R. S. Chung, PhD, Private Bag 58, University of Tasmania, Hobart, Tasmania 7001, Australia Fax: +61 3 62262703 Tel: +61 3 62262657 E-mail: email@example.com
Neuronal growth-inhibitory factor, later renamed metallothionein-3, is one of four members of the mammalian metallothionein family. Metallothioneins are a family of ubiquitous, low-molecular-weight, cysteine-rich proteins. Although neuronal growth-inhibitory factor shares metal-binding and reactive oxygen species scavenging properties with the other metallothioneins, it displays several distinct biological properties. In this review, we examine the recent developments regarding the function of neuronal growth-inhibitory factor within the brain, particularly in response to brain injury or during neurodegenerative disease progression.
Metallothioneins (MTs) are a family of unusual cysteine-rich (30%), 6–7 kDa proteins synthesized predominantly by astrocytes within the brain. The MT3 isoform was first isolated and identified as a neuronal growth-inhibitory factor (GIF) in 1991, a brain-specific protein whose synthesis was notably deficient in the Alzheimer’s disease (AD) brain. It was found to possess a strong ability to impair neurite outgrowth and neuronal survival of cultured neurons, leading to its designation as GIF. It was later discovered that GIF shares approximately 70% amino-acid sequence similarity with the MT family of proteins, leading to its renaming as MT3. Most striking is the conservation within GIF of the unique cysteine motifs found in mammalian MTs. Given that GIF shares a biochemical structure similar to those of the other MT isoforms, it is not surprising that GIF has the characteristic metal-binding and reactive oxygen species (ROS) scavenging capabilities present in all MT isoforms. However, GIF has also been found to exhibit several unique biological properties, suggesting that this MT isoform has different and distinct functions within the brain. Furthermore, the discovery and continued investigation of this brain-specific MT isoform has led to intense interest in the roles of the entire MT family in the brain, with particular focus on the role of these proteins in the injured or neurodegenerative brain.
Discovery of GIF
AD is a neurodegenerative disease that leads to severe dementia and ultimately death. The pathological hallmarks of the disease are intracellular neurofibrillary tangles, dystrophic neurites or curly fibres, and extracellular senile amyloid plaques. These physical alterations are often accompanied by aberrant neurite sprouting and significant neuronal loss, particularly within the cerebral cortex and hippocampus. One explanation for this impaired neuronal survival is that there may be decreased synthesis of a neurotrophic factor that promotes the survival and growth of neurons within the hippocampus and cerebral cortex in the AD brain. However, several studies observed that in fact the AD brain has elevated neurotrophic activity compared with the normal aged brain [1,2]. These studies suggested that a soluble extract from AD brain significantly enhanced neuronal survival and neurite outgrowth of cultured cortical neurons in comparison with aged brain extract.
It was proposed that this increased neurotrophic activity could be a cause of the abnormal neurofibrillary changes that occur within the AD brain. Accordingly, if neurite sprouting was left unchecked, then this process could lead to neuronal exhaustion and eventually cell death, partly explaining the vast neuronal loss in the AD brain. Further investigation of the increased neurotrophic activity of the AD brain revealed that it correlated with the loss of a specific neuroinhibitory factor, rather than the presence of a neurotrophic factor. This factor was later isolated and identified as GIF, its name derived from its ability to significantly impair neuronal survival and neurite outgrowth of cultured neurons . It was postulated that a decrease of GIF in AD may contribute to the aberrant neuronal sprouting characteristic of this disease.
Since its discovery, there has been considerable scientific interest in understanding the expression profile of GIF, and how the biochemical properties of GIF confer neurotrophic functions for this protein. In this minireview we will briefly discuss these studies and describe how GIF may influence neural recovery following brain injury or neurodegenerative disease.
