Hermann M. Schätzl

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.

Individuals with antibodies against hepatitis B core antigen as the only serological marker for hepatitis B infection: high percentage of carriers of hepatitis B and C virus

BACKGROUND/AIMS Several reports have unequivocally demonstrated that some individuals with antibodies against hepatitis B core antigen as the only serological marker for hepatitis B infection are chronic carriers of the hepatitis B virus. Nevertheless, conflicting data exist about the frequency of this phenomenon; its cause is unknown. METHODS In a prospective study we tested individuals who were positive for anti-HBc alone for HBV-DNA as well as for coexisting infections with human immunodeficiency virus and hepatitis C virus. RESULTS Using polymerase chain reaction with primer pairs from three different regions of the hepatitis B virus genome, we found 54 of 164 individuals (32.9%) with anti-HBc alone to be positive for hepatitis B virus, the majority of them showing very low hepatitis B virus concentrations. 14.3% were human immunodeficiency virus positive; half of them were also hepatitis B virus carriers. Surprisingly, 62 of 153 participants (40.5%) in this study showed antibodies against hepatitis C virus, and about two thirds of the latter were also positive for HCV-RNA. This finding could be confirmed by a retrospective analysis of all people tested for hepatitis B virus markers and anti-HCV in our institution during the 2 years before the prospective study was begun. Again, a high correlation was found between the presence of anti-HCV and anti-HBc alone: 49.2% of individuals with anti-HBc only were anti-HCV positive also, compared to 26.8% of HBsAg carriers and only 10% of individuals showing the serological pattern of past hepatitis B. CONCLUSIONS Thus our study of individuals positive for anti-HBc alone revealed a high number of carriers of hepatitis B virus and hepatitis C virus among them; furthermore, we found some evidence that hepatitis C virus infection may favour this unusual hepatitis B virus marker pattern.

Intracellular re‐routing of prion protein prevents propagation of PrPSc and delays onset of prion disease

Prion diseases are fatal and transmissible neurodegenerative disorders linked to an aberrant conformation of the cellular prion protein (PrPc). We show that the chemical compound Suramin induced aggregation of PrP in a post‐ER/Golgi compartment and prevented further trafficking of PrPc to the outer leaflet of the plasma membrane. Instead, misfolded PrP was efficiently re‐routed to acidic compartments for intracellular degradation. In contrast to PrPSc in prion‐infected cells, PrP aggregates formed in the presence of Suramin did not accumulate, were entirely sensitive to proteolytic digestion, had distinct biophysical properties, and were not infectious. The prophylactic potential of Suramin‐induced intracellular re‐routing was tested in mice. After intraperitoneal infection with scrapie prions, peripheral application of Suramin around the time of inoculation significantly delayed onset of prion disease. Our data reveal a novel quality control mechanism for misfolded PrP isoforms and introduce a new molecular mechanism for anti‐prion compounds.

Autophagy induction by trehalose counteracts cellular prion infection

Prion diseases are fatal neurodegenerative and infectious disorders for which no therapeutic or prophylactic regimens exist. In search of cellular mechanisms that play a role in prion diseases and have the potential to interfere with accumulation of intracellular pathological prion protein (PrPSc), we investigated the autophagic pathway and one of its recently published inducers, trehalose. Trehalose, an alpha-linked disaccharide, has been shown to accelerate clearance of mutant huntingtin and α-synuclein by activating autophagy, mainly in an mTOR independent manner. Here, we demonstrate that trehalose can significantly reduce PrPSc in a dose- and time-dependent manner while at the same time it induces autophagy in persistently prion-infected neuronal cells. Inhibition of autophagy, either pharmacologically by known autophagy inhibitors like 3-methyladenine, or genetically by siRNA targeting Atg5, counteracted the anti-prion effect of trehalose. Hence, we provide direct experimental evidence that induction of autophagy mediates enhanced cellular degradation of prions. Similar results were obtained with rapamycin, a known inducer of autophagy, and imatinib, which has been shown to activate autophagosome formation. While induction of autophagy resulted in reduction of PrPSc, inhibition of autophagy increased the amounts of cellular PrPSc, suggesting that autophagy is involved in the physiological degradation process of cellular PrPSc. Preliminary in vivo studies with trehalose in intraperitoneally prion-infected mice did not result in prolongation of incubation times, but demonstrated delayed appearance of PrPSc in the spleen. Overall, our study provides the first experimental evidence for the impact of autophagy in yet another type of neurodegenerative disease, namely prion disease.

