Báo cáo khoa học: Metallothioneins are multipurpose neuroprotectants during brain pathology potx

REVIEW ARTICLE Metallothioneins are multipurpose neuroprotectants during brain pathology Milena Penkowa Section of Neuroprotection, Centre of Inflammation and Metabolism at The Faculty of Health Sciences, University of Copenhagen, Denmark Mammalian metallothioneins (MTs) constitute a super- family of nonenzymatic polypeptides (61–68 amino acids), which are characterized by low molecular weight (6–7 kDa), distinctive amino acid composition (high cysteine content and no or low histidine) and sequence (unique cysteine distribution as Cys-X-Cys), and a high content of sulfur and metals (metal thiolate clusters) [1–3]. In vivo, the metal-binding involves mainly Zn(II), Cu(I), Cd(II), and Hg(II), while in vitro additional and diverse metals such as Ag(I), Au(I), Bi(III), Co(II), Fe(II), Pb(II), Pt(II), and Tc(IV) may be bound to apothionein (the metal-free form) [4,5]. However, during physiological conditions mammalian MTs mostly contain zinc [6,7]. Keywords angiogenesis; antioxidants; apoptosis; defense; inflammation; metalloproteins; neuroregeneration; pharmacology Correspondence M. Penkowa, Section of Neuroprotection, The Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200, Copenhagen, Denmark Fax: +45 3 5327217 Tel: +45 3 5327222 E-mail: M.Penkowa@mai.ku.dk (Received 15 January 2006, revised 23 February 2006, accepted 28 February 2006) doi:10.1111/j.1742-4658.2006.05207.x Metallothioneins (MTs) constitute a family of cysteine-rich metalloproteins involved in cytoprotection during pathology. In mammals there are four isoforms (MT-I ) IV), of which MT-I and -II (MT-I + II) are the best characterized MT proteins in the brain. Accumulating studies have demon- strated MT-I + II as multipurpose factors important for host defense responses, immunoregulation, cell survival and brain repair. This review will focus on expression and roles of MT-I + II in the disordered brain. Initially, studies of genetically modified mice with MT-I + II defi- ciency or endogenous MT-I overexpression demonstrated the importance of MT-I + II for coping with brain pathology. In addition, exogenous MT-I or MT-II injected intraperitoneally is able to promote similar effects as those of endogenous MT-I + II, which indicates that MT-I + II have both extra- and intracellular actions. In injured brain, MT-I + II inhibit macrophages, T lymphocytes and their formation of interleukins, tumor necrosis factor-a, matrix metalloproteinases, and reactive oxygen species. In addition, MT-I + II enhance cell cycle progression, mitosis and cell sur- vival, while neuronal apoptosis is inhibited. The precise mechanisms down- stream of MT-I + II have not been fully established, but convincing data show that MT-I + II are essential for coping with neuropathology and for brain recovery. As MT-I and ⁄ or MT-II compounds are well tolerated, they may provide a potential therapy for a range of brain disorders. Abbreviations AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; 6-AN, 6-aminonicotinamide; ARE, antioxidant response element; BDNF, brain- derived neurotrophic factor; EAE, experimental autoimmune encephalomyelitis; FGF, fibroblast growth factor; FGF-R, FGF-receptor; GDNF, glial-derived neurotrophic factor; IL, interleukin; IL-6KO mice, IL-6 knockout mice (genetic IL-6-deficient mice); M-CSF, macrophage colony- stimulating factor; MMP, matrix metalloproteinase; MRE, metal response elements ; MS, multiple sclerosis; MT, metallothionein; MTF-1, MRE-binding transcription factor-1; MT-KO mice, MT-I + II knock-out mice (genetic MT-I + II deficiency); MT-III ⁄ GIF, metallothionein III ⁄ growth inhibitory factor; NFjB, nuclear factor kappa-B (transcription factor); NGF, nerve growth factor; NT, neurotrophin; PD, Parkinson’s disease; ROS, reactive oxygen species; SOD, superoxide dismutase; TGF-b, transforming growth factor-b;TGF-b-R, TGF-b receptor; TgMT mice, mice with transgenic MT-I overexpression; TNF-a, tumor necrosis factor-a; VEGF, vascular endothelial growth factor. FEBS Journal 273 (2006) 1857–1870 ª 2006 The Author Journal compilation ª 2006 FEBS 1857 In mammals, four major subfamilies exist (MT-I, MT-II, MT-III and MT-IV), of which MT-I and -II (MT-I + II) were discovered in 1957 and are the best described MT proteins. The roles of mammalian MT-I + II in the brain have received mounting scien- tific interest [1,8–10] and are also the focus of this review, which will not address other MT isoforms. MT-I + II are expressed ubiquitously in mammalian tissues, which rapidly increase their mRNA and proteins in response to pathology or administration [1,10]. In rodents, MT-I + II are regulated and produced coordi- nately [2], and they are often described together as one functional entity [4,5]. In mammals, MT-I + II consist of 61 and 62 amino acids, respectively, which are devoid of aromatic amino acids, while one-third of the residues are cysteines (in total 20) that form metal thiolate clus- ters. In the polypeptide chain, cysteines are arranged in series of motifs: Cys-X-Cys, Cys-X-Cys-Cys, C-X-X-C (X is a non-Cys residue), which are absolutely conserved across species [3–6]. The cysteine sulfhydryl groups bind and coordinate 7 moles of divalent metal ions [i.e. Zn(II) or Cd(II)] per mol MT-I + II, while the molar ratio for Cu(I) and Ag(I) is 12. The metal thiolate