Synthesis and secretion of GIF in the brain
Cellular and temporal localization of GIF synthesis
Whilst the MT1 and MT2 isoforms are ubiquitously synthesized throughout most tissues in mammals, GIF is predominantly found within the mammalian central nervous system (CNS) . The less-studied MT4 isoform has a restricted synthesis profile, being found almost exclusively in stratified squamous epithelia . The cell-specific distribution of GIF protein and GIF mRNA has been widely studied, with conflicting results obtained. GIF has been reported at both transcript and protein levels in astrocytes only [1,5], in neurons only [6–8], or in both neurons and astrocytes [9–11]. Despite these conflicting results from a number of studies, the current general consensus is that the GIF protein is primarily synthesized in astrocytes, where it is found predominantly in the soma and fine processes of these cells. Astrocytic synthesis of GIF is mainly found in the cortex, brainstem and spinal cord .
Additionally however, there are also consistent reports of neuronal synthesis of GIF, albeit at seemingly much lower levels than within astrocytes. Neuronal synthesis of GIF is highly localized to specific subsets of cortical and hippocampal neurons, and is found within the axons and dendrites. Neuronal production of GIF is particularly associated with granule cells of the dentate gyrus in the hippocampus, in those neurons that store and release zinc at synapses . While there is substantial evidence for expression of GIF mRNA in neurons from in situ hybridization studies, there is a discrepancy between the co-localization of GIF mRNA and protein, as very few studies are able to demonstrate neuronal localization of GIF at the protein level through immunohistochemistry or similar techniques. Such confirmation at the protein level has been hindered by the availability of appropriate GIF antibodies, and further studies are needed to resolve whether neurons synthesize basal levels of GIF protein.
Neurodegenerative conditions under which GIF levels are altered
The basal level of production of GIF in the CNS is considered to be quite low, particularly in comparison with the MT1 and MT2 isoforms that are more highly expressed by astrocytes in the brain. However, the levels of GIF are altered dramatically in the neurodegenerative or traumatically injured brain. The best-characterized example of this is for AD. Indeed, many studies have analysed the amount of GIF present in AD brain extract compared with non-demented aged controls using both mRNA and protein assays. However, these studies have yielded variable results, with some identifying up-regulation and others down-regulation of GIF levels in the AD brain compared with appropriate age-matched controls [2,12–16]. For instance, immunoreactivity was shown to be reduced in the AD cerebral cortex, specifically in the upper layers of the gray matter, and this correlated with a significantly decreased number of GIF-positive astrocytes . Conversely, other studies have reported elevated GIF protein levels in AD patients when compared with age-matched controls . While it is not clear why GIF levels vary so widely in these studies, it is thought that these results may indicate population-based variations in GIF expression levels. Hence, the expression levels of GIF are significantly reduced in Japanese AD patients [2,12], but do not appear to be reduced in AD patients of North American descent .
By contrast, the synthesis of GIF seems to be down-regulated in most other neurodegenerative diseases, including multiple-system atrophy, Parkinson’s disease, progressive supranuclear palsy and amyotrophic lateral sclerosis, and around senile plaques in Down syndrome . Interestingly, the level of GIF appears to correlate with neuronal loss, as GIF immunoreactivity disappears in areas with vast neuronal loss, but remains in less affected areas.
Given its potent neurite growth-inhibitory properties, it was predicted that the GIF levels might correlate with the failure of the injured brain to regenerate. Indeed, a number of studies have confirmed that levels of GIF protein are up-regulated in reactive astrocytes surrounding cerebral infarcts, stab wounds or excitotoxic brain injuries. For instance, GIF mRNA was substantially up-regulated in reactive astrocytes surrounding degenerated neurons following ventricular injection of kainic acid . Also, GIF was elevated in reactive astrocytes surrounding a stab wound to the brain at 3–4 days post-injury and remained elevated for almost a month [19,20]. The increase in GIF expression in response to these different types of brain injury was often observed in glial cells accumulating at the degenerating area, where the glial scar was beginning to form. Intriguingly, exogenously applied GIF has been shown to induce astrocyte proliferation and migration in vitro . It is possible, then, that the up-regulation and secretion of GIF by astrocytes in the immediate vicinity of a lesion could contribute to the accumulation of reactive astrocytes at the injury site. Interestingly, one study has demonstrated that in the absence of GIF [Gif gene knockout (KO) mice] there were elevated levels of regeneration-associated molecules, such as growth-associated protein 43, following cortical cryolesion . Hence, evidence gathered to date regarding GIF synthesis in response to traumatic brain injury indicates that GIF may be involved in the neuroinhibitory processes that are associated with glial scar formation.
