Metalloproteins and neuronal death

Abstract

Neurodegenerative diseases include Alzheimer’s and Parkinson’s disease that are very common and other diseases that are notorious but occur less often such as Creutzfeldt-Jakob disease. In each case a protein is closely linked to the pathology of these diseases. These proteins include alpha-synuclein, the prion protein and Aβ. Despite first being discovered because of aggregates of these amyloidogenic proteins found in the brains of patients, these proteins all exist in the healthy brain where their normal function involves binding of metals. Recognition of these proteins as metalloproteins implies that the diseases they are associated with are possibly diseases with altered metal metabolism at their heart. This review considers the evidence that cell death in these diseases involves not just the aggregated proteins but also the metals they bind.

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David R. Brown

David R. Brown is Professor of Biochemistry at the Department of Biology and Biochemistry in the University of Bath, UK. Originally from Australia, Professor Brown has worked in the US and Germany before settling in the UK 12 years ago. His research focuses on the metallochemistry of proteins. His group also study regulation at the genetic level of genes encoding these proteins and the role of metals in aging. Professor Brown is a former member of SEAC (UK government advisory board on CJD and BSE) and is both editor and co-author of a number of books including Metallochemistry of Neurodegeneration (RSC Publishing).

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1. Neurodegenerative diseases

First thoughts concerning diseases such as Alzheimer’s disease (AD) and Parkinson’s disease tend to be towards the increased incidence and burden on the health care system due to increased longevity of the populace. For both diseases and others there is a link to trace elements and particularly metals such as copper. There is growing evidence of the importance of the role of metals in these diseases making the study of metals in brain disease one of the most rapidly growing fields in neuroscience.¹ The entire nervous system, including the brain, spinal cord, and peripheral nerves, is rich in metals.² The brain particularly is a specialized organ that concentrates metal ions. Although their importance in metabolism has frequently been marginalized, as illustrated by the use of terms such as “trace elements”, the concentrations of metal ions such as Fe, Cu and Zn in the grey matter of the brain are quite significant, within the range 0.1–0.5 mM.³ Under normal conditions, metal ions interacting with proteins can both generate and defend against ROSs. Therefore with certain metal bindingproteins tightly associated with some neurodegenerative diseases it is important to consider the possible role metals may have in the pathology of these diseases.

The two most common neurodegenerative diseases are AD and Parkinson’s disease.⁴ Other diseases include, Motor-neuron disease, Amyotrophic lateral sclerosis (ALS),⁵ Huntington’s disease, dementia with Lewy bodies, multiple sclerosis, spinocerebellar ataxia,⁶ progressive supranuclear palsy,⁷ frontotemporal dementia⁸ and a variety of less common diseases such as Menke’s disease.⁹ One of the most notorious families of neurodegenerative diseases is Prion disease or transmissible spongiform encephalopathy.¹⁰ These diseases remain quite rare despite the scare of an epidemic of variant Creutzfeldt-Jakob disease (vCJD). The total number of vCJD cases world-wide remains below 200. There are numerous forms of prion diseases in humans and also animals. Bovine spongiform encephalopathy (BSE) and scrapie have become well known animal neurodegenerative diseases while almost all other animal dementias are considered obscure.¹¹

AD is the most common neurodegenerative disease in elderly people and is the cause of 70% of all cases of dementia. It has a frequency of 6% in those 70–74 and this value increases in frequency with age until people over 85 have a 42% chance of developing the disease.¹² Like many similar diseases, there are often early-onset forms where the patients are much younger than the expected age for the disease to occur. Additionally, many rare forms of neurodegeneration are inherited due to gene mutations but Alzheimer’s, Parkinson’s and Prion diseases all have inherited forms as well. Currently, the only known neurodegenerative disease that can be transmitted between individuals is prion disease (vCJD, iatrogenic CJD) where a small number of cases have been transmitted through blood or other products such as growth factors or dura mata.¹³

