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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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Related in: MedlinePlus

MoPrP105-132 is found in lipid rafts. Neuroblastoma cells incubated for 30 minutes with 30 μM rhodamine-labeled MoPrP105-132 and Alexa Fluor 488 labelled CTxB for 30 minutes, the nuclei were revealed using Vectashield with DAPI (blue). (A) Images of MoPrP105-132 (red) and CTxB (green) staining were merged using OpenLab software to show co-localization (yellow). (B) Images of scrambled MoPrP105-132 (red) and CTxB (green) staining showed that there was no co-localization (yellow). (C) FRET analysis showing the raw data between rhodamine-labeled MoPrP105-132 and Alexa Fluor 488 labelled CTxB and (D), the proximity of the donor/acceptor reaction revealed by false-colour intensity. Images of neuroblastoma cells incubated for 30 minutes with 30 μM rhodamine-labeled MoPrP105-132 (red) and stained with FITC-labelled anti-caveolin-1 (green), showing co-localization (yellow). (E) FRET analysis showing the raw data between rhodamine-labeled MoPrP105-132 and FITC-labelled CTxB and (F), the proximity of the donor/acceptor reaction revealed by false-colour intensity. Scale bars, 5 μm. (H), Western-blot analysis of triton X-100 insoluble fractions (lipid rafts) and soluble fractions (non-raft) isolated from neuroblastoma cells incubated with 30 μM MoPrP105-132-FITC for 30 minutes, and probed for MoPrP105-132, caveolin-1, TfR and GM1 (CTxB).
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Figure 1: MoPrP105-132 is found in lipid rafts. Neuroblastoma cells incubated for 30 minutes with 30 μM rhodamine-labeled MoPrP105-132 and Alexa Fluor 488 labelled CTxB for 30 minutes, the nuclei were revealed using Vectashield with DAPI (blue). (A) Images of MoPrP105-132 (red) and CTxB (green) staining were merged using OpenLab software to show co-localization (yellow). (B) Images of scrambled MoPrP105-132 (red) and CTxB (green) staining showed that there was no co-localization (yellow). (C) FRET analysis showing the raw data between rhodamine-labeled MoPrP105-132 and Alexa Fluor 488 labelled CTxB and (D), the proximity of the donor/acceptor reaction revealed by false-colour intensity. Images of neuroblastoma cells incubated for 30 minutes with 30 μM rhodamine-labeled MoPrP105-132 (red) and stained with FITC-labelled anti-caveolin-1 (green), showing co-localization (yellow). (E) FRET analysis showing the raw data between rhodamine-labeled MoPrP105-132 and FITC-labelled CTxB and (F), the proximity of the donor/acceptor reaction revealed by false-colour intensity. Scale bars, 5 μm. (H), Western-blot analysis of triton X-100 insoluble fractions (lipid rafts) and soluble fractions (non-raft) isolated from neuroblastoma cells incubated with 30 μM MoPrP105-132-FITC for 30 minutes, and probed for MoPrP105-132, caveolin-1, TfR and GM1 (CTxB).

Mentions: Initial studies demonstrated that to achieve internalisation and detection of labelled MoPrP105-132 in greater than 50% of the NB4 cells required at least 30 – 90 minutes incubation at 37°C (data not shown). Following this incubation period, 63% ± 8 of rhodamine-labelled MoPrP105-132 co-localised with Alexa Fluor 488 labelled CTxB, which binds to ganglioside-GM1 and identifies lipid rafts [13-15] (Figure 1A). In contrast, no co-localisation between scrambled MoPrP105-132 and CTxB was detected (Figure 1B), indicating that the localisation of MoPrP105-132 in lipid rafts was dependent on the primary sequence of the peptide. To confirm these findings on non-transformed cells, further experiments were conducted using primary cortical neurons; comparable results were obtained (see additional files 1A &1B). A FRET signal generated between acceptor-conjugated MoPrP105-132 and donor conjugated CTxB indicated that the two molecules were in close association, approximately 10–100 Å (Figures 1C, 1D). In contrast, no FRET signal could be detected between scrambled MoPrP105-132 and CTxB (data not shown). To determine whether caveolae played a role in the trafficking of MoPrP105-132, neuroblastoma cells were incubated with rhodamine-labelled MoPrP105-132 for 30 minutes, fixed and probed with FITC labelled anti-caveolin-1. We found 73% ± 3 of MoPrP105-132 co-localised with caveolin-1 (Fig 1E). Furthermore, a FRET signal between MoPrP105-132 and caveolin-1 was also detected confirming the close association of these molecules (Figures 1F &1G). The fluorescence microscopy studies were complemented by lipid rafts isolation studies on MoPrP105-132 treated neuroblastoma cells. Following incubation for 30 minutes, MoPrP105-132 was found in a TfR negative, caveolin-1 and CTxB positive fraction (Figure 1H). These results confirm that MoPrP105-132 can be found in lipid rafts and that caveolin-1 was also present in the lipid rafts that were isolated.


