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Journal of Alzheimer’s Disease 41 (2014) 29–42 DOI 10.3233/JAD-130290 IOS Press

Review

Metallothioneins and the Central Nervous System: From a Deregulation in Neurodegenerative Diseases to the Development of New Therapeutic Approaches Silvia Bolognina,∗ , Bruno Cozzib,∗ , Pamela Zambenedettic and Paolo Zattad a Deparment

of Neurological, Neuropsychological, Morphological and Motor Sciences, Section of Physiology, University of Verona, Verona, Italy b Department of Comparative Biomedicine and Food Science, University of Padova, Padova, Italy c Phatology Division, General Hospital, Dolo-Venezia, Italy d CNR-Institute for Biomedical Technologies, “Metalloproteins” Unit of Padova, Department of Biology, University of Padova, Padova, Italy

Accepted 15 January 2014

Abstract. Metallothioneins (MT) are a family of proteins actively involved in metal detoxification and storage as well as in prevention of free-radical damage. Changes in the levels of MT have been described in a number of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, prion protein disease, Binswanger type of subcortical vascular dementia, and amyotrophic lateral sclerosis. This suggests that MT functions might be more complex and vast than what was initially thought. In this review, we summarize the current knowledge on the potential involvement of MT in the mentioned neurodegenerative diseases while also discussing the emerging evidence proposing MT modulation as a feasible therapeutic approach. Enhancing repair mechanisms after neurological damage and/or protection against oxidative stress through a proper modulation of this family of protein might indeed represent an important avenue to cope neurodegeneration. Keywords: Brain development, metal ions, metallothioneins, neurodegenerative diseases

∗ Correspondence

to: Silvia Bolognin, Departement of Neurological, Neuropsychological, Morphological and Motor Sciences, Section of Physiology, University of Verona, Strada le Grazie 8, 37134 Verona, Italy. Tel.: +39045 8027158; Fax: +39045 8027279; E-mail: [email protected] and Bruno Cozzi, Department of Comparative Biomedicine and Food Science, University of Padova, viale dell’Universit´a 16, 35020 Legnaro-Padova, Italy. Tel.: +39049 8272626; Fax: +39049 8272796; E-mail: bruno.cozzi@ unipd.it.

Metallothioneins (MT) are a family of lowmolecular-weight and cysteine-rich proteins present in all eukaryotes. They were discovered in 1957 in the equine kidney by Margoshe and Vallee and later named MT due to the high metal content and strong affinity for Ib and IIb group transition metals. MT family is comprised of four main members from MT1 to MT4, with multiple isoform sub-classes. MT1 and MT2 are widely expressed in almost all tissues, MT3 mainly in

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the central nervous system (CNS) while MT4 is present in squamous epithelial tissue [1]. The different distribution and regulation pathways of MT suggest that they might exert various biological functions. Nevertheless, despite the extensive studies, their physiological role is still far from being fully understood. Structurally, all MT isoforms are characterized by a conserved array of 20 cysteine residues. When not bound to metals (apo-MT), MT show a quite disordered structure, which becomes ordered upon metal binding [2]. One of the most important characteristics of this family is the presence of two metal-thiolate clusters in two independent domains (␣ and ␤), connected by a flexible polypeptide chain. MT3 possesses 70% sequence identity to the MT1-2 but, as peculiar features, it shows a conserved Cys-Pro-Cys-Pro motif between positions 6 and 9, a Thr at position 5, and an acidic hexapeptide in the C-terminal region. These distinctive chemical features are likely responsible for the different biological functions of MT3 compared to MT1-2 [3]. MT3 is indeed more flexible, especially in the N-terminal ␤-domain [3, 4]. MT1 AND 2 MT1-2 are primarily expressed in astrocytes and, at a lower extent, in neurons [5]. These two isoforms share high sequence homology and similar expression profile. A number of factors has been shown to stimulate their up-regulation including metals, cytokines, hormones, and oxidative molecules [6–8]. Notably, the involvement of MT1-2 in metal metabolism has been extensively investigated and a general consensus exists on their major role in metal detoxification. This was demonstrated using, for instance, MT1-2 knock-out mice, which showed an impairment to recover from heavy metal toxicity compared to controls [9]. Considering that metal metabolism is dysregulated in several neurodegenerative diseases (ND) [10] and in aging [11], it is not surprising that MT expression also varies extensively [12]. Furthermore, MT1-2 seem to also act as radical scavengers. The most convincing results for the antioxidant function of MT have been obtained again in MT knockout mice, which showed greater susceptibility compared to controls toward oxidative stress caused by various factors [13]. A neuroprotective role has been attributed to MT since early studies observed MT expression after bacterial infection. Later investigations have broadened the knowledge on MT1-2 induction after different kinds of brain tissue damage,

