Neurobiology of Disease 4, 137169 (1997)

Article No. NB970163

REVIEW
Zinc Metabolism in the Brain:
Relevance to Human Neurodegenerative Disorders

Math P. Cuajungco* and Gordon J. Lees*,

*Department of Psychiatry and Behavioural Science and Department of Pharmacology
and Clinical Pharmacology, University of Auckland School of Medicine, Private Bag 92019,
Auckland, New Zealand

Received May 12, 1997; accepted August 25, 1997

Zinc is an important trace element in biology. An important pool of zinc in the brain is the one present in
synaptic vesicles in a subgroup of glutamatergic neurons. In this form it can be released by electrical
stimulation and may serve to modulate responses at receptors for a number of different neurotransmit-
ters. These include both excitatory and inhibitory receptors, particularly the NMDA and GABAA
receptors. This pool of zinc is the only form of zinc readily stained histochemically (the chelatable zinc
pool), but constitutes only about 8% of the total zinc content in the brain. The remainder of the zinc is
more or less tightly bound to proteins where it acts either as a component of the catalytic site of enzymes or in
a structural capacity. The metabolism of zinc in the brain is regulated by a number of transport proteins, some
of which have been recently characterized by gene cloning techniques. The intracellular concentration may
be mediated both by efflux from the cell by the zinc transporter ZrT1 and by complexing with apothionein to
form metallothionein. Metallothionein may serve as the source of zinc for incorporation into proteins,
including a number of DNA transcription factors. However, zinc is readily released from metallothionein by
disulfides, increasing concentrations of which are formed under oxidative stress. Metallothionein is a very
good scavenger of free radicals, and zinc itself can also reduce oxidative stress by binding to thiol groups,
decreasing their oxidation. Zinc is also a very potent inhibitor of nitric oxide synthase. Increased levels of
chelatable zinc have been shown to be present in cell cultures of immune cells undergoing apoptosis. This is
very reminiscent of the zinc staining of neuronal perikarya dying after an episode of ischemia or seizure
activity. Thus a possible role of zinc in causing neuronal death in the brain needs to be fully investigated.
Intraventricular injections of calcium EDTA have already been shown to reduce neuronal death after a period
of ischemia. Pharmacological doses of zinc cause neuronal death, and some estimates indicate that
extracellular concentrations of zinc could reach neurotoxic levels under pathological conditions. Zinc is
released in high concentrations from the hippocampus during seizures. Unfortunately, there are contrasting
observations as to whether this zinc serves to potentiate or decrease seizure activity. Zinc may have an
additional role in causing death in at least some neurons damaged by seizure activity and be involved in the
sprouting phenomenon which may give rise to recurrent seizure propagation in the hippocampus. In
Alzheimers disease, zinc has been shown to aggregate b-amyloid, a form which is potentially neurotoxic.
The zinc-dependent transcription factors NF-kB and Sp1 bind to the promoter region of the amyloid
precursor protein (APP) gene. Zinc also inhibits enzymes which degrade APP to nonamyloidogenic peptides
and which degrade the soluble form of b-amyloid. The changes in zinc metabolism which occur during
oxidative stress may be important in neurological diseases where oxidative stress is implicated, such as
Alzheimers disease, Parkinsons disease, and amyotrophic lateral sclerosis (ALS). Zinc is a structural
component of superoxide dismutase 1, mutations in which give rise to one form of familiar ALS. After HIV
infection, zinc deficiency is found which may be secondary to immune-induced cytokine synthesis. Zinc is
involved in the replication of the HIV virus at a number of sites. These observations should stimulate further
research into the role of zinc in neuropathology. r 1997 Academic Press
Key Words: zinc metabolism; metallothionein; zinc neurotoxicity; Alzheimers disease; epilepsy;
cerebral ischemia; neurodegenerative disease.