Lithium induces clearance of protease resistant prion protein in prion-infected cells by induction of autophagy.

Lithium is used for several decades to treat manic‐depressive illness (bipolar affective disorder). Recently, it was found that lithium induces autophagy, thereby promoting the clearance of mutant huntingtin and α‐synucleins in experimental systems. We show here for the first time that lithium significantly reduces the amount of pathological prion protein (PrPSc) in prion‐infected neuronal and non‐neuronal cultured cells by inducing autophagy. Treatment of prion‐infected cells with 3‐methyladenine, a potent inhibitor of autophagy, counteracted the anti‐prion effect of lithium, demonstrating that induction of autophagy mediates degradation of PrPSc. Co‐treatment with lithium and rapamycin, a drug widely used to induce autophagy, had an additive effect on PrPSc clearance compared to treatment with either drug alone. In addition, we provide evidence that the ability to reduce PrPSc and to induce autophagy is common for diverse lithium compounds, not only for the drug lithium chloride, usually administered in clinical therapy. Furthermore, we show here that besides reduction of PrPSc‐aggregates, lithium‐induced autophagy also slightly reduces the levels of cellular prion protein. Limiting the substrate available for conversion of cellular prion protein into PrPSc may provide an additional mechanism for reduction of PrPSc by lithium‐induced autophagy.

Prion-Protein-Specific Aptamer Reduces PrPSc Formation

The critical initial event in the pathophysiology of transmissible spongiform encephalopathies (TSEs) appears to be the conversion of the cellular prion protein (PrPC) into the abnormal isoform PrPSc. This isoform forms high‐molecular‐weight protease K (PK) resistant aggregates that accumulate in the central nervous system of affected individuals. We have selected nuclease‐resistant 2′‐amino‐2′‐deoxypyrimidine‐modified RNA aptamers which recognize a peptide comprising amino acid residues 90–129 of the human prion protein with high specificity. This domain of prion proteins is thought to be functionally important for the conversion of PrPC into its pathogenic isoform PrPSc and is highly homologous among prion proteins of various species including mouse, hamster, and man. Consequently, aptamer DP7 binds to the full‐length human, mouse, and hamster prion protein. At low concentrations in the growth medium of persistently prion‐infected neuroblastoma cells, aptamer DP7 significantly reduced the relative proportion of de novo synthesized PK‐resistant PrPSc within only 16 h. These findings may open the door towards a rational development of a new class of drugs for the therapy or prophylaxis of prion diseases.

Polyclonal anti-PrP auto-antibodies induced with dimeric PrP interfere efficiently with PrPSc propagation in prion-infected cells.

Prion diseases are neurodegenerative infectious disorders for which no prophylactic regimens are known. In order to induce antibodies/auto-antibodies directed against surface-located PrPc, we used a covalently linked dimer of mouse prion protein expressed recombinantly in Escherichia coli. Employing dimeric PrP as an immunogen we were able to effectively overcome autotolerance against murine PrP in PrP wild-type mice without inducing obvious side effects. Treatment of prion-infected mouse cells with polyclonal anti-PrP antibodies generated in rabbit or auto-antibodies produced in mice significantly inhibited endogenous PrPSc synthesis. We show that polyclonal antibodies are binding to surface-located PrPc, thereby interfering with prion biogenesis. This effect is much more pronounced in the presence of full IgG molecules, which, unlike Fab fragments, seem to induce a significant cross-linking of surface PrP. In addition, we found immune responses against different epitopes when comparing antibodies induced in rabbits and PrP wild-type mice. Only in the auto-antibody situation in mice an immune reaction against a region of PrP is found that was reported to be involved in the PrPSc conversion process. Our data point to the possibility of developing means for an active immunoprophylaxis against prion diseases.

