Boon Seng Wong

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.

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.

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

Absence of protease-resistant prion protein in the cerebrospinal fluid of Creutzfeldt-Jakob disease

Creutzfeldt–Jakob disease (CJD), believed to be caused by a protease‐resistant isoform of prion protein (PrPSc), usually manifests itself as a clinically distinctive age‐related dementia because of its rapid progression, occasionally accompanied by cerebellar ataxia. Recently, a variant CJD (vCJD) has been described, which has prominent early psychiatric symptoms and an earlier age of death. Although cerebrospinal fluid (CSF) is part of the extracellular fluid of the central nervous system (CNS), the bulk of its proteins are derived from the plasma and there is increasing concern about possible transmission of prion disease by blood. As investigation of CSF has played a significant role in the diagnosis and management of several neurological diseases, it was decided to characterize PrP present in the CSF of CJD individuals. Significant variation was observed in the level of PrP in the CSF from both non‐CJD and CJD (including vCJD) patients, and the detected PrP forms are protease‐sensitive. Using a conformation‐dependent immunoassay, it was further demonstrated that the PrP detected in the CSF from CJD patients was broadly similar in conformation to that found in non‐CJD patients. Taken together, the results of this study fail to demonstrate any correlation between the presence of protease‐resistant PrP isoform (PrPSc) in the CSF and disease manifestation. Copyright

Impaired neuronal insulin signaling precedes Aβ42 accumulation in female AβPPsw/PS1ΔE9 mice.

Reduced glucose utilization is likely to precede the onset of cognitive deficits in Alzheimers disease (AD). Similar aberrant glucose metabolism can also be detected in the brain of several AD mouse models. Although the cause of this metabolic defect is not well understood, it could be related to impaired insulin signaling that is increasingly being reported in AD brain. However, the temporal relationship between insulin impairment and amyloid-β (Aβ) biogenesis is unclear. In this study using female AβPPsw/PS1ΔE9 mice, we found that the level of Aβ40 was fairly constant in 6- to 15-month-old brains, whereas Aβ42 was only significantly increased in the 15-month-old brain. In contrast, increased levels of IRβ, IGF-1R, IRS1, and IRS-2, along with reduced glucose and insulin content, were detected earlier in the 12-month-old brains of AβPPsw/PS1ΔE9 mice. The reduction in brain glucose content was accompanied by increased GLUT3 and GLUT4 levels. Importantly, these changes precede the significant upregulation of Aβ42 level in the 15-month-old brain. Interestingly, reduction in the p85 subunit of PI3K was only apparent in the 15-month-old AβPPsw/PS1ΔE9 mouse brain. Furthermore, the expression profile of IRβ, IRS-2, and p85/PI3K in AβPPsw/PS1ΔE9 was distinct in wild-type mice of a similar age. Although the exact mechanisms underlining this connection remain unclear, our results suggest a possible early role for insulin signaling impairment leading to amyloid accumulation in AβPPsw/PS1ΔE9 mice.

Altered prion protein glycosylation in the aging mouse brain

The normal cellular prion protein (PrPC) is a glycoprotein with two highly conserved potential N‐linked glycosylation sites. All prion diseases, whether inherited, infectious or sporadic, are believed to share the same pathogenic mechanism that is based on the conversion of the normal cellular prion protein (PrPC) to the pathogenic scrapie prion protein (PrPSc). However, the clinical and histopathological presentations of prion diseases are heterogeneous, depending not only on the strains of PrPSc but also on the mechanism of diseases, such as age‐related sporadic vs. infectious prion diseases. Accumulated evidence suggests that N‐linked glycans on PrPC are important in disease phenotype. A better understanding of the nature of the N‐linked glycans on PrPC during the normal aging process may provide new insights into the roles that N‐linked glycans play in the pathogenesis of prion diseases. By using a panel of 19 lectins in an antibody–lectin enzyme‐linked immunosorbent assay (ELISA), we found that the lectin binding profiles of PrPC alter significantly during aging. There is an increasing prevalence of complex oligosaccharides on the aging PrPC, which are features of PrPSc. Taken together, this study suggests a link between the glycosylation patterns on PrPC during aging and PrPSc.

Chronic exposure to U18666A is associated with oxidative stress in cultured murine cortical neurons

Findings that antioxidant treatment may be beneficial in Alzheimers disease indicate that oxidative stress is an important factor in its pathogenesis. Studies have also suggested that cholesterol imbalance in the brain might be related to the development of neurological disorders. Previously, we have reported that U18666A, a cholesterol transport‐inhibiting agent, leads to apoptosis and intracellular cholesterol accumulation in primary cortical neurons. In this study, we found that neuronal apoptosis mediated by U18666A is associated with oxidative stress in the treated cortical neurons. Cortical neurons treated with U18666A also showed decreased secretion and increased intraneuronal accumulation of β‐amyloid. The association of neuronal apoptosis with oxidative stress and Aβ accumulation may provide clues to the pathogenesis of Alzheimers disease, as well as the role oxidative stress plays in other neurodegenerative diseases.