“Redox control of prion and disease pathogenesis,” Singh et al., Antioxidants and Redox Signalling (2010)

Imbalance of brain metal homeostasis and associated oxidative stress by redox-active metals like iron and copper is an important trigger of neurotoxicity in several neurodegenerative conditions, including prion disorders. Whereas some reports attribute this to end-stage disease, others provide evidence for specific mechanisms leading to brain metal dyshomeostasis during disease progression. In prion disorders, imbalance of brain-iron homeostasis is observed before end-stage disease and worsens with disease progression, implicating iron-induced oxidative stress in disease pathogenesis. This is an unexpected observation, because the underlying cause of brain pathology in all prion disorders is PrP-scrapie (PrPSc), a β-sheet–rich conformation of a normal glycoprotein, the prion protein (PrPC). Whether brain-iron dyshomeostasis occurs because of gain of toxic function by PrPSc or loss of normal function of PrPC remains unclear. In this review, we summarize available evidence suggesting the involvement of oxidative stress in prion-disease pathogenesis. Subsequently, we review the biology of PrPC to highlight its possible role in maintaining brain metal homeostasis during health and the contribution of PrPSc in inducing brain metal imbalance with disease progression. Finally, we discuss possible therapeutic avenues directed at restoring brain metal homeostasis and alleviating metal-induced oxidative stress in prion disorders. Antioxid. Redox Signal. 12, 1271–1294.

Whether the interaction of PrPSc with ferritin is essential for its PK-resistant nature is not entirely clear. However, chelation of iron from prion disease–affected brain homogenates decreases the total amount of PK-resistant PrPSc, suggesting that iron is somehow involved in the stability of PrPSc (12).

These studies suggest that the association of PrPC with copper and iron can have deleterious consequences under certain circumstances because of their redox-active nature. Perhaps the site and nature of PrP–metal interaction and the structure of PrPC itself are important underlying factors in this process, because the formation of β-sheet–rich aggregates on exposure to free radicals has been reported only for a few proteins such as PrPC and α-synuclein (215). Other major iron- and copper-binding proteins do not show this response. It is likely that sequestration of iron in PrPSc aggregates renders them redox active, thus accentuating the associated toxicity. Future investigations are necessary to understand this phenomenon fully.

Table 3. Neurodegenerative Diseases Involving Metal-induced Protein Aggregation
Protein involved Metal Disease
PrPC or PrPSc Cu, Fe, Mn Prion disorders
Cu, Fe, Zn Alzheimer’s disease
α-Synuclein Fe, Cu Parkinson’s disease

A. AntioxidantsStudies on prion-infected cell and mouse models have provided useful information on the therapeutic potential of antioxidants. In cell models, both direct application of cell-permeable antioxidants and indirect methods to restore endogenous antioxidant levels have been tried (24). Flupirtine, a triaminopyridine compound, has become quite popular among researchers because it can act as an N-methyl-d-aspartate (NMDA) antagonist without binding to NMDA receptors. This drug has the exceptional ability to normalize intracellular glutathione levels and restore oxidative balance within the cell, thereby combating accumulation of ROS and other free radicals. The associated upregulation of antiapoptotic protein Bcl-2 and the relatively favorable pharmacokinetic profile of flupirtine make it a promising therapeutic agent to treat prion disorders (151, 161, 168, 194). An equally promising agent is the nonpsychoactive cannabis constituent cannabidiol, by virtue of its antioxidant property, NMDA antagonism, reduction in glutamate release, and blockade of microglial migration and activation, all of which are detrimental factors that aggravate PrPSc-mediated neurotoxicity (55, 138). Similarly, the disaccharide trehalose, known for its ability to reduce Aβ-mediated toxicity by inhibiting its aggregation, also protects prion-infected cells from oxidative damage (18). Another effective agent is EUK-189, a potent Mn SOD/catalase mimetic, that reduces oxidative damage in prion-infected mouse models, as evidenced by reduction in nitrative damage to vital cellular proteins, prolongation of incubation time, and decreased spongiform change in the brains of terminally ill mice (24). Although a clinically viable antioxidant that can alleviate prion disease–associated neurotoxicity is lacking, these observations argue that counteracting oxidative stress may have therapeutic benefit in prion disease and provide the basis for future investigations in this area.