It is important to note that the synthesis of MT1 and MT2 in the injured or diseased brain is quite different from the synthesis of GIF. While GIF synthesis is generally considered to be reduced in neurodegenerative diseases such as AD, amyotrophic lateral sclerosis and Down syndrome, MT1 and MT2 levels are considerably elevated in all of these conditions . In the traumatically injured brain, however, the synthesis of MT1 and MT2, as well as the synthesis of GIF, are up-regulated by reactive astrocytes in the vicinity of the lesion . This suggests that these proteins may have different physiological roles in the stressed brain.
Secretion of GIF
The ability of GIF to inhibit neuronal survival and outgrowth occurs when the protein is applied exogenously to neurons. However, this has raised questions over the physiological relevance of this biological function of GIF, because all MT isoforms have been considered as solely intracellular proteins since their discovery more than 50 years ago. They lack any secretion signal sequences or other such extracellular trafficking signals, and their synthesis has generally been observed within the cytoplasm or nucleus of cells. However, in 2002 it was reported that GIF is secreted by cultured astrocytes . The amount of GIF secreted by the cultured astrocytes was approximately 174 ng·mg−1 of protein, measured using ELISA . This study also determined that the amount of extracellular GIF was around 30% higher than the levels of intracellular GIF in these astrocyte cultures. Furthermore, the absence of cell death suggested that GIF is actively secreted by cells, although the precise mechanism by which GIF is secreted remains unknown. Intriguingly, secretion of the MT1 and MT2 isoforms by cultured astrocytes has also recently been reported. The secretion of MT1 and MT2 by astrocytes appears to be regulated because the basal levels of protein secretion were low and could be stimulated through cytokine-activation of cultured astrocytes . Whether GIF is secreted by a similar mechanism is currently not known. Understanding the specific situations which stimulate GIF secretion by astrocytes will probably reveal important insights into the functional roles of this protein in the brain, and in particular its ability to inhibit neurite outgrowth (as described above).
The functional role of GIF synthesis and secretion in the brain
Taken together, the regulation of GIF synthesis by reactive astrocytes, and its subsequent secretion under certain conditions, could be intimately involved in regulating how the brain responds to and recovers from conditions such as physical or chemical trauma. Based upon the literature referred to above in this minireview, we propose a possible model for how GIF might be involved in the cellular response to brain injury (Fig. 1). As neurons degenerate, the surrounding astrocytes respond by proliferating and assume a reactive phenotype. GIF synthesized by reactive astrocytes may be secreted by these cells to act upon neurons to initially decrease neurite outgrowth in response to the insult. As the condition progresses, perturbation to the interaction between neurons and glial cells may reduce GIF synthesis. Therefore, control over the release of neurotrophic factors, including GIF, by the reactive astrocytes would be compromised. For instance, in the later stages of neurodegenerative diseases the neuronal damage may interfere with the neuroglial interactions, lowering GIF production and secretion in reactive astrocytes. The reduction in GIF would lower the defences against free-radical-mediated attack and protection from neuronal damage. In addition, the reduction in GIF may also allow regenerative sprouting to occur.
Physiological properties of GIF
GIF has several key properties, based upon its unusual biochemical structure, which may influence its function in response to normal or stress-induced conditions. The relationship between protein structure and function is the subject of detailed discussion in two other minireviews in this series [26,27]. In this minireview we will briefly describe the role of GIF in (a) regulation of metal ion homeostasis, (b) free radical scavenging and (c) neurite growth inhibition.
