Disease-associated prion protein oligomers inhibit the 26S proteasome

The mechanism of cell death in prion disease is unknown but is associated with the production of a misfolded conformer of the prion protein. We report that disease-associated prion protein specifically inhibits the proteolytic beta subunits of the 26S proteasome. Using reporter substrates, fluorogenic peptides, and an activity probe for the beta subunits, this inhibitory effect was demonstrated in pure 26S proteasome and three different cell lines. By challenge with recombinant prion and other amyloidogenic proteins, we demonstrate that only the prion protein in a nonnative beta sheet conformation inhibits the 26S proteasome at stoichiometric concentrations. Preincubation with an antibody specific for aggregation intermediates abrogates this inhibition, consistent with an oligomeric species mediating this effect. We also present evidence for a direct relationship between prion neuropathology and impairment of the ubiquitin-proteasome system (UPS) in prion-infected UPS-reporter mice. Together, these data suggest a mechanism for intracellular neurotoxicity mediated by oligomers of misfolded prion protein.     

Disease-associated protein seeding suggests a dissociation between misfolded protein accumulation and neurodegeneration in prion disease

Chronic neurodegenerative diseases, such as prion diseases or Alzheimer's disease, are associated with progressive accumulation of host proteins which misfold and aggregate. Neurodegeneration is restricted to specific neuronal populations which show clear accumulation of misfolded proteins, whilst neighbouring neurons remain unaffected. Such data raise interesting questions about the vulnerability of specific neuronal populations to neurodegeneration and much research has concentrated only on the mechanisms of neurodegeneration in afflicted neuronal populations. An alternative, undervalued and almost completely unstudied question however is how and why neuronal populations are resilient to neurodegeneration. One potential answer is unaffected regions do not accumulate misfolded proteins, thus mechanisms of neurodegeneration do not become activated. In this perspectives, we discuss novel data from our laboratories which demonstrate that misfolded proteins do accumulate in regions of the brain which do not show evidence of neurodegeneration and further evidence that microglial responses may define the severity of neurodegeneration.     

The prion protein M129V polymorphism

The PRNP gene encodes the cellular isoform of prion protein (PrP^c). The M129V polymorphism influences the risk of prion diseases and may modulate the rate of neurodegeneration with age. We present the first study of the polymorphism among Polish centenarians. In the control group (n = 165, ages 18 to 56 years) the observed M129V genotype frequencies agreed with those expected according to the Hardy-Weinberg equilibrium (MM, MV, VV): 43%, 44%, 13% (HWE p > 0.05). Among centenarians (n = 150, ages 100 to 107) both homozygotes were more common than expected and HWE was rejected: 46%, 37%, 17% (expected 42%, 46%, 13%; HWE p = 0.025). This finding is consistent with a higher mortality rate among heterozygotes. However, the observed allele and genotype frequencies did not differ significantly between the oldest-old and the young controls. The genotypic frequencies were not related to severe cognitive impairment among the centenarians.     

Soluble dimeric prion protein binds PrP(Sc) in vivo and antagonizes prion disease

Conversion of cellular prion protein (PrP(C)) into a pathological conformer (PrP(Sc)) is thought to be promoted by PrP(Sc) in a poorly understood process. Here, we report that in wild-type mice, the expression of PrP(C) rendered soluble and dimeric by fusion to immunoglobulin Fcgamma (PrP-Fc(2)) delays PrP(Sc) accumulation, agent replication, and onset of disease following inoculation with infective prions. In infected PrP-expressing brains, PrP-Fc(2) relocates to lipid rafts and associates with PrP(Sc) without acquiring protease resistance, indicating that PrP-Fc(2) resists conversion. Accordingly, mice expressing PrP-Fc(2) but lacking endogenous PrP(C) are resistant to scrapie, do not accumulate PrP-Fc(2)(Sc), and do not transmit disease to others. These results indicate that various PrP isoforms engage in a complex in vivo, whose distortion by PrP-Fc(2) affects prion propagation and scrapie pathogenesis. The unique properties of PrP-Fc(2) suggest that soluble PrP derivatives may represent a new class of prion replication antagonists.     

