Neena Singh

Proteasomal Degradation and N-terminal Protease Resistance of the Codon 145 Mutant Prion Protein

An amber mutation at codon 145 (Y145stop) of the prion protein gene results in a variant of an inherited human prion disease named Gerstmann-Sträussler-Scheinker syndrome. The characteristic features of this disorder include amyloid deposits of prion protein in cerebral parenchyma and vessels. We have studied the biosynthesis and processing of the prion protein containing the Y145stop mutation (PrP145) in transfected human neuroblastoma cells in an attempt to clarify the effect of the mutation on the metabolism of PrP145 and to gain insight into the underlying pathogenetic mechanism. Our results demonstrate that 1) a significant proportion of PrP145 is not processed post-translationally and retains the N-terminal signal peptide, 2) most PrP145 is degraded very rapidly by the proteasome-mediated pathway, 3) blockage of proteasomal degradation results in intracellular accumulation of PrP145, 4) most of the accumulated PrP145 is detergent-insoluble, and both the detergent-soluble and -insoluble fractions are resistant to mild proteinase K (PK) treatment, suggesting that PK resistance is not simply because of aggregation. The present study demonstrates for the first time that a mutant prion protein is degraded through the proteasomal pathway and acquires PK-resistance if degradation is impaired.

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.

Prion Protein Aggregation Reverted by Low Temperature in Transfected Cells Carrying a Prion Protein Gene Mutation

Prion diseases are characterized by the conversion of the normal cellular prion protein (PrPC), a glycoprotein that is anchored to the cell membrane by a glycosylphosphatidylinositol moiety, into an isoform that is protease-resistant (PrPres) and pathogenic. In inherited prion diseases, mutations in the prion protein (PrPM) engender the conversion of PrPM into PrPres. We developed a cell model of Gerstmann-Sträussler-Scheinker disease, a neurodegenerative condition characterized by PrPM-containing amyloid deposits and neuronal loss, by expressing the Gerstmann-Sträussler-Scheinker haplotype Q217R-129V in human neuroblastoma cells. By comparison to PrPC, this genotype results in the following alterations of PrPM: 1) expression of an aberrant form lacking the glycosylphosphatidylinositol anchor, 2) increased aggregation and protease resistance, and 3) impaired transport to the cell surface. Most of these alterations are temperature-sensitive, indicating that they are due to misfolding of PrPM.

Abnormal Brain Iron Homeostasis in Human and Animal Prion Disorders

Neurotoxicity in all prion disorders is believed to result from the accumulation of PrP-scrapie (PrPSc), a β-sheet rich isoform of a normal cell-surface glycoprotein, the prion protein (PrPC). Limited reports suggest imbalance of brain iron homeostasis as a significant associated cause of neurotoxicity in prion-infected cell and mouse models. However, systematic studies on the generality of this phenomenon and the underlying mechanism(s) leading to iron dyshomeostasis in diseased brains are lacking. In this report, we demonstrate that prion disease–affected human, hamster, and mouse brains show increased total and redox-active Fe (II) iron, and a paradoxical increase in major iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) at the end stage of disease. Furthermore, examination of scrapie-inoculated hamster brains at different timepoints following infection shows increased levels of Tf with time, suggesting increasing iron deficiency with disease progression. Sporadic Creutzfeldt-Jakob disease (sCJD)–affected human brains show a similar increase in total iron and a direct correlation between PrP and Tf levels, implicating PrPSc as the underlying cause of iron deficiency. Increased binding of Tf to the cerebellar Purkinje cell neurons of sCJD brains further indicates upregulation of TfR and a phenotype of neuronal iron deficiency in diseased brains despite increased iron levels. The likely cause of this phenotype is sequestration of iron in brain ferritin that becomes detergent-insoluble in PrPSc-infected cell lines and sCJD brain homogenates. These results suggest that sequestration of iron in PrPSc–ferritin complexes induces a state of iron bio-insufficiency in prion disease–affected brains, resulting in increased uptake and a state of iron dyshomeostasis. An additional unexpected observation is the resistance of Tf to digestion by proteinase-K, providing a reliable marker for iron levels in postmortem human brains. These data implicate redox-iron in prion disease–associated neurotoxicity, a novel observation with significant implications for prion disease pathogenesis.

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.

Prion protein (PrP) knock-out mice show altered iron metabolism: a functional role for PrP in iron uptake and transport.