including traumatic brain injury [14] and dopaminergic neurotoxicity [15]. MT3 Data on MT3 are functionally less clear. MT3 was initially discovered while studying the mechanisms underlying Alzheimer’s disease (AD) and found to be down-regulated in AD brains compared to agematched controls, a finding later not reproduced by other laboratories [16–18]. Beside AD, MT3 has been associated with other ND but always with contrasting results according to the models and techniques used [19]. Furthermore, it was initially presumed that MT3 was specific only of the CNS but further studies also showed the expression of MT3 outside the CNS as in the dorsolateral lobe of the prostate, in testis, and in tongue [20, 21]. Despite these conflicting findings, the current general consensus is that MT3 is predominantly expressed within the CNS [17] and is synthesized in astrocytes. It is also present in a specific subset of hippocampal and cortical neurons (for review, see [22]). Interestingly, MT3 seems to play a different role compared to MT1-2, as the transcriptional regulation of MT3 differs from that of MT1-2 [23, 24]. The fact that MT3 synthesis, diversely from MT1-2, is not inducible by metal ions indicates that MT3 does not primarily contribute to sequester toxic or overloaded metals. Most likely, MT3 is primarily involved in the promotion of neuronal survival and repair [25, 26] and in the activation of angiogenic factors that might contribute to the recovery of brain tissue after an insult [27]. Interestingly, the analysis of the inflammatory response and reaction to oxidative stress stimuli in MT3-null mice did not show any relevant difference compared to wild-type mice. In this case, the hypothesis of compensatory mechanisms should be ruled out since the expression of the other brain MT isoforms (MT1-2) was normal [28]. Nevertheless, MT3-null mice were characterized by an age-related increase of reactive astrocytes and were more susceptible to neuron injury induced by kainic acid, a glutamate analogue [29]. Recently, senescence-accelerated mice (SAMP8) were administered intraperitoneally for four weeks with MT3. Under these experimental conditions, MT3 was able to attenuate apoptotic neuronal death in the hippocampus, suggesting that the protein might lessen the development of neurodegeneration [30]. Nevertheless, to further compound the issue MT3 was also found to cause neuronal death when administered at high does on cell cultures [31]. This suggests that the effect of MT3 might be dose-dependent.

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Table 1 MT in neurodegenerative diseases MT Isoform MT 1/2

MT 3

AD ↑in AD brain [62, 63] ALS PrPD

PD BD

↑MT1 mRNA in spinal cord G93A SOD1 Tg mice [52] ↓ in spinal cord astrocytes of ALS patients [50] ↑MT2 mRNA in scrapie-infected hamster brain [106] ↑MT1-2 in a mouse model of BSA [107] ↑MT1-2 in CJD brain [109] ↑in substantia nigra and frontal cortex of PD patients [88] ↑in BD patients [79]

↓in AD brain [16–18] ←→unaltered in AD [5, 73] ↓in Tg2576 mice [75] ↑in spinal cord of SOD mutant mice [51] ↓in CJD brain [109] ↓in PD brain [101] n.r

Alteration of MT distribution in Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), bovine spongiform encephalopathy (BSA), Creutzfeldt–Jakob disease (CJD), prion protein disease (PrPD), Parkinson’s disease (PD), and Binswanger’s disease (BD). n.r., not reported.

