Metallothioneins in Prion- and Amyloid-Related Diseases

Abstract

Prion and other amyloid-forming diseases represent a group of neurodegenerative disorders that affect both animals and humans. The role of metal ions, especially copper and zinc is studied intensively in connection with these diseases. Their involvement in protein misfolding and aggregation and their role in creation of reactive oxygen species have been shown. Recent data also show that metal ions not only bind the proteins with high affinity, but also modify their biochemical properties, making them important players in prion-related diseases. In particular, the level of zinc ions is tightly regulated by several mechanisms, including transporter proteins and the low molecular mass thiol-rich metallothioneins. From four metallothionein isoforms, metallothionein-3, a unique brain-specific metalloprotein, plays a crucial role only in this regulation. This review critically evaluates the involvement of metallothioneins in prion- and amyloid-related diseases in connection with the relationship between metallothionein isoforms and metal ion regulation of their homeostasis.

Keywords

Alzheimer’s disease brain copper metallothionein neurodegenerative disorders prion protein

INTRODUCTION

World Health Organization statistics show that the number of people living with dementia caused by progressive neurodegenerative diseases worldwide is estimated at 35.6 million as of 2010, and there are 7.7 million new cases each year, implying that every four seconds there is a new case of dementia somewhere in the world. These numbers will double by 2030 and more than triple by 2050. Dementia does not just affect individuals, but it also affects and changes society. The huge cost of the disease will challenge health systems to deal with the predicted increase of prevalence. Alzheimer’s disease (AD) and Parkinson’s disease (PD) are probably the most common neurodegenerative diseases associated with advanced age. AD, which is characterized by a loss of cognitive function, is responsible for 60–70% cases of dementia. PD is typically characterized by a loss of motor functions and also cognitive functions, at a later disease stage. AD and PD together possibly contribute to 70–80% of all dementia cases.

Prion diseases are transmissible protein misfolding disorders, in which misfolding of a host-encoded prion protein occurs. Prions are defined as alternatively folded, self-propagating protein conformers, which were discovered in 1982 [1, 2]. Compared to AD and PD, prion diseases are rare with a prevalence of spontaneous and genetic forms at 1-2 cases per 1,000,000 [3]. Self-perpetuating protein isoforms participate in diverse biological processes including translation termination, long-term memory storage, and immune response, suggesting that prion-like alternative folding may be more common than previously thought [4]. All prion diseases share similar features: Extremely long incubation time, slow showing of symptoms, late disease onset and formation of amyloid plaques in the brain, and spongiform changes [3].

These features are also shared in other progressive neurodegenerative disorders, especially AD, PD, Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS) [5, 6]. Another common feature of neurodegenerative diseases is the presence of misfolded protein aggregates in affected regions of the nervous system. Although the major protein component of the pathological aggregation can be unique for each neurodegenerative disease, several proteins misfold and accumulate in multiple diseases (see Table 1) [5].

In prion diseases, according to the seeding-nucleation model, the agent that causes diseases is an abnormal prion protein (PrP^Sc) that catalyses the conversion of normal prion protein (PrP^C) molecules into PrP^Sc (Fig. 1), which is the causative agent of the transmissible spongiform encephalopathies [3]. So the following human prion-related diseases have been reported: Kuru, sporadic, familial/genetic, variant and sporadic Creutzfeld-Jacob diseases (sCJD, f/gCJD, vCJD, iCJD), Gerstmann-Sträussler-Scheinker syndrome, fatal familiar insomnia, sporadic fatal insomnia, and variably protease-sensitive prionopathy [3]. Prion diseases have been identified also in animals (scrapie, mad cow disease, etc.). They are widely discussed in respect to their possible transfer from animals to men.

AD is characterized pathologically by the formation of senile plaques composed of the amyloid-β (Aβ) peptide and neurofibrillary tangles composed of hyperphosphorylated tau. However, the accumulation of Aβ in the brain appears to be critical for the pathogenesis of AD (Fig. 2). The mechanism of initial Aβ aggregation is still not known, and, according to recent articles, prion protein (PrP) may be involved in this process [4, 7]. It has been reported that Aβ aggregates are prions, and this was evidenced by the demonstration that widespread cerebral β-amyloidosis is induced by inoculation of either purified or synthetic Aβ into susceptible transgenic mice [4]. Transmission studies in cellular and animal models of tauopathies [8, 9], synucleinopathies [10, 11], ALS [12], and HD [13] indicate that these diseases are also caused by self-propagating protein aggregates. Evidence indicates that non-prion aggregates (tau, α-synuclein, Aβ, and huntingtin aggregates) can also move between cells and seed the misfolding of their normal conformers. These findings have enormous implications. On the one hand, they question the therapeutic use of transplants, and on the other, they indicate that it may be possible to bring these diseases to an early arrest by preventing cell-to-cell transmission [14].

All proteins involved in the above-mentioned amyloid and prion-related diseases exhibit affinity to heavy metals, which is related to their aggregation (for a summary, see Table 1). Zn(II) and Cu(I, II) and also Al(III), Ca(II), Co(II), Cd(II), Fe(III), Mn(II), Ni(II) ions modulate aggregation of corresponding proteins and are involved in pathogenesis of amyloid and prion-related diseases, as extensively reviewed in [166]. A great piece of information has been gained by investigating a family of small cysteine rich heavy metal binding proteins called metallothioneins (MTs). Level of MTs has been found to be altered in AD, PD, HD, ALS, and CJD [15 –17] and changes in normal homeostasis of essential transition metals such as zinc and copper have been implicated as possible etiological factors [18].