clusters (S cys -M-S cys ) exist in two separate globular domains, the a- and b-domains, which are linked by a small, lysine-rich region, although the domains have few contacts [1,3]. The a-domain in the C-terminus (amino acid residues 33–61 in rat MT-II) contains 11 cysteines and is able to bind four divalent or six monovalent metals, while the N-terminal b-domain (amino acid residues 1–29 in rat MT-II) includes nine cysteines capable of binding three divalent or six monovalent metals [1,4,6] (Fig. 1). These residues are either bridging cysteines, which can bind two divalent ions or they are terminal cysteines that bind only one divalent metal [3,4,11]. When metal ions bind to apothionein, the polypep- tide chain will rapidly fold resulting in the formation of the two native, three-dimensional metal thiolate clusters residing in each domain [3,5]. In the a-domain, the only known MT secondary structure can be found (a short a-helix present in case the protein is fully loa- ded with divalent (not monovalent) metals) [5,6]. The antigenic part (epitope) of the MT-I + II pro- teins is formed by a lysine-rich region, residues 20–25, together with the seven N-terminal residues 1–7, which after protein folding are seen in close proximity in the three-dimensional structure [1,4,6]. The most studied human MT genes are found on chromosome 16, which features very high levels of seg- mentally duplicated sequence among the human auto- somes and abundant genetic polymorphisms, which are also existing in the MT-I + II genes [1,5]. In the chromosome 16 q13 region, MT genes are tightly linked, and as a minimum they consist of 11 MT-I genes (MT-I-A, -B, -E, -F, -G, -H, -I, -J, -K, -L, and -X) encoding functional or nonfunctional RNA, and one gene for the other MT isoforms (the MT-2 A Fig. 1. Schematic drawing of the mammalian MT-II protein showing the two metal-thiolate clusters (C-terminal a-domain and N-terminal b-domain) including the 20 cysteine residues (blue squares) and their sulfur atoms (S), which bind to divalent or monovalent cations (in this case Zn). The domains are linked by a short peptide containing amino acid residues 30–32 in mammalian MT-II (LINK). In the b-domain, three divalent or six monovalent metal ions are coordinated, while in the a-domain four divalent or six monovalent cations can be bound. Both bridging and terminal cysteines are present in mammalian MT-2. The bridging cysteines bind to two separate, divalent cations, while the terminal cysteines chelate one divalent metal. If monovalent metals are bound, all cysteines can chelate two cations. Metallothioneins are neuroprotective factors M. Penkowa 1858 FEBS Journal 273 (2006) 1857–1870 ª 2006 The Author Journal compilation ª 2006 FEBS gene, MT-3 gene, and MT-4 gene) [1,3,4]. However, a gene called MT-like 5 (MTL-5) has been described in the q13 region of chromosome 11, and it encodes a testis-specific MT-like protein named tesmin [3,4,6,7,12]. Compared with the human genes, the mouse MT genes are less complex, as they only have one func- tional gene for each major MT isoform (one gene encoding MT-1, MT-2, MT-3 and MT-4) and these are all located on chromosome 8. As in humans, the mouse genome also contains an MTL-5 gene, which is located on chromosome 19B [1–3,7,12]. However, this review will focus only on mammalian MT-I + II isoforms, while all the other MT isoforms and related MT-like structures (genes or their prod- ucts) will not receive further attention. The major top- ics reviewed here are the in vivo roles of mammalian MT-I + II in immunoregulation, neuroprotection and cerebral regeneration, a field receiving growing scienti- fic interest. Cerebral MT-I + II expression Brain MT-I + II mRNA and proteins are present in low amounts in physiological conditions and are expressed during embryonic development and in neonatals, and with increasing postnatal age MT-I + II immunoreactivity increases and becomes continually more widespread in the CNS [8,9]. In the brain, astrocytes are the main source of MT-I + II, although other cell types, such as choroid plexus epithelia, endothelium and meningeal cells, may also show MT-I + II [1,10]. In neurons the data on MT-I + II expression have been inconsistent, and MT-I + II posi- tive neurons have only been intermittently described [9,13], although MT-I + II were demonstrated to exert direct protective effects upon neurons, as shown in primary neuronal cultures [14,15]. However, it is in general agreed that the levels of MT-I + II are several-fold higher in astrocytes relative to neurons. Thus far, all the brain disorders studied in animals and humans have shown that MT-I + II mRNA and proteins are acutely and highly increased in reactive astroglia as part of the acute inflammation and host defense response [16–20]. To some extent, MT-I + II are also increased in the vascular endothelium, choroid plexus, ependyma, activated microglia ⁄ macrophages, and meninges, while neuronal and oligodendroglial MT-I + II immunoreactivity have not been consis- tently reported [21–23]. MT-I + II mRNA increases are seen within 24 h after an insult to the brain followed by many fold increases in their protein levels as seen typically after 1–3 days postinjury [20–24]. Increased MT-I + II expression is seen in various types of CNS pathology models such as in traumatic, excitotoxic, and ischemic ⁄ hypoxic injury, multiple sclerosis including