The purpose of this review is to present findings on MT (Table 1) relevant to ND mechanisms. Additionally, the rational for their potential use in therapy to counteract neurodegeneration is discussed. Brain MT distribution during development is also described, as it appears that the onset of the MT system is timed to the period immediately preceding the accumulation of trace elements in the developing brain. MT IN BRAIN DEVELOPMENT The tight correlation between MT and essential metals is highlighted by their regulation during development. The balanced acquisition of trace elements is essential for harmonic fetal growth and maternal health. To this effect, the ability of MT to regulate the intracellular concentration of metal ions plays an important role in the developing nervous system. Selected metal ions pass through the placental barrier, which simultaneously inhibits the transfer of excess or unfavorable elements. Thus, the placental barrier is the homeostatic condition that regulates the mother to fetus passage of metal ions, such as Cu and Zn. The ion Zn is particularly important during the late second trimester and especially during the third trimester of pregnancy, starting from gestational week (GW) 27 and 28, up to GW 38–40. During GWs 27–28, the whole fetus contains approximately 18 mg of Zn, a concentration that rises to 58 mg at GW 38–40. Zn deficiency in premature children might be associated with insufficient placental supply of the ion through the placenta [32]. The question is therefore whether the developing brain needs a MT system, and, if the answer is positive, when is the working system required. Based on the data mentioned above, we might expect to find MT-containing cells by the late second trimester, in

relation to the rise of upcoming trace elements that will take place in the last part of the pregnancy. Former studies performed in sheep [33] suggested that the regional expression of MT1-2 in the brain appears around the middle of the pregnancy and progresses during fetal growth. Experiments performed in our laboratory [34] confirmed the temporal sequence and noted that in the developing human brain, the presence of MT1-2-immunoreactive cells (MT1-2-ir) in the neuroepithelium of the ventricular zone (VZ) was not evident before GW 21 (Fig. 1a). Although few sparse MT1-2-ir cells were occasionally detected in the frontal and parietal cortical plates of less developed individuals, a constant network of MT1-2-ir was not recorded in the neuroepithelial VZ. Here, we emphasize that the neuroepithelium of the VZ is the place where neural stem cells descendant of the neural plate divide and proliferate both in sheep and humans. Ependymal and neural cells (including tanycytes, neurons, and glia) originate from the neuroepithelium of the VZ [35, 36]. Rare and sparse, MT1-2 bearing glial cells appear at the end of the second trimester, more frequent in subsequent stages of brain maturation, and finally become a constant presence in the late stages of gestation and early postnatal period. During GWs 21-24, MT1-2-ir cells appear at first in the neuroepithelium of the VZ, and then move to occupy the subventricular zone (Fig. 1b). In fact, stem cells generated here are later found in secondary germinal matrices, including the intermediate plate/subplate (IZ/SP). In the telencephalon of the second trimester, the VZ is the source of neurons and astrocytes directed, through radial migration, to the IZ/SP and the cortical plates. In addition, the presence of MT1-2-ir glial cells is persistent in the frontal and parietal cortex of fetuses at term

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Fig. 1. MT in human fetuses (GW, gestation weeks) and infants (PW, postnatal weeks). SVZ, sub-ventricular zone; VZ, ventricular zone. a) GW 20, topography of germinal areas where no MT-ir elements are evident; b) GW 24, MT elements appear in the VZ and move to the SVZ; c) PW 4, sub-pial layer of the cortex, several neural astrocytes show MT immunoreactivity; d) PW 12, astrocytes in the subcortical white matter. Scale bars: 500 ␮m (a); 200 ␮m (b–d).

and in newborns (Fig. 1c, d). Therefore, the progressive migration of MT1-2-ir follows well the defined migration routes of glial maturation in the human neocortical development [37]. Our data in humans indicated that the MT-ir system starts to organize only at the end of the second trimester, perhaps in relation to cortical growth and increased myelinization [34]. The onset of the MT system appear thus to be timed to the period immediately preceding the accumulation of trace elements in the developing brain. However, we note here that detailed studies performed in mice [38] showed an inverse correlation between MT expression and cadmium accumulation. Discrepancies could be due to specie differences and/or organization of the experimental plan and sampling. Any association of MT expression in the development of the human brain with potential later insurgence of

neurodegenerative pathologies is at the moment uncertain and awaits further studies. MT AND NEURODEGENERATIVE DISEASES MT and amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS) is characterized by the selective loss of motor neurons, resulting in muscular atrophy. Patients die within five years from the onset of the symptoms, mainly because of respiratory failure [39]. Most cases are sporadic, but some patients show a familial pattern of inheritance. The etiology of the disease is unknown but some genetic risk factors have been suggested. Nevertheless, a simple association cannot be made and most authors proposed a complex genetic-environmental interaction

