Squalestatin alters the intracellular trafficking of a neurotoxic prion peptide.
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.


CONCLUSIONS
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.
1Division of Immunology, Infection and Inflammation, Western Infirmary, University of Glasgow, G11 6NT, Glasgow. rkw1m@clinmed.gla.ac.uk
Rona
R
Wilson
Clive
C
Bate
Ronald
R
Boshuizen
Alun
A
Williams
James
J
Brewer
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D051242:Caveolin 1 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism*; D002772:Cholera Toxin / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D051545:Cyclooxygenase 1 / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D009498:Neurotoxins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D054467:Phospholipases A2 / Q000378:metabolism; D011328:Prions / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects*; D014018:Tissue Distribution / Q000187:drug effects; D014233:Tricarboxylic Acids / Q000494:pharmacology*
D015415:Biomarkers; D019086:Bridged Bicyclo Compounds, Heterocyclic; D051242:Caveolin 1; D009498:Neurotoxins; D010446:Peptide Fragments; D011328:Prions; D014233:Tricarboxylic Acids; C419707:prion protein (105-132); C075117:squalestatin 1; D002772:Cholera Toxin; D002784:Cholesterol; D051545:Cyclooxygenase 1; D054467:Phospholipases A2