Tao Pan

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.

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.

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.

An Aggregation-Specific Enzyme-Linked Immunosorbent Assay: Detection of Conformational Differences between Recombinant PrP Protein Dimers and PrPSc Aggregates

ABSTRACT The conversion of the normal cellular prion protein, PrPC, into the protease-resistant, scrapie PrPSc aggregate is the cause of prion diseases. We developed a novel enzyme-linked immunosorbent assay (ELISA) that is specific for PrP aggregate by screening 30 anti-PrP monoclonal antibodies (MAbs) for their ability to react with recombinant mouse, ovine, bovine, or human PrP dimers. One MAb that reacts with all four recombinant PrP dimers also reacts with PrPSc aggregates in ME7-, 139A-, or 22L-infected mouse brains. The PrPSc aggregate is proteinase K resistant, has a mass of 2,000 kDa or more, and is present at a time when no protease-resistant PrP is detectable. This simple and sensitive assay provides the basis for the development of a diagnostic test for prion diseases in other species. Finally, the principle of the aggregate-specific ELISA we have developed may be applicable to other diseases caused by abnormal protein aggregation, such as Alzheimers disease or Parkinsons disease.

Test for Detection of Disease-Associated Prion Aggregate in the Blood of Infected but Asymptomatic Animals

ABSTRACT We have developed a sensitive in vitro assay for detecting disease-associated prion aggregates by combining an aggregation-specific enzyme-linked immunosorbent assay (AS-ELISA) with the fluorescent amplification catalyzed by T7 RNA polymerase technique (FACTT). The new assay, named aggregation-specific FACTT (AS-FACTT), is much more sensitive than AS-ELISA and could detect prion aggregates in the brain of mice as early as 7 days after an intraperitoneal inoculation of PrPSc. However, AS-FACTT was still unable to detect prion aggregates in blood of infected mice. To further improve the detection limit of AS-FACTT, we added an additional prion amplification step (Am) and developed a third-generation assay, termed Am-A-FACTT. Am-A-FACTT has 100% sensitivity and specificity in detecting disease-associated prion aggregates in blood of infected mice at late but still asymptomatic stages of disease. At a very early stage, Am-A-FACTT had a sensitivity of 50% and a specificity of 100%. Most importantly, Am-A-FACTT also detects prion aggregates in blood of mule deer infected with the agent causing a naturally occurring prion disease, chronic wasting disease. Application of this assay to cattle, sheep, and humans could safeguard food supplies and prevent human contagion.

Prion protein is ubiquitinated after developing protease resistance in the brains of scrapie-infected mice

Although the key event in the pathology of prion diseases is thought to be the conversion of cellular prion protein (PrPC) to the protease‐resistant scrapie species termed PrPSc, the factors that contribute to neurodegeneration in scrapie‐infected animals are poorly understood. One probable determinant could be when the accumulation of PrPSc in infected brain overwhelms the ubiquitin–proteasome system and triggers the degenerative cascade. In the present study, it was found that in mouse brains infected with the ME7 scrapie strain, the level of ubiquitin protein conjugates increased significantly at ∼144 days post‐infection (pi) when clinical signs first become apparent. This elevation correlated with the detection of protease‐resistant PrPSc and a decline in two endopeptidase activities associated with proteasome function. However, ubiquitination of PrP was only detected at the terminal stage, 3 weeks after the development of clinical symptoms (∼165 days pi). These results suggest that ubiquitination of PrP is a late event phenomenon and this conjugation occurs after the formation of protease‐resistant PrPSc. Whether this post‐translational modification and the impairment of proteasome function are pivotal events in the pathogenesis of prion diseases remains to be determined. Copyright