To date, a large number of papers have been published making this problem very labyrinthine. A number of studies connected with this topic are sometimes over-interpreted, controversial, and contradictory. Most of the data regarding amyloid diseases and MTs are based on metal binding and metal transfer studies in vitro. Moreover, there are no clear molecular mechanisms for how these proteins work and there is a lot of uncertainty about their metal contents in vivo and expression levels in neurons. The main aim of this review is to highlight the most interesting associations between MTs, prion-related diseases, and other aggregation proteinopathies. In the light of the fact that MTs regulate levels of metal ions that are involved in above-mentioned amyloid and prion-related diseases, its role in pathogenesis of these diseases will be discussed.

LOW MOLECULAR MASS CYSTEINE RICH METALLOTHIONEINS

Metallothioneins (MTs) are small cysteine rich metalloproteins which were discovered in 1957 as cadmium-binding proteins in horse kidneys [19]. Further, this protein and other MT-like proteins were found in tissues of other animal species, yeasts, bacteria, fungi, and plants [20 –22].

MT is currently classified in 15 families [23]. Mammalian MTs are single-chain polypeptides of 61 to 68 amino acid residues. Position and number of the cysteine residues are highly conserved and forms cys-x-cys, cys-x-y-cys, and cys-cys thiolate clusters, where x and y are non-cysteine amino acids. Divalent metals are bound by sulfur atoms in thiolate clusters and there are no free thiol groups in MT structure [24]. MT has two subunits: The more stable α-domain (C-terminal), which incorporates up to four divalent metal atoms, and the more reactive β-domain (N-terminal), which contains only up to three. The exchangeability depends upon the metal species, and, under in vivo conditions, MTs exist mainly in Zn form or as mixed-metal proteins. The binding affinity varies between metals with Cu(I) having the greatest stability constant followed by Cd and Zn. 18 different metal ions may bind to MT, but only Cu(I), Cd(II), Pb(II), Ag(I), Hg(II) and Bi(II) can displace Zn(II) [20, 25]. The tertiary structure of MT is dynamic and metals exchange rapidly within the β-domain, more slowly in the α-domain, and may be also exchanged with other ions bound to intracellular ligands. MT has also been found to donate metal ions to higher-affinity ligands or other proteins [26].

In mammals, four distinct MT isoforms designated MT-1 through MT-4 exist, whereas they are monomeric proteins, containing two metal-thiolate clusters [27]. In humans, at least 10 to 17 MT genes, clustered on chromosome 16, are functional and encode multiple isoforms of MT-1 (MT-1A, – B, – E, – F, – G, – H, – I, – J, – K, – L, and – X) and one isoform of MT-2 (MT-2A), Single genes code for MT-3 and MT-4. Heterogeneity of isoforms results from posttranslational modifications (acetylation) and/or variations in heavy metals content (metalloforms). Isoforms are distributed in various ratios within single tissues and have different rates of degradation. Although the general physicochemical properties of MT isoforms are similar, they have specialized biological functions. The first discovered MT-1/MT-2 are widely expressed isoforms, whose biosynthesis is inducible by a wide range of stimuli, including metals, drugs, and inflammatory mediators. In contrast, MT-3 and MT-4 are non-inducible proteins, with their expression primarily confined to the central nervous system (CNS) and certain squamous epithelia, respectively. MT-1 through MT-3 have been reported to be secreted, suggesting that they may play different biological roles in the intracellular and extracellular space [28]. In the postgenomic era, it is becoming increasingly clear that MTs fulfil multiple functions, including the involvement in zinc and copper homeostasis, protection against heavy metal toxicity. Absence of MTs has been shown to result in impaired recovery from CNS trauma, while overexpression improved recovery. Such neuroprotective functions of MTs have been attributed to their free radical scavenging and heavy metal-binding properties. It is however emerging that an extracellular activity of MTs may also be important since exogenous MT has been shown to interact directly with neurons to promote neuronal survival, neurite outgrowth, and axonal regeneration, thus raising the possibility that MT might be released from astrocytes following CNS injury [29 –31].

METALLOTHIONEINS IN NEURONAL TISSUES

The role of MT is discussed in connection with copper and zinc homeostasis in brain tissue [32]. In addition, displacing Cu (and Fe) ions from MT and other cellular metal-binding proteins may stimulate generation of reactive oxygen species (ROS) via Fenton reaction [33, 34].

Neuronal growth inhibitory factor (metallothionein-3)