its animal model experimental auto- immune encephalomyelitis (EAE), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Pick’s disease, pellagra encephalopathy, immobilization stress, and peripheral nerve injury [1,19,21,25–29]. Cellular MT-I + II distribution MT-I + II have been considered as strictly intracellu- lar proteins [4,29], but in recent years, mounting data indicated that MT-I + II are distributed both intra- and extracellularly [20,30–32]. Inside cells, MT-I + II are distributed in cytoplasm and subcellular organelles like lysosomes and mito- chondria. Depending on the cell cycle phase, differenti- ation or in case of toxicity, MT-I + II are rapidly translocated to the nucleus, as seen during early S-phase and in oxidative stress [4,33–35]. Due to their small size, MT-I + II can diffuse through nuclear pore complexes, although the nuclear trafficking is relying on specific cytosolic partner proteins and the appear- ance of nuclear binding proteins, which in the presence of ROS enhance the nuclear localization of MT-I + II [29,36,37]. Also, perinuclear localization of MT mRNA may contribute to the nuclear import of MT-I + II proteins, as well as some structural altera- tions in the proteins per se (such as lack of post-trans- lational acetylation of lysine and cysteine) are anticipated to regulate the nuclear trafficking [29,36]. Once in the nucleus, MT-I + II are selectively and actively retained by nuclear factors, which are likely to make use of saturable and energy-dependent binding mechanisms, in that elimination of the ATP pool hampers the nuclear translocation and ⁄ or retention of MT-I + II [37]. However, the precise intracellular MT-I + II trafficking system has yet to be clarified. In addition, cells have been demonstrated to actively secrete MT-I + II in vitro, although there is no known signal peptide for cellular export [35,38]. In vivo, MT-I overexpressing transgenic mice display significant MT-I + II immunoreactivity in the brain extracellular space [20]. In the brain, the astrocytes, not the neurons, are the major source of MT-I + II, even though these proteins primarily protect the neurons [9,31,39]. Hence, it is considered that astroglia may secrete MT-I + II to the extracellular space in order for them to protect the surrounding neurons [30]. This is supported by studies of primary cell cultures, which showed that extracellular MT-I + II exert direct effects M. Penkowa Metallothioneins are neuroprotective factors FEBS Journal 273 (2006) 1857–1870 ª 2006 The Author Journal compilation ª 2006 FEBS 1859 upon neurons, as MT-administration enhanced the sur- vival, differentiation and postinjury recovery of cortical, hippocampal, and dopaminergic neurons [14,15]. The experimental data from in vitro and in vivo stud- ies that are reviewed here have shown consistently that intra- and extracellular MT-I + II promote analogous functions [14,28,29,31,35,40–41], which specifies that MT-I + II have roles both in and outside cells. Regulation of MT-I + II MT-I + II are regulated in a coordinate manner [2] and are rapidly increased by various pathological con- ditions [1,20]. However, physiological and lifestyle-rela- ted parameters like nutritional condition and physical activity have also been reported to regulate MT-I + II mRNA and proteins [7,11]. Administration of essential or toxic metals like Zn, Cu, Cd, Hg increase MT-I + II biosynthesis by indu- cing their transcription, for which several cis-acting DNA elements, metal response elements (MREs) in the promoter region are binding sites for trans-acting transcription factors [3,7,43,44]. The MT-I + II gene transcription is initiated when metals occupy the MRE-binding transcription factor-1 (MTF-1), which is a multiple Zn finger protein and the only known medi- ator of the metal responsiveness of MT-I + II [3,44]. Reactive oxygen species (ROS) and oxidative stress also increase expression of MT-I + II, which are highly efficient free radical scavengers in the brain [1,45]. ROS increase the MT-I + II transcriptional response, as shown by exposure to free radicals like superoxide anions and hydroxyl radicals, which rapidly increase MT-I mRNA levels in a dose-dependent man- ner [43,46]. The mechanism involves an antioxidant response element (ARE) in the promoter region, ARE- binding transcription factors, as well as the MTF-1, transcription factors of the basic zipper type (Fos and Fra-1), NF-E2-related factor 2, and the upstream stim- ulatory factor family (USF, a basic helix–loop–helix– leucine zipper protein), although it is likely that other and yet unidentified proteins are involved [7,46]. Thereby, metals and ROS activate MT-I + II gene transcription by different signaling pathways, response elements and transcription factors. In addition, MT-I + II are also increased by gluco- corticoid hormones like corticosterone and dexametha- sone, which signal through glucocorticoid response elements (GREs) present in the gene regulatory region, and also catecholamines (norepinephrine, isoprotere- nol) activate MT-1 + II gene transcription [1,7,47]. During CNS inflammation, major MT-I + II regu- latory factors are proinflammatory cytokines and espe- cially interleukin (IL)-6 [1]. Accordingly, IL-6, IL-3, tumor necrosis factor (TNF)-a, macrophage-colony stimulating factor (M-CSF), and interferons increase brain MT-I + II expression in a cytokine-specific