Outside of clinical aspects of neurodegenerative orders, molecular biology and biochemistry have provided insights into how such diseases develop and the proteins and other molecules associated with disease onset and progress. Although there is no complete picture for any of the diseases, there are many common themes and significant bodies of information on changes associated with these. The most common theme linking the diseases is that there is often a protein or a fragment involved that changes conformation or takes on a new role.¹⁴ Often these proteins are poorly understood in terms of their importance to a cell in health, but it is becoming an exaggeration to say that we do not know their function. The aggregating proteins are termed amyloidogenic and often change conformation to one rich in beta sheet structural elements and/or become resistant to proteases. These proteins can then form aggregates or fibrils that build up in deposits such as plaques which may have an intracellular or extracellular location depending on the disease. This review will look at three of these diseases and look at how metals contribute to the disease process and neuronal loss.

The three diseases that will be discussed are prion disease, AD and Parkinson’s disease. Each of these diseases is associated with at least one protein that aggregates. The prion protein (PrP) is associated with prion diseases and forms extracellular aggregates which include fibrils. Fibrils are long, structured aggregates like rods. The prion protein was not discovered until these aggregates were first isolated.¹⁵ Following this, the normal cellular isoform was isolated.¹⁶ In AD, beta-amyloid (Aβ) was found to be the main component of amyloid plaques. This protein fragment is digested from a larger cellular protein termed APP (amyloid precursor protein) also discovered subsequent to the isolation of Aβ.¹⁷ In Parkinson’s disease, alpha-synuclein (α-Syn) also plays a role and aggregates to form fibrils. However, α-Syn was first termed the non-amyloid component of plaques in AD until the protein was also found to be the main component of Lewy bodies.¹⁸ Again, the identification of this protein resulted in the discovery of a normal cellular protein, of unknown function.

2. Aggregation of metalloproteins

Proteins such as α-Syn, PrP and Aβ have been termed amyloidogenic. Amyloid was originally termed because the protein aggregates were believed to be associated with starch. However, it is now well established that the main component of amyloid deposits such as plaques is protein.¹⁹ In some cases, such as PrP, the protein was first identified from analysis of amyloidogenic deposits in the brain.²⁰ Subsequently, quite a number of these proteins have been shown to be metalloproteins.^1,21 Given that so many proteins first identified as amyloidogenic have turned out to be metalloproteins, it is possibly time to consider a switch in thinking to consider them first as metalloproteins and second as proteins that can form aggregates such as amyloid. This change in thinking then implies that there is a subfamily of metalloproteins that are amyloidogenic and begs the question as to why some metalloproteins can aggregate and cause neurodegeneration. This is especially the case given that almost any protein can be converted to an amyloidogenic protein. While some of these proteins are associated with diseases with no known metal link (e.g. Huntington’s disease), the vast majority have no such association.

While there has been considerable study of mechanisms of aggregation of amyloidogenic proteins and many factors other than metals have been implicated,²² it is important to note that in the case of the three main proteins we are considering, altered metal binding has been shown to play a role. In the case of α-Syn aggregation of the protein can be initiated by binding of metals such as aluminium and copper and to a lesser degree iron.^23–25 This effect is secondary to the protein’s response to increased concentration which appears to be sufficient to induce aggregation.²⁶ Aβ can be inhibited from aggregation by preventing the binding of zinc or copper to the peptide.^27,28 PrP is principally a copper binding protein,²⁹ but in its disease specific isoform, it can be isolated from the brain with another metal bound, manganese.³⁰Manganese binding has been found to cause conversion of PrP to a protease resistant isoform able to induce protein aggregation by a nucleation mechanism.^31,32

3. Metalloproteins associated with diseases

Study of the proteins associated with neurodegeneration has clearly shown similarities between them in terms of their capacity to bind metals. The relationship between disease pathology and metals is emerging. In prion disease there is strong evidence for changes in metal metabolism³³ and evidence for accumulation of large amounts manganese in the brains of patients and animals with the disease.^34,35 In Parkinson’s disease there have been reports of high iron content in patient brains and patients with AD also show changes to metal concentrations in the brain.^36,37 More alarmingly, all three associated proteins (or their disease related fragments) show altered activity in the response to certain metals. Manganese can cause the aggregation of PrP.³¹Copper can cause α-Syn aggregation²⁴ and copper and zinc binding to the Aβ is also associated with its aggregation.²⁸ Clearly, tight regulation of metal interactions with these proteins is essential for preventing or limiting disease.