0969-9961/97 $25.00
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138
INTRODUCTION
Zinc is one of the important trace elements in
biological systems (Bettger & ODell, 1981; Golden,
1989; Mills, 1989; Vallee, 1983). It is redox inert and has
structural, catalytic, and regulatory roles in cellular
biology (Bettger & ODell, 1981; Vallee, 1983; Williams,
1989). Zinc is a small ion (0.65 ) and binds mostly to
nitrogen (N) and sulfur (S) donors, but easily ex-
changes ligands due to its low ligand field stabilization
energy (Williams, 1989). Thus, zinc is exchangeable
between cell compartments. In addition to enzymatic
and structural roles for zinc, several motifs of deoxyri-
bonucleic acid (DNA)-binding proteins have been
identified and characterized as zinc fingers, zinc clus-
ters, and zinc twist (Vallee et al., 1991). Hence, cellular
zinc is also crucial for genetic expression. The biochem-
istry of zinc has been extensively reviewed (Vallee &
Falchuk, 1993).
There is circumstantial evidence suggesting that zinc
might be involved in several neurological dysfunc-
tions. Postmortem studies have revealed significant
variations of zinc content in the pathologic brain
(Andrasi et al., 1995; Constantinidis, 1990, 1991; Corri-
gan et al., 1993; Deibel et al., 1996; Deng et al., 1994;
Dexter et al., 1989a,b, 1991; Jenner et al., 1992; Wenstrup
et al., 1990; Yasui et al., 1993). Further, chelatable zinc is
mobilized and accumulates in degenerating neurons
after transient cerebral ischemia (Koh et al., 1996;
Tonder et al., 1990) and seizure activity (Frederickson et
al., 1989). Some calcium-binding proteins (CBPs) with
helixloophelix motifs (EF-hand motif) contain not
only calcium, but also high-affinity zinc-binding sites
(Baudier et al., 1983; Calissano et al., 1974; Filipek et al.,
1990). Of these proteins, S-100b has been functionally
correlated with various diseases of the brain (Griffin et
al., 1989, 1995; Sheng et al., 1996; Van Eldik & Griffin,
1994). Zinc interacts with the proteins, b-amyloid and
its precursor protein, which are believed to be in-
volved in the causation of degenerative processes in
the brain, particularly in Alzheimers disease (Bush et
al., 1993, 1994a, 1994b, 1995, 1996; Clements et al., 1996;
Esler et al., 1996; Mantyh et al., 1993). Zinc has inhibi-
tory effects on several enzymes critical for normal cell
functioning (Brewer et al., 1979; Donaldson et al., 1971;
Link & von Jagow, 1995; Mustafa et al., 1971; Persechini
et al., 1995; Zhang et al., 1990). This review will
concentrate on both vesicular and ionic zinc contents
in cells that could possibly be perturbed during parox-
ysmal activity and degenerative events in the brain.
We will present and evaluate current understanding
with regard to the role that zinc might play in cellular
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Cuajungco and Lees
degeneration and/or proliferation. We will explore
possible connections between zinc and its effects on
proteinprotein interactions, genetic expression, neuro-
trophic activity, oxidative stress, and programmed cell
death.
1. OVERVIEW OF ZINC METABOLISM IN
THE BRAIN
Localization of Chelatable Zinc in the Central
Nervous System
The content of transition metals in the mammalian
central nervous system (CNS), especially zinc, has
been studied using techniques such as histochemistry
(Danscher, 1981; Danscher et al., 1985; Haug, 1967;
Kozma et al., 1981; Slomianka et al., 1990), histofluores-
cence (Frederickson et al., 1987a), stable-isotope dilu-
tion (Frederickson et al., 1982, 1983), and retrograde/
anterograde techniques (Casanovas-Aguilar et al., 1995;
Christensen et al., 1992; Haug et al., 1971; Howell &
Frederickson, 1989; Kesslak et al., 1987; Long et al.,
1995). There are three main sources of zinc in the brain,
namely: (a) a vesicular pool localized in the synaptic
vesicles of nerve terminals; (b) a membrane-bound,
metalloprotein, or proteinmetal complex pool in-
volved in both metabolic reactions and nonmetabolic
reactions such as structural support for biomembranes
and protein folds; and (c) an ionic pool of free or
loosely bound ions in the cytoplasm (Frederickson,
1989). There are undoubtedly both vesicular and metal-
locomplex pools of zinc in the CNS. However, the
existence of free zinc (Zn21) in the cellular milieu
seems unlikely (Frederickson, 1989) since a number of
zinc-binding proteins are present in brain tissue (Ebadi,
1991; Ebadi & Hama, 1986; Itoh et al., 1983). Neverthe-
less, there is an indication that very low concentrations
of an intracellular ionic pool of zinc exist (Reyes, 1996;
Simons, 1991; Williams, 1989). Chelatable or labile
refers to the ability of complexing agents to abstract
Zn21 from zinc-bound cellular proteins within a given
parameter (e.g., binding affinity of the metalligand
complex). Chelatable zinc is easily detected using the
various histological techniques. The source of the
chelatable zinc pool is believed to be vesicular compart-
ments, loosely bound zinc protein, and ionic zinc.
Among these, the vesicular zinc pool is quantitatively
the most significant (Danscher et al., 1985; Frederick-
son et al., 1983; Haug, 1967; Perez-Clausell & Danscher,
1985). Neurons that have a chelatable zinc pool in their