manner as demonstrated by using transgenic mice with cytokine overexpression [48–51] or cytokine deficiency [29,52–55]. Although the activation of MT-I + II gene tran- scription is by far the best described regulatory mech- anism, repression of MT gene activity has also been reported [4,7]. Hence, during Zn deficiency, MTF-1 may form a complex with a Zn-responsive inhibitor, named MT transcription inhibitor, which prevents MTF-1 from interacting with the MREs, and thereby MT-I + II gene transcription could be negatively con- trolled due to the levels of trace metals [4,7,43]. In human cells, MT-IIA gene activation is inhibited by Zn finger protein PZ120, which interacts with the MT-IIA transcription start site and inhibits gene expression [56]. Also, transcription factors Fos and Fra-1 can inhibit MT-I + II biosynthesis by inter- action with ARE [7]. However, MT-I + II biosynthesis is also affected by post-transcriptional events, since their protein levels do not necessarily reflect the levels of mRNA expression [4,57]. Hence, Cu treatment of adult rats reduced renal MT-I + II mRNA levels while at the same time, the renal MT-I + II protein expression was significantly increased [58], which suggests that post-transcriptional regulation occurs and this may likely affect either the translation and ⁄ or the protein degradation. In fact, MT-I + II are to some degree regulated by means of intracellular protein degradation, which takes place in both lysosomal and nonlysosomal compartments [4,59]. In general, intracellular MT-I + II proteins occur as either metal-containing proteins (MTs) or as apothioneins, and their depletion and ⁄ or restitution may depend on the bound metals, subcellular localiza- tion, and the tissue examined. Hence, turnover rates of cytosolic apothioneins versus lysosomal metal-bound MT-I + II proteins are quite different, in that lyso- somal MT-I + II proteolysis occurs more readily than in the cytosol, although bound metals stabilize MT-I + II proteins and prevent their lysosomal pro- teolysis [59,60]. In the cytoplasm, the 26S proteasome complex degrades apothionein, which due to the lack of metals has a shorter half-life than MTs [4,11]. The type of metal complex may also in itself affect the MT-I + II degradation, as the half-life of Cd-containing proteins is close to 3 days, while Zn-binding reduces half-life to 18–20 h. Also, animal age and the chemical pretreatment may determine the half-life of MT-I + II, as well as MT-I in some cases Metallothioneins are neuroprotective factors M. Penkowa 1860 FEBS Journal 273 (2006) 1857–1870 ª 2006 The Author Journal compilation ª 2006 FEBS has reduced half-life relative to MT-2 [61]. Thus, it is evident that other factors than the metals per se can regulate MT-I + II turnover [4,11,61]. The CNS roles of endogenous MT-I + II Recently, rising interest in MT-I + II neuroprotective functions and therapeutic potential has been evident. During the genomic era it became possible to modify the MT-I + II genes in cultured cells and in animal embryos leading to the generation of MT-I + II knockout (MT-KO) mice [62] and transgenic MT-I overexpressing (TgMT) mice [63]. These genotypes have provided important answers concerning the roles of MT-I + II in the disordered CNS, although at first the data from MT-KO mice were rather disappointing, since these mice developed normally, appeared viable and fertile without any phenotypic changes [62]. Con- sequently, MT-I + II were considered as dispensable factors and ⁄ or proteins that may have abundant com- pensatory backup systems. Years later, this concept was substantially contradic- ted, as it was demonstrated that neuronal survival and brain tissue repair are compromised when MT-I + II are absent. It became clear that during brain disorders, MT-KO mice show significantly enhanced brain tissue destruction, neuronal cell death, and clinical symptoms, when compared with those of wild-type controls [49,64– 66]. Accordingly, even if the MT-I + II proteins may be dispensable during healthy, physiological conditions, they are unquestionably essential for coping with brain damage [1,30,39]. The major histopathological changes seen in brains of MT-KO mice are enhanced inflamma- tory responses including increased recruitment of macro- phages, lymphocytes and their CD34 + hematogenous progenitor cells and enhanced secretion of proinflamma- tory factors like IL-1, IL-3, IL-6, IL-12, TNF-a, lymphotoxin-a (LTa), macrophage activator factor (Mac-1), intercellular adhesion molecule (ICAM-1) and acute phase response gene EB22 [31,49,65–69]. These studies also gave insight into the MT-I + II in vivo antioxidant functions in the brain, as MT-I + II deficiency resulted in amplified ROS formation and oxidative stress including highly increased lipid peroxidation, protein nitrosylation and DNA oxidation, when compared with those of WT controls [19,21,67,70]. In addition, MT-KO mice dis- play significantly increased neurodegeneration and apoptotic cell death relative to WT controls as shown during traumatic brain injury, kainic acid-induced epi- leptic seizures, 6-aminonicotinamide (6-AN)-induced pellagra encephalopathy, ischemia, cytokine-induced meningoencephalitis, peripheral nerve injury, PD, EAE and ALS [23,27,40,62,65,67–71]. During these brain disorders, the MT-KO mice also developed worse clinical symptoms and showed significantly poorer