No superoxide dismutase activity of cellular prion protein in vivo

Prion diseases are characterized by the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(C), which is encoded by the Prnp gene. PrP(C) is highly expressed on neurons and its function is unknown. Recombinant PrP(C) was claimed to possess superoxide dismutase (SOD) activity, and it was hypothesized that abrogation of this function may contribute to neurodegeneration in prion diseases. We tested this hypothesis in vivo by studying copper/zinc and manganese SOD activity in genetically defined crosses of mice lacking the Sod1 gene with mice lacking PrP(C), and with hemizygous or homozygous tga20 transgenic mice overexpressing various levels of PrP(C). We failed to detect any influence of the Prnp genotype and gene dosage on SOD1 or SOD2 activity in heart, spleen, brain, and synaptosome-enriched brain fractions. Control experiments included crosses of mice lacking or overexpressing PrPc with mice overexpressing human Cu2+/Zn2+-superoxide dismutase, and confirmed that SOD enzymatic activity correlated exclusively with the gene dosage of bona fide human or murine SOD. We conclude that PrP(C) in vivo does not discernibly contribute to total SOD activity and does not possess an intrinsic dismutase activity.     

In vitro and in vivo neurotoxicity of prion protein oligomers

The mechanisms underlying prion-linked neurodegeneration remain to be elucidated, despite several recent advances in this field. Herein, we show that soluble, low molecular weight oligomers of the full-length prion protein (PrP), which possess characteristics of PrP to PrPsc conversion intermediates such as partial protease resistance, are neurotoxic in vitro on primary cultures of neurons and in vivo after subcortical stereotaxic injection. Monomeric PrP was not toxic. Insoluble, fibrillar forms of PrP exhibited no toxicity in vitro and were less toxic than their oligomeric counterparts in vivo. The toxicity was independent of PrP expression in the neurons both in vitro and in vivo for the PrP oligomers and in vivo for the PrP fibrils. Rescue experiments with antibodies showed that the exposure of the hydrophobic stretch of PrP at the oligomeric surface was necessary for toxicity. This study identifies toxic PrP species in vivo. It shows that PrP-induced neurodegeneration shares common mechanisms with other brain amyloidoses like Alzheimer disease and opens new avenues for neuroprotective intervention strategies of prion diseases targeting PrP oligomers.     

Tumor-produced secreted form of binding of immunoglobulin protein elicits antigen-specific tumor immunity

Binding of immunoglobulin protein (BiP) is a major molecular chaperone localized in endoplasmic reticulum (ER). It has been demonstrated to interact with nascent Ig. However, contrary to other ER-resident heat shock proteins such as gp96, calreticulin, and ORP150, it is not clear whether tumor-derived BiP plays a role in inducing antitumor immunity. In this study, we show that the tumor-derived secreted form of BiP is capable of inducing antitumor CD8(+) T cell responses. We constructed an ER-retention signal KDEL-deleted mutant of BiP cDNA and transfected it to tumor cells, which resulted in continuous secretion of tumor-derived BiP into the extracellular milieu. We show that this secreted BiP is taken up by bone marrow-derived dendritic cells, and thereafter BiP-associated Ag peptide is cross-presented in association with MHC class I molecules, resulting in elicitation of an Ag-specific CD8(+) T cell response and antitumor effect. This strategy to boost antitumor immune responses shows that a tumor could be its own cellular vaccine via gene modification of the secretion of the tumor Ag-BiP complex.     

Strain-specific kinetics of prion protein formation in vitro and in vivo

The molecular basis of prion strain diversity is proposed to be encoded by distinct conformations of the abnormal scrapie isoform of the prion protein (PrP(Sc)). PrP(Sc) formation for the hyper (HY) and drowsy (DY) strains of the transmissible mink encephalopathy (TME) agent was investigated using the cell-free PrP conversion reaction to determine the role of distinct PrP(Sc) conformations in the rate of in vitro conversion of cellular PrP into protease-resistant PrP. PrP conversion increased at an exponential rate for both TME strains until peak levels were reached at 72-96 h of reaction time. The amount and rate of PrP conversion for HY TME was greater than those for DY TME between 48 h and the peak level of PrP conversion. Between 96 and 120 h, there was a negative rate of PrP conversion; and between 120 and 168 h, the net rate of HY and DY PrP conversion approached zero. These findings suggest that PrP conversion can occur in three distinct stages: an elongation phase, a depolymerization phase, and a steady-state phase. Strain-specific properties between the TME strains were identified only during the elongation phase. The steady-state phase could be disrupted by the addition of PrP(Sc) to, or by sonication of, the cell-free PrP conversion reaction. These treatments resulted in an increase in the amount of PrP conversion that was equal to or greater than that found during the peak level of PrP conversion for both TME strains, indicating that the steady-state phase was in dynamic equilibrium. In a related study, the rate of accumulation of HY and DY PrP(Sc) in hamster brain exhibited a strain-specific pattern that had similarities to the strain-specific PrP conversion reaction during the elongation phase. These results suggest that strain-specific conformations of PrP(Sc) have the ability to influence the rate of additional PrP(Sc) formation from cellular PrP both in vitro and in vivo.     

