Yaping Gu

Protease-Resistant Human Prion Protein and Ferritin Are Cotransported across Caco-2 Epithelial Cells: Implications for Species Barrier in Prion Uptake from the Intestine

Foodborne transmission of bovine spongiform encephalopathy (BSE) to humans as variant Creutzfeldt-Jakob disease (CJD) has affected over 100 individuals, and probably millions of others have been exposed to BSE-contaminated food substances. Despite these obvious public health concerns, surprisingly little is known about the mechanism by which PrP-scrapie (PrPSc), the most reliable surrogate marker of infection in BSE-contaminated food, crosses the human intestinal epithelial cell barrier. Here we show that digestive enzyme (DE) treatment of sporadic CJD brain homogenate generates a C-terminal fragment similar to the proteinase K-resistant PrPSc core of 27-30 kDa implicated in prion disease transmission and pathogenesis. Notably, DE treatment results in a PrPSc-protein complex that is avidly transcytosed in vesicular structures across an in vitro model of the human intestinal epithelial cell barrier, regardless of the amount of endogenous PrPC expression. Unexpectedly, PrPSc is cotransported with ferritin, a prominent component of the DE-treated PrPSc-protein complex. The transport of PrPSc-ferritin is sensitive to low temperature, brefeldin-A, and nocodazole treatment and is inhibited by excess free ferritin, implicating a receptor- or transporter-mediated pathway. Because ferritin shares considerable homology across species, these data suggest that PrPSc-associated proteins, in particular ferritin, may facilitate PrPSc uptake in the intestine from distant species, leading to a carrier state in humans.

Identification of cryptic nuclear localization signals in the prion protein

Abnormal transport of C-terminally truncated prion protein (PrP) to the nucleus has been reported in cell models of familial prion disorders associated with a stop codon mutation at residues 145 or 160 of the PrP. In both cases, PrP is translocated to the nucleus in an energy-dependent fashion, implying the presence of cryptic nuclear localization signal(s) in this region of PrP. In this report, we describe the presence of two independent nuclear localization signals (NLS) in the N-terminal domain of PrP that differ in the efficiency of nuclear targeting. When acting independently, each NLS sequence mediates the transport of tagged bovine serum albumin into the nucleus of permeabilized cells. When acting together, the two NLS sequences complement each other in transporting the N-terminal fragment of PrP to the nucleus of transfected cells, where it accumulates at steady state. Interestingly, nuclear translocation of PrP is blocked completely if the N-terminal fragment is extended to include one or two N-glycans. The glycosylated PrP fragment, instead, accumulates in the endoplasmic reticulum. Extension of the N-terminal fragment to include both N-glycans and the glycosyl phosphatidylinositol anchor, as expected, directs PrP to the plasma membrane. These observations hold implications for the pathogenesis of familial prion disorders, where truncated and abnormally glycosylated mutant PrP forms may accumulate in the nucleus and initiate neurotoxicity through novel mechanisms.

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.

Mutant prion protein-mediated aggregation of normal prion protein in the endoplasmic reticulum: implications for prion propagation and neurotoxicity

Familial prion disorders are believed to result from spontaneous conversion of mutant prion protein (PrPM) to the pathogenic isoform (PrPSc). While most familial cases are heterozygous and thus express the normal (PrPC) and mutant alleles of PrP, the role of PrPC in the pathogenic process is unclear. Plaques from affected cases reveal a heterogeneous picture; in some cases only PrPM is detected, whereas in others both PrPC and PrPM are transformed to PrPSc. To understand if the coaggregation of PrPC is governed by PrP mutations or is a consequence of the cellular compartment of PrPM aggregation, we coexpressed PrPM and PrPC in neuroblastoma cells, the latter tagged with green fluorescent protein (PrPC–GFP) for differentiation. Two PrPM forms (PrP231T, PrP217R/231T) that aggregate spontaneously in the endoplasmic reticulum (ER) were generated for this analysis. We report that PrPC–GFP aggregates when coexpressed with PrP231T or PrP217R/231T, regardless of sequence homology between the interacting forms. Furthermore, intracellular aggregates of PrP231T induce the accumulation of a C‐terminal fragment of PrP, most likely derived from a potentially neurotoxic transmembrane form of PrP (CtmPrP) in the ER. These findings have implications for prion pathogenesis in familial prion disorders, especially in cases where transport of PrPM from the ER is blocked by the cellular quality control.

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.

Paradoxical Role of Prion Protein Aggregates in Redox-Iron Induced Toxicity

Background Imbalance of iron homeostasis has been reported in sporadic Creutzfeldt-Jakob-disease (sCJD) affected human and scrapie infected animal brains, but the contribution of this phenotype to disease associated neurotoxicity is unclear. Methodology/Principal Findings Using cell models of familial prion disorders, we demonstrate that exposure of cells expressing normal prion protein (PrPC) or mutant PrP forms to a source of redox-iron induces aggregation of PrPC and specific mutant PrP forms. Initially this response is cytoprotective, but becomes increasingly toxic with time due to accumulation of PrP-ferritin aggregates. Mutant PrP forms that do not aggregate are not cytoprotective, and cells show signs of acute toxicity. Intracellular PrP-ferritin aggregates induce the expression of LC3-II, indicating stimulation of autophagy in these cells. Similar observations are noted in sCJD and scrapie infected hamster brains, lending credence to these results. Furthermore, phagocytosis of PrP-ferritin aggregates by astrocytes is cytoprotective, while culture in astrocyte conditioned medium (CM) shows no measurable effect. Exposure to H2O2, on the other hand, does not cause aggregation of PrP, and cells show acute toxicity that is alleviated by CM. Conclusions/Significance These observations suggest that aggregation of PrP in response to redox-iron is cytoprotective. However, subsequent co-aggregation of PrP with ferritin induces intracellular toxicity unless the aggregates are degraded by autophagosomes or phagocytosed by adjacent scavenger cells. H2O2, on the other hand, does not cause aggregation of PrP, and induces toxicity through extra-cellular free radicals. Together with previous observations demonstrating imbalance of iron homeostasis in prion disease affected brains, these observations provide insight into the mechanism of neurotoxicity by redox-iron, and the role of PrP in this process.