Aβ and amyloid precursor protein

There is a commonly held view that AD results when a specific protein in the brain, the Amyloid Precursor Protein (APP), is metabolised incorrectly to β-amyloid (Aβ), a 39–49 amino acid peptide. The disease is characterised by the deposition of Aβ as diffuse extracellular plaques or intracellular, neuritic plaques with dense cores. The deposition of Aβ is found predominantly in the hippocampus and temporal lobe cortex and is probably closely related to the primary pathogenesis of AD, with consequent neuronal death and increase in oxidative stress.^38–40 Many studies have confirmed that Aβ is neurotoxic in cell culture. There is an obvious similarity between AD and prion diseases, in that both disease are characterised by the deposition of an abnormal form of a normal cellular protein. Unlike in prion diseases, the length of the Aβ species is considered to be the important factor in AD pathogenesis as Aβ is a proteolytically cleaved version of the normal APP.

The metallochemistry of Aβ has been investigated in some detail.²¹ Aβ can be rapidly precipitated by Zn²⁺ ions at low physiological concentrations and it was recently reported that other metal ions like Cu²⁺, Fe³⁺, unlike Zn²⁺, produced a greater aggregation of Aβ at pH 6.8–7.9.⁴¹ Such an environment probably resembles conditions occurring in the brain. The significance of these in vitro studies with Aβ and metal ions is emphasised by other data showing that the homeostasis of Zn, Cu and Fe, are significantly altered in AD brain.³⁸ Experiments utilizing microparticle-induced X-ray emission (PIXE) analysis of cortical and accessory basal nuclei of the amygdala showed that these metals accumulated in the neurophil of AD brain, and that their concentrations were increased 3–5 fold compared with age matched controls. The metals were found to be particularly high in the Aβ deposits.^3,42Zn²⁺ in Aβ amyloid deposits was recently detected by histological fluorescent techniques in human brain.⁴³ It was also noted that the apolipoprotein E4 (apoE4) allele, which commonly appears in late onset AD cases, is associated with increased serum levels of Cu²⁺ and Zn²⁺ in AD. This suggests that the underlying perturbations in metal homeostasis associated with AD are systemic and not just confined to the brain.⁴⁴

Zn/Cu-selective chelators reportedly enhanced the resolubilization of pathological Aβ deposits from post-mortem AD brain samples, suggesting that Cu and Zn ions may play a role in assembling these deposits.⁴⁵ The metals could also play a more significant role other than purely facilitating fibril formation. In vitro work from Ashley Bush’s lab reported that Aβ is a redox active protein, which reduces Cu(II) or Fe(III) and then produces H₂O₂ by electron transfer to O₂.^46,47 Aβ cytotoxicity was shown to be mostly mediated by H₂O₂ produced directly by the Aβ variant, as the toxicity of the peptide was augmented by Cu²⁺ correlating with the degree of metal reduction by the same peptide. Aβ is very vulnerable to Cu(II)-mediated auto-oxidation, which leads to oxidative effects such as carbonyladduct formation, histidine modification and dityrosine crosslinking and such modifications have been located on the Aβ deposits extracted from AD amyloid.⁴⁸

The metal mediated redox activity of Aβ may well play a significant role in the pathogenesis of AD in vivo, though this still remains to be shown. The affinities of the Zn²⁺ binding sites on Aβ1-40 were measured as 100 nM and 5 μM, indicating that they may be occupied under physiological conditions.⁴⁹ The highest affinity Cu²⁺ binding site on Aβ1-42 has a measured association constant (Ka) of 10⁻¹⁵ M.⁵⁰ With such strong affinity for Cu²⁺, Aβ species like Aβ1-42 are likely to bind Cu²⁺in vivo. The increase in Cu²⁺ affinity of Aβ1-42 over the normal APP is related to APP proteolysis. APP is a membrane spanning protein and the Cu²⁺ binding site is probably hidden within the protein, becoming exposed in the proteolysed Aβ fragment. Also the peptide Aβ1-42 has a higher β-sheet content, and these structures are frequently found in the tertiary structure of Cu²⁺ catalytic sites.