neurological outcome relative to WT controls (Fig. 2). In contrast to brain disordered MT-KO mice, the TgMT mice showed significantly less neuropathological damage, while their tissue repair and neurological out- come were improved relative to WT control mice [13,20,28,31,40,55,72,73]. Thus, TgMT mice subjected to diverse brain disorders display reduced inflammatory responses of macrophages and lymphocytes including significantly decreased levels of proinflammatory cytokines, matrix metallo- proteinases (MMPs), and ROS. Also, the amounts of delayed brain tissue damage consisting of oxidative stress, neurodegeneration and apoptotic cell death were radically reduced in TgMT mice relative to wild-type controls [13,20,28,31,55,72]. To this end, comparisons of the MT-I + II containing cells in the brain with the cell populations suffering from oxidative stress and apopto- tic death showed clearly that damaged and ⁄ or dying cells are devoid of MT-I + II expression, which are confined to surviving cells, and this likely reflects the cytoprotection conferred by MT-I + II [19,72]. In addition, MT-I overexpression after brain injury stimulates the astroglial responses including the expres- sion of anti-inflammatory cytokines, growth factors, neurotrophins and their receptors, such as IL-10, fibro- blast growth factor (FGF), FGF-receptor (FGF-R), transforming growth factor (TGF)-b, TGF- b-receptor (TGF-b-R), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), neurotrophin (NT)-3–5, brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF) [13,20,28,31, 72,73]. Concomitantly, MT-I overexpression improves brain tissue repair including neuronal regrowth and vascular remodeling by angiogenesis, as well as the TgMT mice show improved clinical outcome, when compared with those of the wild-type control mice [13,15,28,31,72]. To study brain restoration and tissue repair, a suit- able model is the traumatic, focal brain injury to the cortex, which results in a cortical necrotic cavity with- out viable cells that gradually will be replaced with glial scar tissue, vascular network and extracellular matrix [10,24,54,74]. These processes are significantly enhanced by MT-I + II, which are essential for the CNS wound repair to occur [30,31,72,64]. Hence, in the injured MT-KO mice the lesion cavity persists after 3 months, by which severe inflammation is ongoing; while in wild- type controls, the necrotic cavity is usually replaced with a scar after 30 days [65,66], while in MT-Tg mice this M. Penkowa Metallothioneins are neuroprotective factors FEBS Journal 273 (2006) 1857–1870 ª 2006 The Author Journal compilation ª 2006 FEBS 1861 scar tissue is established before day 20 postinjury [31,72]. Moreover, following brain pathology MT-I + II are essential for the recruitment of neuroglial precursor cells [19,22,75] and their migration towards the site of injury. Hence, the increased tissue repair promoted by MT-I + II after injury is likely mediated in part by regeneration, where newly formed cells repopulate the tissue and in part by regrowth and sprouting of survi- ving cells. The roles of exogenous MT-I + II Shortly after the first data emerged from genetically modified MT-KO and TgMT mice subjected to neuro- pathology, new studies were conducted focusing on the potential therapeutic use of MT-I + II proteins. For this, adult rodents were injected intraperitoneally with exogenous MT-I and ⁄ or MT-II (MT-I ⁄ II) proteins during healthy conditions and neuropathological dis- orders like brain injury, pellagra encephalopathy and EAE [9,22,28,30,31,75]. The used MT-I ⁄ II proteins contained metals, which were mainly Zn (the Zn con- tent was approximately 7%) and small amounts of Cd (the Cd content was < 0.5%). Therefore, these metals were included in the control treatment regimen. The intraperitoneal administration of exogenous MT-I ⁄ II modulates immunoregulation and improves neuroprotection and CNS recovery in vivo during brain pathology, reflecting that MT-I + II have extracellular roles. This was far from anticipated at the time of the first publication (2000), as MT-I + II had been con- sidered as strictly intracellular proteins [4]. At first, exogenous MT-I and ⁄ or MT-II proteins were injected intraperitoneal in rats with EAE that were evaluated clinically and histopathologically. In a dose- and time-dependent manner, the MT-I ⁄ II treat- ment reduced the severity of neurological symptoms and the mortality relative to placebo control groups. MT-I ⁄ II treatment in EAE reduced significantly the activation and recruitment of macrophages and T lymphocytes including levels of IL-1b, IL-6, IL-12, Inhibition Stimulation MT-I+II Oxidative stress Microgliosis Macrophages & T cells Cytokine release Oxidative stress Tissue Loss Neurological symptoms Apoptosis BBB disruption Cerebral ECs Neurons Neurodegeneration Fig. 2. Schematic drawing of the main anti- inflammatory, antioxidant and anti-apoptotic actions of MT-I + II leading to neuroregen- eration, angiogenesis and repair. MT-I + II modulate an array of vital cellular functions that involve cytoprotection, angiogenesis, DNA repair and the maintenance of tissue homeostasis. During pathology, MT-I + II inhibit inflammation and cytokines and pro- tect against oxidative stress, degeneration, and apoptosis. Metallothioneins are neuroprotective factors M. Penkowa 1862 FEBS Journal 273 (2006) 1857–1870 ª 