Abstract
BackgroundNeurotoxic 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.ResultsFluorescence 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.ConclusionAs 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.
Highlights
Neurotoxic peptides derived from the protease-resistant core of the prion protein are used to model the pathogenesis of prion diseases
63% ± 8 of rhodamine-labelled MoPrP105-132 co-localised with Alexa Fluor 488 labelled cholera toxin subunit B (CTxB), which binds to ganglioside-GM1 and identifies lipid rafts [13,14,15] (Figure 1A)
To determine whether caveolae played a role in the trafficking of MoPrP105-132, neuroblastoma cells were incubated with rhodamine-labelled MoPrP105132 for 30 minutes, fixed and probed with FITC labelled anti-caveolin-1
Summary
Neurotoxic peptides derived from the protease-resistant core of the prion protein are used to model the pathogenesis of prion diseases. BMC Neuroscience 2007, 8:99 http://www.biomedcentral.com/1471-2202/8/99 of PrPSc which accumulate in the brain of infected animals [4,5,6] It remains unclear if PrPSc causes neuronal damage itself, or acts via other molecular forms of PrP that have been suggested as causative agents in prion disease [7,8,9]. The process of neuronal loss can be investigated in vitro using highly defined synthetic peptides derived from the protease-resistant core of PrPSc. The majority of neurotoxicity studies have employed a peptide consisting of amino acids 106–126 of the human prion protein (HuPrP106126) which possesses many of the properties of the PrPSc isoform, notably a high β-pleated sheet content, fibril formation and toxicity for neurons in vitro [7]. We demonstrate that in neuroblastoma cells, MoPrP105-132 co-localises with cholera toxin subunit B (CTxB), which binds to the ganglioside GM1 [12,13] and caveolin-1 [14,15], markers of specialised microdomains called lipid rafts
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