Prion diseases are a group of invariably fatal neurodegenerative disorders, for which there is not cure. The molecular mechanisms of these diseases involve a complex variety of processes operating simultaneously and synergistically, including: (i) protein aggregation; (ii) oxidative stress; (iii) reduced levels of potent free-radical scavengers; (iv) unbalance of metal ions. Therefore, it is extremely challenging to develop therapies for prion diseases and other major neurodegenerative diseases. The dominant drug discovery paradigm is one disease, one target, one molecule, which ignores the polyetiological nature of prion diseases and similar maladies. Thus, some researchers have suggested this paradigm as one possible factor in the ongoing failure of current neurotherapeutic drugs. An alternative approach involves compound(s) that interact simultaneously with multiple targets. This polypharmacological approach is more promising because it is better at addressing the multifactorial and progressive pathophysiological processes involved in prion diseases.
Last Updated (Tuesday, 26 July 2011 14:45)
Read more...
The cellular prion protein (PrP), PrPC, is a ubiquitous glycoprotein that is predominantly expressed in the developing and mature nervous system. It can be found at the cell surface linked to lipid rafts via a glycosylphosphatidylinositol anchor. Despite PrP represents one of the most intensively studied mammalian proteins, its physiological function still remains unclear. The interest in studying PrPC lies in some important characteristics of this protein. In fact, PrP has at least two stable conformations and, according to the protein-only hypothesis, the infectious agent of TSEs (Transmissible Spongiform Encephalopaties) is the prion that is predominantly composed of a conformational variant of PrPC, known as PrPSc. Therefore, an elucidation of the physiological function of PrPC has the potential to help understanding the mechanisms involved in prion-induced neurodegeneration. In order to unveil its function, part of our laboratory undertook a detailed analysis of the spatial and temporal expression of PrPC during CNS development in mouse and subsequently in the opossum.
Last Updated (Tuesday, 26 July 2011 14:45)
Read more...
Prion diseases, or transmissible spongiform encephalopathies (TSE), are neurodegenerative disorders, which affect human and several animals. The incubation time of the diseases is generally quite long, and once symptoms appear, the diseases progress rapidly, leading to brain damage and death. Till now, prion diseases are invariably fatal with no effective treatment. The causing agent of prion disease is known as a prion (acronysm for proteinaceous infectious particle), which cause disease within the central nervous system disrupting the normal tissue. Neuropathology of the disease is featured by neuronal loss, gliosis and spongiform changes. The prion protein (PrP) is highly conserved among species. The putative functions of normal cellular form, PrPC and the mechanism of prion replication- a process in which PrPC converts into the pathological form of the prion or PrPSc, have been intensively studied so far.
In 2004 the production of synthetic prions via in vitro induction of misfolding and aggregation of bacterially expressed recombinant prion protein (recPrP) was introduced. The material was formed from amyloidal mouse recPrP compose residues 89-230, demonstrating that conversion after polymerization of purified recPrP into amyloidal fibrils, that represent a subset of β sheet-rich structures, is sufficient for the generation of infectivity. The first mouse synthetic prion strain inoculated into transgenic (Tg) 9949 mouse line expressing N-terminal truncated mouse recPrP(∆23-88) showed clinical disease. Furthermore, brain tissue from these sick mice contained prions that caused TSE disease when inoculated into wild-type mice and Tg mice overexpressing PrP. Neuropathological findings suggested that a novel prion strain was created. The incubation time, neuropathological lesion profiles and conformation stability (expressed as Gdn1/2 values) indicated that synthetic prions differ from RML and many other prion strains such as scrapie or bovine spongiform encephalopathy.
Read more...
One of the strongest arguments supporting the “protein-only hypothesis” is the link between prion diseases and inherited human (Hu) mutations in the PRNP gene. Several point mutations leading to familial Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker (GSS) disease, fatal familial insomnia have been identified. Our understanding of the mechanisms by which mutations cause disease remains limited. We believe that high-resolution 3D structures of the mutated PrP, more susceptible for spontaneous conversion into the pathogenic form, will help us to understand the molecular mechanism at early stages of the disease. Recently, we determined the NMR structure of the truncated recombinant HuPrP containing the GSS-related Q212P mutation (Ilc, Giachin et al., 2010). This structure revealed unique conformational features compared to the known structures of other mammalian PrP. The most remarkable differences involve the C-terminal end of the protein and the β2–α2 loop region. To provide new clues on the role of pathological point mutations on PrP structure, we have been investigating the high-resolution 3D structure of different disease-linked HuPrP mutants.
