Metallothionein in the central nervous system: Roles in protection, regeneration and cognition

Abstract

Metallothionein (MT) is an enigmatic protein, and its physiological role remains a matter of intense study and debate 50 years after its discovery. This is particularly true of its function in the central nervous system (CNS), where the challenge remains to link its known biochemical properties of metal binding and free radical scavenging to the intricate workings of brain. In this compilation of four reports, first delivered at the 11th International Neurotoxicology Association (INA-11) Meeting, June 2007, the authors present the work of their laboratories, each of which gives an important insight into the actions of MT in the brain. What emerges is that MT has the potential to contribute to a variety of processes, including neuroprotection, regeneration, and even cognitive functions. In this article, the properties and CNS expression of MT are briefly reviewed before Dr Hidalgo describes his pioneering work using transgenic models of MT expression to demonstrate how this protein plays a major role in the defence of the CNS against neurodegenerative disorders and other CNS injuries. His group's work leads to two further questions, what are the mechanisms at the cellular level by which MT acts, and does this protein influence higher order issues of architecture and cognition? These topics are addressed in the second and third sections of this review by Dr West, and Dr Levin and Dr Eddins, respectively. Finally, Dr Aschner examines the ability of MT to protect against a specific toxicant, methylmercury, in the CNS.

Introduction

Fifty years ago, an unusual cadmium-binding protein was isolated from horse kidney (Margoshes and Vallee, 1957). Due to its high content of metals and cysteine residues, this protein was named metallothionein (MT) (Kagi and Vallee, 1960, Kagi and Vallee, 1961). In these 50 years, an overwhelming flow of information about these proteins has been published. It is clear that they constitute a superfamily of proteins, widely distributed in the animal kingdom as well as other phylogenetic groups (Andrews, 2000, Bremner, 1987a, Coyle et al., 2002, Ghoshal and Jacob, 2001, Hamer, 1986, Vasak and Hasler, 2000). In humans, the MT genes are tightly clustered in the q13 region of chromosome 16 (Karin et al., 1984a, Palmiter et al., 1992, Quaife et al., 1994, West et al., 1990), consisting of seven functional MT-I genes (MT-1A, -B, -E, -F, -G, -H and -X) and a single gene encoding each of the other MT isoforms, namely MT-II (the MT-2A gene), MT-III and MT-IV. Mice have a much simpler MT gene structure, with only one functional gene for each isoform MT-I–MT-IV, all located on chromosome 8 (Cox and Palmiter, 1983, Palmiter et al., 1992, Quaife et al., 1994, Searle et al., 1984). The genes Mt1 and Mt2 are expressed coordinately in most tissues including the central nervous system (CNS) (Campagne et al., 2000, Searle et al., 1984, Yagle and Palmiter, 1985), while Mt3 and Mt4 show a much more restricted tissue expression (mainly CNS and stratified squamous epithelia, respectively). The evolutionary retention of MT genes argues that they contribute important physiological properties; nonetheless these remain elusive to some extent.

All CNS MTs are composed of a single polypeptide chain of 61–68 amino acids, 20 of which are highly conserved cysteine residues, and there are no disulfides, aromatic amino acids or histidine. A major feature of their amino acid sequences is the occurrence of Cys-Xaa-Cys, Cys-Xaa-Yaa-Cys and Cys-Cys motifs, where Xaa and Yaa stands for an amino acid residue other than Cys (Kagi and Schaffer, 1988). These proteins usually bind 7 divalent metal ions (e.g. Zn(II)) and up to 12 monovalent copper ions, partitioned into two metal-thiolate clusters (Bogumil et al., 1998, Faller et al., 1999, Vasak and Kaji, 1994). Each cluster is located in a separate protein domain designated α (residues 32–61) and β (residues 1–31). When compared with the classical 61–62 amino acid sequences of MT-I and -II (hereafter referred to generically as MT-I/II, unless otherwise indicated) the sequence of MT-III (68 amino acids) shows two inserts: a single Thr in the N-terminal region and an acidic hexapeptide in the C-terminal region (Uchida et al., 1991).

It is generally accepted that the expression of MT-I/II proteins is highly inducible in response to a range of stimuli, including metals, hormones, cytokines, oxidative agents, inflammation and stress (Bremner, 1987b, Sato and Bremner, 1993). Mt-1 and Mt-2 genes are co-ordinately regulated in mice by metals and glucocorticoids (Searle et al., 1984, Yagle and Palmiter, 1985). Metal-induced synthesis is mediated through the action of short cis-acting DNA sequences known as metal-responsive elements (MREs), which are present in the promotor region of all mammalian MT genes (Karin et al., 1984b, Radtke et al., 1993), and is mediated mainly by metal response element-binding transcription factor (MTF-1), a zinc-sensitive trans-acting factor (Andrews, 2000, Radtke et al., 1993, Westin and Schaffner, 1988). Similarly, glucocorticoid-responsive elements (GREs) are responsible for expression of some MT genes in response to glucocorticoids (Karin et al., 1984b, Kelly et al., 1997). MT gene expression is also regulated by antioxidant-response elements (AREs), although some MREs also respond to oxidants, again MTF-1 being involved (Samson and Gedamu, 1998). Increased levels of MT-I/II in response to inflammatory factors is very likely to be influenced by cytokines such as interleukin (IL-6) through signal transducer and activator of transcription (STAT) factors (Lee et al., 1999). Nevertheless, it is feasible that multiple signals participate in stress, inflammation and oxidative stress induction of the MT genes (Fig. 1). The regulation of MT-III and MT-IV gene expression is poorly known.

