Manganese chelation therapy extends survival in a mouse model of M1000 prion disease.

Previous in vitro and in vivo investigations have suggested manganese (Mn(2+)) may play a role in pathogenesis through facilitating refolding of the normal cellular form of the prion protein into protease resistant, pathogenic isoforms (PrP(Sc)), as well as the subsequent promotion of higher order aggregation of these abnormal conformers. To further explore the role of Mn(2+) in pathogenesis, we undertook a number of studies, including an assessment of the disease modifying effects of chelation therapy in a well-characterized mouse model of prion disease. The di-sodium, calcium derivative of the chelator, cyclohexanediaminetetraacetic acid (Na(2)CaCDTA), was administered intraperitoneally to mice inoculated intra-cerebrally with either high or low-dose inocula, with treatment beginning early (shortly after inoculation) or late (at the usual mid-survival point of untreated mice). Analyses by inductively coupled plasma-mass spectrometry demonstrated brain Mn(2+) levels were selectively reduced by up to 50% in treated mice compared with untreated controls, with copper, iron, zinc and cobalt levels unchanged. In mice administered high-dose inocula, none of the treatment groups displayed an increase in survival although western blot analyses of early intensively treated mice showed reduced brain PrP(Sc) levels; mice infected using low-dose inocula however, showed a significant prolongation of survival (p = 0.002). Although our findings support a role for Mn(2+) in prion disease, further studies are required to more precisely delineate the extent of pathogenic involvement.

A manganese-superoxide dismutase/catalase mimetic extends survival in a mouse model of human prion disease.

Animal models, and human postmortem studies, of prion disease have demonstrated the presence of heightened oxidative stress in the brain, with additional findings supporting the likelihood that the normal isoform of prion protein directly contributes to neuronal antioxidant defences. Although such data are consistent with the postulate that oxidative stress plays a salient pathogenic role in prion disease, it remains possible that oxidative damage represents a secondary or relatively less important phenomenon in neurons already rendered dysfunctional from other primary insults. To provide further insights into the relative pathogenic importance of oxidative stress, we employed a potent manganese-superoxide dismutase/catalase mimetic, EUK-189, as a therapeutic in our mouse model of human prion disease. A significant but relatively modest prolongation of survival in EUK-189-treated mice was observed, which correlated with reductions in oxidative, especially nitrative, damage to proteins when compared to untreated disease controls. Lesion profiling also revealed reductions in spongiform change in specific brain regions of terminally sick EUK-189-treated mice. Our results are consistent with heightened oxidative stress playing a pathogenic role in prion disease but underscore the need for more biologically potent and, most likely, broader spectrum antioxidant treatments if more successful amelioration is to be achieved.

NMR characterization of the pH 4 beta-intermediate of the prion protein: the N-terminal half of the protein remains unstructured and retains a high degree of flexibility.

Prion diseases are associated with the misfolding of the PrP (prion protein) from a largely alpha-helical isoform to a beta-sheet-rich oligomer. CD has shown that lowering the pH to 4 under mildly denaturing conditions causes recombinant PrP to convert from an alpha-helical protein into one that contains a high proportion of beta-sheet-like conformation. In the present study, we characterize this soluble pH 4 folding intermediate using NMR. (15)N-HSQC (heteronuclear single-quantum correlation) studies with mPrP (mouse PrP)-(23-231) show that a total of 150 dispersed amide signals are resolved in the native form, whereas only 65 amide signals with little chemical shift dispersion are observable in the pH 4 form. Three-dimensional (15)N-HSQC-TOCSY and NOESY spectra indicate that the observable residues are all assigned to amino acids in the N-terminus: residues 23-118. (15)N transverse relaxation measurements indicate that these N-terminal residues are highly flexible with additional fast motions. These observations are confirmed via the use of truncated mPrP-(112-231), which shows only 16 (15)N-HSQC amide peaks at pH 4. The loss of signals from the C-terminus can be attributed to line broadening due to an increase in the molecular size of the oligomer or exchange broadening in a molten-globule state.

Prion protein does not redox-silence Cu2+, but is a sacrificial quencher of hydroxyl radicals.

