Mutant Prion Protein Expression Is Associated with an Alteration of the Rab GDP Dissociation Inhibitor (GDI)/Rab11 Pathway*□S

The prion protein (PrP) is a glycosylphosphatidylinositolanchored membrane glycoprotein that plays a vital role in prion diseases, a class of fatal neurodegenerative disorders of humans and animals. Approximately 20% of human prion diseases display autosomal dominant inheritance and are linked to mutations in the PrP gene on chromosome 20. PrP mutations are thought to favor the conformational conversion of PrP into a misfolded isoform that causes disease by an unknown mechanism. The PrP mutation D178N/Met-129 is linked to fatal familial insomnia, which causes severe sleep abnormalities and autonomic dysfunction. We showed by immunoelectron microscopy that this mutant PrP accumulates abnormally in the endoplasmic reticulum and Golgi of transfected neuroblastoma N2a cells. To investigate the impact of intracellular PrP accumulation on cellular homeostasis, we did a two-dimensional gel-based differential proteomics analysis. We used wide range immobilized pH gradient strips, pH 4–7 and 6–11, to analyze a large number of proteins. We found changes in proteins involved in energy metabolism, redox regulation, and vesicular transport. Rab GDP dissociation inhibitor (GDI) was one of the proteins that changed most. GDI regulates vesicular protein trafficking by acting on the activity of several Rab proteins. We found a specific reduction in the level of functional Rab11 in mutant PrP-expressing cells associated with impaired post-Golgi trafficking. Our data are consistent with a model by which mutant PrP induces overexpression of GDI, activating a cytotoxic feedback loop that leads to protein accumulation in the secretory pathway. Molecular & Cellular Proteomics 9:611–622, 2010. Familial Creutzfeldt-Jakob disease (fCJD), GerstmannSträussler-Scheinker syndrome, and fatal familial insomnia (FFI) are dominantly inherited degenerative disorders of the central nervous system (CNS) linked to mutations in the prion protein (PrP) gene on chromosome 20 (1). The pathogenic mutations favor conversion of PrP into a misfolded pathogenic isoform that accumulates in the CNS, ultimately leading to neuronal dysfunction and degeneration by a mechanism still unknown (2). A mutation at PrP codon 178, resulting in the substitution of aspartic acid for asparagine is linked to two different inherited prion diseases, depending on the amino acid specified at the polymorphic site 129 of the mutant allele where either methionine or valine can be present. The D178N/ Val-129 haplotype is linked to fCJD, whereas D178N/Met-129 is associated with FFI (3). PrP is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein of uncertain function (4). Like other membrane proteins, PrP is synthesized in the rough endoplasmic reticulum (ER), transits the Golgi, and is delivered to the cell surface where it resides in lipid rafts (5, 6). Several mutant PrP molecules, in contrast, misfold soon after synthesis in the ER (7), accumulate in the secretory pathway, and are less efficiently delivered to the cell surface (8–15). Mutant PrPs expressed in transfected cells and primary neurons from transgenic mice acquire biochemical properties of pathogenic PrP, including insolubility in non-denaturing detergents and protease resistance (14, 16–19). These observations suggest that mutant PrP misfolding and abnormal intracellular localization may trigger pathogenic processes (2, 20). In line with this view, ER accumulation of mutant PrP and alteration of ER morphology have been found