Metal-binding properties of GIF and role in synaptic activity
MTs are generally considered to have an important role in maintaining the homeostasis of key metal ions within the body. Like other MT isoforms, GIF binds both monovalent and divalent metal ions with high affinity. They have the ability to inhibit heavy metal toxicity [i.e. Cd(II), Hg(II)] and regulate the transport and storage of essential heavy metals [Cu(I) and Zn(II)]. However, GIF does not seem to be essential for metal-handling within the body, because studies have reported that GIF-deficient mice were not more prone to Zn or Cd toxicity compared with control animals . However, as GIF has been localized both intracellularly and extracellularly, it may have an important role in metal-related extracellular neurochemistry. It is important to note that the GIF is able to bind Cu(I) and Zn(II) concurrently. For example, GIF isolated from the human brain has been found to contain both Zn(II) and Cu(I), in a Cu4Zn3-GIF structure . The ability of GIF to bind redox-active metal ions, such as Cu(I), may suggest a crucial mechanism of how the protein is able to reduce oxidative stress. The metal-binding properties of GIF are discussed in greater detail in the two other minireviews of this series [26,27].
A clear indication of the physiological functions of GIF and its metal-binding properties has been gained from studies carried out under stress-induced conditions. For example, GIF KO mice are highly susceptible to kainic acid-induced seizures. This was localized to seizure-induced injury to CA3 hippocampal neurons, resulting in the GIF KO mice experiencing more convulsions and greater mortality than their wild-type littermates . GIF-overexpressing mice experience much less kainic acid-induced brain damage compared with controls , implying that GIF may play an important protective role in response to kainic acid-induced seizures.
One explanation of how GIF can protect against epileptic seizures is through its regulation of Zn(II) levels. Synaptically released zinc is thought to play a key role in neuronal signalling, and in response to kainic acid insult, neurons release Zn(II), as well as glutamate, from the synaptic cleft. Interestingly, neurons with a high GIF mRNA content are those known to store zinc in their axon terminals . In addition, the synthesis of GIF within cultured cells led to a significant increase in intracellular Zn(II) stores within those cells . GIF, through its zinc-binding properties, may act to recover synaptically released Zn(II) and enable its recycling into synaptic vesicles. Hence, the absence of GIF would lead to an accumulation of extrasynaptic zinc, which would cause inappropriate synaptic firing and subsequent seizures. Interestingly, GIF may also have a role in regulating synaptic levels of zinc, as the GIF-deficient mice had a reduced content of Zn(II) within a number of brain regions .
ROS scavenging ability of GIF
The coordination of redox-active metal ions to GIF may indirectly prevent ROS generation through metal chelation. Conversely, all MT isoforms have the ability to directly scavenge harmful ROS within the CNS and other tissues. ROS are highly reactive chemical compounds that are constantly generated by metabolic processes in vivo and cause serious damage to DNA, protein and membranes containing polyunsaturated fatty acids. ROS production is a normal by-product of metabolic activity, and under normal conditions, the ROS are effectively quenched before being liberated to cause damage. Regulation of ROS occurs through several ROS scavengers, or antioxidants. Antioxidants can be both enzymatic and non-enzymatic, and function to protect cells against oxidative damage. Specific antioxidants include superoxide dismutase for superoxide, catalase for hydrogen peroxide, and glutathione peroxidase for hydrogen peroxide and lipid peroxide. In addition, there are many other non-specific antioxidants, such as glutathione. Another efficient general free-radical scavenger is MT. MT is a strong nucleophil, because of its high cysteine content, enabling it to efficiently bind reactive ROS .
MT can function as a potent scavenger of hydrogen peroxide, hydroxyl, nitric oxide (NO) and superoxide radicals. Studies have shown that extracellular and intracellular GIF can protect cells from oxidative stress. There have been a number of studies investigating the specific free-radical scavenging ability of GIF. One such study reported that GIF was able to directly scavenge hydroxyl radicals generated in a Fenton-type reaction or by photolysis of hydrogen peroxide. In the same study, GIF was unable to scavenge superoxide (generated in the hypoxanthine oxidase reaction) or NO (generated from NOC-7) . The neuroprotection provided by extracellular GIF against free-radical-mediated attack was subsequently explored in vitro. To induce a condition indicative of oxidative stress, neurons were grown in a hyperoxic environment. Hyperoxia stimulates neuronal cells to differentiate or undergo cell death. Exogenous GIF prevented the induction of neuronal differentiation, quantified by the level of neurite outgrowth in young cortical neuronal cultures. The protein also prevented neuronal cell death in older cultures . GIF not only protected neurons from oxidative stress, but was also shown to directly quench ROS within the cultured neurons. Using an indicator for intracellular ROS, GIF was shown to reduce ROS production in neurons.