Oxidative stress is believed to play a central role in the pathogenesis of prion diseases, a group of fatal neurodegenerative disorders associated with a conformational change in the prion protein (PrP(C)). The precise physiological function of PrP(C) remains uncertain; however, Cu(2+) binds to PrP(C) in vivo, suggesting a role for PrP(C) in copper homeostasis. Here we examine the oxidative processes associated with PrP(C) and Cu(2+). (1)H NMR was used to monitor chemical modifications of PrP fragments. Incubation of PrP fragments with ascorbate and CuCl(2) showed specific metal-catalyzed oxidation of histidine residues, His(96/111), and the methionine residues, Met(109/112). The octarepeat region protects His(96/111) and Met(109/112) from oxidation, suggesting that PrP(90-231) might be more prone to chemical modification. We show that Cu(2+/+) redox cycling is not 'silenced' by Cu(2+) binding to PrP, as indicated by H(2)O(2) production for full-length PrP. Surprisingly, although detection of Cu(+) indicates that the octarepeat region of PrP is capable of reducing Cu(2+) even in the absence of ascorbate, H(2)O(2) is not generated unless ascorbate is present. Full-length PrP and fragments cause a dramatic reduction in detectable hydroxyl radicals in an ascorbate/Cu(2+)/O(2) system; however, levels of H(2)O(2) production are unaffected. This suggests that PrP does not affect levels of hydroxyl radical production via Fentons cycling, but the radicals cause highly localized chemical modification of PrP(C).

High affinity copper binding by stefin B (cystatin B) and its role in the inhibition of amyloid fibrillation.

We show that human stefin B, a protease inhibitor from the family of cystatins, is a copper binding protein, unlike stefin A. We have used isothermal titration calorimetry to directly monitor the binding event at pH 7 and pH 5. At pH 7 stefin B shows a picomolar affinity for copper but at pH 5 the affinity is in the nanomolar range. There is no difference in the affinity of copper between the wildtype stefin B (E31 isoform) and a variant (Y31 isoform), whereas the mutant (P79S), which is tetrameric, does not bind copper. The conformation of stefin B remains unaltered by copper binding. It is known that below pH 5 stefin B undergoes a conformational change and amyloid fibril formation. We show that copper binding inhibits the amyloid fibril formation and, to a lesser degree, the initial aggregation. Similarities to and differences from other copper binding amyloidogenic proteins are discussed.

Correlative studies support lipid peroxidation is linked to PrP(res) propagation as an early primary pathogenic event in prion disease.

To assess whether heightened oxidative stress plays an early and primary pathogenic role in transmissible spongiform encephalopathies (TSE), we undertook detailed correlative studies using a mouse-adapted model of human disease. The spatio-temporal evolution of the abnormal, protease-resistant isoform of the prion protein (PrP(res)) and neuropathological changes were correlated with the occurrence and type of oxidative stress. Heightened oxidative stress was demonstrated, but restricted to elevated levels of free aldehydic breakdown products of lipid peroxidation, affecting all brain regions to varying extents. The increase in lipid peroxidation was highest over the mid-incubation period, with the onset showing close temporal and general topographical concordance with the first detection of PrP(res) with both pre-empting the typical neuropathological changes of spongiform change, gliosis and neuronal loss. Further, prion propagation over the disease course was assessed using murine bioassay. This revealed that the initial rapid increase in infectivity titres was contemporaneous with the abrupt onset and maximisation of lipid peroxidation. The present results are an important extension to previous studies, showing that heightened oxidative stress in the form of lipid peroxidation is likely to constitute an early primary pathogenic event in TSE, associated temporally with the integral disease processes of prion propagation and PrP(res) formation, and consistent with causal links between these events and subsequent typical neuropathological changes.

Diverse fibrillar peptides directly bind the Alzheimer's amyloid precursor protein and amyloid precursor-like protein 2 resulting in cellular accumulation.