terminal boutons and release zinc during synaptic
Zinc Metabolism in the Brain
activity are termed zinc-enriched neurons (ZEN)
(Danscher, 1996; Frederickson, 1989). The identifica-
tion of ZEN in the mammalian brain has been previ-
ously reviewed (Frederickson & Moncrieff, 1994; Slomi-
anka, 1992). The metabolic or structural pool of zinc
may not be chelatable due to its strong complexation
with proteins and/or location of zinc deep within the
protein structure (Frederickson, 1989). In the CNS,
labile zinc is mainly detected in the telencephalon,
with particularly high concentrations in the vesicular
compartments of the hippocampal mossy fiber termi-
nals (Danscher et al., 1985; Frederickson et al., 1983;
Haug, 1967; Perez-Clausell & Danscher, 1985; Slomi-
anka et al., 1990). The neo-Timms sulfide/silver tech-
nique is a histochemical staining method used to
detect heavy metals, has a high selectivity for zinc, and
is the most common method used to detect zinc in
mammalian brain (e.g. see Danscher, 1981, 1996; Haug,
1967; Sloviter, 1982). The TimmDanscher reaction
products label only the chelatable pool of zinc (Dan-
scher, 1981, 1996; Danscher et al., 1985; Perez-Clausell
& Danscher, 1985).
Cellular Zinc Levels
The tissue zinc content in the gray matter is between
150 and 200 M (Ebadi, 1991; Sato et al., 1984), while a
220300 M concentration of terminal bouton zinc is
estimated to be present in the vesicles of the mossy
fibers (Frederickson et al., 1983).
Zn21 is crucial to over 200 proteins and enzymes
(Prasad, 1996; Vallee & Falchuk, 1993). Zn21 concentra-
tions range from #1029 M within the cytoplasm in
most cells to $1023 M in some vesicles (Williams,
1989). In transgenic baby hamster kidney (BHK) cells
that express zinc influx transporter proteins, it was
estimated that vesicular zinc concentrations reach up
to 14 mM when these cells are exposed to high levels of
exogenous zinc (Palmiter et al., 1996a). Whether these
vesicular zinc concentrations can be attained by the
synaptic vesicles of ZENs under pathological condi-
tions in vivo is currently unknown.
Cell survival in vitro is compromised when cells are
exposed to extracellular zinc concentrations ([Zn21]e)
between 225 and 1000 M in neuronal cells (Choi et al.,
1988; Koh & Choi, 1994; Yokoyama et al., 1986) and
between 7.5 and 200 M in nonneuronal cells (Provin-
cialli et al., 1995; Telford & Fraker, 1995). [Zn21]e may
reach 300 M during seizure activity (Assaf & Chung,
1984). Hence, it is essential that cells regulate their
intracellular zinc concentrations ([Zn21]i) through ma-
139
nipulation of ion influx and efflux. In addition, physi-
ological chelation of Zn21 to apothionein or other
zinc-sequestering proteins, and amino acids ensures
homeostatic control (Ebadi, 1991; Palmiter & Findley,
1995; Palmiter et al., 1992, 1996a,b).
Zinc Release and Uptake
Zn21 is released in a calcium-dependent manner
from the hippocampal mossy fiber terminals during
spontaneous activity (Charton et al., 1985), after stimu-
lation evoked electrically (Aniksztejn et al., 1987; How-
ell et al., 1984), after administration of potassium ions
(Aniksztejn et al., 1987; Assaf & Chung, 1984; Charton
et al., 1985), or after administration of kainic acid (KA)
(Assaf & Chung, 1984; Charton et al., 1985). Whether
the liberated zinc is in an ionic form or in a low-affinity
zinc complex remains to be determined. The amount of
zinc that may be released from synaptic vesicles of the
mossy fiber terminals is approximately 8% of the total
hippocampal zinc (Frederickson et al., 1983). It was
suggested that the concentration of zinc released may
reach 300 M at the peak of convulsive activity (Assaf
& Chung, 1984). This concentration was determined
based on the accumulated Zn21 in the perfusing
medium of hippocampal tissue slices. The actual con-
centration within the synaptic cleft may be even
greater as the estimated value from the perfusate is an
average concentration of Zn21 which has been diluted
by the surrounding extracellular fluid.
Neuronal presynaptic terminals sequester Zn21 from
the extracellular space (Howell et al., 1984; Perez-
Clausell & Danscher, 1986; Wensink et al., 1988). High-
and low-affinity uptake mechanisms for zinc were
found by Howell and colleagues (1984) with KD > 15
M and KD > 361 M, respectively; Wensink et al.
(1988) reported an even higher binding affinity con-
stant of KD > 0.25 M. Zinc uptake is saturable and
unaffected by adenosine triphosphate (ATP) levels or
the concentration gradient of sodium ions (Wensink et
al., 1988).
Very recently, three homologous mammalian zinc
transporters (ZnTs) involved in the sequestration
(ZnT-2/ZnT-3) and extrusion (ZnT-1) of zinc from
transfected BHK cells were cloned and functionally
identified (Palmiter & Findley, 1995; Palmiter et al.,
1996a,b). Interestingly, mammalian ZnTs have se-
quence homology with ZnT genes identified in yeast
(Kamizono et al., 1989; Zhao & Eide, 1996). ZnT-1
appears to be mostly localized in the plasma mem-
brane (Palmiter & Findley, 1995) and is expressed in
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140
the CNS and other organs (Palmiter et al., 1996b).
According to Palmiter and Findley (1995), the rate of
zinc efflux increases as the extracellular concentration