Intracellular GIF has been shown to protect against free-radical-mediated attack. The knocking down of endogenous GIF by antisense oligodeoxynucleotides in cultured cortical neurons was used to examine the neuroprotection provided by intracellular GIF against oxidative stress. The reduction in intracellular GIF increased the rate of cortical neuronal death in a hyperoxic environment . In a similar study, it was reported that fibroblasts expressing GIF were more resistant to cellular cytotoxicity induced by hydrogen peroxide . Therefore, both intracellular and extracellular GIF demonstrates a strong ROS-scavenging property that is likely to protect neurons against oxidative damage in stress-induced conditions.
Neurite growth-inhibitory property of GIF
As previously discussed, GIF displays both neurotoxic and neuroinhibitory actions. The neuronal growth-inhibitory properties of GIF may have an important influence on the neural recovery from brain injury or neurodegenerative diseases. The presence of GIF was neurotoxic to cultured rat cortical neurons, inhibited neurite formation in developing neurons and delayed neurite elongation [2,24,31]. Importantly, GIF inhibited regenerative sprouting following axonal transection of mature neurons . However, in the absence of brain extract (either AD or from the normal brain) GIF promotes neuron survival of cultured cortical neurons .
The neuroinhibitory action of GIF is very specific to this MT isoform and can be attributed to the small differences in the protein sequence of this isoform compared with all other mammalian MTs. The human GIF amino acid sequence is 70% similar to human MT2, retaining all the 20 conserved cysteine residues . The only structural differences between GIF and MT1 and MT2 include a Cys-Pro-Cys-Pro(6–9) motif and Thr5 in the β-domain of GIF and a different amino acid structure in the α-domain . Structural and cell biology studies have revealed that the Cys-Pro-Cys-Pro(6–9) motif seems to be critical for the biological activity of GIF. Mutations in this motif, specifically changing the two Pro residues to Ala and Ser (found in human MT2) abolished the neuroinhibitory activity of GIF . Evidence from studies using the inactive mutant of Zn7GIF also liberated similar results, showing that despite the contrasting biological activities of GIF compared with MT1 and MT2, the metal-binding affinities remain comparable. However, the precise spatial structure and the mechanism behind the unique biological activity of GIF remain to be elucidated.
Subsequent studies have investigated whether GIF acts in a neuroinhibitory manner when administered directly into the brain following traumatic brain injury. In one such study, the injection of GIF into the site of a stab wound promoted tissue repair, and the area of the stab wound treated with GIF was significantly smaller than those of control rats . Interestingly, GIF showed dose-dependent activity, with a high dose being detrimental to tissue repair . In addition, motoneurons transfected with GIF prevented loss of injured facial motoneurons . It remains to be elucidated why GIF has neuroprotective functions when administered to the injured brain, versus its neuroinhibitory actions upon cultured neurons.
Current thoughts on the role of GIF in AD
Since its discovery in 1991 as a factor that might be deficient in the AD brain, a number of studies have investigated the role of GIF in the pathogenesis of AD. While this has focussed primarily on the neuronal inhibitory properties of GIF, several recent studies have provided new insight into other mechanisms through which GIF might influence the disease process in AD.
Role of the neurite growth-inhibitory properties of GIF in AD
There have been extensive studies examining the role of GIF in AD, linked to its initial discovery as a factor deficient in the AD brain. There remains no clear consensus on the role of GIF in the pathogenesis of AD, and therefore we will briefly review some of the current thoughts on the role of GIF in AD. There are numerous pathological hallmarks in AD that are commonly accompanied by neuronal alterations. These physical alterations include aberrant neurite sprouting and significant neuronal loss.