The Alzheimer's disease Abeta peptide can increase the levels of cell-associated amyloid precursor protein (APP) in vitro. To determine the specificity of this response for Abeta and whether it is related to cytotoxicity, we tested a diverse range of fibrillar peptides including amyloid-beta (Abeta), the fibrillar prion peptides PrP106-126 and PrP178-193 and human islet-cell amylin. All these peptides increased the levels of APP and amyloid precursor-like protein 2 (APLP2) in primary cultures of astrocytes and neurons. Specificity was shown by a lack of change to amyloid precursor-like protein 1, tau-1 and cellular prion protein (PrP(c)) levels. APP and APLP2 levels were elevated only in cultures exposed to fibrillar peptides as assessed by electron microscopy and not in cultures treated with non-fibrillogenic peptide variants or aggregated lipoprotein. We found that PrP106-126 and the non-toxic but fibril-forming PrP178-193 increased APP levels in cultures derived from both wild-type and PrP(c)-deficient mice indicating that fibrillar peptides up-regulate APP through a non-cytotoxic mechanism and irrespective of parental protein expression. Fibrillar PrP106-126 and Abeta peptides bound recombinant APP and APLP2 suggesting the accumulation of these proteins was mediated by direct binding to the fibrillated peptide. This was supported by decreased APP accumulation following extensive washing of the cultures to remove fibrillar aggregates. Pre-incubation of fibrillar peptide with recombinant APP18-146, the putative fibril binding site, also abrogated the accumulation of APP. These findings show that diverse fibrillogenic peptides can induce accumulation of APP and APLP2 and this mechanism could contribute to pathogenesis in neurodegenerative disorders.

Antioxidant and Metal Chelation-Based Therapies in the Treatment of Prion Disease.

Many neurodegenerative disorders involve the accumulation of multimeric assemblies and amyloid derived from misfolded conformers of constitutively expressed proteins. In addition, the brains of patients and experimental animals afflicted with prion disease display evidence of heightened oxidative stress and damage, as well as disturbances to transition metal homeostasis. Utilising a variety of disease model paradigms, many laboratories have demonstrated that copper can act as a cofactor in the antioxidant activity displayed by the prion protein while manganese has been implicated in the generation and stabilisation of disease-associated conformers. This and other evidence has led several groups to test dietary and chelation therapy-based regimens to manipulate brain metal concentrations in attempts to influence the progression of prion disease in experimental mice. Results have been inconsistent. This review examines published data on transition metal dyshomeostasis, free radical generation and subsequent oxidative damage in the pathogenesis of prion disease. It also comments on the efficacy of trialed therapeutics chosen to combat such deleterious changes.

Immunotherapeutic approaches in prion disease: progress, challenges and potential directions.

Therapeutic trials utilizing animal models of prion disease have explored a variety of compounds and a number of approaches with varying success, including several immunotherapeutic strategies, such as passive immunization through the delivery of viruses carrying nucleic acid inserts encoding prion protein-specific immunoglobulin. Targeted, organ-specific cellular production of therapeutic proteins is a relatively unexplored approach in the treatment of neurodegeneration despite many successful experimental outcomes in animal models and human trials of other diseases of the CNS. Emphasizing studies utilizing mouse models of disease, this review outlines developments and limitations of immunological approaches to the treatment of prion diseases. In addition, the authors discuss the potential of an experimental therapeutic strategy, utilizing hybridoma cells injected directly into the CNS to establish long-term production of anti-prion antibodies in vivo within the organ associated with the greatest pathogenic change in prion disease, the brain.

Metal attenuating therapies in neurodegenerative disease.

The clinical and pathological spectrum of neurodegenerative diseases is diverse, although common to many of these disorders is the accumulation of misfolded proteins, with oxidative stress thought to be an important contributing mechanism to neuronal damage. As a corollary, transition metal ion dyshomeostasis appears to play a key pathogenic role in a number of these maladies, including the most common of neurodegenerative diseases. In this review, studies spanning a wide variety of neurodegenerative disorders are presented with their involvement of transition metals compared and contrasted, including more detailed treatise in relation to Alzheimer's disease, Parkinson's disease and prion diseases. For each of these diseases, a discussion of the evolving scientific rationale for the development of therapies aimed at ameliorating the detrimental effects of transition metal dysregulation, including results from various human trials, is then provided.