MT-3 represents a unique metalloprotein also called neuronal growth inhibitory factor because of the ability to inhibit outgrowth of neuronal cells [35, 36]. Although MT-3 was discovered in the brain, it was presumed to be brain-specific; however, in following years, MT-3 has also been isolated in other tissues like salivary glands [37], reproductive organs, kidneys [38 –40], and several tumors [41 –43]. From a structural point of view, compared to other MT isoforms, MT-3 contains single-residue insertion (Thr) at position 5 and an acidic hexapeptide insertion in positions 56 –61 in the α-domain. It has been shown that these two insertions are essential for biological activity of MT-3 and heavy metals binding [44, 45]. Even the first studies were focused on metal binding capacity of MT-3. Initially, it has been isolated from the human brain with a metal content of four Cu(I) and three Zn(II) ions per one MT-3 molecule [46, 47]. The higher affinity of recombinant E. coli MT-3 than of MT-1 and MT-2 for Cu⁺ [48], together with the relevance of the presence of Cu in the neuronal system, prompted a detailed study of the MT-3 Cu-binding behavior. However, whereas Cu(I) binding capacity of MT-3, features of the in vivo folded MT-3 complexes and Cd^2 + or Cu⁺ exchange reactions are still discussed and remains questionable, models describing its binding capacity for Zn(II) have been established [49]. The analyses of the Zn^2 +– Cd^2 + and Zn^2 +– Cu⁺ replacement processes revealed that this mammalian MT isoform is best suited for functional handling of the Zn– Cu equilibrium and exchange reactions [44]. As for copper-MT-3 (Cu-MT), its exact role has been elusive so far, but it has been strongly supported that it may act as depository for copper transfer into copper apo-proteins, and/or copper apo-chaperones, involved in copper trafficking [50, 51]. Moreover, a role in copper detoxification has been shown in yeast and mammals [29]. As follows from above-mentioned facts, MT-3 plays crucial role in metals homeostasis due to the ability to bind both monovalent and divalent metal ions concurrently making it important factor for homeostasis of two essential heavy metals, Cu(I) and Zn(II) in the brain [44, 52]. Otherwise, the homeostasis of copper and zinc is maintained by MT-1 and MT-2, the most abundant MTs expressed predominately in astrocytes and to a lesser extent in neurons, present in tissues and cells of brain tissue including choroid plexus epithelium, ependymal cells, endothelium, meningeal cells, and microglia.

MTs in ROS and heavy metals homeostasis

Another potential role of MTs is protection against oxidative stress by chelation of free metal ions and by their scavenging. As a matter of fact, Zn₇MT-3 could be shown to act as a scavenger for free Cu(II) ions through their reduction to Cu(I) and binding to the protein [53]. During the reaction cysteinate ligands are oxidized to disulfides with concomitant release of Zn(II). As a consequence Zn₇MT-3, in the presence of ascorbate, acts as a scavenger for the copper-catalysed hydroxyl radicals [29]. MT also acts as a potent scavenger of hydrogen peroxide, hydroxyl radical, nitric oxide, and superoxide anion radicals, so, it is reasonable to assume that MT-3 acts as a neuroprotective compound, eventhough certain controversy remains.

Intracellular Zn(II) signaling in cognition has been reviewed by Takeda et al. [54]. MT-3, together with MT-1 and MT-2, also serves as a source of labile zinc in the brain. Zinc ions can be bound or released dependent on pH and redox conditions and administered to other zinc-dependent proteins, as shown for ferritin, where interaction with MT-1, MT-2, and MT-3 lead to simultaneous Fe(II) and Zn(II) release [55]. The kinetics of zinc ions transfer from Cu₄Zn₃MT-3 to apo-carbonic anhydrase (apoCA) was studied, and zinc ions transfer rate constants and thermodynamic parameters were obtained. It is found that like other MTs, porcine Cu₄Zn₃MT-3 can also transfer its zinc atom to apoCA, even easier than MT-1 and MT-2 [56]. It has been shown that S-nitrosothiols that have been suggested to be means of storage and transport of nitric oxide, react preferentially with zinc-thiolate clusters of MT-3 compared to MT-1 and MT-2, which results in release of Zn(II) from MT-3 structure. This indicates that MT-3 is biologically specific in converting NO signals to zinc signals [57]. It has been suggested that Zn₇MT-3 actively participates in a synaptic cycle of zinc vesicles, synaptic vesicles harboring vesicular zinc [58], and was also found in the extracellular space. In this case, MT-3 is isolated from such a place as a Cu(I)₄,Zn_3–4MT-3 species. This feature joined with its downregulation in AD [59].

Zinc ions play a crucial role in lysosomal changes and cell death in neurons and astrocytes under oxidative stress, thus MT-3 plays an important role in normal lysosomal function. This fact was demonstrated by Lee at al. [60]. Excess of zinc ions in in vitro cultured cell lines (cortical cells, C6 rat glioma cells) induced necrotic and programmed cell death, respectively [61, 62]. Similar results were shown also in vivo and are relevant for hippocampus, where an excess of zinc ions released during some pathological conditions may contribute to the selective death of neuronal cells [63 –65]. At present, it is known that not only excess, but also critical zinc ions depletion, induce apoptosis via increased creation of ROS and subsequently increased oxidative damage and activity of pro-apoptotic enzymes [66 –68].

MTs in regulation

MT-3 has both neurotoxic and neuroinhibitory actions that are involved in its regulatory role in cell growth and death, as shown on different cell models [69]. Neuronal growth inhibitory properties affect neural recovery from brain injury or neurodegenerative diseases. However, MT-3 is able to regulate cell fate in tissues other than brain. This fact has been shown in various in vitro systems. Somji et al. investigated the expression of MT-3 in cell cultures derived from the human proximal tubule (HPT). They showed that mortal HPT cells expressed MT-3, while the HPV-immortalized HK-2 cells had no expression of MT-3. In further studies, the authors investigated the effect of MT-3 expression on Cd(II)-induced cytotoxicity on the HK-2 cell line transfected by MT-3 coding sequence. The results demonstrated that HK-2 cells stably transfected with MT-3 were more sensitive to the cytotoxic effects of Cd(II). Furthermore, increase in Cd(II)-induced cytotoxicity was correlated to an alteration in the mechanism of cell death, being changed from an apoptotic mechanism in cells not expressing the MT-3 gene to a necrotic mechanism in cells expressing the MT-3 gene [70]. All these processes are very specific to this type of MT and are probably based on the small differences in its primary structure in comparison with other MTs.