is increased, which initially indicates that efflux is an
energy-dependent mechanism and that ZnT-1 is not a
channel or a facilitated transporter. However, it was
found that efflux was unaffected by metabolic inhibi-
tors of oxidative phosphorylation (sodium azide or
potassium cyanide). A similar resistance to azide or
cyanide was observed with the zinc effluxer in fungi
(Vepachedu & Mohan, 1996), indicating functional
homology with the mammalian ZnT-1 transporter.
Thus, ZnT-1 was postulated to be some type of second-
ary active transporter (Palmiter & Findley, 1995). Mean-
while, ZnT-2 was suggested to be a component protein
of vesicular (acidic) intracellular compartments which
facilitates zinc accumulation, but has a low affinity for
zinc and is hardly detectable in the brain (Palmiter et
al., 1996a,b). On the other hand, ZnT-3, another zinc
transporter that may be involved in zinc uptake into
synaptic vesicles of ZENs was very recently cloned by
Palmiters laboratory (Palmiter et al., 1996b). ZnT-3 is
expressed exclusively in murine brain and testis. ZnT-3
messenger ribonucleic acid (mRNA) is detected most
strongly in neurons of the hippocampal dentate gran-
ule cells and pyramidal cells of the CA1 and CA3
regions, as well as in the cerebral cortex. Immunolabel-
ing of ZnT-3 produced staining patterns similar to that
shown by neo-Timms sulfide/silver method in detect-
ing vesicular zinc in the CNS.
Zinc uptake from the blood to the brain is under
homeostatic control (Blair-West et al., 1990; Kasarskis,
1984; Pullen et al., 1995). However, homeostatic control
of intracellular zinc by vesicular sequestration in the
CNS has never been evaluated.
Zinc and Metallothionein
The importance of zinc in cellular functions makes
metallothionein (MT) of critical importance in the
control of its bioavailability (Vallee & Falchuk, 1993).
Metallothionein proteins are a group of low-molecular-
weight (Mr 5 67 kDa), single-polypeptide chains of
60 or more amino acid residues. Currently, there are
only four identified functional mammalian isoforms
(MT-I, -II, -III, and -IV), although an additional six
human genes coding for MT have been characterized
(Stennard et al., 1994). MT has relatively high cysteine
(2530%) and serine/lysine (1218%) contents with a
complete absence of heterocyclic and aromatic amino
acids. It is able to bind up to a total of 7 g atom
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Cuajungco and Lees
zinc/molecule of thionein protein. The reader is re-
ferred to a recent review on the functions of MT
(Vallee, 1995).
MT is ubiquitous in most organs and can be induced
by different endogenous or exogenous stimuli includ-
ing heavy metals and glucocorticoids (Kagi & Schaffer,
1988; Vallee, 1995). Studies have found MT to modu-
late the transfer and homeostasis of metal ions (Cano-
Gauci & Sarkar, 1996; Ebadi, 1991; Ebadi et al., 1995,
1996; Masters et al., 1994; Zeng et al., 1991a,b). MT
prevents zinc deficiency and toxicity in vivo (Kelly et
al., 1996). MT has also been proposed to function as a
detoxifying agent of reactive metals and free radicals
(Sato & Bremner, 1993; Thornalley & Vasak, 1985).
Furthermore, it also regulates gene expression in vitro
via interactions with transcription factors (Cano-Gauci
& Sarkar, 1996; Zeng et al., 1991a,b). In cultured Ehrlich
cells, the cellular concentration of zinc-bound MT was
determined to be approximately 13 M (Krezoski et al.,
1988). MT, however, is not a long-term storage for zinc
as it has a short biological half-life in these cells
(Krezoski et al., 1988). Hence, both vesicular storage
and the control of ZnTs also provide important contri-
butions to zinc homeostasis (Fig. 1).
Expression of MT isoforms is tissue-specific. For
example, MT-I and MT-II are expressed in a zinc-
dependent manner (Atar et al., 1995; Durnam & Pal-
miter, 1981) and are detectable in most mammalian
organs including the brain (Ebadi, 1991; Ebadi et al.,
1995, 1996; Palmiter et al., 1992). Investigations on the
mechanism of convulsive seizures and the consequent
observations of changes in zinc staining in the hippo-
campus led to the discovery of MT in the brain (Ebadi,
1991; Ebadi et al., 1995; Itoh et al., 1983). Two recently
discovered isoforms, MT-III and MT-IV, were observed
to be expressed mainly in brain (Palmiter et al., 1992;
Pountney et al., 1994; Tsuji et al., 1992; Uchida et al.,
1991), and in squamous epithelia, respectively (Quaife
et al., 1994). MT-III has 7 additional amino acids, giving
a total of 68 residues (Tsuji et al., 1992; Uchida et al.,
1991). MT-III is localized among a subset of astrocytes
within the vicinity of neuronal perikarya and den-
drites (Tsuji et al., 1992; Uchida et al., 1991) and is
expressed independently of the common potent induc-
ers of MT-I and MT-II (Palmiter et al., 1992). Recently,
MT-III was detected in ZENs of the hippocampal
mossy fiber in vivo (Erickson et al., 1997; Masters et al.,
1994). It was observed that cultured brain cells express-
ing MT-III sequester exogenously added zinc as evi-
denced by an increased intensity of Timms stain.
MT-III is unique among the other known isoforms in
that it has an inhibiting effect on neuronal growth;
Zinc Metabolism in the Brain 141