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as a cause of the motor neuron degeneration [40]. Early epidemiological studies hypothesized that sporadic ALS could be related to exposure to several heavy metals. Although later studies failed to confirm this connection [41], considering the importance of MT in reducing metal cellular uptake, several laboratories investigated whether MT genes were inappropriately silenced in ALS. No evidence of genetic changes was reported neither for MT3 nor for MT2A, apparently excluding that abnormal gene silencing might be responsible for a lack in metal detoxification [42, 43]. A clear genetic-based association with the pathology has been made only for mutations of the gene encoding for Cu/Zn-superoxide dismutase 1 (SOD1), a cytosolic enzyme with a Zn and Cu binding site that catalyzes the conversion of superoxide radicals to hydrogen peroxide. A potential mechanism of toxicity might be related to the reduced ability of the mutant SOD1 to interact properly with the Cu chaperone resulting in pathogenic protein misfolding [44]. Mutations of SOD1 gene resulted in decreased affinity of the protein for Zn [45] and increased affinity for Cu [45, 46]. SOD1-decreased affinity for Zn caused an increment in nitrotyrosine formation [47] while Cu increased affinity enhanced Cu-mediated oxidative stress [48], which both led to neuronal death. On the basis of these studies, MT have been linked to the pathology due to the ability to maintain transition metal homeostasis. Furthermore, the involvement of MT in ALS pathogenesis was suggested by data showing a decrease of MT1-2 immunoreactivity in astrocytes of ALS patient lumbar spinal cord compared to age-matched controls. MT3 was also reduced in both cervical and lumbar spinal cords of ALS patients, although the difference was not statistically significant [49]. Tokuda et al. [50] showed an increase of MT1-2 in the spinal cord of young SOD mutant mice, when the motor paralysis was not evident yet. On the contrary, MT3 became altered at adult age when the motor dysfunction was clearly manifested. Similarly, in a different mouse model for ALS, MT1 mRNA expression was significantly upregulated in the spinal cord even before the animals exhibited signs of motor paralysis, while the level of MT3 tended to be elevated only in the later stage [51]. According to this observation, we might hypothesize that MT1 isoform is more directly linked to motor neuron death in ALS than MT3. ALS has also been associated with free radical toxicity as a consequence of redox metal dysmetabolism [52, 53]. The contribution of these and other factors to the development of ALS clearly awaits further investigations.

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MT and Alzheimer’s disease AD is the most common form of senile dementia in the elderly. The AD brain is characterized by the extracellular deposition of the amyloid-␤ peptide (A␤) in senile plaques, and by the intraneuronal aggregates of paired helical filaments of the hyperphosphorylated tau protein [54, 55]. Many studies have implicated biometals in the development and/or progression of AD [19, 56–59]. Plasma Cu/MT ratio was found indicative of disease progression in AD patients [60]. This association hints at a possible link between some pathological features of the disease and the MT expression. Several studies demonstrated that MT1-2 are upregulated in AD [61–63] but contrasting findings are reported for MT3 (see below). Among many hypotheses trying to explain the pathological alterations underlying AD, it has been suggested that activated microglia may release proinflammatory cytokines and oxidative species that may contribute to neuronal damage [17, 64, 65]. Indeed, immunohistochemical evaluation of AD brain revealed high expression of MT1-2 in astrocytes and in microcapillaries compared to control patients [61]. MT1-2 expression is also enhanced in reactive astrocytes in a transgenic mouse model of AD [66]. Astrocyte-derived MT seem to exert neuroprotection by modulating multiple events associated with A␤ pathology such as inflammation, oxidative stress, and apoptosis [66]. In particular, extracellular MT1 attenuated inflammatory activation of microglia following A␤ stimulation [66, 67]. This suggests that MT1 might improve the pathological intracellular environment by scavenging reactive oxygen species and activating anti-apoptotic pathways. MT1 and MT2 deficiency in Tg2576 reduced A␤ plaque burden in the cortex and hippocampus [68], suggesting that both MT1 and 2 deficiency might delay some of the human A␤PPinduced pathological alteration. Diversely from MT1-2, the role of MT3 in AD is more uncertain. Many studies indicate that MT3 mRNA is down-regulated in AD brains [16–18, 69] and this might, therefore, contribute to the increase of aberrant neuronal sprouting associated with the disease. An alternative explanation is that the decrease of MT3 induced by A␤ has a neuroprotective function and counteracts AD progression [70]. In addition, Martin et al. [71] showed that in Tg2576 transgenic mouse model of AD, MT3 was reduced compared to wild-type mice. The pathological changes that develop in this mode might be responsible for the degrada-