The biological activity of MT-3 as a GIF involves interacting protein partners in the brain. The subcellular localization of MT-3 in rat brain astrocytes showed an association with organelles involved in the secretion pathway-free ribosomes, rough endoplasmic reticulum, small vesicles, the outer mitochondrial membrane, plasma membrane, and also around the blood vessels. The localization in neurons was mostly found in the axons, dendrites, the synaptic vesicles, and the postsynaptic densities [71]. It has been shown that MT-3 directly interacts with a G-protein Rab3A [58], and the association of mouse MT-3 with heat-shock protein-84 and heat-shock protein-70 (Hsp-84, Hsp-70 respectively), dihydropyrimidinase-like protein 2, creatin-kinase and β-actin has also been published [72]. New proteins have been reported to interact with MT-3. These new partner proteins – Exo84p, 14-3-3 Zeta, α- and β-Enolase, Aldolase C, Malate dehydrogenase, ATP synthase, and Pyruvate kinase were involved in transport, chaperoning, scaffold, glycolytic metabolism, and neuronal growth [73].

MT-3 can also probably regulate psychological behavior; however, this process remains almost unknown. This phenomenon has been demonstrated on MT-3 deficient mice, where MT-3 knock-out mice had significantly shorter social interactions [74]. Obesity and downregulated hypothalamic leptin receptors have been reported in male, but not female MT-3 knockout mice. Under conditions of MT-3 deficiency the leptin receptor (LepR) was downregulated and levels of phosphorylated extracellular signal-regulated kinase (p-Erk-1/2) were also reduced in the hypothalamus. A possibility of zinc ions administration to Erk-1/2 by MT-3 was suggested, as after exposure of MT-3 null hypothalamic cells to zinc activated Erk-1/2 and induced LepR expression. They demonstrated that MT-3 may be involved in central leptin signaling in the brain of male mice, particularly in the hypothalamus, and the consequent increase in peripheral energy expenditure, but not appetite suppression [75].

Dou et al. found that psychological stress induced hippocampus zinc dyshomeostasis and depression-like behavior in rats. Compared to control group, the rats exposed to psychological stress showed decreased total zinc levels and increased free zinc levels in hippocampus together with decreased expression of zinc transporters ZnT1, ZnT3, ZIP1, and MTs of unspecified isoform [76]. In humans, the decreased level of total thiol compound was found in blood plasma of cognitive-impaired individuals with recurrent depressive disorder [77].

Eugenol, a candidate antidepressant compound of plant origin [78] exhibited an antidepressant-like activity in mice and induced hippocampal expression of MT-3. This antidepressant activity was comparable to that of imipramine. Both eugenol and imipramine induced brain-derived neurotrophic factor in the hippocampus with and without induction of MT-3, respectively. It may be possible that MT-3 expression is involved in the exhibition of antidepressant-like activity of eugenol, but not of imipramine [79].

METALLOTHIONEINS, METALS, AND AMYLOID-RELATED DISEASES

The role of MTs was investigated in some metal-linked neurodegenerative diseases such as PD, prion, and prion-related diseases [15, 80]. Due to the affinity of MTs to heavy metals, it is necessary to discuss connection between MTs, especially its role in metal homeostasis and metals that are involved in this group of diseases [15, 17]. In a normal brain, a high concentration of essential heavy metals like zinc, copper, and iron is present. Dysregulated metals homeostasis, abnormal metal– protein interactions, and the associated oxidative stress, protein misfolding, and aggregation are critical common pathological hallmarks of the progression of several metal-linked neurodegenerative disorders [18 , 82].

Studies conducted on hemiparkinsonian rats suggest that the free-radical scavenging potency, including that of MT-3, is reduced in a PD brain [15]. Nicotine-encapsulated nanoparticles improved neuroprotective efficacy against MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced parkinsonism in culture of mouse dopaminergic neurons. The nicotine released into the cells led to increasing of MT-3 synthesis [83]. Generally, proposed models show that MT-3 is able to bind Cu(I) when Cu homeostasis is disrupted and Cu together with Zn ions are involved in the formation of amyloids by modulating the aggregation of Aβ peptides [84]. Aβ, particularly in aggregated forms, triggers pro-inflammatory reactions of microglia and astrocytes, which usually surround the amyloid deposits [85 –88]. Evidence exists which shows that metal ions, especially Zn(II) and Cu(II), modulate the aggregation pathways of Aβ upon binding was accumulated [89 –91]. Interestingly, the roles of Cu(II) and Zn(II) in AD pathology appear to be distinct. Cu(II) induced Aβ aggregation is associated with neurotoxicity, whereas Zn(II) induced aggregation is reported to be neuroprotective or neurotoxic depending on the conditions. The difference in neurotoxicity of Zn(II) and Cu(II) may be related to their difference in production of ROS [92]. While the redox cycling of Cu-Aβ complexes can promote ROS generation, Zn(II) is redox silent. Cu(II) promotes Aβ-mediated neurotoxicity by inducing the formation of amyloid fibrils and protofibrils, while Zn(II) binding is associated with more amorphous aggregates that appears to be less neurotoxic [84]. The role of MT-3 in an animal model of AD was also studied. Manso et al. found that in MT-3 deficient mice which overexpress human AβPP (hAβPP) and show symptoms of AD pathology, the amyloid plaque burden and/or hAβPP expression were decreased and exogenously administered Zn₇-MT-3 increased soluble Aβ₄₀ and Aβ₄₂ and amyloid plaque gliosis. The treated mice exhibited changes in behavior (increased deambulation and exploration and decreased anxiety) [93].

