Man Sun Sy

A single amino acid alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy

A mutation equivalent to P102L in the human PrP gene, associated with Gerstmann–Straussler syndrome (GSS), has been introduced into the murine PrP gene by gene targeting. Mice homozygous for this mutation (101LL) showed no spontaneous transmissible spongiform encephalopathy (TSE) disease, but had incubation times dramatically different from wild‐type mice following inoculation with different TSE sources. Inoculation with GSS produced disease in 101LL mice in 288 days. Disease was transmitted from these mice to both wild‐type (226 days) and 101LL mice (148 days). In contrast, 101LL mice infected with ME7 had prolonged incubation times (338 days) compared with wild‐type mice (161 days). The 101L mutation does not, therefore, produce any spontaneous genetic disease in mice but significantly alters the incubation time of TSE infection. Additionally, a rapid TSE transmission was demonstrated despite extremely low levels of disease‐associated PrP.

Chronic Wasting Disease of Elk: Transmissibility to Humans Examined by Transgenic Mouse Models

Chronic wasting disease (CWD), a prion disease affecting free-ranging and captive cervids (deer and elk), is widespread in the United States and parts of Canada. The large cervid population, the popularity of venison consumption, and the apparent spread of the CWD epidemic are likely resulting in increased human exposure to CWD in the United States. Whether CWD is transmissible to humans, as has been shown for bovine spongiform encephalopathy (the prion disease of cattle), is unknown. We generated transgenic mice expressing the elk or human prion protein (PrP) in a PrP-null background. After intracerebral inoculation with elk CWD prion, two lines of “humanized” transgenic mice that are susceptible to human prions failed to develop the hallmarks of prion diseases after >657 and >756 d, respectively, whereas the “cervidized” transgenic mice became infected after 118–142 d. These data indicate that there is a substantial species barrier for transmission of elk CWD to humans.

Increased levels of oxidative stress markers detected in the brains of mice devoid of prion protein

Although minor abnormalities have been reported in prion protein (PrP) knock‐out (Prnp−/–) mice, the normal physiological function of PrP, the causative agent implicated in transmissible spongiform encephalopathies (TSE), remains unresolved. Since there are increasing correlations between oxidative stress and amyloidoses, we decided to investigate whether PrP plays a role in oxidative modulation. We found higher levels of oxidative damage to proteins and lipids in the brain lysates of Prnp−/– as compared to wild‐type (WT) mice of the same genetic background. These two indicators, protein oxidation and lipid peroxidation, are hallmarks of cellular oxidative damage. Elevated levels of ubiquitin‐protein conjugates were also observed in Prnp−/– mice, a probable consequence of cellular attempts to remove the damaged proteins as indicated by increased proteasome activity. Taken together, these findings are indicative of a role for PrP in oxidative homeostasis in vivo.

Oxidative impairment in scrapie-infected mice is associated with brain metals perturbations and altered antioxidant activities.

Prion diseases are characterized by the conversion of the normal cellular prion protein (PrPC) into a pathogenic isoform (PrPSc). PrPC binds copper, has superoxide dismutase (SOD)‐like activity in vitro, and its expression aids in the cellular response to oxidative stress. However, the interplay between PrPs (PrPC, PrPSc and possibly other abnormal species), copper, anti‐oxidation activity and pathogenesis of prion diseases remain unclear. In this study, we reported dramatic depression of SOD‐like activity by the affinity‐purified PrPs from scrapie‐infected brains, and together with significant reduction of Cu/Zn‐SOD activity, correlates with significant perturbations in the divalent metals contents. We also detected elevated levels of nitric oxide and superoxide in the infected brains, which could be escalating the oxidative modification of cellular proteins, reducing gluathione peroxidase activity and increasing the levels of lipid peroxidation markers. Taken together, our results suggest that brain metal imbalances, especially copper, in scrapie infection is likely to affect the anti‐oxidation functions of PrP and SODs, which, together with other cellular dysfunctions, predispose the brains to oxidative impairment and eventual degeneration. To our knowledge, this is the first study documenting a physiological connection between brain metals imbalances, the anti‐oxidation function of PrP, and aberrations in the cellular responses to oxidative stress, in scrapie infection.