It has been demonstrated that MT-I/II occur throughout the brain and spinal cord, and that the main cell expressing these MT isoforms is the astrocyte, especially the reactive astrocyte (Holloway et al., 1997). Nevertheless, MT-I/II expression is also found in ependymal cells, epithelial cells of choroid plexus, meningeal cells of the pia mater and endothelial cells of blood vessels. Neurons appear to express MT-I/II to a much lower extent than astrocytes, while in the normal brain, oligodendrocytes and microglia are essentially devoid of MT-I/II, but the latter cells do upregulate MT-I/II expression in response to injury (Hidalgo et al., 2001). MT-III was discovered as a putative factor important in Alzheimer disease (Uchida et al., 1991). In contrast to MT-I/II, there is still significant uncertainty regarding the cellular source of MT-III in the CNS, and astrocytes as well as neurons have been suggested to be the main cellular sources (Kobayashi et al., 1993, Masters et al., 1994b, Uchida, 1994, Hidalgo et al., 2001).

It is clear from the literature that MT-I/II and MT-III not only show a distinct pattern of expression in the CNS, but also respond differently to a number of insults. MT-I/II are typically upregulated in response to tissue injury, even to subtle insults. For instance, brain MT-I/II are known to be upregulated by psychogenic stress (Hidalgo et al., 1990) or by the administration of bacterial endotoxin (Palmiter et al., 1992, Searle et al., 1984), glutamate analogues (Acarin et al., 1999b, Dalton et al., 1995), cryogenic injury (Penkowa et al., 1999a, Penkowa et al., 1999c), or by stroke/ischemia (Campagne et al., 1999, Campagne et al., 2000, Neal et al., 1996, Tang et al., 2002, Trendelenburg et al., 2002). It is thus clear that MT-I/II may play an important role in the overall response of the brain to damage, a response that is thought to be orchestrated by a number of cytokines in a complex manner (Allan and Rothwell, 2001). Results obtained with microarrays and transgenic mice with null mutations have demonstrated that IL-6 (Poulsen et al., 2005) and TNF-α (Quintana et al., 2007) play major roles in the response of the cortex to injury (Fig. 2). These studies also demonstrated that both cytokines regulate MT-I/II expression, which is in line with results obtained with transgenic mice overexpressing either IL-6 (Carrasco et al., 1999, Hernandez et al., 1997) or TNF-α (Carrasco et al., 2000a) and with the presence of STAT response elements in the promoters (Lee et al., 1999). In contrast to MT-I/II, MT-III expression shows up- or downregulation depending on the model, time, etc. in animal models of brain injury. MT-III expression has been shown to be increased by stab wounds (Anezaki et al., 1995, Hozumi et al., 1995, Hozumi et al., 1996) and kainic acid administration (Anezaki et al., 1995), but decreased by cortical ablation of the somatosensory cortex (Yuguchi et al., 1995a), facial nerve transection (Yuguchi et al., 1995b), and middle cerebral artery occlusion (Inuzuka et al., 1996). A biphasic response of MT-III to CNS injury, with initial downregulation followed by upregulation, was observed in response to N-methyl-d-aspartate (NMDA) (Acarin et al., 1999a) or to a cryolesion (Penkowa and Hidalgo, 2000, Penkowa et al., 1999c).

Tissue injury elicits an inflammatory response and oxidative stress. There is increasing experimental evidence that oxidative stress contributes significantly to the neuropathology of several adult neurodegenerative disorders as well as to stroke, trauma, seizures and neuronal degeneration caused, among other reasons, by persistent activation of glutamate-gated ion channels (Coyle and Puttfarcken, 1993). As could be expected, brain MT-I/II levels have been consistently reported to be increased in Alzheimer's disease (Duguid et al., 1989, Uchida, 1994, Adlard et al., 1998, Chuah and Getchell, 1999, Zambenedetti et al., 1998), Pick's disease (Duguid et al., 1989) short-course Creutzfeld-Jakob disease (Kawashima et al., 2000), amyotrophic lateral sclerosis (Sillevis Smitt et al., 1992, Sillevis Smitt et al., 1994, Blaauwgeers et al., 1996), multiple sclerosis (Lock et al., 2002, Penkowa et al., 2003b), and aging (Suzuki et al., 1994).

Taken together, the above studies strongly suggest a significant role of MT-I/II and MT-III during neurodegenerative diseases and in response to brain injury, but demonstration of their involvement in specific processes in the CNS has been problematical, due in large part to the broader lack of a definitive understanding of the physiological role for MT in any tissue. In the sections to follow, four research groups present their investigations into the involvement of MT in physiological and protective CNS processes. It is interesting, and consistent with the long history of MT investigation, that convincing arguments can be made for a multi-faceted role of MT, spanning normal physiological processes and also a major reactive role in the face of injurious events such as toxicity or lesion.