Pathogenic mutations in the glycosylphosphatidylinositol signal peptide of PrP modulate its topology in neuroblastoma cells.

Point mutations M232R (PrP(232R)), M232T (PrP(232T)), and P238S (PrP(238S)) in the glycosylphosphatidylinositol signal peptide (GPI-SP) of the prion protein (PrP(C)) segregate with familial Creutzfeldt-Jakob disease (CJD). However, the mechanism by which these mutations induce cytotoxicity is unclear since the GPI-SP is replaced by a GPI anchor within 5 min of PrP synthesis and translocation into the endoplasmic reticulum (ER). To examine if mutations in this region interfere with translocation of nascent PrP into the ER or anchor addition, the metabolism of PrP(232R) and PrP(232T) was investigated in transfected human neuroblastoma cells. In this report, we demonstrate that PrP mutations M232R and M232T do not interfere with GPI anchor addition. Instead, these mutations increase the stability and transport of GPI-SP mediated post-translationally translocated PrP to the plasma membrane, where it is linked to the lipid bilayer in a potentially neurotoxic C-transmembrane ((Ctm)PrP) orientation. Furthermore, we demonstrate that the GPI-SP of PrP functions as an efficient co-translational and inefficient post-translational ER translocation signal when tagged to an unrelated protein, underscoring the functional versatility of this peptide. These data uncover an alternate pathway of ER translocation for nascent PrP, and provide information on the possible mechanism(s) of neurotoxicity by mutations in the GPI-SP.

Mutant prion protein D202N associated with familial prion disease is retained in the endoplasmic reticulum and forms 'curly' intracellular aggregates.

Transmissible Spongiform Encephalopathies are fatal neurodegenerative disorders of humans and animals that are familial, sporadic, and infectious in nature. Familial disorders of humans include Gerstmann-Straussler-Scheinker disease (GSS), familial Creutzfeldt-Jakob disease (CJD), and fatal familial insomnia, and result from point mutations in the prion protein gene. Although neurotoxicity in familial cases is believed to result from a spontaneous change in conformation of mutant prion protein (PrP) to the pathogenic PrP-scrapie (PrPSc) form, emerging evidence indicates otherwise. We have investigated the processing and metabolism of mutant PrP D202N (PrP202N) in cell models to elucidate possible mechanisms of cytotoxicity. In this report, we demonstrate that PrP202N expressed in human neuroblastoma cells fails to achieve a mature conformation following synthesis and accumulates in the endoplasmic reticulum as 'curly' aggregates. In addition, PrP202N cells show increased sensitivity to free radicals, indicating that neuronal susceptibility to oxidative damage may account for the neurotoxicity observed in cases of GSS resulting from PrP D202N mutation.

Aggresome formation by mutant prion proteins: the unfolding role of proteasomes in familial prion disorders.

Although familial prion disorders are a direct consequence of mutations in the prion protein gene, the underlying mechanisms leading to neurodegeneration remain unclear. Potential pathogenic mechanisms include abnormal cellular metabolism of the mutant prion protein (PrP(M)), or destabilization of PrP(M) structure inducing a change in its conformation to the pathogenic PrP-scrapie (PrP(Sc)) form. To further clarify these mechanisms, we investigated the biogenesis of mutant PrP V203I and E211Q associated with Creutzfeldt-Jakob disease, and PrP Q212P associated with Gerstmann-Straussler-Scheinker syndrome in neuroblastoma cells. We report that all three PrP(M) forms accumulate similarly in the cytosol in response to proteasomal inhibition, and finally assemble as classical aggresomes. Since the three PrP(M) forms tested in this report are distinct, we propose that sequestration of misfolded PrP(M) into aggresomes is likely a general response of the cellular quality control that is not specific to a particular mutation in PrP. Moreover, since PrP has the remarkable ability to refold into PrP(Sc) that can subsequently replicate, PrP(M) sequestered in aggresomes may cause neurotoxicity by both direct and indirect pathways; directly through PrP(Sc) aggregates, and indirectly by depleting normal PrP, through induction of a cellular stress response, or by other undefined pathways. On the other hand, sequestered PrP(M) may be relatively inert, and cellular toxicity may be mediated by early intermediates in aggresome formation. Taken together, these observations demonstrate the role of proteasomes in the pathogenesis of familial prion disorders, and argue for further explanation of its mechanistic details.

