Therapeutic targeting of cellular prion protein: toward the development of dual mechanism anti-prion compounds

PrP Sc , a misfolded, aggregation-prone isoform of the cellular prion protein (PrP C ), is the infectious prion agent responsible for fatal neurodegenerative diseases of humans and other mammals. PrP Sc can adopt different pathogenic conformations (prion strains), which can be resistant to potential drugs, or acquire drug resistance, posing challenges for the development of effective therapies. Since PrP C is the obligate precursor of any prion strain and serves as the mediator of prion neurotoxicity, it represents an attractive therapeutic target for prion diseases. In this minireview, we briefly outline the approaches to target PrP C and discuss our recent identification of Zn(II)-BnPyP, a PrP C -targeting porphyrin with an unprecedented bimodal mechanism of action. We argue that in-depth understanding of the molecular mechanism by which Zn(II)-BnPyP targets PrP C may lead toward the development of a new class of dual mechanism anti-prion compounds

The Central Region of the Cellular Prion Protein Attenuates the Intrinsic Toxicity of N-Terminus

The misfolding of the cellular prion protein (PrPC) into the aggregate prone conformer (PrPSc) is at the heart of a class of neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). These diseases affect humans, bovine, sheep, and other mammals. PrPC is a well conserved mammalian protein and is highly expressed in the brain. PrPC is composed of two main domains consisting of the unstructured N-terminus and the structured C-terminus. The two main domains are connected by a short linker called the central region (CR). Previous studies have shown internal amino-proximal deletions spanning the CR generate a profound neurotoxicity in mice. The toxicity generated by these deletions have been shown to parallel bona fide prion diseases, and thus making them a good model system for studying prion diseases. The neurotoxicity elicited by CR deletion mutants is thought to originate from a misregulation of the extreme N-terminus of PrPC. However, these deletion mutants make two major changes that could potentially lead to the misregulation of the N-terminus. These include: 1) weakening of a protective metal-driven cis-interaction, and 2) deletion of the locus important in a regulatory cleavage event. This study used designed mutations to allow for one of these changes at a time while not altering the other. We find that neither blocking the metal-driven cis-interaction nor cleavage generates toxicity. Conversely, we show that the WT PrPC CR sequence is necessary for regulating the toxicity. Moreover, we find that the WT PrPC CR sequence is necessary for PrPC homodimerization on the cell surface, which could be a protective mechanism from the deleterious effects of a misregulated extreme N-terminus. Additionally, we found that pathological mutations in the CR causing a certain set of prion diseases in humans do not affect the metal-driven cis-interaction. Furthermore, these result suggests a difference in the origin of neurotoxicity between CR pathological mutations and C-terminal pathological mutations

A cationic tetrapyrrole inhibits toxic activities of the cellular prion protein

Prion diseases are rare neurodegenerative conditions associated with the conformational conversion of the cellular prion protein (PrPC) into PrPSc, a self-replicating isoform (prion) that accumulates in the central nervous system of affected individuals. The structure of PrPSc is poorly defined, and likely to be heterogeneous, as suggested by the existence of different prion strains. The latter represents a relevant problem for therapy in prion diseases, as some potent anti-prion compounds have shown strain-specificity. Designing therapeutics that target PrPC may provide an opportunity to overcome these problems. PrPC ligands may theoretically inhibit the replication of multiple prion strains, by acting on the common substrate of any prion replication reaction. Here, we characterized the properties of a cationic tetrapyrrole [Fe(III)-TMPyP], which was previously shown to bind PrPC, and inhibit the replication of a mouse prion strain. We report that the compound is active against multiple prion strains in vitro and in cells. Interestingly, we also find that Fe(III)-TMPyP inhibits several PrPC-related toxic activities, including the channel-forming ability of a PrP mutant, and the PrPC-dependent synaptotoxicity of amyloid-beta (A beta) oligomers, which are associated with Alzheimer's Disease. These results demonstrate that molecules binding to PrPC may produce a dual effect of blocking prion replication and inhibiting PrPC-mediated toxicity

Prion protein-specific antibodies-development, modes of action and therapeutics application

Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are lethal neurodegenerative disorders involving the misfolding of the host encoded cellular prion protein, PrPC. This physiological form of the protein is expressed throughout the body, and it reaches the highest levels in the central nervous system where the pathology occurs. The conversion into the pathogenic isoform denoted as prion or PrPSc is the key event in prion disorders. Prominent candidates for the treatment of prion diseases are antibodies and their derivatives. Anti-PrPC antibodies are able to clear PrPSc from cell culture of infected cells. Furthermore, application of anti-PrPC antibodies suppresses prion replication in experimental animal models. Major drawbacks of immunotherapy are immune tolerance, the risks of neurotoxic side effects, limited ability of compounds to cross the blood-brain barrier and their unfavorable pharmacokinetic. The focus of this review is to recapitulate the current understanding of the molecular mechanisms for antibody mediated anti-prion activity. Although relevant for designing immunotherapeutic tools, the characterization of key antibody parameters shaping the molecular mechanism of the PrPC to PrPSc conversion remains elusive. Moreover, this review illustrates the various attempts towards the development of anti-PrP antibody compounds and discusses therapeutic candidates that modulate PrP expression

