The transmissible spongiform encephalopathies or prion diseases are a group of fatal neurodegenerative disorders that affect animals and humans and can exhibit sporadic, inherited, or infectious presentations. Propagation of the disease requires the expression of the cellular prion protein (PrPC), a glycosyl-phosphatidylinositol (GPI)-anchored cell surface sialoglycoprotein. The protein is converted into an abnormal form, called PrPSc, through a major conformational change. In contrast to PrPC, the pathogenic PrPSc isoform has a relatively high content of β-sheet structure, which is partially resistant to proteolytic digestion, is insoluble in non-ionic detergents, and has a tendency to aggregate into amyloid-like fibrils and plaques.
According to the protein-only hypothesis, transmission of these diseases does not require nucleic acids; PrPSc itself is the infectious prion pathogen. Accumulation of the toxic insoluble PrPSc has been assumed to be the most probable cause of neuronal death in prion diseases. However, given that clinical manifestations may occur either before or without characteristic PrPSc deposits, it has been suggested that neurotoxicity is unlikely to be the only factor in the pathogenesis of such diseases. In view of the evidence that PrPSc's neurotoxic properties are not sufficient to understand the neurodegeneration associated with these devastating diseases, we have been working to uncover the biological functions of PrPC and loss-of-function mechanisms in prion diseases.
Strong evidence for a neuroprotective PrPC function derives from our description of a putative PrPC p66 ligand, which was later identified as the stress-inducible protein 1 (STI-1). In collaboration with Rafael Linden (Federal University of Rio de Janeiro, Brazil) and Ricardo Brentani (A.C. Camargo Cancer Hospital, Brazil), we demonstrated that the interaction between PrPC and STI-1 prevented the programmed cell death of undifferentiated postmitotic retinal cells and hippocampal neurons and also induced neuronal differentiation. STI-1 raised intracellular cAMP levels and activated the Erk pathway. The neuroprotective effect required the activity of cAMP-dependent protein kinase , whereas neuronal differentiation depended on MAP kinase. The STI-1 binding site is located at the PrPC fragment 113–128, which overlaps with the so-called “neurotoxic” peptide PrP106–126, a peptide that mimics the physicochemical properties and putative neurotoxicity of PrPSc. Our data showed that this peptide was able to inhibit PrPC binding to STI-1, thus abolishing the neuroprotective effect triggered by this interaction. Thus, it is possible to speculate that, during the development of prion diseases, changes in the PrPC molecule result in a lowering of affinity for STI-1 to the threshold for programmed cell death.
It is well known that PrPC strongly binds to Cu2+ and thus may be involved in both copper metabolism and protection against oxidative stress. In collaboration with Nibaldo Inestrosa (Catholic University of Chile), we verified that PrPC gene expression is upregulated by copper in neuronal cells. Thus, copper may also modulate PrPC functions by regulating its cellular expression levels.
Important clues about PrPC's functional properties were also provided by our observation that PrPC is a cell-surface ligand for laminin. PrPC interacts with a laminin decapeptide (RNIAEIIKDI) located at the C-terminus of the laminin γ-1 chain. In addition, we mapped the laminin binding site of PrPC to within amino acids 173–182. Using primary hippocampal neuron cultures, we observed that the PrPC-laminin γ-1 chain interaction induces neuronal adhesion and neurite maintenance and extension.
It has been shown that degradation of laminin by plasminogen activator, induced by the glutamate analogue kainic acid, leads to neuronal death. It is conceivable that PrPc may be one of the receptors involved with laminin signaling for neuroprotection. Indeed, we showed that PrPc-null mice are more sensitive than wild-type animals to seizures induced by kainic acid.
Intracellular trafficking of PrPC is important for generating protease-resistant PrP species, but little is known about how endocytosis affects PrPC function. In collaboration with Marco Antonio Prado (Federal University of Minas Gerais, Brazil), we showed that, contrary to what would be expected for a GPI-anchored protein, clathrin-mediated endocytosis and classical endocytic organelles participate in PrPC trafficking. Moreover, the N-terminal domain of PrPC is involved in sorting events that can direct the protein during its intracellular journey. Cellular signaling can be triggered or regulated by PrPC, and we suggest that endocytosis of PrPC may influence signaling in several ways. Definition of the processes that participate in PrPC endocytosis and intracellular trafficking can have a major impact on our understanding of the mechanisms involved in PrPC function and conversion to protease-resistant conformations.
To obtain our most recent data, which we generated in collaboration with Ivan Izquierdo and Martin Cammarota (Catholic University of Rio Grande do Sul, Brazil), we used rats in an aversively motivated test of learning to investigate whether laminin or STI1 engagement to PrPC affects memory consolidation. Inhibition of PrPC–STI1 or PrPC–laminin interaction, which we achieved by infusing into the hippocampus specific antibodies or peptides mimicking the binding sites within PrPC, abrogated memory consolidation. Strikingly, STI1 peptide 230–245, the PrPc binding site, had a potent enhancing effect on memory formation and consolidation, suggesting a potential use of ST11 230–245 peptide as a pharmacological agent.
More than 20 mutations in the human PrPC gene have been shown to segregate with inherited Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, and fatal familial insomnia. In this context, although inherited forms represent only a fraction of the prion diseases, mutated PrPC proteins are ideal models for studying loss of PrPC function.
We are testing for physiological roles of PrPC in neurons and glial cells and how these roles are altered in mutated PrPC proteins associated with inherited forms of prion diseases. To understand loss-of-function mechanisms, we are comparing wild-type and mutated PrPC proteins with regard to their interaction with laminin and STI-1, cellular trafficking, signaling, and participation in neuronal survival, differentiation, and regeneration. Understanding the physiological role of PrPC, coupled with experimental models related to prion pathology such as disease-associated PrPC mutations, is likely to help in the development of proper therapeutic measures for these devastating diseases.
Since 1995, human prion diseases have been under compulsory notification in Brazil, and we are working with the Brazilian Ministry of Heath to screen for them in the Brazilian population.
Our work is also supported by the Ludwig Institute for Cancer Research and the State of São Paulo Research Foundation (FAPESP).
Last updated August 2008