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tion of MT3. However, other studies did not confirm the down-regulation [72, 73], and others showed that MT3 remained essentially unaltered in AD compared to control patients [5, 74]. MT and Binswanger type of subcortical vascular dementia Progressive subcortical vascular encephalopathy of Binswanger’s type, so-called Binswanger’s disease (BD), is a common form of vascular dementia. It is characterized by lacunar infarcts and diffused white matter lesions [75, 76]. The pathologic hallmarks of BD have been ascribed to cerebral hypoperfusion associated with cerebrovascular risk factors, hypertension in particular. Although the pathogenesis of these subcortical white matter lesions remains unclear, there is a general agreement on considering cortical and/or white matter astrocytosis and fibrillar gliosis as peculiar pathologic features of BD [75]. It has been shown that oligodendrocytes could be sensitive and vulnerable to experimental ischemia or hypoperfusion in the cerebral white matter [77]. Immunohistochemical analysis of BD brains revealed that MT1-2 expression was higher when compared to control patients [78]. This increase was evident both in the white as well as in the grey matter [78]. MT1-2 high positivity might be correlated with the high statistically significant oxidation of ␣-tocopherol-quinone as observed in BD [79]. This provides evidence of the occurrence of oxidative stress phenomena in this disorder. Alteration of the blood-brain barrier (BBB) in BD brains may contribute to the diffuse degeneration of the white matter [80], with a consequent enhanced expression of MT. Moreover, Masumura et al. [81] observed that the increase in TUNEL-positive oligodendrocytes and the reduction of astrocytes might be both possible causes of white matter rarefaction. Numerous reactive astrocytes in pathological samples were positive also for endothelin-1, a vasoconstrictor peptide detected in the plasma of BD patients [82]. Like many other cytokines and interleukins, endothelin-1 was able to induce the expression of MT [83]. However, the potential protecting role of MT toward reactive astrocytosis requires further experimental support. MT and Parkinson’s disease The progressive degeneration of nigrostriatal dopaminergic neurons is the main feature characteriz-

ing Parkinson’s disease (PD). As for the others ND, the cause of the disease is still unknown. Among the potential contributing factors, Cu-mediated oxidative stress and altered Cu homeostasis have been both hypothesized [84]. Consequently, the involvement of MT with the disease has been strongly suggested. An early study reported that MT1-2 expression was unchanged in the astrocytes of substantia nigra of PD patients [85]. A more recent study confirmed this finding, but it also showed increased MT transcript expression in cerebral cortex and putamen [86]. On turn, Micheal et al. found increased expression of several MT1 isoforms (e.g., MT1E, MT1F) and MT2A in PD substantia nigra and frontal cortex [87] pinpointing at inter-individual variation of MT1-2 isoform expression. The expression of megalin, the neuronal MT receptor, was also significantly increased [87]. The number of cases in the first study was very small (n = 5) and an individual variation was observed. The more recent and detailed characterization of the second study might partly account for the discrepancy. Data on transgenic mice demonstrated that brain regional induction of MT might be helpful in the prevention of PD in the aged brain [88]. According to one hypothesis, MT might provide neuroprotection from nitrative stress by increasing the rate of coenzyme Q10 (CoQ10 ) synthesis [89]. Indeed, MT gene overexpression in the brain of MT transgenic mice inhibited nitration of ␣-synuclein and preserved mitochondrial CoQ10 levels [88–91]. Another important aspect is that the neuronal death occurring in PD can be correlated to inflammatory reactions characterized by the activation of microglia cells [92]. This is confirmed by the elevated levels of cytokines found in the cerebrospinal fluid of PD patients [93]. Interestingly, MT1-2 were not upregulated in astrocytes of the substantia nigra of PD patients [85]. On the contrary, microglial cells appeared to be consistently activated [94]. Other authors proposed that MT may scavenge free radicals by releasing Zn to the neuronal membrane [95], thus supporting an antioxidant role for both Zn and MT [96, 97]. A shift in the glutathione redox balance, as a consequence of oxidative stress events, can accelerate Zn release from MT. Dyshomeostasis of Zn has been indeed observed in PD [98, 99]. An oral Zn test was used to assess Zn status in 25 controls and 100 PD patients and it showed a significantly Zn decreased in the PD group compared to the controls [98]. Zn concentration was also measured by atomic absorption spectrophotometry in serum and cerebrospinal fluid of 37 PD patients and 37 age-matched controls [99].