Cell-surface prion protein interacts with glycosaminoglycans

We used ELISA and flow cytometry to study the binding of prion protein PrP to glycosaminoglycans (GAGs). We found that recombinant human PrP (rPrP) binds GAGs including chondroitin sulphate A, chondroitin sulphate B, hyaluronic acid, and heparin. rPrP binding to GAGs occurs via the N-terminus, a region known to bind divalent cations. Additionally, rPrP binding to GAGs is enhanced in the presence of Cu2+ and Zn2+, but not Ca2+ and Mn2+. rPrP binds heparin strongest, and the binding is inhibited by certain heparin analogues, including heparin disaccharide and sulphate-containing monosaccharides, but not by acetylated heparin. Full-length normal cellular prion protein (PrPC), but not N-terminally truncated PrPC species, from human brain bind GAGs in a similar Cu2+/Zn2+-enhanced fashion. We found that GAGs specifically bind to a synthetic peptide corresponding to amino acid residues 23-35 in the N-terminus of rPrP. We further demonstrated that while both wild-type PrPC and an octapeptide-repeat-deleted mutant PrP produced by transfected cells bound heparin at the cell surface, the PrP N-terminal deletion mutant and non-transfectant control failed to bind heparin. Binding of heparin to wild-type PrPC on the cell surface results in a reduction of the level of cell-surface PrPC. These results provide strong evidence that PrPC is a surface receptor for GAGs.

Identification of Novel Proteinase K-resistant C-terminal Fragments of PrP in Creutzfeldt-Jakob Disease

The central event in the pathogenesis of prion diseases, a group of fatal, transmissible neurodegenerative disorders including Creutzfeldt-Jakob disease (CJD) in humans, is the conversion of the normal or cellular prion protein (PrPC) into the abnormal or scrapie isoform (PrPSc). The basis of the PrPC to PrPSc conversion is thought to involve the diminution of α-helical domains accompanied by the increase of β structures within the PrP molecule. Consequently, treatment of PrPSc with proteinase K (PK) generates a large PK-resistant C-terminal core fragment termed PrP27-30 that in human prion diseases has a gel mobility of ∼19-21 kDa for the unglycosylated form, and a ragged N terminus between residues 78 and 103. PrP27-30 is considered the pathogenic and infectious core of PrPSc. Here we report the identification of two novel PK-resistant, but much smaller C-terminal fragments of PrP (PrP-CTF 12/13) in brains of subjects with sporadic CJD. PrP-CTF 12/13, like PrP27-30, derive from both glycosylated as well as unglycosylated forms. The unglycosylated PrPCTF 12/13 migrate at 12 and 13 kDa and have the N terminus at residues 162/167 and 154/156, respectively. Therefore, PrP-CTF12/13 are 64-76 amino acids N-terminally shorter than PrP27-30 and are about half of the size of PrP27-30. PrP-CTF12/13 are likely to originate from a subpopulation of PrPSc distinct from that which generates PrP27-30. The finding of PrP-CTF12/13 in CJD brains widens the heterogeneity of the PK-resistant PrP fragments associated with prion diseases and may provide useful insights toward the understanding of the PrPSc structure and its formation.

Anti-prion antibodies for prophylaxis following prion exposure in mice

Prion disease is characterized by a conformational change of the normal form of the prion protein (PrP(C)) to the scrapie-associated form (PrP(Sc)). Since the emergence of new variant Creutzfeldt-Jakob disease a potentially large human population is at risk for developing prion disease. Currently, no effective treatment or form of post-exposure prophylaxis is available for prion disease. We recently showed that active immunization with recombinant PrP prolongs the incubation period of scrapie. Here we show that anti-PrP antibodies following prion exposure are effective at increasing the incubation period of the infection. Stimulation of the immune system is an important therapeutic target for the prion diseases, as well as for other neurodegenerative illnesses characterized by abnormal protein conformation.