B.  Metal chelatorsMost of the strategies aimed at metal chelation are targeted toward copper because the association of iron with prion-disease pathogenesis is a relatively new observation (203). Contradictory observations have been reported for copper, in which both increased and reduced levels of brain copper have been implicated in disease-associated neurotoxicity. In an experimental paradigm in which the loss of neurons and astrogliosis was induced by introduction of copper into the dorsal hippocampus of rats, co-injection of a synthetic peptide corresponding to the octapeptide repeat domain of PrP (PrP59-91) that binds copper reduced neuronal death (42). With a similar premise, chelation of copper with d-penicillamine, a drug used routinely for treating Wilson disease, decreased brain-copper content of prion-infected mice by 30% and increased the incubation period, supporting the idea that increased levels of brain copper promote diseases (58, 201). However, contradictory reports suggest a protective role for copper in prion disorders. It was observed that neuroblastoma cells cultured in the presence of copper ions lost the ability to bind and internalize PrPSc, thereby evading infection and toxicity. A significant delay in the onset of clinical disease also was observed in scrapie-infected hamsters given a dietary supplement of copper (91), supporting these observations. It is likely that the protective effect of copper reflects internalization and degradation of PrPC on exposure to copper, the substrate for PrPSc generation, although a direct effect on the generation of PrPSc cannot be ruled out because inhibition of PrPSc accumulation is observed after the addition of copper in vitro to PMCA reactions, a procedure used to amplify PrPSc (39, 159).

The involvement of redox iron and imbalance of brain-iron homeostasis in prion disease–associated neurotoxicity is a relatively new observation, and the effect of iron chelation on disease pathogenesis has not been tried in cell or mouse models. However, chelation of iron from prion disease–affected human and mouse brain homogenates in vitro reduces the amount of disease-associated PrPSc, suggesting that this method may be used prophylactically to decrease prion infectivity in consumable products (12). A similar reduction of PrPSc levels in vivo may prove useful in decreasing PrPSc load, although optimal iron chelators that are nontoxic at therapeutic doses and can cross the blood–brain barrier effectively have not been developed. Studies in MPTP mouse models of Parkinson disease report significant benefit from the concomitant administration of blood–brain barrier–permeable iron chelator VK-28 [5-(4-(2-hydroxyethyl) piperazin-1-yl (methyl)-8-hydroxyquinoline] and its derivative M30 [5-(N-methyl-N-propargyaminomethyl)-8-hydroxyquinoline], providing direct evidence for the involvement of iron in disease pathogenesis (253). However, the applicability of these compounds in prion disease–associated neurotoxicity is yet to be investigated. Although apparently encouraging, the reduction of brain iron may aggravate the disease by increasing iron uptake by surviving cells, warranting caution in using such compounds. Restoring brain-iron homeostasis in diseased brains is therefore a daunting task, because complex biochemical pathways are involved in iron metabolism.

However, iron chelation as a means to reduce the toxicity associated with its redox-active nature has been pursued actively in diseases like AD and PD. Several chelators have been tried, the most prominent ones being desferrioxamine (DFO) and 5-chloro-7-iodo-quinolin-8-ol (Clioquinol) (90). Although DFO showed some success in studies with AD patients, it does not cross the blood–brain barrier effectively and is toxic in therapeutic doses, making it an unsuitable drug for the treatment of AD or prion disorders. Clioquinol is an antibiotic that binds to Zn, Cu, and iron, and crosses the blood–brain barrier effectively (90). Structurally, Clioquinol is related to quinacrine analogues that have been used effectively in other studies on prion disease–affected experimental models and in human trials and could be a safe drug for in vivo use (152). The use of Clioquinol in scrapie-infected hamsters increases the  incubation time modestly, suggesting a future potential for the use of this drug in humans (175). Likewise, promising results were observed when Clioquinol was administered orally to mouse models of AD and PD (38, 105).

The use of antioxidants and metal ion chelators are the major approach toward developing therapies for prion disorders; however, most of the drugs are effective when administered at a very early stage of the disease. 

“Copper Redox Cycling in the Prion Protein Depends Critically on Binding Mode”, Lin Liu, Dianlu Jiang, Alex McDonald, Yuanqiang Hao, Glenn L. Millhauser, Feimeng Zhou
Journal of the American Chemical Society. Jul 2011: 110718073144042


“Change in the characteristics of ferritin induces iron imbalance in prion disease affected brains,”Ajay Singh, Liuting Qing, Qingzhong Kong, Neena Singh
Neurobiology of Disease. Dec 2011


“Neurodegeneration and oxidative stress: prion disease results from loss of antioxidant defense,” Brown,  Folia Neuropathol (2005)


Prion diseases or transmissible spongiform encephalopathies (TSEs) are rare neurodegenerative disorders that can be acquired either by direct transmission, inherited through dominant mutations in the prion protein gene or via an unknown sporadic cause. This latter group constitutes the vast majority of cases. Like many neurodegenerative diseases the hallmarks of oxidative damage can be readily detected throughout the brain of the affected individual. However, unlike most other neurodegenerative diseases, prion diseases are connected with a dramatic loss of antioxidant defence. As abnormal protein accumulates in the diseased brain there is both an increase of oxidative substances and a loss of the defences that keep them in check. In particular the normal cellular prion protein has been shown to be an antioxidant. Conversion of this protein to the protease resistant isoform is accompanied by a loss of this antioxidant activity. This change creates a paradox as the loss of activity is not accompanied by a loss of protein expression. It is likely that this prevents other cellular defences from responding sufficiently to protect neurons from the heightened oxidative burden. Recent experiments with transgenic mice have shown that when prion protein expression is switched off during the course of prion disease, cell death is dramatically halted and the mouse recovers from the disease. This result clearly illustrates that the continued expression of non-function prion protein is essential for disease progression. This implies that the presence of this abnormal protein during prion disease causes a failure of cellular antioxidant defence. This failed defence is the fundamental cause of the massive neurodegeneration that results in the fatal nature of TSEs. The role of oxidative stress in TSEs and other neurodegenerative disorders are discussed in this review.