Last Updated (Tuesday, 26 July 2011 14:58)
Read more...
Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases, called Transmissible Spongiform Encephalopathies (TSEs) (Prusiner, 1982). They can manifest as infectious, sporadic or genetic disorders involving posttranslational modifications of the cellular prion protein (PrPC). Epidemiological studies carried out on Creutzfeldt-Jakob disease revealed that 87% of cases were sporadic, 8% genetic and 5% iatrogenic (Will, Alperovitch et al., 1998). The molecular mechanisms by which PrPC is converted into its pathological isoform have not yet been established. While point mutations and seeds represent a trigger to cross the energy barriers for genetic and infectious forms, respectively, there are no indications regarding sporadic forms. (Benetti and Legname, 2009).
To dissect the molecular mechanisms underlying the conversion, we are focusing on two different aspects: tertiary structure of natural prions as well as their properties and the intrinsic features of recombinant prion proteins. To these aims we are using several biophysical techniques such as circular dichroism, fluorescence, differential scanning calorimetry, NMR, small angle X-ray scattering, atomic force microscopy, single molecule force spectroscopy and transmission electron microscopy.
Last Updated (Monday, 01 August 2011 15:07)
Read more...
Despite the optimism regarding treatment and prevention of brain amyloidoses by immunotherapy, no effective immunotherapy exists for prion disease. Early indications of the potential of antibody therapy for prion disease came from in vitro studies, showing reduction in infectivity of prions after incubation with an anti-PrP antibody. Recently, various studies have been carried out to identify successful strategies for antibody–related therapies; in particular, monoclonal antibodies that bind PrPC are promising candidates to antagonize prion infection either in vitro or in vivo. An important obstacle in the development of efficacious regimens for active immunization is host tolerance to endogenous PrPC: so far, the therapeutic efficacy of these immunization approaches has been limited. On the other hand, passive immunization suffers from the intrinsic problem of poor antibody diffusion from vessels into tissues, especially in the nervous tissue: administration of monoclonal antibodies has been shown to prevent the pathogenesis only when applied simultaneously, or shortly after, peripheral prion infection. Moreover, production of large amounts of monoclonal antibodies for therapy is technically challenging and expensive. In 2001 Peretz et al. analyzed the ability of some recombinant antibody antigen–binding fragment (Fabs) to inhibit prion propagation in a cultured mouse neuroblastoma cell line infected with PrPSc. The most effective binder, denominated D18, was found to abolish prion replication and to clear pre-exiting PrPSc, eliminating 50% of PrPSc from the cells within about 24 hours. The activity of D18 seemed to be due to its ability to specifically recognize the total population of PrPC molecules on the cell surface. In PrPC, D18 epitope spans from residues 132-156 and incorporates helix A of PrPC. This sequence lays within the region of the protein thought to bind PrPSc, an essential step for prion propagation: therefore, it can be argued that D18 operates mechanistically by directly blocking or modifying interaction of PrPC with PrPSc. Although this study showed that Fab fragments against PrPC are able to cure scrapie-infected cells, their effectiveness in prion disease therapy in vivo has not yet been evaluated. In this respect, Fabs are smaller molecules compared to full-length antibodies, and their monovalent nature may be beneficial. In fact, intracerebral injection of some anti-PrP immunoglobulin G (IgG) antibodies seemed to provoke neurotoxicity. Thus, we are currently studying novel in vivo gene therapy approaches for efficient delivery of antibody fragments in the brain.
Peretz D., Williamson R.A., Kaneko K., Vergara J., Leclerc E., Schmitt- Ulms G., Mehlhorn I.R., Legname G., Wormald M.R., Rudd P.M., Dwek R.A., Burton D.R., Prusiner S.B. (2001) Antibodies inhibit prion propagation and clear cell cultures of prion infectivity. Nature, 412 (6848), 739–43. Last Updated (Monday, 01 August 2011 15:08)
|
|
Research Lines
Tuesday, 26 July 2011 14:23 | 
Written by Giuseppe Legname | 