Cellular and subcellular morphological localization of normal prion protein in rodent cerebellum

Normal cellular prion protein, a necessary protagonist in fatal neurodegenerative prion diseases, was mapped in rodent cerebellum to establish its cellular and ultrastuctural localization. Existing morphological data about native prion protein distribution in brain tissues remain, indeed, contradictory and do not fit with biochemical and cell biological results. Using ultrastructural preembedding immunocytochemistry and a monoclonal anti‐mouse prion protein antibody, this report shows that cellular prion protein is present in all cortico‐cerebellar and deep nuclei neuronal cell types, as well as in all glial cell types. The heaviest expression appears on parallel fibres and astrocytic processes. The protein is exclusively located on the outer cell membrane and in Golgi and endosomal intracytoplasmic organelles, with no cytoplasmic or synaptic vesicle labelling. Most important, and in contrast with previous ultrastructural data, cellular prion protein is shown to be distributed on all portions of neurons, without any preferential synaptic targeting. The present morphological report shows, for the first time in vivo, that the cellular prion protein is present on the entire cell surface membrane of all neuronal and glial cell types of the rat cerebellum. This ubiquitous presence supports the notion that prion protein has a generalized cellular function in brain tissue rather than a specialized role restricted to synaptic transmission.

Clearance and prevention of prion infection in cell culture by anti-PrP antibodies.

Prion diseases are transmissible and invariably fatal neurodegenerative disorders associated with a conformational transformation of the cellular prion protein (PrPC) into a self‐replicating and proteinase K (PK)‐resistant conformer, scrapie PrP (PrPSc). Humoral immunity may significantly prolong the incubation period and even prevent disease in murine models of prionoses. However, the mechanism(s) of action of anti‐PrP monoclonal antibodies (Mabs) remain(s) obscure. The murine neuroblastoma N2a cell line, infected with the 22L mouse‐adapted scrapie strain, was used to screen a large library of Mabs with similar binding affinities to PrP, to identify those antibodies which could clear established infection and/or prevent infection de novo. Three Mabs were found capable of complete and persistent clearing of already‐infected N2a cells of PrPSc. These antibodies were 6D11 (generated to PK‐resistant PrPSc and detecting PrP residues 93–109), and 7H6 and 7A12, which were raised against recombinant PrP and react with neighbouring epitopes of PrP residues 130–140 and 143–155, respectively. Mabs were found to interact with PrPSc formation both on the cell surface and after internalization in the cytosol. Treatment with Mabs was not associated with toxicity nor did it result in decreased expression of PrPC. Both preincubation of N2a cells with Mabs prior to exposure to 22L inoculum and preincubation of the inoculum with Mabs prior to infecting N2a cells resulted in a significant reduction in PrPSc levels. Information provided in these studies is important for the rational design of humoral immune therapy for prion infection in animals and eventually in humans.

Heterogeneity of normal prion protein in two-dimensional immunoblot: presence of various glycosylated and truncated forms

The common use of one‐dimensional (1‐D) immunoblot with a single monoclonal antibody (Mab) engenders the notion that the normal or cellular prion protein (PrPC) comprises few and simple forms. In this study we used two‐dimensional (2‐D) immunoblot with a panel Mabs to various regions of the prion protein to demonstrate the complexity of the PrPC present in human brain. We distinguished over 50 immunoblot spots, each representing a distinct PrPC species based on combinations of different molecular weights and isoelectric points (pIs). The PrPC heterogeneity is due to the presence of a full‐length and two major truncated forms as well as to the diversity of the glycans linked to most of these forms. The two major truncated forms result from distinct cleavage sites located at the N‐terminus. In addition, enzymatic removal of sialic acid and lectin binding studies indicate that the glycans linked to the full‐length and truncated PrPC forms differ in their structure and ratios of the glycoforms. The truncation of PrPC and the heterogeneity of the linked glycans may play a role in regulating PrPC function. Furthermore, the presence of relatively large quantities of different PrPC species may provide additional mechanisms by which the diversity of prion strains could be generated.