2006 The Author Journal compilation ª 2006 FEBS TNF-a and ROS, which was seen in brain, spleen, and bone marrow [9,42]. The EAE lesions (plaques) with demyelination, apoptotic cell death, axonal degener- ation and transection were radically reduced by MT-I ⁄ II administration relative to control treatment [22]. Concomitantly, MT-I ⁄ II-treated animals dis- played improved remyelination, regeneration and clin- ical recovery from EAE relative to the placebo groups. This therapeutic effect was due to MT-I + II-activa- tion of oligodendroglial progenitors ⁄ stem cells and enhanced expression of growth and trophic factors (FGF, TGF-b, NT-3–5 and NGF), which were signifi- cantly enhanced by MT-I + II in EAE and even more during the recovery phases, when compared with those of the placebo controls [22,76]. In later studies, exogenous MT-I ⁄ II proteins were administered during experimental models of traumatic brain injury (freeze lesion with dry ice) and pellagra encephalopathy (induced by administration of 6-AN). The acute (primary) injuries (the trauma- or 6-AN- induced necrosis) were comparable in the treatment groups, but in the following days, some pronounced differences in the responses to pathology appeared. Thus, animals receiving MT-I ⁄ II-treatment showed sig- nificantly less oxidative stress, neurodegeneration and apoptotic cell death (delayed damage) in the days ⁄ weeks following the primary injuries [28–31,75]. In these studies, the MT-I ⁄ II treatment also enhanced repair responses including expression of growth ⁄ trophic factors, astrogliosis, angiogenesis, neuronal regrowth [75], and particularly after the traumatic brain injury, it was evident that MT-I ⁄ II enhance reorganization of the necrotic lesion cavity [31]. The metal bound state of MT was preferred because the metalloform is likely to be the more physiological relevant form of the protein, and also because it is significantly less susceptible to degradation than apothionein. However, none of the effects of the MT-I ⁄ II treatment were seen after administration of the metals per se, but the latter may still be important as MT-I ⁄ +II adopt their tertiary structure upon chelation of metal ions [1,3,4]. However, the mole- cular mechanisms by which the MT-I ⁄ II treatment promoted neuroprotection and repair remain to be fully clarified (Fig. 3). The MT-I + II molecular mechanisms To clarify the specific functions of the MT-I + II pro- teins, many different approaches and techniques have been applied throughout thousands of studies. Although they described the MT-I + II structure, chemical characteristics, regulation, expression, distribution, degradation and the consequences of reducing or increasing MT-I + II in cells; they have not yet clarified the precise signaling and mechanisms by which MT-I + II exert immunoregulatory and neuro- protective actions. However, many possibilities are likely, since MT-I + II are indeed multipurpose proteins involved in a broad range of functions, which include, but are not restricted to metal ion homeostasis, scavenging of ROS, redox status, immune defense responses, pro- tein–protein and protein–nucleotide interactions, regu- lation of Zn fingers and Zn-containing transcription factors, mitochondrial respiration, thermogenesis, body energy metabolism, angiogenesis, cell cycle progression, and cell survival and differentiation [1,6,29,30,33, 39,77,78]. Some of these MT-I + II actions may have therapeutic relevance in a range of acute and chronic neurological disorders, in which inflammation and oxidative stress are central in the pathophysiology [79–84]. Accordingly, MT-I + II may signal through diverse molecular pathways. The immunomodulatory actions of MT-I + II reduce proinflammatory mediators including cytokines, MMPs, and adhesion molecules [20,32,72]. The reduction of brain IL-1, IL-6, IL-12 and TNF-a could be a central mechanism in the MT-I + II anti- inflammatory effects, since these cytokines are major immune activators that increase leukocyte activation, transendothelial migration, and chemoattraction, thereby leading to neuroinflammatory infiltrates [79–81]. Hence, genetic deficiency or overexpression of these cytokines or their receptors will diminish or enhance the brain inflammatory leukocytes [74,81,84,85]. Thus, IL-6 knockout mice (genetic IL-6-deficient mice) (IL-6KO) mice are resistent to EAE sensibilization, while IL-6 overexpressors show spontaneous chronic Fig. 3. Summary of the major biological functions of MT-I + II. M. Penkowa Metallothioneins are neuroprotective factors FEBS Journal 273 (2006) 1857–1870 ª 2006 The Author Journal compilation ª 2006 FEBS 1863 neuroinflammation and degeneration [73,86], which reflects that IL-6 is activating hematopoiesis, acute phase responses, and inflammation. IL-1 and IL-12 are also central pro-inflammatory cytokines that are cru- cial in the development of Th1 cells and initiation of autoimmune attacks, demyelination and neurodegener- ative diseases and neuronal cell death by apoptosis and necrosis [49,80,82,84,85]. As the pro-inflammatory cytokines mediate significant neurotoxicity and chronic pathology, the MT-I + II-inhibition of their mRNA and protein biosynthesis [66] will likely contribute to improved neuroprotection. It was recently shown that MT-I + II share certain structural and functional similarities with beta- and delta-chemokines CCL-17 and CX3CL-1 