Studies made with the complex of Cu²⁺ with Aβ1-42 showed that it had a very strong reduction activity (+550 mVs with respect to Ag/AgCl). This value is comparable with type II copper proteins such as SOD and is likely to denote a biological purpose for the metal binding.²¹ Furthermore, Aβ binds both Cu²⁺ and Zn²⁺. Thus the oxidative damage induced by Aβ might be mechanistically related to the oxidative stress induced by abnormal SOD activity.

In addition to beta amyloid, APP, the precursor to Aβ, also binds copper, but at an alternative site. The copper binding domain is found in the N-terminus of APP within amino acid residues 135–175.⁵¹ The copper binding domain contains the typical His-X-His motif of a type II site, similar to that seen for superoxide dismutase and lysyl oxidase copper homeostasis protein groups. APP reduces the bound copper to Cu(I) suggesting the protein could have a copper reductase activity.⁵² This reduction leads to oxidation of cystines 144 and 158 and resulting in the formation of a new disulfide bridge.⁵³ The dissociation constant for Cu binding to APP has been suggested to be approximately 10 nM.⁵⁴ Binding of the Cu to this domain involves the imidazole rings of three histidines supporting the notion that this site has higher specificity for Cu then other type II sites.⁵⁵

Copper binding to APP has been shown to reduce formation of Aβ.⁵⁶ One hypothesised mechanism for this is inhibition of APP dimerisation. It has been suggested that dimerisation of the protein is a requirement for cleavage by the beta-secretase that is necessary for Aβ formation.⁵⁷Copper binding at the Cu-binding domain of APP may prevent dimerisation. Thus the availability of copper may alter the dimer/monomer equilibrium sufficiently to inhibit access of the beta secretase.⁷⁷ It is interesting to note that chelation therapy is an ongoing possibility for the treatment of AD. A copper chelator, clioquinol prevents the formation of Aβ deposits in transgenic mice.⁵⁸ Clinical trials investigating the possibility to use analogues of this agent in humans continue at the present time.⁵⁹

Prion protein

The prion protein exists in a number of different forms.⁶⁰ The first of these is the cellular isoform (PrP^c) expressed in cells such as neurons. This form is protease sensitive, monomeric, has two asparagine-linked polysaccharide chains and is tethered to the outside by a glycosylphopotidyinositol anchor. There has been considerable study of PrP^c as a copper bindingprotein. PrP^c binds four to five copper atoms.⁶¹ Four of these are bound to an octameric repeat region in the N-terminus while the fifth binding site lies also in the unstructured N-terminus but distal to the octameric repeats. The affinity of copper for the protein has been quite controversial^29,62–66 but is now largely agreed to be at least in the low nanomolar range.⁶⁷ This affinity is not affected by glycosylation as has recently been demonstrated with purified native protein from mouse brain.⁶⁷ The second isoform of PrP is the disease related isoform, PrP^Sc. This isoform is protease resistant, rich in beta-sheet structure, an absence of bound copper and is associated with disease transmission through experimental infection.⁶⁰

Alterations to PrP expression have significant effects on copper metabolism. A recent study of a very large range of copper bindingproteins showed that altered expression of PrP causes changes in the expression of other copper bindingproteins such as APP, α-Syn, MNK-ATPase, Cu/Zn SOD, extracellularSOD, the copper chaperone Atox1 and the copper transporting proteins CTR2 and DMT-1.⁶⁸ Similarly, in mouse scrapie a similar set of proteins are altered. Changes in copper alter PrP expression in the brain and similarly altering PrP expression changes the amount of copper in cells.⁶⁹ Altogether, the evidence that PrP is an important part of copper homeostasis is overwhelming.⁶¹