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Zn level was unchanged in the serum but was significantly decreased in the cerebrospinal fluid of PD patients compared to controls, suggesting that it could be related to the risk of developing PD. These data underline that oxidative stress is part of the pathological events leading to PD, and the increase of MT1-2 in PD brain may be a useful tool to counteract brain damage. The role of MT3 in the pathology is less clear: a down-regulation of MT3 has been observed [100]. MT3 mRNA was down regulated in the striatum of a model of hemi-parkinsonian rat induced by injection with 6-hydroxydopamine. Intraperitoneal levodopa treatment almost restored the MT3 level. However, it has also been suggested that MT3 can be a source of toxic Zn in neurons, confirming that MT3 may not always have a protective role [101]. The authors observed that with kainate-induced seizures or intracerebral injections of sodium nitroprusside, MT3 null mice and Zn transporter 3 (Znt3)/MT3 double-null mice exhibited less neuronal death in the CA1 of the hippocampus compared to control and Znt3- null mice, respectively. MT and prion protein disease Prion protein disease (PrPD) belongs to a group of fatal ND whose clinical symptoms include tremor, behavioral impairment, and ataxia. The common feature shared by these disorders is the accumulation in the CNS of the abnormally folded isoform (PrPSc ) of the physiological cellular prion protein (PrPC ). PrPC is an ubiquitous glycoprotein which consists mainly of an ␣-helix structure, while the converted PrPSc is predominantly formed by ␤-sheet-rich insoluble aggregates, which are resistant to degradation [102]. Despite the wide number of studies published, the mechanism through which PrPC is involved in PrPD development, and more generally its physiological role, still remain unclear. The range of functions attributed to PrP varies from Fe uptake and transport [103] to the involvement in Cu homeostasis [104]. An increased level of MT2 mRNA has been reported in scrapie-infected hamster brain [105] as well as an immunohistochemical overexpression of MT1-2 in the glia associated with spongiosis in a mouse model of bovine spongiform encephalopathy [106]. Marked astrocytic MT1-2 immunolabeling was also seen in bovine spongiform encephalopathy-affected cattle compared to control animals [107]. Coming back to the human variants of the disease, Kawashima et al. [108] showed, by immunohistochemistry and western blot analysis, that in the brain of Creutzfeldt–Jakob dis-

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ease (CJD) cases with a disease course shorter that 15 months, MT1-2 increased compared to controls. Activation of microglia has been recognized as a feature related to PrPD [109, 110]. It is conceivable that accumulation of PrPSc might produce microglia activation due to the insoluble nature of the mutant isoform [109]. On the other hand, MT3 is apparently decreased in the CJD brain with long disease duration compared to control brains [108]. However, it is not clear whether the different expression of MT is involved in the abnormal deposition of PrP or vice versa if the mutant PrP influences the expression of MT in astrocytes and neurons. A link between PrPD and MT might lie in the ability of PrPC to selectively bind Cu [111], a necessary step for the normal protein folding [112]. The potential involvement of PrPC in Cu metabolism was first proposed by Pattison and Jebbett [113] with the observation that the histopathology of mouse scrapie resembled that induced by the Cu chelator cuprizone. When chronically administered to mice, cuprizone induced myelin loss. Demyelination occurred in deeper layers of the cortex and showed a pattern of rounded spots with variable diameter [114]. It has also been suggested that PrPC might play an important role in reducing oxidative stress and that its enzymatic function depends on Cu incorporation [115]. Indeed, Cu was found to induce expression of cellular PrP in primary cell cultures, upregulating the expression of this protein both at the cell surface as well as within intracellular compartments [116]. Cu promotes the aggregation of human PrP [117]. These findings stimulated the investigations on metal ion alteration in PrD human brains. Changes in the levels of Cu but also manganese bound to PrPSc were found in sporadic CJD compared to PrPC in normal subjects [118]. Besides, antioxidant activity of purified PrPSc was dramatically reduced up to 85% in the sporadic CJD, with a consequent increase in oxidative stress markers.