Cell surface accumulation of a truncated transmembrane prion protein in Gerstmann-Straussler-Scheinker disease P102L.

A familial prion disorder with a proline to leucine substitution at residue 102 of the prion protein (PrP(102L)) is typically associated with protease-resistant PrP fragments (PrP(Sc)) in the brain parenchyma that are infectious to recipient animals. When modeled in transgenic mice, a fatal neurodegenerative disease develops, but, unlike the human counterpart, PrP(Sc) is lacking and transmission to recipient animals is questionable. Alternate mice expressing a single copy of PrP(102L) (mouse PrP(101L)) do not develop spontaneous disease, but show dramatic susceptibility to PrP(Sc) isolates from different species. To understand these discrepant results, we studied the biogenesis of human PrP(102L) in a cell model. Here, we report that cells expressing PrP(102L) show decreased expression of the normal 18-kDa fragment on the plasma membrane. Instead, a 20-kDa fragment, probably derived from transmembrane PrP ((Ctm)PrP), accumulates on the cell surface. Because the 20-kDa fragment includes an amyloidogenic region of PrP that is disrupted in the 18-kDa form, increased surface expression of 20-kDa fragment may enhance the susceptibility of these cells to PrP(Sc) infection by providing an optimal substrate, or by amplifying the neurotoxic signal of PrP(Sc). Thus, altered susceptibility of PrP(101L) mice to exogenous PrP(Sc) may be mediated by the 20-kDa (Ctm)PrP fragment, rather than PrP(102L) per se.

Prion peptide 106-126 as a model for prion replication and neurotoxicity.

Prion diseases or transmissible spongiform encephalopathies are neurodegenerative disorders that are genetic, sporadic, or infectious. The pathogenetic event common to all prion disorders is a change in conformation of the cellular prion protein (PrPC) to the scrapie isoform (PrPSc), which, unlike PrPC, aggregates easily and is partially resistant to protease digestion. Although PrPSc is believed to be essential for the pathogenesis and transmission of prion disorders, the mechanism by which PrPSc deposits cause neurodegeneration is unclear. It has been proposed that in some cases of prion disorders, a transmembrane form of PrP, termed CtmPrP may be the mediator of neurodegenerative changes rather than PrPSc per se. In order to understand the underlying cellular processes by which PrPSc mediates neurodegeneration, we have investigated the mechanism of neurotoxicity by a beta-sheet rich peptide of PrP in a cell model. We show that exposure of human neuronal cell lines NT-2 and M17 to the prion peptide 106-126 (PrP106-126) catalyzes the aggregation of endogenous cellular prion protein (PrPC) to an amyloidogenic form that shares several characteristics with PrPSc. Intracellular accumulation of these PrPSc-like forms upregulates the synthesis of CtmPrP, which is proteolytically cleaved in the endoplasmic reticulum and the truncated C-terminal fragment is transported to the cell surface. In addition, we have isolated mutant NT-2 and neuroblastoma cells that are resistant to toxicity by PrP106-126 to facilitate further characterization of the biochemical pathways of PrP106-126 neurotoxicity. The PrP106-126-resistant phenotype of these cells could result from aberrant binding or internalization of the peptide, or due to an abnormality in the downstream pathway(s) of neuronal toxicity. Thus, our data suggest that PrPSc aggregation occurs by a process of 'nucleation' on a pre-existing 'seed' of PrP. Furthermore, the PrP106-126-resistant cells reported here will provide a unique opportunity for identifying the cellular and biochemical pathways that mediate neurotoxicity by PrPSc.