Disease-associated F198S mutation increases the propensity of the recombinant prion protein for conformational conversion to scrapie-like form

The critical step in the pathogenesis of transmissible spongiform encephalopathies (prion diseases) is the conversion of a cellular prion protein (PrP(c)) into a protease-resistant, beta-sheet rich form (PrP(Sc)). Although the disease transmission normally requires direct interaction between exogenous PrP(Sc) and endogenous PrP(C), the pathogenic process in hereditary prion diseases appears to develop spontaneously (i.e. not requiring infection with exogenous PrP(Sc)). To gain insight into the molecular basis of hereditary spongiform encephalopathies, we have characterized the biophysical properties of the recombinant human prion protein variant containing the mutation (Phe(198) --> Ser) associated with familial Gerstmann-Straussler-Scheinker disease. Compared with the wild-type protein, the F198S variant shows a dramatically increased propensity to self-associate into beta-sheet-rich oligomers. In a guanidine HCl-containing buffer, the transition of the F198S variant from a normal alpha-helical conformation into an oligomeric beta-sheet structure is about 50 times faster than that of the wild-type protein. Importantly, in contrast to the wild-type PrP, the mutant protein undergoes a spontaneous conversion to oligomeric beta-sheet structure even in the absence of guanidine HCl or any other denaturants. In addition to beta-sheet structure, the oligomeric form of the protein is characterized by partial resistance to proteinase K digestion, affinity for amyloid-specific dye, thioflavine T, and fibrillar morphology. The increased propensity of the F198S variant to undergo a conversion to a PrP(Sc)-like form correlates with a markedly decreased thermodynamic stability of the native alpha-helical conformer of the mutant protein. This correlation supports the notion that partially unfolded intermediates may be involved in conformational conversion of the prion protein.     

Protein conformation significantly influences immune responses to prion protein

In prion diseases, such as variant Creutzfeldt-Jakob disease normal cellular prion protein (PrPC), a largely alpha-helical structure is converted to an abnormal conformational isoform (PrPSc) that shows an increase in beta-sheet content. Similarly, the recombinant form of PrPC (ralpha-PrP) can be converted to a conformation dominated by beta-sheet (rbeta-PrP) by reduction and mild acidification in vitro, a process that may mimic in vivo conversion following PrPC internalization during recycling. Despite PrPSc accumulation and prion propagation in the lymphoreticular system before detectable neuroinvasion, no Ab response to PrP has been detected, probably due to immune tolerance. To investigate how the immune system may respond to alpha- and beta-PrP, we immunized Prnp(0/0) mice that are not tolerant of PrP with ralpha-PrP and rbeta-PrP. In this study, we show that although T cells stimulated by these differently folded conformers PrP recognize similar immunodominant epitopes (residues 111-130 and 191-210) the cytokine profile in response to ralpha- and rbeta-PrP was different. Challenge with ralpha-PrP elicited a strong response of IL-5 and IL-10, whereas rbeta-PrP led to an early increased production of IFN-gamma. In addition, immunization with ralpha-PrP led to production of predominantly IgG1 isotype Ab in the sera, whereas after immunization with rbeta-PrP, IgG2b was significantly produced. Thus, both humoral and cellular responses to these differently folded isoforms of the same protein are different, indicating a possible involvement of Th1 and Th2 pathway activation. These differences may be exploitable diagnostically and therapeutically for prion diseases, such as variant Creutzfeldt-Jakob disease.     

Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo

Proteins are often made in more than one form, with alternate versions sometimes residing in different cellular compartments than the primary species. The mammalian prion protein (PrP), a cell surface GPI-anchored protein, is a particularly noteworthy example for which minor cytosolic and transmembrane forms have been implicated in disease pathogenesis. To study these minor species, we used a selective labeling strategy in which spatially restricted expression of a biotinylating enzyme was combined with asymmetric engineering of the cognate acceptor sequence into PrP. Using this method, we could show that even wild-type PrP generates small amounts of the (Ctm)PrP transmembrane form. Selective detection of (Ctm)PrP allowed us to reveal its N-terminal processing, long half-life, residence in both intracellular and cell surface locations, and eventual degradation in the lysosome. Surprisingly, some human disease-causing mutants in PrP selectively stabilized (Ctm)PrP, revealing a previously unanticipated mechanism of (Ctm)PrP up-regulation that may contribute to disease. Thus, spatiotemporal tagging has uncovered novel aspects of normal and mutant PrP metabolism and should be readily applicable to the analysis of minor topologic isoforms of other proteins.     