The aggregation of α-synuclein (α-Syn), the major component of intracellular Lewy body inclusions in dopaminergic neurons of the substantia nigra, plays a critical role in PD etiology [94, 95]. Aberrant tight binding of one Cu(II) to α-Syn and associated oxidative stress appears to contribute to the degeneration of dopaminergic neurons through the abnormal aggregation of this protein [96 –98]. This process is closely connected with the misbalance of Cu ions. Meloni and Vasak established that α-Syn-Cu(II) possesses catalytic oxidase activity, so it can promote production of hydroxyl radicals, α-Syn oxidation, and oligomerization. Zn₇MT-3, through Cu(II) removal from the α-Syn-Cu(II) complex, efficiently prevents its deleterious redox activity [99]. In the light of the role of zinc ions in the induction of caspase-3, MT-3 prevents oxidative stress via Zn/Cu homeostasis and has significant neuroprotective effect [60, 100]. A protective role of extracellular Zn₇MT-3 from Cu(II) toxicity has been suggested on the basis of investigations of its reactivity toward free Cu(II) ions [53]. Zn₇MT-3 efficiently scavenged and redox-silenced the free Cu(II) ions through Cu(II) reduction to Cu(I) by thiolate ligands and binding to the protein, forming an air stable Cu(I)₄Zn₄MT-3 species [53]. ROS-quenching by resulting Cu(I)₄Zn₄MT-3 might be beneficial for neural tissues [53]. Studies aimed at understanding the protective effect of human Zn₇MT-3 against Aβ_1–40 toxicity showed that the protein efficiently removes copper not only from soluble Aβ_1–40– Cu(II) oligomers, but also from insoluble aggregates. In this process, Cu(II) is reduced by protein thiolates, forming the stable Cu(I)₄Zn₄MT-3 species described above and the non-redox-active Aβ_1–40– Zn(II). This metal swap completely quenches the ROS production mediated by Cu(II) bound to Aβ_1–40 and occurs not only in vitro, but also in human neuroblastoma cell culture, whereby the toxic effect of Aβ_1–40– Cu(II) is abolished [101]. Pedersen et al. investigated the mechanism of exchange of metal ions between Zn₇-MT-3 and Cu-Aβ_1–40. It was shown that the exchange of metal ions occurs via free Cu(II) and that metal ion exchange induces time-dependent amyloidogenic structural and morphological changes in Aβ_1–40 in a time scale of hours. Moreover, the morphological changes were due to binding of Zn(II) to Aβ_1–40 aggregates [84]. The protective effect of human Zn₇MT-2A against Aβ_1–40– Cu(II) toxicity was also investigated and compared with that of Zn₇MT-3 [102] in rat cortical neurons and neuronal cell culture. The authors of that study have shown that Zn₇MT-2A blocked Cu(II)-Aβ induced changes in ionic homeostasis and subsequent neurotoxicity of cultured cortical neurons and MT-2A can represent a crucial player in protection against Aβ aggregation and toxicity. However, the ensuing redox cycling of copper in oxygen-sensitive Cu(I), ZnMTs can change their properties from antioxidant to prooxidant. In this context it may be noted that although the MT-1/2 isoforms have been found to be significantly upregulated in regions of Aβ plaques in the AD brain, the presence of substantial concentrations of Cu(II) in these plaques (0.3 mM) has been shown [103 –105]. Clearly, more studies regarding the stability of brain MTs formed in the reaction between Zn₇MTs and different concentrations of Cu(II) to molecular oxygen are needed.

The expression of MTs was diminished in patients with sporadic ALS. The immunoreactivities of both MT-1/2 and MT-3 stained dominantly in glial cells and were decreased in the spinal cords of patients with ALS [106]. Those findings also align with the finding that familial ALS (FALS) model mice (G93A SOD1) crossed with MT-1/2 or MT-3 knock-out mice had accelerated expression of ALS [107]. Judged from these findings, both MT-1/2 and MT-3 play important roles in the progression of ALS.

Prions, metals, and aggregation

Prions are ubiquitous cell surface glycoproteins. Cellular prion proteins connect with cholesterol- and glycosphingolipid-rich lipid rafts through association of their glycosyl-phosphatidylinositol anchor with saturated raft lipids and interaction of their N-terminal regions [108]. They are expressed in CNS within synaptic membranes. While there is an established role for PrP^C in transmissible spongiform encephalopathies (TSEs), the physiological role of PrP^C has still not been fully determined. The wide distribution of PrP^C among mammalian species and the high conservation of this protein indicate a role of general importance [109]. Proteomic studies show that PrP^C belongs among the most important Cu-chelating proteins in brain and its role in metal homeostasis, neuroprotective signaling, lymphocyte activation, neurite growth, synaptogenesis, cellular signaling, cell viability and in the cellular response to oxidative stress have all been proposed [110, 111]. In prion diseases, the transition from natively folded PrP^C to misfolded PrP^Sc is a crucial pathogenic event [81, 112]. PrP^Sc generates soluble oligomers that, in turn, aggregate into amyloid fibers [113] by still not fully understood mechanism. High concentration of precursors, inflammation, oxidative stress, metals binding, and viral and microbial infections are possible triggers [114, 115]. According to seeding-nucleation model, the pre-existing or acquired PrP^Sc oligomers catalyse the conversion of PrP^C molecules into PrP^Sc fibrils; the breakage of which provides more PrP^Sc templates for the conversion process [4]. Compared to wild-type, mutant forms of prion and prion-like proteins contain more N-terminal amino acid repetitions and are less glycosylated, which makes them more susceptible to misfolding and oligomerization. Lack of glycosylation also affects the transmembrane transport and metals interaction [93]. All prions and prion-like proteins contain metal in their structure and their conformation is dependent on metals binding [116]. N-terminal highly conserved octapeptide repeat sequences (PHGGGWGQ) in the protein play a crucial role in the promoting of the structure and further arrangement of PrP^C; these sequences have a high affinity for various divalent cations and the binding sites appear to play a role in the pathogenesis of prion diseases (Fig. 3).