Despite overwhelming evidence implicating the prion protein (PrP) in prion disease pathogenesis, the normal function of this cell surface glycoprotein remains unclear. In previous reports we demonstrated that PrP mediates cellular iron uptake and transport, and aggregation of PrP to the disease causing PrP-scrapie (PrPSc) form results in imbalance of iron homeostasis in prion disease affected human and animal brains. Here, we show that selective deletion of PrP in transgenic mice (PrPKO) alters systemic iron homeostasis as reflected in hematological parameters and levels of total iron and iron regulatory proteins in the plasma, liver, spleen, and brain of PrPKO mice relative to matched wild type controls. Introduction of radiolabeled iron (59FeCl3) to Wt and PrPKO mice by gastric gavage reveals inefficient transport of 59Fe from the duodenum to the blood stream, an early abortive spike of erythropoiesis in the long bones and spleen, and eventual decreased 59Fe content in red blood cells and all major organs of PrPKO mice relative to Wt controls. The iron deficient phenotype of PrPKO mice is reversed by expressing Wt PrP in the PrPKO background, demonstrating a functional role for PrP in iron uptake and transport. Since iron is required for essential metabolic processes and is also potentially toxic if mismanaged, these results suggest that loss of normal function of PrP due to aggregation to the PrPSc form induces imbalance of brain iron homeostasis, resulting in disease associated neurotoxicity.

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.

Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

Converging evidence leaves little doubt that a change in the conformation of prion protein (PrPC) from a mainly α-helical to a β-sheet rich PrP-scrapie (PrPSc) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrPSc, nor the normal function of PrPC is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrPC mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrPC increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrPC on ferritin iron content is enhanced by stimulating PrPC endocytosis, and reversed by cross-linking PrPC on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP102L decreases ferritin iron content significantly relative to PrPC expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrPC nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrPC in cellular iron uptake and transport to ferritin, and dysfunction of PrPC as a significant contributing factor of brain iron imbalance in prion disorders.

Binding of pro-prion to filamin A disrupts cytoskeleton and correlates with poor prognosis in pancreatic cancer

The cellular prion protein (PrP) is a highly conserved, widely expressed, glycosylphosphatidylinositol-anchored (GPI-anchored) cell surface glycoprotein. Since its discovery, most studies on PrP have focused on its role in neurodegenerative prion diseases, whereas its function outside the nervous system remains unclear. Here, we report that human pancreatic ductal adenocarcinoma (PDAC) cell lines expressed PrP. However, the PrP was neither glycosylated nor GPI-anchored, existing as pro-PrP and retaining its GPI anchor peptide signal sequence (GPI-PSS). We also showed that the PrP GPI-PSS has a filamin A-binding (FLNa-binding) motif and interacted with FLNa, an actin-associated protein that integrates cell mechanics and signaling. Binding of pro-PrP to FLNa disrupted cytoskeletal organization. Inhibition of PrP expression by shRNA in the PDAC cell lines altered the cytoskeleton and expression of multiple signaling proteins; it also reduced cellular proliferation and invasiveness in vitro as well as tumor growth in vivo. A subgroup of human patients with pancreatic cancer was found to have tumors that expressed pro-PrP. Most importantly, PrP expression in tumors correlated with a marked decrease in patient survival. We propose that binding of pro-PrP to FLNa perturbs FLNa function, thus contributing to the aggressiveness of PDAC. Prevention of this interaction could provide an attractive target for therapeutic intervention in human PDAC.

Brain iron homeostasis: from molecular mechanisms to clinical significance and therapeutic opportunities.

Iron has emerged as a significant cause of neurotoxicity in several neurodegenerative conditions, including Alzheimers disease (AD), Parkinsons disease (PD), sporadic Creutzfeldt-Jakob disease (sCJD), and others. In some cases, the underlying cause of iron mis-metabolism is known, while in others, our understanding is, at best, incomplete. Recent evidence implicating key proteins involved in the pathogenesis of AD, PD, and sCJD in cellular iron metabolism suggests that imbalance of brain iron homeostasis associated with these disorders is a direct consequence of disease pathogenesis. A complete understanding of the molecular events leading to this phenotype is lacking partly because of the complex regulation of iron homeostasis within the brain. Since systemic organs and the brain share several iron regulatory mechanisms and iron-modulating proteins, dysfunction of a specific pathway or selective absence of iron-modulating protein(s) in systemic organs has provided important insights into the maintenance of iron homeostasis within the brain. Here, we review recent information on the regulation of iron uptake and utilization in systemic organs and within the complex environment of the brain, with particular emphasis on the underlying mechanisms leading to brain iron mis-metabolism in specific neurodegenerative conditions. Mouse models that have been instrumental in understanding systemic and brain disorders associated with iron mis-metabolism are also described, followed by current therapeutic strategies which are aimed at restoring brain iron homeostasis in different neurodegenerative conditions. We conclude by highlighting important gaps in our understanding of brain iron metabolism and mis-metabolism, particularly in the context of neurodegenerative disorders.