Differential contribution of superoxide dismutase activity by prion protein in vivo

Normal prion protein (PrP(C)) is a copper binding protein and may play a role in cellular resistance to oxidative stress. Recently, copper-bound recombinant PrP(C) has been shown to exhibit superoxide dismutase (SOD)-like activity. However, as PrP(C) affinity for copper is low in comparison to other cupro-proteins, the question remains as to whether PrP(C) could contribute SOD activity in vivo. To unravel this enigma, we compared the SOD activity in lysates extracted from different regions of the brain from wild-type mice before and after the depletion of PrP(C). We found that removal of PrP(C) from the brain lysates reduced the levels of total SOD activity. The level of contribution to the total SOD activity was correlated to the level of PrP expressed and to the predominant form of PrP present in the specific brain region. Collectively, these results provide strong evidence that PrP(C) differentially contributes to the total SOD activity in vivo.     

Flexible N-terminal region of prion protein influences conformation of protease-resistant prion protein isoforms associated with cross-species scrapie infection in vivo and in vitro

Transmissible spongiform encephalopathy (TSE) diseases are characterized by the accumulation in brain of an abnormal protease-resistant form of the host-encoded prion protein (PrP), PrP-res. PrP-res conformation differs among TSE agents derived from various sources, and these conformational differences are thought to influence the biological characteristics of these agents. In this study, we introduced deletions into the flexible N-terminal region of PrP (residues 34-124) and investigated the effect of this region on the conformation of PrP-res generated in an in vitro cell-free conversion assay. PrP deleted from residues 34 to 99 generated 12-16-kDa protease-resistant bands with intact C termini but variable N termini. The variable N termini were the result of exposure of new protease cleavage sites in PrP-res between residues 130 and 157, suggesting that these new cleavage sites were caused by alterations in the conformation of the PrP-res generated. Similarly truncated 12-16-kDa PrP bands were also identified in brain homogenates from mice infected with mouse-passaged hamster scrapie as well as in the cell-free conversion assay using conditions that mimicked the hamster/mouse species barrier to infection. Thus, by its effects on PrP-res conformation, the flexible N-terminal region of PrP seemed to influence TSE pathogenesis and cross-species TSE transmission.     

In vivo cytotoxicity of the prion protein fragment 106-126

Transmissible spongiform encephalopathies are fatal neurological diseases characterized by astroglyosis, neuronal loss, and by the accumulation of the abnormal isoform of the prion protein. The amyloid prion protein fragment 106-126 (P106-126) has been shown to be toxic in cultured hippocampal neurons (). Here, we show that P106-126 is also cytotoxic in vivo. Taking advantage of the fact that retina is an integral part of the central nervous system, the toxic effect of the peptide was investigated by direct intravitreous injection. Aged solutions of P106-126 induced apoptotic-mediated retinal cell death and irreversibly altered the electrical activity of the retina. Neither apoptosis nor electroretinogram damages were observed with freshly diluted P106-126, suggesting that the toxicity is linked to the aggregation state of the peptide. The retina provides a convenient in vivo system to look for potential inhibitors of cytotoxicity associated with spongiform encephalopathies.     

Cytosolic prion protein toxicity is independent of cellular prion protein expression and prion propagation

Prion diseases are transmissible neurodegenerative diseases caused by a conformational isoform of the prion protein (PrP), a host-encoded cell surface sialoglycoprotein. Recent evidence suggests a cytosolic fraction of PrP (cyPrP) functions either as an initiating factor or toxic element of prion disease. When expressed in cultured cells, cyPrP acquires properties of the infectious conformation of PrP (PrP(Sc)), including insolubility, protease resistance, aggregation, and toxicity. Transgenic mice (2D1 and 1D4 lines) that coexpress cyPrP and PrP(C) exhibit focal cerebellar atrophy, scratching behavior, and gait abnormalities suggestive of prion disease, although they lack protease-resistant PrP. To determine if the coexpression of PrP(C) is necessary or inhibitory to the phenotype of these mice, we crossed Tg1D4(Prnp(+/+)) mice with PrP-ablated mice (TgPrnp(o/o)) to generate Tg1D4(Prnp(o/o)) mice and followed the development of disease and pathological phenotype. We found no difference in the onset of symptoms or the clinical or pathological phenotype of disease between Tg1D4(Prnp(+/+)) and Tg1D4(Prnp(o/o)) mice, suggesting that cyPrP and PrP(C) function independently in the disease state. Additionally, Tg1D4(Prnp(o/o)) mice were resistant to challenge with mouse-adapted scrapie (RML), suggesting cyPrP is inaccessible to PrP(Sc). We conclude that disease phenotype and cellular toxicity associated with the expression of cyPrP are independent of PrP(C) and the generation of typical prion disease.     