As mentioned above, PrP can also bind manganese. The affinity of PrP is similar to that of other recognised manganese bindingproteins.³² Alteration in PrP expression changes the expression of manganese bindingproteins such as MnSOD⁷⁰ and manganese transporters NRAMP1 and DMT1.⁶⁸ DMT-1 is also increased in expression in the brains of scrapie infected mice. Increased expression of DMT-1 also increased the expression of proteinase K resistant PrP in scrapie infected cells. In parallel the manganese levels in scrapie infected cells is greatly elevated when compared to uninfected controls.⁶⁸ These findings parallel findings found from studying manganese levels in the brains of animals with BSE or scrapie and patients with sporadic CJD or variant CJD. In all cases so far examined, prion infection results in an elevation in the level of manganese in specific brain regions.^30,34,35,71 Given that manganese can cause conversion of PrP to a protease resistant isoform with many characteristics of PrP^Sc, the potential of these changes to either initiate or accelerate PrP^Sc formation is clear. What remains to be determined is what comes first, the formation of PrP^Sc or the increase in neuronal manganese levels. However, as cells grown in the presence of high levels of manganese can form protease resistant PrP³¹ there is a distinct possibility that any alteration in cellular manganese metabolism could be sufficient to trigger protein conversion (Fig. 1). Sporadic CJD is a disease of people of around 60 and occurs at a very low frequency, therefore it remains a distinct possibility that altered manganese metabolism could be sufficient to initiate prion disease. However, given the extreme low incidence (1 in a million) it may be extremely difficult to verify this experimentally.

Fig. 1 Metals and PrP. The prion protein interacts with both copper and manganese. Interactions with copper appear to be necessary for the normal activity of the protein while those associated with manganese are largely associated with changes that occur in prion disease such as protein aggregation, change in protein structure and toxicity of the protein.

Alpha-synuclein

α-Syn is the major fibrillar component of Lewy bodies and Lewy neurites.^18,72 Recombinant α-Syn readily assembles into filaments in vitro, which are similar in morphology, staining and structure to α-Syn filaments extracted from diseased brains.^73,74 Kinetics of fibrillation are in favour of a nucleation-dependent mechanism.⁷⁵ Analysis of α-Syn in Lewy bodies from patients, shows an increase in beta sheet content in the protein.⁷⁶ The central NAC region of α-Syn is responsible for the conformational change from random coil to β-sheet.⁷⁷ The missense mutations associated with the disease (A30P, A53T and E46K) lead to an increased rate of oligomerisation.^78,79

It is only recently that α-Syn has emerged as a metalloprotein. Initially, the only relation noted between α-Syn and metals was the potential of metals to trigger aggregation.²⁴ An earlier study had noted that lack of the C-terminus of α-Syn resulted in less aggregation of the protein.⁸⁰ A more recent study has suggested that α-Syn has two sites that bind copper.²⁵ This study suggested that there was a site centred on the single histidine at amino acid residue 50 with a dissociation constant in the 0.1–50 μM range, while the other site at the C-terminus had a much lower affinity (400–500 μM). Following this, some other studies did not support the histidine as the major copper binding site.⁸¹ Since then, further analysis has greatly clarified the picture suggested that there are at least three domains in the protein contribute to copper binding.^82–84 While one site is the histidine, there are also contributions from the C-terminus (residues 119–126) and the N-terminus (residues 1–5). These studies have culminated in an in depth EPR analysis of binding modes.⁸⁵ The study shows that there are multiple co-ordination modes for the two main copper binding sites on the protein which are pH dependent. The mode of binding varies in terms of the involvement or not of the histidine 50. Thus two binding modes with and without involvement of this histidine occur, explaining some of the discrepancies between earlier findings. While these two binding modes at the N-terminus are present at pH 7.4, a third binding mode was seen at pH 5 with a fourth present in the C-terminus also at pH 5. Thus copper binding by α-Syn seems quite dynamic. A study with peptides clearly showed that copper binding requires interaction between separate domains of the protein.⁸⁶ This also creates the possibility that copper binding sites could be formed intermolecularly as well as within a single peptide. Given that molecular crowding can induce intermolecular interactions, this may explain how copper can accelerate aggregation of the α-Syn.