Essential Role of the Prion Protein N Terminus in Subcellular Trafficking and Half-life of Cellular Prion Protein

Aberrant metabolism and conformational alterations of the cellular prion protein (PrPc) are the underlying causes of transmissible spongiform encephalopathies in humans and animals. In cells, PrPc is modified post-translationally and transported along the secretory pathway to the plasma membrane, where it is attached to the cell surface by a glycosylphosphatidylinositol anchor. In surface biotinylation assays we observed that deletions within the unstructured N terminus of murine PrPc led to a significant reduction of internalization of PrP after transfection of murine neuroblastoma cells. Truncation of the entire N terminus most significantly inhibited internalization of PrPc. The same deletions caused a significant prolongation of cellular half-life of PrPc and a delay in the transport through the secretory pathway to the cell surface. There was no difference in the glycosylation kinetics, indicating that all PrP constructs equally passed endoplasmic reticulum-based cellular quality control. Addition of the N terminus of the Xenopus laevis PrP, which does not encode a copper-binding repeat element, to N-terminally truncated mouse PrP restored the wild type phenotype. These results provide deeper insight into the life cycle of the PrPc, raising the novel possibility of a targeting function of its N-proximal part by interacting with the secretory and the endocytic machinery. They also indicate the conservation of this targeting property in evolution.

The prion protein requires cholesterol for cell surface localization

The cellular prion protein PrP(c) is attached to the plasma membrane by a glycosyl-phosphatidyl-inositol (GPI-) anchor and is localized in lipid rafts, membrane microdomains characterized by a high content of sphingolipids and cholesterol. Previous studies revealed that perturbation of cholesterol synthesis prevents prion conversion, explained by redistribution of PrP(c) at the plasma membrane. We investigated the influence of inhibition of cholesterol synthesis by the HMG-CoA-reductase inhibitor mevinolin on the trafficking of PrP(c) in neuronal cells. Treatment with mevinolin significantly reduces the amount of surface PrP(c) and leads to its accumulation in the Golgi compartment. Analysis of mutant PrPs highlights the importance of the GPI-anchor for raft localization and provides information about domains implicated in lipid raft association of PrP in the secretory pathway. Our data show that cholesterol is essential for the cell surface localization of PrP(c), known to be necessary for prion conversion.

Glycosylation deficiency at either one of the two glycan attachment sites of cellular prion protein preserves susceptibility to bovine spongiform encephalopathy and scrapie infections.

The conversion into abnormally folded prion protein (PrP) plays a key role in prion diseases. PrPC carries two N-linked glycan chains at amino acid residues 180 and 196 (mouse). Previous in vitro data indicated that the conversion process may not require glycosylation of PrP. However, it is conceivable that these glycans function as intermolecular binding sites during the de novo infection of cells on susceptible organisms and/or play a role for the interaction of both PrP isoforms. Such receptor-like properties could contribute to the formation of specific prion strains. However, in earlier studies, mutations at the glycosylation sites of PrP led to intracellular trafficking abnormalities, which made it impossible to generate PrP glycosylation-deficient mice that were susceptible to bovine spongiform encephalopathy (BSE) or scrapie. We have now tested more than 25 different mutations at both consensus sites and found one nonglycosylated (T182N/T198A) and two monoglycosylated (T182N and T198A) mutants that rather retained authentic cellular trafficking properties. In vitro all three mutants were converted into PrPres. PrP mutant T182N/T198A also provoked a strong dominant-negative inhibition on the endogenous wild type PrP conversion reaction. By using the two monoglycosylated mutants, we generated transgenic mice overexpressing PrPC in their brains at levels of 2–4 times that of nontransgenic mice. Most interestingly, such mice proved readily susceptible to a challenge with either scrapie (Chandler and Me7) or with BSE. Incubation times were comparable or in some instances even significantly shorter than those of nontransgenic mice. These data indicate that diglycosylation of PrPC is not mandatory for prion infection in vivo.