The prion protein

Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders represented principally by Creutzfeldt-Jakob disease in humans, and by scrapie and bovine spongiform encephalopathy in animals. Also called prion diseases, these disorders have the property of being infectious, sporadic or genetic in origin. Although the nature of the responsible agent of these diseases is uncertain, it is clear that a protein called PrPSc has a central role in their pathology. PrPSc is a conformational variant of a normal protein called PrPC. PrPC is a glycoprotein expressed by most tissues and is attached on the cell membrane by a glycosyl-phosphatidylinositol anchor which would be consistent with roles in cell adhesion, ligand uptake, or transmembrane signaling. NMR studies revealed that the protein has a globular domain and a long amino-terminal tail that contains repeated octapeptide domains which bind metal ions with high affinities. PrPC is localized on the cell membrane in detergent resistant microdomains and may be part of functional complexes with other molecules. This is particularly relevant, knowing the possible role of the molecule in signal transduction, resistance to oxidative stress and neuronal survival. In conclusion, it appears that the understanding of the biology of PrP is essential for the understanding of the physiological function of the protein as well as for its pathological conversion considering that trafficking of this molecule governs generation of PrPSc.     

A deleted prion protein that is neurotoxic in vivo is localized normally in cultured cells

The prion protein (PrP) possesses sequence-specific domains that endow the molecule with neuroprotective and neurotoxic activities, and that may contribute to the pathogenesis of prion diseases. To further define critical neurotoxic determinants within PrP, we previously generated Tg(ΔCR) mice that express a form of PrP harboring a deletion of 21 amino acids within the central domain of the protein [ Li et al., EMBO J . 26 (2007), 548 ]. These animals exhibit a neonatal lethal phenotype that is dose-dependently rescued by co-expression of wild-type PrP. In this study, we examined the localization and cell biological properties of the PrP(ΔCR) protein in cultured cells to further understand the mechanism of PrP(ΔCR) neurotoxicity. We found that the distribution of PrP(ΔCR) was identical to that of wild-type PrP in multiple cell lines of both neuronal and non-neuronal origin, and that co-expression of the two proteins did not alter the localization of either one. Both proteins were found in lipid rafts, and both were localized to the apical surface in polarized epithelial cells. Taken together, our results suggest that PrP(ΔCR) toxicity is not a result of mislocalization or aggregation of the protein, and more likely stems from altered binding interactions leading to the activation of deleterious signaling pathways.     

Disease-associated amyloid and misfolded protein aggregates activate the inflammasome

The pathogenesis of type 2 diabetes, Alzheimer's disease and amyotrophic lateral sclerosis continues to be debated. Recently, the inflammasome protein complex has been shown to be a key regulator of IL-1β, a cytokine implicated in each of these diseases. In all three cases, it is now apparent that unique protein aggregates caused by inappropriate oligomerization or misfolding are sensed by the inflammasome, providing a unifying mechanism for this IL-1β production. What evolved as an innate defense against infection-related particles, therefore, now seems to be a driving force for inflammation in these diseases. This review discusses the basic research behind these findings and the potential for new therapeutic interventions this affords.     

In vivo aggregation of the HET-s prion protein of the fungus Podospora anserina

We have proposed that the [Het-s] infectious cytoplasmic element of the filamentous fungus Podospora anserina is the prion form of the HET-s protein. The HET-s protein is involved in a cellular recognition phenomenon characteristic of filamentous fungi and known as heterokaryon incompatibility. Under the prion form, the HET-s protein causes a cell death reaction when co-expressed with the HET-S protein, from which it differs by only 13 amino acid residues. We show here that the HET-s protein can exist as two alternative states, a soluble and an aggregated form in vivo. As shown for the yeast prions, transition to the infectious prion form leads to aggregation of a HET-s--green fluorescent protein (GFP) fusion protein. The HET-s protein is aggregated in vivo when highly expressed. However, we could not demonstrate HET-s aggregation at wild-type expression levels, which could indicate that only a small fraction of the HET-s protein is in its aggregated form in vivo in wild-type [Het-s] strains. The antagonistic HET-S form is soluble even at high expression level. A double amino acid substitution in HET-s (D23A P33H), which abolishes prion infectivity, suppresses in vivo aggregation of the GFP fusion. Together, these results further support the model that the [Het-s] element corresponds to an abnormal self-perpetuating aggregated form of the HET-s protein.