Tài liệu Báo cáo khoa học: Neuronal growth-inhibitory factor (metallothionein-3): evaluation of the biological function of growth-inhibitory factor in the injured and neurodegenerative brain pdf

MINIREVIEW
Neuronal growth-inhibitory factor (metallothionein-3):
evaluation of the biological function of growth-inhibitory
factor in the injured and neurodegenerative brain
Claire Howells, Adrian K. West and Roger S. Chung
Menzies Research Institute, University of Tasmania, Hobart, Australia
Introduction
Metallothioneins (MTs) are a family of unusual cyste-
ine-rich (30%), 6–7 kDa proteins synthesized predomi-
nantly 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 approx-
imately 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 proper-
ties, suggesting that this MT isoform has different and
distinct functions within the brain. Furthermore, the
discovery and continued investigation of this brain-spe-
cific 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 neurofibril-
lary tangles, dystrophic neurites or curly fibres, and
Keywords
brain injury; metals; neuronal growth-
inhibitory factor (GIF); neurodegenerative
disease; oxidative stress
Correspondence
R. S. Chung, PhD, Private Bag 58,
University of Tasmania, Hobart, Tasmania
7001, Australia
Fax: +61 3 62262703
Tel: +61 3 62262657
E-mail:
(Received 27 November 2009, revised
13 March 2010, accepted 19 May 2010)
doi:10.1111/j.1742-4658.2010.07718.x
Neuronal growth-inhibitory factor, later renamed metallothionein-3, is one
of four members of the mammalian metallothionein family. Metallothione-
ins are a family of ubiquitous, low-molecular-weight, cysteine-rich proteins.
Although neuronal growth-inhibitory factor shares metal-binding and reac-
tive 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-inhibi-
tory factor within the brain, particularly in response to brain injury or

during neurodegenerative disease progression.
Abbreviations
AD, Alzheimer’s disease; Ab, b-amyloid; CNS, central nervous system; GIF, neuronal growth-inhibitory factor; KO, knockout;
MT, metallothionein; NO, nitric oxide; ROS, reactive oxygen species.
FEBS Journal 277 (2010) 2931–2939 ª 2010 The Authors Journal compilation ª 2010 FEBS 2931
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 neu-
rons 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 neurofibril-
lary changes that occur within the AD brain. Accord-
ingly, if neurite sprouting was left unchecked, then this
process could lead to neuronal exhaustion and eventu-
ally 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 fac-

tor. This factor was later isolated and identified as
GIF, its name derived from its ability to significantly
impair neuronal survival and neurite outgrowth of cul-
tured neurons [2]. 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 sci-
entific 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 fol-
lowing 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) [3]. The less-studied MT4 iso-
form has a restricted synthesis profile, being found
almost exclusively in stratified squamous epithelia [4].
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 [6].
Additionally however, there are also consistent
reports of neuronal synthesis of GIF, albeit at seem-
ingly much lower levels than within astrocytes. Neuro-
nal synthesis of GIF is highly localized to specific
subsets of cortical and hippocampal neurons, and is
found within the axons and dendrites. Neuronal pro-
duction 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 [7].
While there is substantial evidence for expression of
GIF mRNA in neurons from in situ hybridization
studies, there is a discrepancy between the co-localiza-
tion 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 appropri-
ate 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 neurode-
generative 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. How-
ever, these studies have yielded variable results, with
some identifying up-regulation and others down-regu-
lation 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 lay-
ers of the gray matter, and this correlated with a sig-
nificantly decreased number of GIF-positive astrocytes
[2]. Conversely, other studies have reported elevated
Function of GIF in injured and degenerative brain C. Howells et al.
2932 FEBS Journal 277 (2010) 2931–2939 ª 2010 The Authors Journal compilation ª 2010 FEBS
GIF protein levels in AD patients when compared
with age-matched controls [14]. 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
[15].
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 syn-
drome [17]. 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 proper-
ties, it was predicted that the GIF levels might corre-
late 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 excito-
toxic brain injuries. For instance, GIF mRNA was
substantially up-regulated in reactive astrocytes sur-
rounding degenerated neurons following ventricular
injection of kainic acid [18]. 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 begin-
ning to form. Intriguingly, exogenously applied GIF
has been shown to induce astrocyte proliferation and
migration in vitro [14]. 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 mole-
cules, such as growth-associated protein 43, following
cortical cryolesion [21]. 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 gen-
erally considered to be reduced in neurodegenerative
diseases such as AD, amyotrophic lateral sclerosis and
Down syndrome, MT1 and MT2 levels are consider-
ably elevated in all of these conditions [22]. In the trau-
matically injured brain, however, the synthesis of MT1
and MT2, as well as the synthesis of GIF, are up-regu-
lated by reactive astrocytes in the vicinity of the lesion
[23]. 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 exoge-
nously to neurons. However, this has raised questions
over the physiological relevance of this biological func-
tion of GIF, because all MT isoforms have been con-
sidered as solely intracellular proteins since their
discovery more than 50 years ago. They lack any secre-
tion signal sequences or other such extracellular traf-
ficking 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 [24]. The amount of GIF
secreted by the cultured astrocytes was approximately
174 ngÆmg
)1
of protein, measured using ELISA [24].
This study also determined that the amount of extra-
cellular GIF was around 30% higher than the levels of
intracellular GIF in these astrocyte cultures. Further-
more, the absence of cell death suggested that GIF is
actively secreted by cells, although the precise mecha-
nism 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 [25]. Whether
GIF is secreted by a similar mechanism is currently
not known. Understanding the specific situations
which stimulate GIF secretion by astrocytes will prob-
ably 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
C. Howells et al. Function of GIF in injured and degenerative brain
FEBS Journal 277 (2010) 2931–2939 ª 2010 The Authors Journal compilation ª 2010 FEBS 2933
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 reac-
tive 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 pro-
tection from neuronal damage. In addition, the reduc-
tion 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 func-
tion 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 ani-
mals [26]. However, as GIF has been localized both
intracellularly and extracellularly, it may have an
important role in metal-related extracellular neuro-
chemistry. 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 Cu
4
Zn
3
-GIF struc-
ture [2]. 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 condi-
tions. For example, GIF KO mice are highly suscepti-
ble to kainic acid-induced seizures. This was localized
Neuron
GIF
GIF
Low GIF
Reactive astrocyte
Astrocyte
ROS
GIF GIF
ROS
Axon injury
ABC
Fig. 1. Diagrammatic model describing the
possible physiological roles of GIF at differ-
ent stages following a traumatic brain injury.
(A) Under normal conditions there is a low
level of GIF synthesis within astrocytes and
neurons. (B) Following brain injury, astro-
cytes assume the reactive phenotype and
increase their synthesis of GIF. Reactive
astrocytes secrete GIF, which then acts to
scavenge harmful ROS and blocks axon