“Oxidative stress and the prion protein in transmissible spongiform encephalopathies,” Milhavet et al., Brain Res Brain Res Rev (2002)


Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic or genetic in origin. These diseases are believed to be the consequence of the conformational conversion of the prion protein into an abnormal isoform. Their exact pathogenic mechanism remains uncertain, but it is believed that oxidative stress plays a central role. In this article, we will first review in detail the data supporting the latter hypothesis. Subsequently, we will discuss the relationship between the prion protein and the cellular response to oxidative stress, attempting ultimately to link PrP function and neurodegeneration in these disorders.


“Oxidative stress and neurodegeneration in prion diseases,” Kim et. al., Ann N Y Acad Sci (2001)


Transmissible spongiform encephalopathies (TSEs), also termed prion diseases, are a group of fatal neurodegenerative diseases that affect humans and a number of other animal species. The etiology of these diseases is thought to be associated with the conversion of a normal protein, PrPC, into an infectious, pathogenic form, PrPSc. The PrPSc form shows greater protease resistance than PrPC and accumulates in affected individuals, often in the form of extracellular plaques. The pathogenesis and the molecular basis of neuronal cell death in these diseases are not well understood. Oxidative stress has been proposed to play an important role in the pathogenesis of several neurodegenerative disorders. In the present study, evidence of oxidative stress in scrapie, the archetype disease of the TSEs, is discussed. In addition, the mechanisms whereby oxidative stress could lead to neuronal degeneration are described.


“The Use of Antioxidants in transmissible spongiform encephalopathies: a case report,” Drisko, Journal of the American College of Nutrition (2002)


It is possible that strategies blocking the effect of proinflammatory cytokines and the resulting oxidative damage may stem the progressive damage to the neuropil that occurs in spongiform encephalopathies. In fact, similar benefits have been described when antioxidants are used adjunctively in Alzheimer’s disease [22]. The reported case points to beneficial effects when antioxidant therapies are used in transmissible spongiform encephalopathies. The case revealed an early reversal in cognitive decline and subsequent improvements in myoclonus, apnea and rigidity. Although death was the ultimate outcome, the patient succumbed to the illness over 22 months after the onset of symptoms when the early rapid decline predicted demise within a few months. Further investigation into the use of antioxidants and other types of agents quelling inflammation needs to be undertaken. If antioxidants could be combined with treatments for the inciting infective agent, a new direction could be taken in the outcome of transmissible spongiform encephalopathies including CJD and vCJD.

During the course of her hospitalization, the family administered a mixture of antioxidants including NADH, vitamin E (mixed tocopherols), alpha lipoic acid, multivitamin and a fresh fruit and vegetable puree, which was taken over several days. After this administration, the patient became more responsive and began to speak in appropriate although short sentences. The patient also demonstrated less rigidity and stated that she felt better. Because of the improvement, the family decided to continue antioxidant and nutrient treatment at home after discharge. Appropriate dialogue with palliative care was initiated, and the family was made aware of the severity of her diagnosis.


“Functional implications of multistage copper binding to prion protein,” Hodak et al., PNAS (2009)


The prion protein (PrP) is responsible for a group of neurodegenerative diseases called the transmissible spongiform encephalopathies. The normal function of PrP has not yet been discovered, but indirect evidence suggests a linkage to its ability to bind copper. In this article, low-copper-concentration bindings of Cu2+ to PrP are investigated by using a recently developed hybrid density functional theory (DFT)/DFT method. It is found that at the lowest copper concentrations, the binding site consists of 4 histidine residues coordinating the copper through ε imidazole nitrogens. At higher concentrations, 2 histidines are involved in the binding, one of them in the axial position. These results are in good agreement with existing experimental data. Comparison of free energies for all modes of coordination shows that when enough copper is available, the binding sites will spontaneously rearrange to accommodate more copper ions, despite the fact that binding energy per copper ion decreases with concentration. These findings support the hypothesis that PrP acts as a copper buffer in vivo, protecting other proteins from the attachment of copper ions. Using large-scale classical molecular                      dynamics, we also probe the structure of full-length copper-bound PrP, including its unfolded N-terminal domain. The results show that copper attachment leads to rearrangement of the structure of the Cu-bonded octarepeat region and to development of turns in areas separating copper-bound residues. These turns make the flexible N-terminal domain more rigid and thus more resistant to misfolding. The last result suggests that copper binding plays a beneficial role in the initial stages of prion diseases.