Ruliang Li

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.

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.

Protease-Resistant Human Prion Protein and Ferritin Are Cotransported across Caco-2 Epithelial Cells: Implications for Species Barrier in Prion Uptake from the Intestine

Foodborne transmission of bovine spongiform encephalopathy (BSE) to humans as variant Creutzfeldt-Jakob disease (CJD) has affected over 100 individuals, and probably millions of others have been exposed to BSE-contaminated food substances. Despite these obvious public health concerns, surprisingly little is known about the mechanism by which PrP-scrapie (PrPSc), the most reliable surrogate marker of infection in BSE-contaminated food, crosses the human intestinal epithelial cell barrier. Here we show that digestive enzyme (DE) treatment of sporadic CJD brain homogenate generates a C-terminal fragment similar to the proteinase K-resistant PrPSc core of 27-30 kDa implicated in prion disease transmission and pathogenesis. Notably, DE treatment results in a PrPSc-protein complex that is avidly transcytosed in vesicular structures across an in vitro model of the human intestinal epithelial cell barrier, regardless of the amount of endogenous PrPC expression. Unexpectedly, PrPSc is cotransported with ferritin, a prominent component of the DE-treated PrPSc-protein complex. The transport of PrPSc-ferritin is sensitive to low temperature, brefeldin-A, and nocodazole treatment and is inhibited by excess free ferritin, implicating a receptor- or transporter-mediated pathway. Because ferritin shares considerable homology across species, these data suggest that PrPSc-associated proteins, in particular ferritin, may facilitate PrPSc uptake in the intestine from distant species, leading to a carrier state 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.

Aggresome formation by mutant prion proteins: The unfolding role of proteasomes in familial prion disorders

Although familial prion disorders are a direct consequence of mutations in the prion protein gene, the underlying mechanisms leading to neurodegeneration remain unclear. Potential pathogenic mechanisms include abnormal cellular metabolism of the mutant prion protein (PrP(M)), or destabilization of PrP(M) structure inducing a change in its conformation to the pathogenic PrP-scrapie (PrP(Sc)) form. To further clarify these mechanisms, we investigated the biogenesis of mutant PrP V203I and E211Q associated with Creutzfeldt-Jakob disease, and PrP Q212P associated with Gerstmann-Straussler-Scheinker syndrome in neuroblastoma cells. We report that all three PrP(M) forms accumulate similarly in the cytosol in response to proteasomal inhibition, and finally assemble as classical aggresomes. Since the three PrP(M) forms tested in this report are distinct, we propose that sequestration of misfolded PrP(M) into aggresomes is likely a general response of the cellular quality control that is not specific to a particular mutation in PrP. Moreover, since PrP has the remarkable ability to refold into PrP(Sc) that can subsequently replicate, PrP(M) sequestered in aggresomes may cause neurotoxicity by both direct and indirect pathways; directly through PrP(Sc) aggregates, and indirectly by depleting normal PrP, through induction of a cellular stress response, or by other undefined pathways. On the other hand, sequestered PrP(M) may be relatively inert, and cellular toxicity may be mediated by early intermediates in aggresome formation. Taken together, these observations demonstrate the role of proteasomes in the pathogenesis of familial prion disorders, and argue for further explanation of its mechanistic details.

Differential expression of cellular prion protein in mouse brain as detected with multiple anti-PrP monoclonal antibodies

The normal cellular prion protein (PrP(C)) plays an essential role in the development of prion diseases. Indirect evidence has suggested that different PrP(C) glycoforms may be expressed in different brain regions and perform distinct functions. However, due to a lack of monoclonal antibodies (Mabs) that are specific for mouse PrP(C), the expression of PrP(C) in the mouse brain has not been studied in great detail. We used Mabs specific for either the N-terminus or the C-terminus of the mouse PrP(C) to study its expression in the mouse brain by immunoblotting and immunohistochemistry. Immunoblotting studies demonstrated that the expression of PrP(C) differed quantitatively as well as qualitatively in different regions of the brain. The anti-C-terminus Mabs reacted with all three molecular weight bands of PrP(C); the anti-N-terminus Mabs only reacted with the 39-42 kDa PrP(C). The results from immunohistochemical staining revealed the spatial distribution of PrP(C) in the mouse brain, which were consistent with that from immunoblotting. Although expression of PrP(C) has been reported to be required for long-term survival of Purkinje cells, we were unable to detect PrP(C) in the Purkinje cell layer in the cerebellum with multiple anti-PrP Mabs. Our findings suggest that PrP(C) variants, i.e. various glycoforms and truncated forms, might be specifically expressed in different regions of mouse brain and might have different functions.

Induction of HO-1 and NOS in doppel-expressing mice devoid of PrP: Implications for doppel function

Ectopic expression of the doppel (Dpl) protein, a homologue of the prion protein (PrP), was recently associated with cerebellar Purkinje cell degeneration observed in two aging prion protein knock-out (Prnp(0/0)) mouse lines. We investigated the possible role of Dpl in oxidative metabolism. Two Prnp(0/0) mouse lines of similar genetic background were studied. One line expresses Dpl in the brain and displays Dpl-associated cerebellar abnormalities. The other has no elevated expression of Dpl and no cerebellar abnormalities. We observed a correlation between Dpl expression and the induction of both heme oxygenase 1 (HO-1) and nitric oxide synthase systems (nNOS and iNOS). These responses are suggestive of increased oxidative stress in the brains of the Dpl-expressing Prnp(0/0) mice. No induction was observed with Hsp-60, indicating a specific response by the HO/NOS system. We proposed that Dpl expression exacerbates oxidative damage that is antagonistic to the protective function of wild-type PrP.

Normal Cellular Prior Protein Is Preferentially Expressed on Subpopulations of Murine Hemopoietic Cells

We studied the expression of normal cellular prion protein (PrPC) in mouse lymphoid tissues with newly developed mAbs to PrPC. Most of the mature T and B cells in the peripheral lymphoid organs do not express PrPC. In contrast, most thymocytes are PrPC+. In the bone marrow, erythroid cells and maturing granulocytes are PrPC+. Approximately 50% of the cells in the region of small lymphocytes and progenitor cells also express PrPC. Most of these PrPC+ cells are CD43+, but B220−, surface IgM− (sIgM−), and IL-7R−, a phenotype that belongs to cells not yet committed to the B cell lineage. Another small group of the PrPC+ cell are B220+, and some of these are also sIgM+. The majority of the B220+ cells, however, are PrPC−. Therefore, PrPC is preferentially expressed in early bone marrow progenitor cells and subsets of maturing B cells. Supporting this interpretation is our observation that stimulation of bone marrow cells in vitro with PMA results in a decrease in the number of PrPC+B220− cells with a corresponding increase of sIgM+B220high mature B cells. This result suggests that the PrPC+B220− cells are potential progenitors. Furthermore, in the bone marrow of Rag-1−/− mice, there are an increased number of PrPC+B220− cells, and most of the developmentally arrested pro-B cells in these mice are PrPC+. Collectively, these results suggest that PrPC is expressed preferentially in immature T cells in the thymus and early progenitor cells in the bone marrow, and the expression of PrPC is regulated during hemopoietic differentiation.