regeneration during the initial responses to
the injury. (C) At later stages, interference in
neuroglial interactions may decrease GIF
production and secretion by surrounding
astrocytes. The decreased levels of extra-
cellular GIF would allow ROS-mediated
attack on astrocytes and neurons, and may
also promote excessive neurite sprouting.
Function of GIF in injured and degenerative brain C. Howells et al.
2934 FEBS Journal 277 (2010) 2931–2939 ª 2010 The Authors Journal compilation ª 2010 FEBS
to seizure-induced injury to CA3 hippocampal neu-
rons, resulting in the GIF KO mice experiencing more
convulsions and greater mortality than their wild-type
littermates [28]. GIF-overexpressing mice experience
much less kainic acid-induced brain damage compared
with controls [26], 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) lev-
els. 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 gluta-
mate, from the synaptic cleft. Interestingly, neurons
with a high GIF mRNA content are those known to
store zinc in their axon terminals [7]. In addition, the
synthesis of GIF within cultured cells led to a signifi-
cant increase in intracellular Zn(II) stores within those
cells [7]. 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 extrasynap-
tic 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 [28].
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 com-
pounds 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 sev-
eral ROS scavengers, or antioxidants. Antioxidants
can be both enzymatic and non-enzymatic, and func-
tion to protect cells against oxidative damage. Specific
antioxidants include superoxide dismutase for superox-
ide, catalase for hydrogen peroxide, and glutathione
peroxidase for hydrogen peroxide and lipid peroxide.
In addition, there are many other non-specific antioxi-
dants, such as glutathione. Another efficient general
free-radical scavenger is MT. MT is a strong nucleo-
phil, because of its high cysteine content, enabling it to

efficiently bind reactive ROS [29].
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 investi-
gating 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 scav-
enge superoxide (generated in the hypoxanthine oxi-
dase reaction) or NO (generated from NOC-7) [24].
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 [24]. GIF not
only protected neurons from oxidative stress, but was
also shown to directly quench ROS within the cul-
tured 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 neu-
roprotection provided by intracellular GIF against oxi-
dative stress. The reduction in intracellular GIF
increased the rate of cortical neuronal death in a
hyperoxic environment [24]. In a similar study, it was
reported that fibroblasts expressing GIF were more
resistant to cellular cytotoxicity induced by hydrogen
peroxide [30]. Therefore, both intracellular and extra-
cellular GIF demonstrates a strong ROS-scavenging
property that is likely to protect neurons against oxi-
dative 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
C. Howells et al. Function of GIF in injured and degenerative brain
FEBS Journal 277 (2010) 2931–2939 ª 2010 The Authors Journal compilation ª 2010 FEBS 2935
transection of mature neurons [31]. However, in the
absence of brain extract (either AD or from the nor-
mal brain) GIF promotes neuron survival of cultured
cortical neurons [31].
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
[2]. The only structural differences between GIF and
MT1 and MT2 include a Cys-Pro-Cys-Pro(6–9) motif
and Thr5 in the b-domain of GIF and a different
amino acid structure in the a-domain [32]. 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 neuroinhibi-
tory activity of GIF [33]. Evidence from studies using
the inactive mutant of Zn
7
GIF 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 [34]. Interestingly,