in vitro [87], whereby MT-I + II may regulate leukocyte chemo- taxis, although this has yet to be confirmed in vivo. Other cell culture studies showed that MT-I + II may inhibit monocytic activation and invasion including secretion of cytokines [88–90]. Moreover, MT-I + II inhibit macrophage-induced T cell proliferation and the activation of cytotoxic T cells and antigen-specific B cells [91–94]. To this end, MT-I + II may also reduce inflamma- tion by interfering directly with cell–cell interactions as MT-I + II were demonstrated to bind specifically to the membranes of macrophages, T and B cells, which thereby are inactivated [91–95]. These MT-I + II anti-inflammatory effects can also be seen in humans, as patients with autoimmune and allergic diseases show depletion of systemical MT-I + II and occurrence of anti-MT-I + II auto- IgGs against MT-I + II, an alteration that is most pronounced during clinical exacerbations [96,97]. How- ever, as in animals, the human MT-I + II levels can be fully replenished by various agents, among which steroids like glucocorticoid and cortisone can be used; and interestingly, steroids in general increase MT-I + II levels right before the patients show signifi- cant clinical improvements [97,98]. Also in MS patients, the MT-I + II expression levels are highest during the recovery and remission of disease [76]. In fact, the molecular mechanism of steroid-medi- ated immuno suppression could be a steroid-caused MT-I + II augmentation, given that glucocorticoid- treated patients show significant MT-II increases in their peripheral leukocytes shortly before the therapeu- tic effect of steroid commenced [98]. In support of this, dexamethasone-induced MT-II can be used as an indi- cator of glucocorticoid sensitivity [99]. This correlation between steroids and MT-I + II also exists in the brain, where MT-I + II mRNA and proteins are enhanced significantly by glucocorticoids [100]. In case MT-I + II are central mechanisms of steroid therapeutic effects, then MT-I + II might be used as a more specific anti-inflammatory agent likely having less side-effects than steroids. As proinflammatory cytokine profiles are associated with development of human type-2 diabetes, which also affects the brain, we recently examined MT-I + II in such patients. Interestingly, systemical MT-I + II expression and function are depleted in type-2 diabet- ics relative to healthy subjects [101]. Hence, both con- stitutive and stress-related MT-I + II were deficient in the patients versus the healthy control subjects, which suggests that an absence of MT-I + II may have a key role in the pathogenesis of type-2 diabetes [101]. Indeed, in a following study of experimental diabetes, it was shown that diabetic MT-I + II depletion can be fully restored by medication, and such MT-I + II replenishment is associated with disease remission [102]. In addition, the MT-I + II inhibition of MMPs, which are Zn-dependent endopeptidases produced by inflammatory cells, may also contribute to amelior- ation of a number of human autoimmune diseases, where MMPs are involved in pathophysiological events like diapedesis of infiltrating cells, tissue degradation and blood–brain barrier breakdown [103,104]. Furthermore, MT-I + II stimulate astroglial res- ponses including expression of anti-inflammatory signals, growth ⁄ trophic factors [72,66,68]. Although astrogliotic scarring traditionally has been considered as inhibitors of neuroregeneration, mounting and con- vincing data have now shown that reactive astrocytes provide essential neuroprotection and recovery. Hence, astrocytes endow neurons with antioxidants, energy substrates, anti-inflammatory and trophic ⁄ growth fac- tors; and they improve neurogenesis and neurological outcome [70,105,106]. Hence, ablation of astroglia during brain pathology leads to massive increases in neurodegeneration, de- myelination, infiltration by leucocytes, and cell death [106]. Thus, astroglial responses activated by MT-I + II may contribute to increased neuron survi- val, regeneration and CNS recovery. Also, MT-I + II increase expression of IL-10, FGF, TGF-b, VEGF, NGF, NT-3–5, BDNF, GDNF and their receptors; and this could in itself mediate neuroprotection as well as contribute to the MT-I + II-mediated repair, angi- ogenesis and vascular remodeling [19,20,22,30,32,39]. Together, these actions of MT-I + II can contribute to overall improvements in CNS cell survival and recovery [1,27,28,31,72,64–66,76]. Taken as a whole, these effects upon cerebral inflam- mation suggest that MT-I + II could be causing a Metallothioneins are neuroprotective factors M. Penkowa 1864 FEBS Journal 273 (2006) 1857–1870 ª 2006 The Author Journal compilation ª 2006 FEBS general shift in the balance between pro- and anti- inflammatory molecules. The actual mechanisms through which MT-I + II inhibit neurodegeneration and cell death remain to be fully described, although a range of anti-apoptotic effects have been shown in animals and humans. First, the anti-inflammatory effects as well as the antioxidant properties of MT-I + II could each contribute to decreased neurodegeneration and cell loss [79–83], although it is unlikely that these MT-I + II effects are the only responsible mechanisms. Hence, when the MT-I + II anti-inflammatory actions are counterbalanced, as done by using double transgenic mice overexpressing both MT-I and