Copper has emerged as a strongly interacting divalent metal ion to PrP^C. Prions cooperatively bind Cu ions [117, 118], resulting in a stabilized structure [119] and an acquired Cu-dependent superoxide dismutase (SOD)-like activity [120, 121]. On the other hand, Younan et al. have established that a β-sheet-like transition is observed when Cu ions are bound to the amyloidogenic fragment of PrP and is due only to local Cu(II) coordination to the individual binding sites centered at His95 and His110. In conclusion, Cu(II) ions destabilize the native fold of PrP^C and may accomplish the transition to a misfolded state [122]. PrP^C can bind from one to four Cu(II) ions in vivo while retaining its soluble form. Six binding domains have been established in its N-terminal domain. One Cu(II) ion is bound to each of four octapeptide sequences. Two additional Cu(II) binding sites are located between the OR region and the C-terminal domain [123]. Other metals, especially Zn(II), Fe(II) and Mn(II) also bind to PrP, although with lower affinity. Zinc ions bind preferentially histidine ligands; there are four metal binding sites within the octapeptide region. His ligands promote PrP-PrP interactions and lead to protein dimerization and oligomerization [124]. Another role of other heavy metals has been investigated. Kanthasamy et al. investigated the effect of Mn(II) and Cd(II) on the PrP degradation and aggregation in mouse neuronal cells expressing PrP. Cd(II) was more neurotoxic after 24 h of exposure, though it is difficult to rule out the possibility that the neurotoxic effects of Cd were linked to the presence of MT-3 in cells. In addition, Cd(II) profoundly inhibited proteasomal activity, which resulted in greatly increased formation of high molecular weight ubiquitinated PrPs and dramatically increased formation of PrP oligomers but not proteinase-K resistant PrP [116]. Potentiometric and nuclear magnetic resonance studies revealed that Cd(II) is coordinated by the His residue in PrP [125]. This fact indicates a possible role of metal ions in neurotoxicity due to the coordinatory role of His residue in PrP^C [116]. Similar results have also been demonstrated for other prion-related diseases. It has been established that trace metal contamination initiates the apparent auto-aggregation, amyloidosis, and oligomerization of Alzheimer’s Aβ peptides [126]. Mn(II) binding resulted in an altered conformation of PrP, displacement of Cu(II), and altered redox chemistry of the metal-protein complex [127] and infectious prion protein alters manganese transport and neurotoxicity in a cell culture model of prion disease [128].

There are a number of neuropathological similarities and genetic links between AD and prion diseases. The coexistence of AD pathology in CJD has been reported [129] and PrP^C has been shown to co-localize with Aβ in plaques. PrP^C-Aβ plaques have been shown to be present in most CJD patients with associated AD-type pathology, and it has been proposed that PrP^C may promote Aβ plaque formation. A genetic correlation between PrP^C and AD has also been reported. Systematic meta-analysis of AD genetic association studies revealed that the gene encoding PrP^C (PRNP) is a potential AD susceptibility gene [130] and the Met/Val 129 polymorphism in PRNP has been reported to be a risk factor for early-onset AD [131 , 133].