4. Neuronal death in prion disease

In prion diseases an abnormal isoform of PrP (PrP^Sc) is generated in excess. There are clear links between the production of this protein and neuronal loss as seen in the disease. Continued research over the last 15 years has attempted to understand the mechanism of cell death. Without being conclusive the majority of data points to a direct toxic effect.⁸⁷ One of the central findings of the study of neuronal death in prion disease is the necessity for cellular expression of the normal host isoform (PrP^c). Originally, this finding arose from in vitro experiments where toxic forms of PrP were applied to cultured neurones lacking the expression of PrP^c.^88,89 The finding was subsequently verified in vivo using a conditional knockout mouse model where expression of PrP^c was switched off during the course prion disease and this prevented subsequent cell loss.⁹⁰ It is important to note that the presence of PrP^Sc alone is not sufficient to cause neuronal destruction.⁹¹ In addition, co-expression of a different form of PrP that cannot be converted to PrP^Sc has a protective effect preventing neuronal damage.⁹² What is unknown in these transgenic mouse systems is what other changes occur when PrP is switched off. Studies on traditional knockout mice have shown alterations in antioxidant defence⁸⁷ that would suggest alternative defence mechanisms are more active when PrP is not expressed. The implication of this is that the presence of non-functional PrP inhibits the activity of oxidative defence mechanisms. Cell lines can be generated that are constitutively infected with TSEs. These cell lines do not show significant cell death but they do show decreased levels of a number of antioxidants.⁹³ This supports the hypothesis that cell death in prion disease results from a failure in antioxidant defence rather than oxidative damage in excessive levels alone. This has an immediate implication relating to the function of PrP^c. This implies that the protein can act as a sensor for oxidative stress or could in itself be an antioxidant. There is strong evidence that PrP^c has an activity like a superoxide dismutase.⁹⁴

Oxidative stress has been a measure in prion disease models.⁹⁵ Markers of oxidative stress have been shown to be present in the brains of mice infected with scrapie and the brains of patients with CJD.^96,97 The source of the oxidative damage is possibly as a result of activated glia. Both in vitro and in vivo models have suggested that activation of microglia occurs prior to neuronal loss.^24,98,99 The activation of the microglia is a result of the presence of PrP^Sc. This change in itself is insufficient. Activation of wild-type microglia in the presence of neurons from PrP-knockout mice was shown not to result in significant cell death.¹⁰⁰ The implication is that PrP expression must occur in prion disease for a failure of oxidative defence to be sufficient for cell death to occur. The immediate implication is that PrP is inherently part of the oxidative defence mechanism of a cell. While in the absence of PrP expression other protective mechanisms are activated, the presence of abnormal PrP suggests the expression of PrP without the possible protective effects of the normal isoform.¹⁰¹ Support for this notion comes from data that shows PrP is upregulated when other antioxidants are down regulated or knocked out. Knockout of any of the three major SODs causes dramatic upregulation of PrP and a double knockout of Cu/Zn SOD and extracellularSOD results in a greater increase in PrP expression.⁶⁸

As mentioned above, prion disease is accompanied by a change in manganese. This is a metal usually found at very low levels in mammalian cells but which has a very high potential to cause oxidative stress. While most models of prion toxicity have used peptides, a new model using recombinant PrP has been developed. This model is based on refolding the recombinant protein in the presence of manganese, allowing the formation of a toxic, non-fibrillar form of PrP.¹⁰² This model has the advantage of generating highly toxic protein allowing low concentrations to be used. Until this time in vitro models using peptides or recombinant protein have required high concentrations making it unlikely that the models are an accurate reflection of what occurs in vivo. It is becoming increasingly accepted that fibrillar forms of amyloidogenic proteins are not the toxic form. The same is true for PrP.¹⁰³ The toxicity of Mn-PrP requires manganese, but the amount of manganese bound to the protein is insufficient to cause toxicity on its own.¹⁰² PrP with manganese bound can be isolated from the brain of patients with CJD and experimental scrapie infected mice.^30,71 This demonstrates that this neurotoxic species possibly exists in vivo. However, there is still a need to demonstrate that this species is responsible for neuronal loss in vivo.