pro- inflammatory cytokine IL-6, it was evident that MT-I + II still reduce neurodegeneration and cell death significantly [32,72,73]. However, the MT-I + II antioxidant effects likely contribute to neuroprotection, as the cerebral ROS formation and oxidative stress are inversely related to the MT-I + II levels but not to the expression of other antioxidants such as Cu ⁄ Zn-super oxide dismutase (Cu ⁄ Zn-SOD), Mn-SOD, and catalase [32,40,65,72]. During mitochon- dria-specific oxidative stress, MT-I + II are indispens- able and have key roles in the mitochondrial protection, which did not relate to other antioxidants like glutathione peroxidase, catalase, Mn-SOD, and Cu ⁄ Zn-SOD [45]. MT-I + II may also prevent neuronal damage by having critical roles in metal ion homeostasis. Partic- ularly the Zn regulation by MT-I + II may have major importance, since Zn is central for a broad range of functions. However, tight control of the Zn levels is necessary as an overload or deficiency of this metal leads to severe neurotoxicity [1,107]. Also, MT-I + II transfer Zn directly to mitochondrial fac- tors, Zn-finger proteins and transcription factors, which also are essential for several signaling pathways and cell fate [4,34,78]. Along with Zn, various metal ions with a neurotoxic potential are bound and released by MT-I + II, which can thereby influence a range of cellular metabolites and pathways in the brain. A disrupted metal ion homeostasis causes oxida- tive stress, degeneration and neuronal cell death, and accordingly, dysregulation of metals has been associ- ated with many pathologies including stroke, epilepsy, PD, AD, and traumatic brain injury [6,10,21,44,107]. Hence, the MT-I + II regulation of metal ion availab- ility and levels in the CNS is most likely to contribute to the MT-I + II protective functions. Besides having roles in metal ion regulation, MT-I + II proteins also obtain their tertiary structure and enhanced molecular stability from their chelation of metals [4,6,11]. To this end, it is important that the different MT-I ⁄ II treatments injected into animals were all fully loaded Zn 7 –MT complexes, as the metal ensures pro- tein stability, folding and longer half-life [3,4]. However, MT-I + II interact and modulate many intracellular messengers that are directly or indirectly regulating the apoptotic cascade, and therefore MT-I + II may affect additional pathways during their responses to damage and promotion of tissue repair. The nucleotides ATP and GTP [5,34,108] bind to MT-I + II proteins, whereby both structural and functional changes are seen in the proteins [108]. Also, the MT-I + II and ATP levels inside cells are interre- lated, which in itself could affect cell loss or survival, since ATP depletion is part of the apoptotic cascade [40]. The MT-I + II and ATP connection may also be implicated in other actions, such as the MT-I + II- caused stabilization and rejuvenation of the ageing mitochondrial genome [40] and MT-I + II-regulation of energy balance and metabolism [77,78]. To this end, MT-I + II can donate Zn directly to mitochondrial aconitase (m-aconitase) by means of direct protein– protein interaction [109]. In addition, MT-I + II regulate the levels, activity and cellular localization of the transcription factor NFjB [10,70,95], which is involved in cell fate during neuropathology. Besides, MT-I + II induce a range of common proto-oncogenes (like bcl-2 and c-myc) whilst pro-apoptotic proteins (like p53 and caspase-3) are inhibited [11,20,32,33,41,74]. The roles of MT-I + II in cell fate and the MT-I + II connection to other factors involved in cell cycle regulation have led to many studies of MT-I + II roles in cancer. It is not surprising that MT-I + II may prevent tumor cell death by protecting against pro-apoptotic treatment regimes [11,33]. How- ever, when the cancer is located in ectodermal tissues (such as colon, bladder and skin), a positive correla- tion exists between increased MT-I + II levels and an improved prognosis [11]. Final comments This review summarizes the current knowledge and advances in the understanding of MT-I + II roles in immunomodulation and neuroprotection. The findings indicate that MT-I + II inhibit efficiently proinflam- matory cytokines, ROS, MMPs and pro-apoptotic sig- nals, which all may cause a broad range of brain disorders. As shown by many independent groups, the MT-I + II levels are inversely related to the degree of brain damage observed after traumatic injury, EAE, epileptic seizures, ischemia, and neurodegenerative M. Penkowa Metallothioneins are neuroprotective factors FEBS Journal 273 (2006) 1857–1870 ª 2006 The Author Journal compilation ª 2006 FEBS 1865 diseases like PD, ALS and pellagra [9,15,20,22,27,30– 32,40,69–71]. Consequently, MT-I + II might provide new drug targets against neurological disorders, especi- ally those containing autoimmunity, neurodegeneration and neuron loss. As MT-I + II compounds are in gen- eral well tolerated, they may be used in the future as therapeutic and ⁄ or preventive medications. 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REVIEW ARTICLE Metallothioneins are multipurpose neuroprotectants during brain pathology Milena Penkowa Section of Neuroprotection,. and brain tissue repair are compromised when MT-I + II are absent. It became clear that during brain disorders, MT-KO mice show significantly enhanced brain