Therapeutic interventions ameliorating prion disease.

Of the many unresolved issues in relation to prion diseases, effective treatments remain an elusive exigency, although some progress has been made. This review describes disease-ameliorating therapeutic strategies reported to date in animal models of prion disease, as well as providing a brief overview of selected completed human treatment trials. Included in vivo studies have been broadly dichotomized according to the time of introduction of the treatment in relation to animal inoculation and also according to their possible principal mechanism of action, although the latter is not always entirely clear, and often there is likely to be more than one mechanism. Consequent to the pathogenic primacy of cellular prion protein (PrP(c))-to-scrapie PrP(c) (PrP(sc)) conversion, most reported treatments appear to directly target this replication process, although various other strategies, such as depletion of reaction substrates and abrogation of downstream effector pathways, have been utilized. Many factors, including experimental design, militate against reliable extrapolation of study results to the routine clinical setting or limit easy translational application to human disease. Notably problematic are approaches wherein benefit has been shown but the treatment was initiated before, at or soon after inoculation of experimental animals.

Dynamics of a truncated prion protein, PrP(113-231), from (15)N NMR relaxation: order parameters calculated and slow conformational fluctuations localized to a distinct region.

Prion diseases are associated with the misfolding of the prion protein (PrP(C)) from a largely alpha-helical isoform to a beta-sheet rich oligomer (PrP(Sc)). Flexibility of the polypeptide could contribute to the ability of PrP(C) to undergo the conformational rearrangement during PrP(C)-PrP(Sc) interactions, which then leads to the misfolded isoform. We have therefore examined the molecular motions of mouse PrP(C), residues 113-231, in solution, using (15)N NMR relaxation measurements. A truncated fragment has been used to eliminate the effect of the 90-residue unstructured tail of PrP(C) so the dynamics of the structured domain can be studied in isolation. (15)N longitudinal (T(1)) and transverse relaxation (T(2)) times as well as the proton-nitrogen nuclear Overhauser effects have been used to calculate the spectral density at three frequencies, 0, omega(N,) and 0.87omega(H). Spectral densities at each residue indicate various time-scale motions of the main-chain. Even within the structured domain of PrP(C), a diverse range of motions are observed. We find that removal of the tail increases T(2) relaxation times significantly indicating that the tail is responsible for shortening of T(2) times in full-length PrP(C). The truncated fragment of PrP has facilitated the determination of meaningful order parameters (S(2)) from the relaxation data and shows for the first time that all three helices in PrP(C) have similar rigidity. Slow conformational fluctuations of mouse PrP(C) are localized to a distinct region that involves residues 171 and 172. Interestingly, residues 170-175 have been identified as a segment within PrP that will form a steric zipper, believed to be the fundamental amyloid unit. The flexibility within these residues could facilitate the PrP(C)-PrP(Sc) recognition process during fibril elongation.

Manganese binding to the prion protein.

There is considerable evidence that the prion protein binds copper. However, there have also been suggestions that prion protein (PrP) binds manganese. We used isothermal titration calorimetry to identify the manganese binding sites in wild-type mouse PrP. The protein showed two manganese binding sites with affinities that would bind manganese at concentrations of 63 and 200 mum at pH 5.5. This indicates that PrP binds manganese with affinity similar to other known manganese-binding proteins. Further study indicated that the main manganese binding site is associated with His-95 in the so-called "fifth site" normally associated with copper binding. Additionally, it was shown that occupancy by copper does not prevent manganese binding. Under these conditions, manganese binding resulted in an altered conformation of PrP, displacement of copper, and altered redox chemistry of the metal-protein complex. Cyclic voltammetric measurements suggested a complex redox chemistry involving manganese bound to PrP, whereas copper-bound PrP was able to undergo fully reversible electron cycling. Additionally, manganese binding to PrP converted it to a form able to catalyze aggregation of metal-free PrP. These results further support the notion that manganese binding could cause a conformation change in PrP and trigger changes in the protein similar to those associated with prion disease.

Mouse galectin-1 inhibits the toxicity of glutamate by modifying NR1 NMDA receptor expression.