POTENTIAL THERAPEUTIC USE OF MT: A RATIONAL APPROACH The neuroprotective properties of MT have been highlighted by several authors [5, 119–121]. The precise molecular mechanism responsible for this neuroprotection is still partially obscure, although the activation of heterotrimeric G-protein pathways, with a consequent increase in phospholipase C, has been hypothesized [66]. The rationale underlying the potential use of MT in ND is multiple.

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First, the generation of reactive oxygen species and oxidative damage is a common feature of ND, indicating that defects in the oxidative stress handling machinery contribute to the pathogenesis of these disorders. It is worth mentioning that the entity of this contribution to the development of neurodegeneration has been a matter of intense debate. For instance, several authors pointed to oxidative stress as a primary etiological factor for AD [122] or for PD [123], while others suggested that this is a consequence of protective mechanisms. Aside from the causes of the different ND, protection against oxidative stress might help vulnerable neurons to counteract neurodegenerative mechanisms. Second, late-life ND are intimately linked to aging and MT showed strong anti-aging effects [124, 125]. Dietary compounds combined with genetically increased MT1 have been demonstrated to increase lifespan in mice [126]. Coherently, some polymorphisms of MT genes have been related to increased lifespan and lower inflammatory status in humans [127]. Clearly, the possibility to activate longevityassociated pathways to predispose individuals toward healthy aging [128], eventually counteracting neurodegeneration, is a very attractive option. Several studies have clearly reported that MT enhanced cell survival and tissue regeneration. Lanza et al. [129] found significantly increased MT3 mRNA level after nerve injury in rats. Remarkably, neurons treated with exogenous MT1-2 showed improved neuronal survival and axonal outgrowth in cortical, hippocampal, and dopaminergic cultures [130]. MT1-2 knockout mice showed impaired axonal regeneration after sciatic nerve crush [131] and MT2A treatment promoted neurite elongation and post-injury reactive neurite growth [130]. To the best of our knowledge, no pharmacological compound has been reported to specifically induce MT synthesis. Furthermore, MT showed scarce ability to cross the BBB as they form polymers in solution. Besides, Chung’s laboratory was unable to detect MT in the brains of MT1-2 knockout mice following MT-1/2 intraperitoneal injection, despite the fact they were found in kidney and urine as discussed in [132]. More recently, the result was confirmed after MT2A intramuscular or intraperitoneal single injection or one-daily injection for 3 days [133]. MT2A did not cross the intact BBB. The design of a synthetic peptide can be a solution to overcome this issue. Ambjørn and coworkers [134] demonstrated that EmtinB, a peptide modeled after the ␤-domain of MT, had on neuronal cultures similar effects to those of the native pro-