5. Neuronal death in Parkinson’s disease

The prevalence of fibrillar aggregates of α-Syn associated with these neurodegenerative diseases has led many authors to hypothesise that they cause cell death.^72,104,105 However, the survival of neurons with intracellular Lewy bodies shows that the presence of intracytoplasmic α-Syn aggregates are not toxic to all cells.¹⁸ Considerable evidence suggests that oligomers, formed as prefibrillar intermediates, may be the toxic component.^106–109 The most common hypothesis for the mechanism of α-Syn oligomer toxicity is that oligomers can create pores in membranes, which increases cell permeability to ions from the extracellular space leading to cell death.^106,108 This hypothesis is largely based on some species of α-Syn oligomers that are annular or pore-like with outer diameters of 10–12 nm and inner diameters of 2 nm.^109,110 These features may lead to the increased but non-specific ion permeability observed with α-Syn over-expressing cells.¹¹¹

The presence and relevance of extracellular α-Syn has recently gained attention. There are two main hypotheses regarding the source of extracellular α-Syn. It may be released upon cell death⁷⁴ or secreted from neuronal cells, as evidenced in culture.^112–114 The exocytosis of α-Syn is thought to be linked to a preferential secretion of damaged proteins from the cell.^113,115 Accordingly, α-Syn is present in the blood and cerebrospinal fluid (CSF) of both normal and PD-affected individuals.^116–118 The fate of extracellular α-Syn appears to depend on its form, such that α-Syn monomers are degraded by extracellularproteases, such as MMP3,¹¹⁴ whereas fibrils and oligomers of α-Syn are cleared by endocytosis and lysosomal degradation.¹¹⁹ However, the toxic species and mechanism of toxicity are still unclear.

In PD brains, high levels of metals are also found in specific regions of the CNS such as the substantia nigra.³⁶ The relationship between α-Syn and metals is still being characterised. As mentioned above, α-Syn binds to copper and iron^83–85 and α-Syn aggregation is stimulated in the presence of these metals.^21,82 This has led us to examine whether the toxicity of extracellular synuclein proteins was exacerbated in the presence of metals. Oligomerisation of α-Syn combined with a loss of metal homeostasis may be a key to the neurodegeneration observed with these diseases. Our initial studies looked at the toxicity of peptides based on the human α-Syn sequence.⁸⁶ Copper was found to increase the toxicity of a peptide equivalent to the C-terminus of α-Syn. This toxicity was further increased with addition of an N-terminal peptide. Our data suggested that simultaneous copper binding to both the N- and C-termini simultaneous could generate oligomeric α-Syn. Co-ordination of copper by α-Syn as a result of residues at distant sites has been suggested previously.⁸⁵ On the basis of these results we extended our study to the toxicity of full length α-Syn and its relation to copper. Our recently published work¹²⁰ indicates that a unique oligomeric species of α-Syn forms in the presence of copper and this form of α-Syn may represent the toxic form of the protein. We generated aggregated α-Syn by rapid shaking and applied the resulting mixture of fibrils, oligomers and monomeric protein to SH-SY5Y cells. On the aggregated protein generated in the presence of copper was toxic. The copper bound was not responsible for the toxicity on its own. Fractionation of the toxic aggregates proved that neither the fibrils nor the monomeric species were responsible for the toxicity in the presence of copper. The remaining oligomeric component was quite uniform and consisted of spherical, stellate oligomers (spikey balls). Analysis with electron microscopy demonstrated that these oligomers were only present when α-Syn was bound to copper. This clearly demonstrates the potentially central role of copper in the toxicity of α-Syn (Fig. 2). It is therefore imperative to determine the relevance of this form of α-Syn to Parkinson’s disease and other synucleinopthies and fully characterise the oligomer.