One of the major causes of neuronal death in neurodegenerative disease is excitotoxicity from the neurotransmitter glutamate. This form of cell death could arise from either excess levels of glutamate due to decreased astrocyte clearance or due to increased susceptibility. We have identified galectin-1, a galactose-binding lectin, as a potential neuroprotective factor secreted by astrocytes. Our results show that both native and recombinant galectin-1 protects mouse and rat cerebellar neurons from the toxic effects of glutamate. Galectin-1 applied to neurons increased their expression of the NMDA receptor NR1 and increased the proportion of the NR1a subunit subtype while antisense knockdown of the NR1a receptor blocked the neuroprotective effect of galectin-1. This effect of the protein was dependent upon it carbohydrate recognition domain, suggesting that the protein acts in a reduced dimerized form. In addition, galectin-1 caused a decreased expression of PKC associated with increased resistance to glutamate toxicity. These results suggest that the astrocytic lectin galectin-1 could protect neurons against the effects of excitotoxicity as seen in stroke and ischemic injury.

Extended period of asymptomatic prion disease after low dose inoculation: assessment of detection methods and implications for infection control.

We used quantal dose-titration of a mouse-adapted human transmissible spongiform encephalopathy strain (M470) to compare different analytical methods for their ability to detect asymptomatic brain prion infection after low dose inoculation. At a time point approximately 2.5-fold beyond the mean incubation period of high dose inocula, asymptomatic brain infection was commonly observed using histologic examination, Western blot, and "blind" bioassay following intracerebral inoculation with low titer inocula. At this time point, when a clinical end-point titration would usually be determined, evidence of infection was seen in all healthy animals inoculated with up to 100-fold lower inoculation doses than the lowest causing consistent clinical disease. For the assessment of the presence of asymptomatic infection, we compared different Western immunoblot and histopathological methods in relation to "blind" bioassay using transgenic Tga/20 mice overexpressing mouse prion protein (PrP). Sodium phosphotungstic acid (NaPTA) precipitation of protease-resistant PrP isoforms (PrP(res)) prior to Western blotting was found to approach the sensitivity of the Tga/20 bioassay and was superior to conventional Western blot and histopathological methods, wherein infectivity was commonly found when both of the latter were negative. Re-scaling the original titer by incorporating "blind" transmission data from surviving asymptomatic mice revises the estimate two orders of magnitude higher than the value derived using the conventional clinical disease outcome approach. We also found that the sensitivity of the NaPTA Western blot technique, if used with a diluent such as PBS compared with 10% normal brain homogenate, is adversely affected by up to around 20-fold. We postulate that infectious titer estimates based on more sensitive detection systems such as we report provide a more accurate indication of ultimate transmission risk.

Detection of prion epitopes on PrP and PrP of transmissible spongiform encephalopathies using specific monoclonal antibodies to PrP.

Amino acid residues 90-120 of the prion protein (PrP) are likely to be critical for the conversion of PrP(c) to PrP(sc) in the transmissible spongiform encephalopathies. We raised 10 monoclonal antibodies against the 90-120 amino acid region, mapped the epitope specificity of these anti-PrP antibodies, and investigated the expression of epitopes recognized by the antibodies in both PrP(c) and PrP(sc). Four out of five of the anti-PrP antibodies raised in a prion knockout mouse immunized with the linear peptide of PrP90-120 could detect PrP(sc) in 'native' and denatured forms and PrP(c) in normal cells, as well as recognize epitopes within PrP93-112 residues. In contrast, the other six anti-PrP reagents, including five raised from the two knockout mice immunized with conformationally modified PrP90-120 peptide, could detect PrP(c) and recognize epitopes within PrP93-107 residues. Four of these reagents could also detect denatured PrP(sc) on western blots but not PrP(sc) plaques in brain tissue. The results indicate that residues PrP93-102 are exposed in PrP(c) but are buried upon conversion to the PrP(sc) isoform. Furthermore, PrP103-107 residues are partially buried in PrP(sc) while only the PrP107-112 epitope remains exposed, suggesting that the region PrP93-112 undergoes conformational changes during its conversion to PrP(sc).