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.

Cell-Specific Metabolism and Pathogenesis of Transmembrane Prion Protein

ABSTRACT The C-transmembrane form of prion protein (CtmPrP) has been implicated in prion disease pathogenesis, but the factors underlying its biogenesis and cyotoxic potential remain unclear. Here we show that CtmPrP interferes with cytokinesis in cell lines where it is transported to the plasma membrane. These cells fail to separate following cell division, assume a variety of shapes and sizes, and contain multiple nuclei, some of which are pyknotic. Furthermore, the synthesis and transport of CtmPrP to the plasma membrane are modulated through a complex interaction between cis- and trans-acting factors and the endoplasmic reticulum translocation machinery. Thus, insertion of eight amino acids before or within the N region of the N signal peptide (N-SP) of PrP results in the exclusive synthesis of CtmPrP regardless of the charge conferred to the N region. Subsequent processing and transport of CtmPrP are modulated by specific amino acids in the N region of the N-SP and by the cell line of expression. Although the trigger for CtmPrP upregulation in naturally occurring prion disorders remains elusive, these data highlight the underlying mechanisms of CtmPrP biogenesis and neurotoxicity and reinforce the idea that CtmPrP may serve as the proximate cause of neuronal death in certain prion disorders.

Processing and Mis-Processing of the Prion Protein: Insights into the Pathogenesis of Familial Prion Disorders

The cellular prion protein (PrPC), though apparently innocuous, is the main agent responsible for infectious, familial, and sporadic prion disorders. Through its remarkable ability to undergo a change in conformation from a mainly α-helical to a β-sheet rich conformation commonly referred to as PrP-scrapie (PrPSc), PrPC becomes infectious and pathogenic, a feature that is unique to this glycoprotein1–4. Over the years, most studies have focused on the mechanism of PrPC to PrPSc conversion and its subsequent transmission to susceptible hosts, ignoring the less common familial prion disorders that result from point mutations in the prion protein gene (PRNP). In these disorders, mutant PrP (PrPM) is presumed to undergo a spontaneous change in conformation to PrPSc without participation from an exogenous source of in-fectious PrPSc. Once initiated, the process proceeds exponentially, and deposits of mutant PrPSc are believed to result in the neurotoxicity ob-served in familial cases of prion disorders5,6. However, prion-specific neuropathology is often observed in the absence of detectable PrPSc, indicating the presence of alternative pathways of neurotoxicity incertain cases of prion disorders7. Recent studies on the processing of normal and mutant PrP underscore the importance of abnormal metabolism and various topological forms of PrP in prion disease pathogenesis8. In this chapter, we will review information on the complex pathways of intracellular trafficking and metabolism of normal and various mutant PrP forms, and highlight some of the abnormal pathways that may contribute to neurotoxicity in familial prion disorders.

Isolation of human neuronal cells resistant to toxicity by the prion protein peptide 106-126

Prion diseases or transmissible spongiform encephalopathies, are neurodegenerative disorders that are genetic, sporadic, or infectious. The pathogenetic event common to all prion disorders is the conformational transformation of the cellular prion protein (PrP^C) to the scrapie form (PrP^Sc), that deposits in the brain parenchyma and induces neuronal death. Infectious prion disorders are caused by exogenously introduced PrP^Sc that acts as a template in the conversion of endogenous PrP^C to nascent PrP^Sc, and subsequently the process becomes autocatalytic. To understand the process of cellular uptake of PrP^Sc and its mechanism of cellular toxicity, previous studies have used a PrP fragment spanning residues 106-126 (PrP^Tx) that is toxic to primary neurons in culture, and mimics PrP^Sc in its biophysical properties [9,11,14]. Several possible mechanisms of cell death by PrP^Tx have been proposed [2,3,10,11,18], but the existing data are unclear. To identify the biochemical pathways of neurotoxicity by this fragment, we have isolated mutant neuroblastoma and NT-2 cells that are resistant to toxicity by PrP^Tx. We show that these cells bind and internalize PrP^Tx in a temperature dependent fashion, and the peptide accumulates in intracellular compartments, probably lysosomes, where it has an unusually long half-life. The PrP^Tx-resistant phenotype of the cells reported in this study could result from aberrant binding or internalization of the peptide, or due to an abnormality in the downstream pathway(s) of neuronal toxicity. The PrP^Tx-resistant cells are therefore a useful tool for evaluating the cellular and biochemical pathways that lead to cell death by this peptide, and will provide insight into the mechanism(s) of neurotoxicity by PrP^Sc.