One hypothesis for why there is pronounced neural dysfunction and death is the presence of increased neurotrophic activity in the AD brain. It was on this assumption that GIF was first discovered as a neuronal growth-inhibitory factor, and a lack of GIF might contribute to the generation of neurofibrillary changes within the AD brain, including inappropriate neurite sprouting. Abnormal neurite sprouting might then lead to neuronal exhaustion and eventually cell death. In vitro studies have clearly demonstrated the ability of GIF to block neurite outgrowth, supporting the initial proposal of Uchida et al.  that a lack of GIF might be involved in AD pathogenesis. However, postmortem human studies have provided mixed results on the level of GIF present in the AD brain. The generation of GIF KO or GIF overexpressing mice with tau-based experimental mouse models of AD might provide considerable insight into the potential role of GIF in neurofibrillary changes associated with AD.
Potential role of metal-binding/exchange properties of GIF in AD
One of the primary pathological hallmarks of AD is the formation of β-amyloid (Aβ) plaques, composed primarily of Aβ(1–40) and Aβ(1–42) peptides, which associate to form abnormal extracellular deposits of fibrils and amorphous aggregates . Aβ is a metalloprotein that possesses high- and low-affinity Cu(I)- and Zn(II)-binding sites. Furthermore, metal ion homeostasis within the AD brain is dramatically unbalanced and is proposed to be involved in the pathology of Aβ. For example, within the amyloid plaques the Cu(I) and Fe(III)/Zn(II) concentrations were ∼ 0.4 and ∼ 1 mm respectively, compared with 70 and 340–350 μm, respectively, in healthy neocortical parenchyma . The close association between metal ions and Aβ has prompted many groups to investigate numerous metal chelators to establish whether they have the potential to modify Aβ pathogenesis in AD.
Intriguingly, GIF has strong Cu(I)- and Zn(II)-binding efficiencies, suggesting that it may be able to modify metal-induced Aβ aggregation in AD. In this context, it has recently been reported that Zn7GIF was shown to remove Cu from aggregated Aβ(1–40)-Cu(II) with the concomitant binding of Zn, leading to the formation of SDS-soluble monomeric zinc-bound Aβ and soluble copper-bound GIF . Therefore, the metal swap led to simultaneous modification of the final form of Aβ(1–40) and suggests that it caused the de-aggregation of Aβ. In a therapeutic context, this indicates that MT might potentially promote the clearance of Aβ plaques. However, it is important to note that recent therapeutic approaches have been successful in clearing Aβ plaques in clinical trials, but not for reducing neurodegeneration in the AD brain .
It is important to note that while Aβ aggregation can be induced by both Cu(II) and Zn(II), only the Cu(II)-induced aggregation of Aβ is toxic. The neurotoxicity of Cu(II)-induced Aβ aggregation is related to the generation of ROS through the redox cycling of Cu. In this regard, GIF has been demonstrated to not only prevent Aβ aggregation, but also block the generation of ROS by Aβ-Cu(II) [27,38]. Therefore, the ability of GIF to redox-silence metal ions and directly scavenge ROS (discussed above) provide two distinct mechanisms through which GIF can act to protect against Aβ pathogenesis and neurotoxicity in AD (Fig. 2).
The discovery of a brain-specific MT isoform, GIF, in 1991, sparked considerable interest in understanding the role of all MTs in the brain, and particularly within the injured or diseased brain. In the case of GIF, the protein has many neuroprotective properties that could provide vital protection during stress-induced conditions. Studies have already focused on the ability of GIF to protect against Aβ pathology and neurotoxicity in AD, with promising results. By contrast, GIF also demonstrates a unique neuroinhibitory property, which may promote cellular survival and repair following injury, but could also be detrimental in neurodegenerative diseases. The studies to date have only just started to investigate and understand the physiological roles of GIF within the brain. Further studies are required to determine the function of GIF within the normal, injured and neurodegenerative brain, and to establish the therapeutic potential of the protein.
This work was supported by an Australian Research Council (ARC) Discovery Project Grant (DP0556630; RSC), and funding from the Jack & Ethel Goldin Foundation (Alzheimer’s Australia). RSC holds an ARC Research Fellowship.