GIF showed dose-dependent activity, with a high dose
being detrimental to tissue repair [34]. In addition,
motoneurons transfected with GIF prevented loss of
injured facial motoneurons [35]. It remains to be eluci-
dated why GIF has neuroprotective functions when
administered to the injured brain, versus its neuro-
inhibitory 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 con-
sensus 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 numer-
ous pathological hallmarks in AD that are commonly
accompanied by neuronal alterations. These physical
alterations include aberrant neurite sprouting and sig-
nificant neuronal loss.
One hypothesis for why there is pronounced neural

dysfunction and death is the presence of increased neu-
rotrophic activity in the AD brain. It was on this
assumption that GIF was first discovered as a neuro-
nal 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. [2] that a lack of GIF might
be involved in AD pathogenesis. However, postmor-
tem 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 pro-
vide 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 b-amyloid (Ab) plaques, composed
primarily of Ab(1–40) and Ab(1–42) peptides, which
associate to form abnormal extracellular deposits of
fibrils and amorphous aggregates [36]. Ab is a metallo-
protein 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 Ab. For example, within the amyloid pla-

ques the Cu(I) and Fe(III) ⁄ Zn(II) concentrations were
 0.4 and  1mm respectively, compared with 70 and
340–350 lm, respectively, in healthy neocortical paren-
chyma [37]. The close association between metal ions
and Ab has prompted many groups to investigate
numerous metal chelators to establish whether they
have the potential to modify Ab pathogenesis in AD.
Function of GIF in injured and degenerative brain C. Howells et al.
2936 FEBS Journal 277 (2010) 2931–2939 ª 2010 The Authors Journal compilation ª 2010 FEBS
Intriguingly, GIF has strong Cu(I)- and Zn(II)-bind-
ing efficiencies, suggesting that it may be able to mod-
ify metal-induced Ab aggregation in AD. In this
context, it has recently been reported that Zn
7
GIF was
shown to remove Cu from aggregated Ab(1–40)-Cu(II)
with the concomitant binding of Zn, leading to the for-
mation of SDS-soluble monomeric zinc-bound Ab and
soluble copper-bound GIF [38]. Therefore, the metal
swap led to simultaneous modification of the final
form of Ab(1–40) and suggests that it caused the
de-aggregation of Ab. In a therapeutic context, this
indicates that MT might potentially promote the clear-
ance of Ab plaques. However, it is important to note
that recent therapeutic approaches have been success-
ful in clearing Ab plaques in clinical trials, but not for
reducing neurodegeneration in the AD brain [39].
It is important to note that while Ab aggregation can
be induced by both Cu(II) and Zn(II), only the Cu(II)-
induced aggregation of Ab is toxic. The neurotoxicity of

Cu(II)-induced Ab aggregation is related to the genera-
tion of ROS through the redox cycling of Cu. In this
regard, GIF has been demonstrated to not only prevent
Ab aggregation, but also block the generation of ROS
by Ab-Cu(II) [27,38]. Therefore, the ability of GIF to
redox-silence metal ions and directly scavenge ROS (dis-
cussed above) provide two distinct mechanisms through
which GIF can act to protect against Ab pathogenesis
and neurotoxicity in AD (Fig. 2).
Conclusion
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 Ab pathology and neurotoxic-
ity 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 neurodegenera-
tive 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.
Acknowledgements
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.
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Cu-GIF
+
Reducing
agent
Reducing
agent
Zn-GIF
+
+
Aβ-Zn
i)
Aβ
β
-Cu(II)
A
β
-Cu(I) A

β
-Cu(II) A
β
-Cu(II)A
β
-Cu(I)A
β
-Cu(II)
O
2
ROS O
2
ROS
+
ii
)
Zn-GIF
Cu-GIF
)
AB
Fig. 2. A diagrammatic model describing the possible protective properties of GIF against Ab pathogenesis and neurotoxicity in Alzheimer’s
disease. (A) In the presence of a reducing agent, the copper coordinated to the Ab can undergo redox cycling to produce harmful ROS that
damage the neurons. (B) MT is likely to protect against Ab-Cu(II) by a number of mechanisms: Zn
7
GIF is proposed to undergo a transmetalla-
tion event with the Ab-Cu(II), which removes the redox-active Cu(II) from the Ab, coordinating it to MT as redox-stable Cu(I), while simulta-
neously Ab coordinates redox-inert Zn. This event would prevent the generation of ROS. ZnGIF or CuGIF could also directly scavenge ROS.
C. Howells et al. Function of GIF in injured and degenerative brain
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