132-biotin for 90 minutes, then fixed and stained with Texas Red-streptavidin (red) and with anti-GM130, anti-Grp78, anti-TfR-FITC or anti-LAMP-1-FITC (all green) (A) Co-localisation between MoPrP105-132 and GM130, or (B) between MoPrP105-132 and Grp78. (C) In squalestatin treated cells co-localisation was observed between MoPrP105-132 and TfR and (D) between MoPrP105-132 and LAMP-1. Scale bars, 5 μm. (E) In squalestatin-treated cells, following fractionation of whole microsomal extracts (MEx) using iron dextran and density gradient centrifugation, MoPrP105-132 was detected in the lysosome fraction (F3).

Mentions: Further analysis of the trafficking of MoPrP105-132 in squalestatin-treated neuroblastoma cells showed that only 7% ± 2 of MoPrP105-132 co-localised with the Golgi (Figure 4A) and 3% ± 2 of MoPrP105-132 co-localised with the ER (Figure 4B), suggesting that in these cells MoPrP105-132 does not undergo retrograde trafficking. We found that 68% ± 8 of MoPrP105-132 co-localised with early endosomes (Figure 4C) and 40% ± 6 of MoPrP105-132 co-localised with lysosomes (Figure 4D). These observations suggest that in squalestatin-treated cells the MoPrP105-132 peptide is diverted away from retrograde transport to the Golgi/ER and directed into the classical endosome/lysosomal degradative pathway. A summary of the differences in the trafficking pathways of MoPrP105-132 in untreated and squalestatin-treated neuroblastoma cells is presented as Table 1. The re-routing of MoPrP105-132 to the classical endocytic pathway in squalestatin-treated cells was confirmed by analysis of endosomal fractions. In endosomal fractions isolated from squalestatin-treated neuroblastoma cells pulsed with MoPrP105-132 for 1 hour, MoPrP105-132 was detected in the LAMP-1 positive, TfR negative, lysosomal fraction (Figure 4E).


Squalestatin alters the intracellular trafficking of a neurotoxic prion peptide.

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

Squalestatin reroutes MoPrP105-132 into lysosomes. Neuroblastoma cells were incubated with 1 μM squalestatin for 24 hours before incubation with 30 μM MoPrP105-132-biotin for 90 minutes, then fixed and stained with Texas Red-streptavidin (red) and with anti-GM130, anti-Grp78, anti-TfR-FITC or anti-LAMP-1-FITC (all green) (A) Co-localisation between MoPrP105-132 and GM130, or (B) between MoPrP105-132 and Grp78. (C) In squalestatin treated cells co-localisation was observed between MoPrP105-132 and TfR and (D) between MoPrP105-132 and LAMP-1. Scale bars, 5 μm. (E) In squalestatin-treated cells, following fractionation of whole microsomal extracts (MEx) using iron dextran and density gradient centrifugation, MoPrP105-132 was detected in the lysosome fraction (F3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2131757&req=5

Figure 4: Squalestatin reroutes MoPrP105-132 into lysosomes. Neuroblastoma cells were incubated with 1 μM squalestatin for 24 hours before incubation with 30 μM MoPrP105-132-biotin for 90 minutes, then fixed and stained with Texas Red-streptavidin (red) and with anti-GM130, anti-Grp78, anti-TfR-FITC or anti-LAMP-1-FITC (all green) (A) Co-localisation between MoPrP105-132 and GM130, or (B) between MoPrP105-132 and Grp78. (C) In squalestatin treated cells co-localisation was observed between MoPrP105-132 and TfR and (D) between MoPrP105-132 and LAMP-1. Scale bars, 5 μm. (E) In squalestatin-treated cells, following fractionation of whole microsomal extracts (MEx) using iron dextran and density gradient centrifugation, MoPrP105-132 was detected in the lysosome fraction (F3).
Mentions: Further analysis of the trafficking of MoPrP105-132 in squalestatin-treated neuroblastoma cells showed that only 7% ± 2 of MoPrP105-132 co-localised with the Golgi (Figure 4A) and 3% ± 2 of MoPrP105-132 co-localised with the ER (Figure 4B), suggesting that in these cells MoPrP105-132 does not undergo retrograde trafficking. We found that 68% ± 8 of MoPrP105-132 co-localised with early endosomes (Figure 4C) and 40% ± 6 of MoPrP105-132 co-localised with lysosomes (Figure 4D). These observations suggest that in squalestatin-treated cells the MoPrP105-132 peptide is diverted away from retrograde transport to the Golgi/ER and directed into the classical endosome/lysosomal degradative pathway. A summary of the differences in the trafficking pathways of MoPrP105-132 in untreated and squalestatin-treated neuroblastoma cells is presented as Table 1. The re-routing of MoPrP105-132 to the classical endocytic pathway in squalestatin-treated cells was confirmed by analysis of endosomal fractions. In endosomal fractions isolated from squalestatin-treated neuroblastoma cells pulsed with MoPrP105-132 for 1 hour, MoPrP105-132 was detected in the LAMP-1 positive, TfR negative, lysosomal fraction (Figure 4E).

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