Limits...
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.

Show MeSH

Related in: MedlinePlus

MoPrP105-132 associates with cPLA2 and COX-1. Neuroblastoma cells were incubated with 30 μM MoPrP105-132-biotin for 5 minutes or 20 minutes at 37°C then fixed and stained with Texas Red-streptavidin (red), anti-cPLA2-FITC (green) or anti-COX-1-FITC (green). Nuclei were revealed using Vectashield with DAPI (blue). (A) Co-localisation (yellow) between MoPrP105-132 (red) and cPLA2 (green) after 5 minutes, but not after 20 minutes (B). (C) Co-localisation (yellow) between MoPrP105-132 (red) and anti-COX-1-FITC (green) after 5 minutes, but not after 20 minutes (D). Scale bars, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2131757&req=5

Figure 5: MoPrP105-132 associates with cPLA2 and COX-1. Neuroblastoma cells were incubated with 30 μM MoPrP105-132-biotin for 5 minutes or 20 minutes at 37°C then fixed and stained with Texas Red-streptavidin (red), anti-cPLA2-FITC (green) or anti-COX-1-FITC (green). Nuclei were revealed using Vectashield with DAPI (blue). (A) Co-localisation (yellow) between MoPrP105-132 (red) and cPLA2 (green) after 5 minutes, but not after 20 minutes (B). (C) Co-localisation (yellow) between MoPrP105-132 (red) and anti-COX-1-FITC (green) after 5 minutes, but not after 20 minutes (D). Scale bars, 5 μm.

Mentions: Since a close association exists between PG's and prion-induced neurotoxicity [28-31], the relationship between MoPrP105-13 and enzymes involved in the production of PGs was examined. Neuroblastoma cells were incubated with MoPrP105-132 for either 5 minutes or 20 minutes at 37°C, fixed, and stained with antibodies to either cPLA2 or COX-1. After 5 minutes 38% ± 7 of MoPrP105-132 co-localised with cPLA2 (Figure 5A) and 70% ± 5 of MoPrP105-132 co-localised with COX-1 (Figure 5C). After 20 minutes only 2% ± 1 of MoPrP105-132 co-localised with cPLA2 (Figure 5B) and no co-localisation was observed between MoPrP105-132 and COX-1 (Figure 5D), indicating that the association between MoPrP105-132 and cPLA2 or COX-1 was short lived.


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 associates with cPLA2 and COX-1. Neuroblastoma cells were incubated with 30 μM MoPrP105-132-biotin for 5 minutes or 20 minutes at 37°C then fixed and stained with Texas Red-streptavidin (red), anti-cPLA2-FITC (green) or anti-COX-1-FITC (green). Nuclei were revealed using Vectashield with DAPI (blue). (A) Co-localisation (yellow) between MoPrP105-132 (red) and cPLA2 (green) after 5 minutes, but not after 20 minutes (B). (C) Co-localisation (yellow) between MoPrP105-132 (red) and anti-COX-1-FITC (green) after 5 minutes, but not after 20 minutes (D). Scale bars, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: MoPrP105-132 associates with cPLA2 and COX-1. Neuroblastoma cells were incubated with 30 μM MoPrP105-132-biotin for 5 minutes or 20 minutes at 37°C then fixed and stained with Texas Red-streptavidin (red), anti-cPLA2-FITC (green) or anti-COX-1-FITC (green). Nuclei were revealed using Vectashield with DAPI (blue). (A) Co-localisation (yellow) between MoPrP105-132 (red) and cPLA2 (green) after 5 minutes, but not after 20 minutes (B). (C) Co-localisation (yellow) between MoPrP105-132 (red) and anti-COX-1-FITC (green) after 5 minutes, but not after 20 minutes (D). Scale bars, 5 μm.
Mentions: Since a close association exists between PG's and prion-induced neurotoxicity [28-31], the relationship between MoPrP105-13 and enzymes involved in the production of PGs was examined. Neuroblastoma cells were incubated with MoPrP105-132 for either 5 minutes or 20 minutes at 37°C, fixed, and stained with antibodies to either cPLA2 or COX-1. After 5 minutes 38% ± 7 of MoPrP105-132 co-localised with cPLA2 (Figure 5A) and 70% ± 5 of MoPrP105-132 co-localised with COX-1 (Figure 5C). After 20 minutes only 2% ± 1 of MoPrP105-132 co-localised with cPLA2 (Figure 5B) and no co-localisation was observed between MoPrP105-132 and COX-1 (Figure 5D), indicating that the association between MoPrP105-132 and cPLA2 or COX-1 was short lived.

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