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 is found in lipid rafts. Neuroblastoma cells incubated for 30 minutes with 30 μM rhodamine-labeled MoPrP105-132 and Alexa Fluor 488 labelled CTxB for 30 minutes, the nuclei were revealed using Vectashield with DAPI (blue). (A) Images of MoPrP105-132 (red) and CTxB (green) staining were merged using OpenLab software to show co-localization (yellow). (B) Images of scrambled MoPrP105-132 (red) and CTxB (green) staining showed that there was no co-localization (yellow). (C) FRET analysis showing the raw data between rhodamine-labeled MoPrP105-132 and Alexa Fluor 488 labelled CTxB and (D), the proximity of the donor/acceptor reaction revealed by false-colour intensity. Images of neuroblastoma cells incubated for 30 minutes with 30 μM rhodamine-labeled MoPrP105-132 (red) and stained with FITC-labelled anti-caveolin-1 (green), showing co-localization (yellow). (E) FRET analysis showing the raw data between rhodamine-labeled MoPrP105-132 and FITC-labelled CTxB and (F), the proximity of the donor/acceptor reaction revealed by false-colour intensity. Scale bars, 5 μm. (H), Western-blot analysis of triton X-100 insoluble fractions (lipid rafts) and soluble fractions (non-raft) isolated from neuroblastoma cells incubated with 30 μM MoPrP105-132-FITC for 30 minutes, and probed for MoPrP105-132, caveolin-1, TfR and GM1 (CTxB).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: MoPrP105-132 is found in lipid rafts. Neuroblastoma cells incubated for 30 minutes with 30 μM rhodamine-labeled MoPrP105-132 and Alexa Fluor 488 labelled CTxB for 30 minutes, the nuclei were revealed using Vectashield with DAPI (blue). (A) Images of MoPrP105-132 (red) and CTxB (green) staining were merged using OpenLab software to show co-localization (yellow). (B) Images of scrambled MoPrP105-132 (red) and CTxB (green) staining showed that there was no co-localization (yellow). (C) FRET analysis showing the raw data between rhodamine-labeled MoPrP105-132 and Alexa Fluor 488 labelled CTxB and (D), the proximity of the donor/acceptor reaction revealed by false-colour intensity. Images of neuroblastoma cells incubated for 30 minutes with 30 μM rhodamine-labeled MoPrP105-132 (red) and stained with FITC-labelled anti-caveolin-1 (green), showing co-localization (yellow). (E) FRET analysis showing the raw data between rhodamine-labeled MoPrP105-132 and FITC-labelled CTxB and (F), the proximity of the donor/acceptor reaction revealed by false-colour intensity. Scale bars, 5 μm. (H), Western-blot analysis of triton X-100 insoluble fractions (lipid rafts) and soluble fractions (non-raft) isolated from neuroblastoma cells incubated with 30 μM MoPrP105-132-FITC for 30 minutes, and probed for MoPrP105-132, caveolin-1, TfR and GM1 (CTxB).
Mentions: Initial studies demonstrated that to achieve internalisation and detection of labelled MoPrP105-132 in greater than 50% of the NB4 cells required at least 30 – 90 minutes incubation at 37°C (data not shown). Following this incubation period, 63% ± 8 of rhodamine-labelled MoPrP105-132 co-localised with Alexa Fluor 488 labelled CTxB, which binds to ganglioside-GM1 and identifies lipid rafts [13-15] (Figure 1A). In contrast, no co-localisation between scrambled MoPrP105-132 and CTxB was detected (Figure 1B), indicating that the localisation of MoPrP105-132 in lipid rafts was dependent on the primary sequence of the peptide. To confirm these findings on non-transformed cells, further experiments were conducted using primary cortical neurons; comparable results were obtained (see additional files 1A &1B). A FRET signal generated between acceptor-conjugated MoPrP105-132 and donor conjugated CTxB indicated that the two molecules were in close association, approximately 10–100 Å (Figures 1C, 1D). In contrast, no FRET signal could be detected between scrambled MoPrP105-132 and CTxB (data not shown). To determine whether caveolae played a role in the trafficking of MoPrP105-132, neuroblastoma cells were incubated with rhodamine-labelled MoPrP105-132 for 30 minutes, fixed and probed with FITC labelled anti-caveolin-1. We found 73% ± 3 of MoPrP105-132 co-localised with caveolin-1 (Fig 1E). Furthermore, a FRET signal between MoPrP105-132 and caveolin-1 was also detected confirming the close association of these molecules (Figures 1F &1G). The fluorescence microscopy studies were complemented by lipid rafts isolation studies on MoPrP105-132 treated neuroblastoma cells. Following incubation for 30 minutes, MoPrP105-132 was found in a TfR negative, caveolin-1 and CTxB positive fraction (Figure 1H). These results confirm that MoPrP105-132 can be found in lipid rafts and that caveolin-1 was also present in the lipid rafts that were isolated.

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