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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 reduces the distribution of MoPrP105-132 and CTxB in neuroblastoma cells. Neuroblastoma cells were incubated with 1 μM squalestatin for 24 hours and subsequently incubated with MoPrP105-132-rhodamine (red) and CTxB-Alexa Fluor 488 (green), nuclei were revealed using Vectashield with DAPI (blue). (A) Lack of co-localisation was apparent between MoPrP105-132 and CTxB in squalestatin treated cells compared with (B) untreated cells. Scale bar, 5 μm. (C) Isolation of lipid raft/non-raft membranes from neuroblastoma cells treated with 1 μM squalestatin for 24 hours. In squalestatin-treated cells the majority of MoPrP105-132 was present in the non-raft fraction.
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Figure 3: Squalestatin reduces the distribution of MoPrP105-132 and CTxB in neuroblastoma cells. Neuroblastoma cells were incubated with 1 μM squalestatin for 24 hours and subsequently incubated with MoPrP105-132-rhodamine (red) and CTxB-Alexa Fluor 488 (green), nuclei were revealed using Vectashield with DAPI (blue). (A) Lack of co-localisation was apparent between MoPrP105-132 and CTxB in squalestatin treated cells compared with (B) untreated cells. Scale bar, 5 μm. (C) Isolation of lipid raft/non-raft membranes from neuroblastoma cells treated with 1 μM squalestatin for 24 hours. In squalestatin-treated cells the majority of MoPrP105-132 was present in the non-raft fraction.

Mentions: Previous studies have shown that depletion of cholesterol in neurones protects against the neurotoxicity induced by prion peptides [23], including MoPrP105-132 (see additional data 3). To determine whether squalestatin was simply altering the quantity of MoPrP105-132 ingested, squalestatin treated or untreated neuroblastoma cells were incubated with 30 μM MoPrP105-132 conjugated to FITC. The levels of cell associated fluorescence in each condition was determined after 30 minutes incubation by FACS analysis of cells and expressed as arbitrary units of mean fluorescence intensity. There was no significant difference between the mean fluorescence intensity for untreated and squalestatin treated cells (23.2 ± 2 compared to 25.3 ± 4, n = 6, P > 0.05). As a number of studies have demonstrated that lipid rafts involved in internalisation and trafficking are cholesterol sensitive, the effect of squalestatin on the intracellular trafficking of MoPrP105-132 was investigated. Following pre-treatment with squalestatin, neuroblastoma cells were incubated with CTxB and MoPrP105-132 for 30 minutes at 37°C. Fluorescence microscopy revealed that in squalestatin-treated cells only 5% ± 1 of the MoPrP105-132 co-localised with CTxB (Figure 3A), whereas 75% ± 7 of MoPrP105-132 co-localised with CTxB in untreated neuroblastoma cells (Figure 3B). When lipid rafts were isolated from neuroblastoma cells that had been pre-treated with squalestatin, most of the MoPrP105-132 was detected in the non-raft fraction, while CTxB and caveolin-1 were present in both raft and non-raft fractions (Figure 3C). Such findings suggest that the localisation of MoPrP105-132 to lipid rafts is sensitive to cholesterol depletion.


Squalestatin alters the intracellular trafficking of a neurotoxic prion peptide.

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

Squalestatin reduces the distribution of MoPrP105-132 and CTxB in neuroblastoma cells. Neuroblastoma cells were incubated with 1 μM squalestatin for 24 hours and subsequently incubated with MoPrP105-132-rhodamine (red) and CTxB-Alexa Fluor 488 (green), nuclei were revealed using Vectashield with DAPI (blue). (A) Lack of co-localisation was apparent between MoPrP105-132 and CTxB in squalestatin treated cells compared with (B) untreated cells. Scale bar, 5 μm. (C) Isolation of lipid raft/non-raft membranes from neuroblastoma cells treated with 1 μM squalestatin for 24 hours. In squalestatin-treated cells the majority of MoPrP105-132 was present in the non-raft fraction.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Squalestatin reduces the distribution of MoPrP105-132 and CTxB in neuroblastoma cells. Neuroblastoma cells were incubated with 1 μM squalestatin for 24 hours and subsequently incubated with MoPrP105-132-rhodamine (red) and CTxB-Alexa Fluor 488 (green), nuclei were revealed using Vectashield with DAPI (blue). (A) Lack of co-localisation was apparent between MoPrP105-132 and CTxB in squalestatin treated cells compared with (B) untreated cells. Scale bar, 5 μm. (C) Isolation of lipid raft/non-raft membranes from neuroblastoma cells treated with 1 μM squalestatin for 24 hours. In squalestatin-treated cells the majority of MoPrP105-132 was present in the non-raft fraction.
Mentions: Previous studies have shown that depletion of cholesterol in neurones protects against the neurotoxicity induced by prion peptides [23], including MoPrP105-132 (see additional data 3). To determine whether squalestatin was simply altering the quantity of MoPrP105-132 ingested, squalestatin treated or untreated neuroblastoma cells were incubated with 30 μM MoPrP105-132 conjugated to FITC. The levels of cell associated fluorescence in each condition was determined after 30 minutes incubation by FACS analysis of cells and expressed as arbitrary units of mean fluorescence intensity. There was no significant difference between the mean fluorescence intensity for untreated and squalestatin treated cells (23.2 ± 2 compared to 25.3 ± 4, n = 6, P > 0.05). As a number of studies have demonstrated that lipid rafts involved in internalisation and trafficking are cholesterol sensitive, the effect of squalestatin on the intracellular trafficking of MoPrP105-132 was investigated. Following pre-treatment with squalestatin, neuroblastoma cells were incubated with CTxB and MoPrP105-132 for 30 minutes at 37°C. Fluorescence microscopy revealed that in squalestatin-treated cells only 5% ± 1 of the MoPrP105-132 co-localised with CTxB (Figure 3A), whereas 75% ± 7 of MoPrP105-132 co-localised with CTxB in untreated neuroblastoma cells (Figure 3B). When lipid rafts were isolated from neuroblastoma cells that had been pre-treated with squalestatin, most of the MoPrP105-132 was detected in the non-raft fraction, while CTxB and caveolin-1 were present in both raft and non-raft fractions (Figure 3C). Such findings suggest that the localisation of MoPrP105-132 to lipid rafts is sensitive to cholesterol depletion.

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