tein in terms of neuritogenic and survival promoting effects. The same group showed that in vivo treatment with EmtinB significantly attenuated seizures in C57BL/6J mice exposed to kainic acid doses and reduced kainic acid-induced neurodegeneration in the CA1 region of the hippocampus. Importantly, EmtinB was able to cross the BBB [135]. The binding of MT and EmtinB to megalin and to the low-density lipoprotein receptor-related protein 1 (LRP), activated the extracellular signal-regulated kinases (ERK), Akt, and cAMP response element-binding (CREB) [134] (Fig. 2). Since Akt and ERK are involved in the promotion of neuronal survival and differentiation [136], these signaling cascades might underlie the survivalpromoting effect of MT and EmtinB in vitro and, at least partially, the neuroprotective effect of EmtinB in vivo. Several reports suggest that MT can be released from cultured cells under various conditions [137–139] and internalized by target cells through lipid-raftdependent endocytosis [140]. Kim et al. [62] found that MT1 was released from astrocytes and internalized by neurons in vitro and suggested that astrocytic MT1 might act on neurons in a paracrine manner to exert neuroprotective effects against A␤ toxicity. We think one of the most interesting recent findings on MT is that they might be intermediary in the stress-induced immunomodulatory response to neuronal injury. MT1-2 were initially assigned purely intracellular functions. However, detection of MT1-2 in the extracellular environment in vivo and the secretion of MT1-2 by cultured cortical astrocytes upon exposure to Zn and interleukin-1 highlighted their role as important extracellular agents [121]. Intraperitoneally administered Zn-MT2 following cortical brain injury (cryolesion) resulted in a significant reduction of proinflammatory cytokine, neuronal apoptosis, and reactive gliosis [141]. Exogenous MT2A administration was shown to increase neurite elongation and to promote axonal growth in embryonic rat cortical neurons [130]. Chung and collaborators suggested that neuronal damage induced the expression and secretion of MT by astrocytes [142]. Moreover, cell surface receptors for MT1-2 have been identified and found expressed by neuronal cells [134]. Nevertheless, the molecules involved in this signaling are still unknown. It is not yet clear whether the extracellular accumulation of MT following brain injury is due to the release from dead cells or it implies an active release mechanism. Whether MT represent a target for exogenous therapeutic intervention, either via the promotion

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Fig. 2. A simplified model of MT signaling in the brain. Upon different kinds of stress factors MT can be released from astrocytes triggering neuronal MT receptor upregulation (megalin and LRP). According to some hypotheses, MT might trigger activation of multiple downstream proteins including Akt and CREB and thus, it may promote survival and neurogenesis [134]. MT can also bind metal ions reducing potentially toxic overload and/or counteracting oxidative stress mechanisms. ROS, reactive oxygen species.

of neuronal regeneration [121] or through the activation of antioxidant mechanisms, awaits further investigation. CONCLUSIONS Starting from the last century, human lifespan has markedly increased, thereby creating new challenges for biomedical sciences. ND in particular have attracted significant attention, due to the exponential rise of their incidence. The identification of effective therapeutic strategies for the prevention and treatment of these diseases it is hindered by the still poor understanding of the etiological factors associated with these pathologies. Indeed, the fact that technological innovations and more sophisticated studies have not translated into successful pharmacological options might suggest that we have to somehow reconsider the nature of neurodegeneration as a complex process in which overlapping mechanisms may underpin neuronal loss and clinical manifestation. In this perspective, it is likely that the development of cutting edge strategies will

rely on a multi-targeted approach. The potential use of MT in therapy would fit in this framework as it has become clear that the physiological functions of MT are relevant for multiple brain functions. Therefore, deregulation in the distribution of this family of protein may contribute to the age-associated cognitive decline and eventually cognitive impairment. On the other hand, MT enhancement may improve the resistance of vulnerable neurons, delaying the onset/progression of ND. MT have a role in regulating several pathways connected to neurodegeneration, such as metal homeostasis, reactive oxygen species production, and oxidative/nitrative stress response. However, many questions still remain unanswered. The hypothesis that MT are specifically involved in the etiology of the neurodegenerative process in the brain needs further investigations. More likely, deregulation of MT is several steps downstream to the crucial primary etiological factors causing different ND. In fact, MT deregulation might be a reactionary or protective mechanism to cope neurodegenerative mechanisms. Indeed, their alteration in ND could be only a consequence of neu-

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ronal degeneration as, for instance, oxidative stress and inflammation which occur in almost all the diseases, not only those related to the brain. Most of the studies here presented are descriptive and do not allow to convincingly speculate into the molecular mechanisms causally driving MT deregulation. Despite these still open issues, the evidence supporting the use of MT as a tool to stimulate repair and protection in the brain are encouraging, if not utterly convincing. However, we are aware that aging per se is able to cause an increase in brain MT levels [143–145]. This increase has been correlated to a physiological attempt to counteract oxidative stress phenomena and to preserve brain functioning. This natural response seems to be enhanced in pathological conditions such as ND. Therefore, a selective modulation of a specific isoform might be needed. Further studies are required to determine the function of MT in physiological and pathological conditions and to establish their therapeutic potential. The data available so far are encouraging but warrant further research.

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DISCLOSURE STATEMENT Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=2108). [18]

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