Fig. 2 Copper interaction with alpha-synuclein generates toxic oligomers. Recombinant α-Syn can be induced to aggregate either with or without the addition of copper. In general, presence of copper increase the rate of aggregation as measure by standard flurometric assays such as the ThT assay . Aggregated α-Syn is toxic to neurones only in the presence of copper. Electron microscopy indicated that the aggregates solution was a mixture of fibrils, oligomers and monomer. Purification of both the oligomers and fibrils was carried out and examples of electron microscopy images are shown. Only the oligomers showed significant toxicity. Oligomers such as those shown only occurred in the presence of copper.

6. Neuronal loss in Alzheimer’s disease

Neuronal death in AD undoubtedly has a complex mechanism. A myriad of factors have been investigated to come up with the best possibility for a mechanism. However, in these cases there are often conflicting data and an information over-load makes it difficult to identify the true mechanism. Three main hypotheses exist. One suggestion is that the accumulation of phosphorylated tau in paired helical filaments (tangles) causes cell death.¹²¹ The second hypothesis is that there is specific dysfunction of cholinergic neurons. Many elegant studies support the cholinergic hypothesis,¹²² showing that a dysfunctional cholinergic system is sufficient to produce memory deficit in animal models that are analogous to Alzheimer’s dementia. Brains from AD patients show degeneration of cholinergic neurons of the basal fore brain.¹²³ A marked decline in cholinergic markers, choline acetyltransferase and acetyl cholinesterase has been reported in the cerebral cortex of AD brain.¹²⁴ Although cholinergic deficits cannot fully account for the overall neuropathological features observed in AD, it represents a significant part of AD etiology and further research regarding the preferential vulnerability of this system in AD is warranted. The third, but most commonly accepted hypothesis is that Aβ is directly toxic to neurons. There have been many studies looking at cellular changes induced by Aβ and the possible way it could induce changes to cells to trigger an apoptotic cascade. The changes suggested include both oxidative and nitrative stress as well as decreased membrane fluidity, alteration of the cytoskeleton and nucleus, redox-active iron, inflammatory or autoimmune processes.^125–127

There is strong evidence that the formation of Aβ aggregates requires interaction with metals (see above). This implies that neuronal death caused by Aβ requires metals as well. Therefore metal binding to Aβ is indirectly involved in the mechanism of cell death in AD as initiated by the aggregation of Aβ. Evidence for this comes from studies using metal chelators that alter disease progression in experimental models.⁵⁸Clioquinol and its analogues can clear Aβplaques and abrogate behavioural changes in rodent models. These compounds have been less successful in clinical trials but this does not detract from the clear relevance of considering metal interaction with Aβ when considering neuronal loss in AD (Fig. 3).

Fig. 3 Metals and beta-amyloid. Copper plays numerous contradictory roles in the formation of Aβ. BACE-1 is the enzyme that cleaves APP to form Aβ and the enzyme binds copper. It is unclear at present if this metal is a cofactor necessary for the protein’s activity or not. However, there is evidence that copper does inhibit the formation of Aβ. The aggregation of Aβ to form plaque has been shown to require both copper and zinc. Additional copper chelators have been effective at reducing plaque load in animal models. The consequence of this is reduced symptoms. The potential benefits for chelation therapy for Alzheimer’s disease are still unclear.

7. Conclusions

The three diseases considered here are all similar in that they involve metalloproteins. That these proteins are metalloproteins has taken a backseat to the fact that they are amyloidogenic. In each case the biological role of the protein is uncertain and in each case the disease specific form was identified both before the normal cellular isoform was identified and before they were known to be metalloproteins. Given that all three proteins have been shown to be neurotoxic, and that in each case the toxicity may involve an interaction with metal, then there is reason to consider that the metal binding is a unifying element when considering these diseases. The time has come to consider these diseases to be “metalloprotein-dependent neurodegenerative diseases”. This would place consideration of the central proteins as metalloproteins first. The advantage of this would be to change global thinking about these diseases to consider a fundamental and common underlying mechanism to these diseases, namely disturbance to cellular metallochemistry.

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