Role of MT in prion aggregation and prion disease

The possible connection between prion disease and MTs has been shown in the work of Kawashima et al., who investigated brain tissue of prion diseased patients with and without prion protein mutation and polymorphism [17]. In CJD patients with a relatively long disease course, the immunoreaction for both MT-1/2 and MT-3 in the astrocytes was significantly reduced, and this finding was not modified by the genotypes of the patients. MT-1/2 proteins were accumulated in the CJD brain with short disease duration, whereas MT-3 in the CJD brain with long disease duration was significantly reduced compared to normal brains. These findings indicated possible involvement of MT proteins in progression of prion diseases. Varela-Nallar et al. indicated upregulation of PrP expression by copper in neuronal cells by an MTF-1-(metal-regulatory transcription factor)-independent mechanism, and suggested a metal-specific modulation of PrP in neurons [134]. The mainly unstructured N-terminal part of the PrP^C structure can bind up to six Cu(II) ions [81]. Also, in this case oxidative stress, associated with the copper catalysed transformations of prion protein, plays an important role in the disease progression. In view of widely different Cu(II) binding motifs in Aβ, α-Syn, and prion proteins, a general protective role of Zn₇MT-3 against Cu(II) toxicity in the brain can be envisaged. Also, oxidative stress associated with the copper catalysed transformations of prion protein plays an important role in the disease progression. It is not only copper that is studied in connection with PrP. Rachidi et al. studied regulation of PrP^C expression by Zn(II) ions [135]. They studied the relationship between PrP^C and zinc ions intracellular homeostasis using a cell line expressing a doxycycline-inducible PrP^C gene. PrP^C-expressing cells were more resistant to zinc-induced toxicity, suggesting there is an adaptative mechanism induced by PrP^C. These authors also observed significant re-localization of intracellular exchangeable zinc in vesicles after PrP^C expression. This fact is supported by the work of Watt et al., who observed enhancement of zinc uptake to neuronal cells by prion protein [136]. Also Pushie et al. determined that PrP^C regulates brain metal homeostasis and metal distribution. Brain sections from wild-type, prion gene knockout, and PrP^C overexpressing mice revealed striking variation in the levels of iron, copper, and even zinc in specific brain regions as a function of PrP^C expression [137]. It has been established that prion protein-mediated zinc uptake is ablated in cells expressing familial associated mutants of the protein and in prion-infected cells. These data suggest that alterations in the cellular prion protein-mediated zinc uptake may contribute to neurodegeneration in prion- and other neurodegenerative diseases [138]. Kwahara et al. found that co-presence of Zn(II) or Cu(II) during aging inhibited β-sheet formation by PrP(106-126) fragment and attenuated its neurotoxicity on primary cultured rat hippocampal neurons [139]. In addition, PrP^C expression induces MT expression, a zinc-upregulated zinc-binding protein. These facts indicate that in prion diseases, the conversion of PrP^C to PrP^Sc deregulates MT-mediated zinc homeostasis. On the other hand, the deregulation of zinc homeostasis may induce changes in PrP^C folding and may be involved in the prion diseases, especially in the light of the fact that zinc ions drive a tertiary fold in prion protein [140]. PrP^C folding is physiologically regulated by reversible binding of Cu(II) ions. On the other hand, presence of Fe(II) and Mn(II) leads to the conformational changes to a protease-resistant β-sheet rich structure and finally amorphous aggregates. In the case of Zn(II), no intermediate has been detected before the formation of the amorphous aggregates. MT-3 with affinity to copper and zinc ions regulates catalytic redox properties of PrP by an unusual redox-dependent metal-swap reaction. Copper-catalysed transformations of PrP that lead to the production of ROS, PrP oxidation, and cleavage and aggregation has been established in transmissible spongiform encephalopathies [141]. ROS-mediated β-cleavage of PrP^C at the cell surface, was inhibited following hydroxyl radical quenching and has a prerequisite for the octarepeat region [142]. These results suggest that β-cleavage of PrP^C is an initial consequence following exposure to ROS in the extracellular environment contributing to a pathway involved in antioxidant protection of neuronal cells [143]. The protective role of MT-3 has been demonstrated in the case of Aβ, where prevention against its neuronal toxicity is based on the preventing copper-mediated Aβ aggregation, abolishing the production ROS and the related cellular toxicity (Fig. 4) [144, 145].

A different role of human MT-2 and MT-3 in Aβ binding by transthyretin (TTR) was studied. Within the CNS, TTR is primarily synthesized and secreted into the cerebrospinal fluid by the epithelial cells of choroid plexus in the brain. TTR expression is induced in response to the overproduction of Aβ peptides and overexpressed TTR forms stable complexes with Aβ, a key protein on the pathophysiology of AD, sequestering it and preventing its aggregation and/or fibril formation [146]. TTR cleaves full-length Aβ, generating smaller peptides with lower amyloidogenic properties, and it is also able to degrade aggregated Aβ [147, 148]. TTR, an Aβ scavenger protein interacting with MT-2 has been found to interact also with MT-3. While MT-2 diminished TTR-Aβ binding, MT-3 had the opposite effect [149]. Thus, a less efficient removal of Aβ would be expected when MT-3 expression is decreased and MT-2 levels are increased, and this appears to be the case in AD.