Antibody binding increases the flexibility of the prion protein

Prion diseases are associated with the conversion of the cellular prion protein (PrP) into a pathogenic conformer (PrPSc). A proposed therapeutic approach to avoid the pathogenic transformation is to develop antibodies that bind to PrP and stabilize its structure. POM1 and POM6 are two monoclonal antibodies that bind the globular domain of PrP and have different biological responses, i.e., trigger neurotoxicity mimicking prion infections (POM1) or prevent neurotoxicity (POM6). The crystal structures of PrP in complex with the two antibodies show similar epitopes which seems inconsistent with the opposite phenotypes. Here, we investigate the influence of the POM1 and POM6 antibodies on the flexibility of the mouse PrP by molecular dynamics simulations. The simulations reveal that the POM6/PrP interface is less stable than the POM1/PrP interface, ascribable to localized polar mismatches at the interface, despite the former complex having a larger epitope than the latter. In the presence of any of the two antibodies, the flexibility of the globular domain increases everywhere except for the β1-α1 loop in the POM1/PrP complex which suggests the involvement of this loop in the pathological conversion. The secondary structure of PrP is preserved whereas the polar interactions involving residues Glu146, Arg156 and Arg208 are modified upon antibody binding

Prion strains viewed through the lens of cryo-EM

Mammalian prions are lethal transmissible pathogens that cause fatal neurodegenerative diseases in humans and animals. They consist of fibrils of misfolded, host-encoded prion protein (PrP) which propagate through templated protein polymerisation. Prion strains produce distinct clinicopathological phenotypes in the same host and appear to be encoded by distinct misfolded PrP conformations and assembly states. Despite fundamental advances in our understanding of prion biology, key knowledge gaps remain. These include precise delineation of prion replication mechanisms, detailed explanation of the molecular basis of prion strains and inter-species transmission barriers, and the structural definition of neurotoxic PrP species. Central to addressing these questions is the determination of prion structure. While high-resolution definition of ex vivo prion fibrils once seemed unlikely, recent advances in cryo-electron microscopy (cryo-EM) and computational methods for 3D reconstruction of amyloids have now made this possible. Recently, near-atomic resolution structures of highly infectious, ex vivo prion fibrils from hamster 263K and mouse RML prion strains were reported. The fibrils have a comparable parallel in-register intermolecular β-sheet (PIRIBS) architecture that now provides a structural foundation for understanding prion strain diversity in mammals. Here, we review these new findings and discuss directions for future research

Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells

Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumorinitiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype

Involvement of PrPC in kainate-induced excitotoxicity in several mouse strains

The cellular prion protein (PrPC) has been associated with a plethora of cellular functions ranging from cell cycle to neuroprotection. Mice lacking PrPC show an increased susceptibility to epileptic seizures; the protein, then, is neuroprotective. However, lack of experimental reproducibility has led to considering the possibility that other factors besides PrPC deletion, such as the genetic background of mice or the presence of so-called "Prnp flanking genes", might contribute to the reported susceptibility. Here, we performed a comparative analysis of seizure-susceptibility using characterized Prnp(+/+) and Prnp(0/0) mice of B6129, B6.129, 129/Ola or FVB/N genetic backgrounds. Our study indicates that PrPC plays a role in neuroprotection in KA-treated cells and mice. For this function, PrPC should contain the aa32-93 region and needs to be linked to the membrane. In addition, some unidentified "Prnp-flanking genes" play a role parallel to PrPC in the KA-mediated responses in B6129 and B6.129 Prnp(0/0) mice

Prion Protein as a Toxic Acceptor of Amyloid-β Oligomers

The initial report that cellular prion protein (PrPC) mediates toxicity of Amyloid-β (Aβ) species linked to Alzheimer’s disease was initially treated with scepticism, but growing evidence supports this claim. That there is a high-affinity interaction is now clear and its molecular basis is being unravelled whilst recent studies have identified possible down-stream toxic mechanisms. Determination of the clinical significance of such interactions between PrPC and disease-associated Aβ species will require experimental medicine studies in humans. Compounds that inhibit PrP-dependent Aβ toxicity are starting to be trialled in humans and, although it is clear that only a fraction of Alzheimer’s disease toxicity could be governed by PrPC, a partial but still therapeutically useful role in human disease may soon be testable