Metallothionein-1 and 2 and prion-related diseases

Changes in MTs (MT-1/2) in connection with neurodegenerative diseases such as AD, PD, ALS, and spinocerebellar degeneration have been published and reviewed several times [80 , 151]. Overexpression of MT-1 H and G isoform has been found by microarray analysis in PD brains compared to normal aging brains [152] and peptide derived from MT-3 has been identified as candidate cerebrospinal fluid biomarker of AD [153]. Regarding prion diseases, a number of publications demonstrate an upregulation of MT-1/2 in the brains of TSE affected cattle, humans, and experimentally inoculated rodents. Since the prion protein also binds copper, and oxidative stress is one of the events presumably triggered by PrP^Sc deposition, it seems plausible that MTs have a relevant role in the outcome of these neurodegenerative processes. To gain knowledge of the role of MTs in TSE pathogenic, and particularly of that of MT-1/2, a transgenic MT-1/2 knockout mouse model (MT-1/2 KO) was intracerebrally inoculated with the mouse-adapted strain of scrapie. The incubation period showed neither significant differences between MT-1/2 KO and wild-type mice nor the development of neurological signs. Upon neuropathological characterization of the brains, moderate differences were observed in astroglial and microglial response, spongiosis score and PrP^Sc deposition, particularly in brain regions to which the studied strain showed a stronger tropism (i.e., hippocampus). Results showed that the brain defense mechanisms against PrP^Sc deposition involve, aside from MT-1/2, other molecules, such as heat shock proteins (Hsp), namely Hsp25, which are capable of compensating for the lack of MT-1/2 [154]. Expression of small Hsps proteins (HspB1, HspB5, HspB6, HspB8, Hsp27, heme oxygenase 1 (Hsp32)) and metallothioneins MT-1, MT-2, and MT-3 connected with AD, PD, and HD in the CNS has been found in several papers [155–157 , 158]. The authors show the stress induction of these proteins during chronic neurodegeneration due to protein misfolding in age-related diseases. However, chronic and acute damage of brain tissue must be carefully separated in this case. This evidence is discussed with respect to potential brain region and cell-type specific responses and vulnerability to stress. Tortosa et al. evaluated immunohistochemically the stress-related heat shock protein 25 (Hsp25) and MT-1/2 in the brains of a murine model of bovine spongiform encephalopathy (BSE) and revealed involvement of MT-1/2 and Hsp25 in BSE pathogenesis [159]. These proteins are expressed in low levels under physiological conditions and may act as molecular chaperones and are involved also in cellular processes occurring after exposure to oxidative stress. A protective effect of Hsp proteins was observed in PD and AD, as well as in the diseases where expanded polyglutamine tract occurs [160, 161]. Although the basal expression of all these proteins is well documented in neurons and astrocytes, their physiological expression in vivo in oligodendroglia and microglia is less elucidated. A clear functional role in neuroprotection against ischemia has been shown only for HspB1 using genetic knockout approaches. HspB1-inducing drugs and viral delivery in rodent models is paving the way to potential therapeutic treatments during stroke in humans. Such mechanistic studies have not been performed in models of protein misfolding diseases (proteinopathies such as AD, PD, polyglutamine, and prion diseases), but a key similarity is the increased expression of small stress proteins in glial cells, especially associated with an astrocytic stress response. This likely affords cytoprotection through a sustained defense against oxidative stress and modulation of neuroinflammation. The apparent redistribution of many small stress proteins to extracellular protein deposits, and in some cases to intracellular protein inclusion bodies points to a further modulatory function during protein aggregation and/or degradation that could be linked to the neuroprotective activities of these proteins in both neurons and glial cells. Given the chronic nature of proteinopathies and the complex interdependence between protein aggregation, imbalance of the cellular redox-homeostasis and inflammatory and other signaling events, it will be difficult to detail the neuroprotective role(s) of each stress protein in vivo. Nevertheless the induction and manipulation of multiple stress proteins in different cell types and cell compartments has to be considered for the development of effective neuroprotective strategies in the CNS [158].

To define genes associated with or responsible for the neurodegenerative changes observed in TSEs, gene expression in scrapie-infected mouse brain using “mRNA differential display” has been analysed. The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. The authors found increased expression of MT-2 that has been previously been reported to occur in experimental scrapie, where increased expression of MT-2 mRNA was found the terminal stage of the disease [162].

An increase in MT-1/2 mRNA concentrations has been reported in the CNS of scrapie-infected rodents. In this study Hanlon et al. compared cattle with BSE, cattle affected by neurological disease other than BSE, and clinically healthy cattle in respect of MT-1/2 immunoreactivity in brainstem medullary tissue. Marked astrocytic MT-1/2 immunolabeling was seen in all affected animals, in contrast to clinically healthy cases. In BSE, MT-1/2 immunoreactive astrocytes were confined specifically to areas of vacuolation or abnormal prion protein deposition, or both. MT-1/2 immunolabeling was also seen in a small number of animals with a neurological disease other than BSE. These findings complement previous studies by demonstrating increased levels of MT-1/2 in TSE-infected brain tissue, indicating that MT-1/2 may play some as yet unidentified role in the response to TSE infection [163].

Although the immunoreactivities for MTs in CJD brains varied from case to case, they were generally dependent upon the disease duration. In CJD patients with a relatively long disease course, the immunoreaction for both MT-1/2 and MT-3 in the astrocytes was reduced, and this finding was not modified by the patients’ genotypes. On the other hand, in patients with Gerstmann-Straussler-Scheinker syndrome, MT-1/2 immunoreactivity in the astrocytes was exclusively reduced, while the immunoreaction for MT-3 was relatively well preserved. Especially the astrocytes in the vicinities of the kuru plaques exhibited a weak or no immunoreaction even for MTs. Quantitative western blot analysis also revealed MT-1/2 protein accumulation in CJD brain with short disease duration, whereas MT-3 in CJD brains with long disease duration was reduced in comparison to normal brains.

CONCLUSION

Copper and zinc ions are essential to normal cell functions. However, they are intimately involved in amyloid-forming diseases. They have an effect on conformation of certain proteins and could participate in their aggregation. They promote folding into aggregation prone conformers or stabilize non-amyloidogenic conformations that which have increased toxicity. MT-3 is a unique protein that was first discovered in normal human brains. Expression of MT-3 has been found to be downregulated or altered in AD, PD, HD, ALS, and CJD. MT-3 is involved in homeostasis of essential transition metals such as zinc and copper, but, moreover, it can exert neurotoxic effects. Indeed, redox-active copper aberrantly bound to amyloidogenic proteins can react with molecular oxygen, resulting in ROS generation.

Many studies have been published in which MT-1 and/or MT-2 have been proposed to play a role. These isoforms are of great importance for maintaining homeostasis of metal ions in many tissues, except for the brain. This tissue can be considered as a MT-3-specific and the role of the other two isoforms in brain metal homeostasis seems to be less important.

Footnotes

ACKNOWLEDGMENTS

Financial support from CEITEC CZ.1.05/1.1.00/02.0068 is highly acknowledged. CMG is a recipient of a Consolidation level Investigador FCT, from the Fundação para a Ciência e a Tecnologia, Portugal (IF/01046/2014). The authors would like to acknowledge the contribution of the COST Action TD1304 The Network for the Biology of Zinc (Zinc-Net).

Authors’ disclosures available online ().

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