FIG. 1. Zinc metabolism cycle. The diagram illustrates cell regulation of intracellular zinc following uptake or permeation through ionotropic
glutamate receptors. Homeostatic control of zinc levels is accomplished via vesicular-mediated and membrane protein-transporter-mediated
sequestration of ions (ZnTs). Zinc buffering is also accomplished by metallothionein protein (MT). MT has a and b clusters that can bind up to a
total of seven zinc atoms. MT synthesis is induced by zinc (and other agents) by liberating the transcription factor MTF-1 from MT transcription
inhibitor (MTI). MTF1 is believed to bind to the metal responsive element (MRE) of the MT gene promoter, thus initiating transcription.
However, the mechanism that controls the expression of the MTF-1 gene or the ZnT gene is not known. TF, transcription factor.

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142 Cuajungco and Lees

hence, it is also known as growth inhibitory factor Zinc Involvement in Cytokine Function
(Erickson et al., 1994; Pountney et al., 1994; Tsuji et al.,
1992; Uchida et al., 1991). Cytokines are various polypeptides that mediate
cellular responses in immunity and inflammation in
both the peripheral nervous system and the CNS
(Hopkins & Rothwell, 1995; Rothwell & Hopkins,
Zinc, Metallothionein, and Gene Expression
1995). Inflammatory and immune responses are known
to deplete serum zinc bioavailability (Bremner & May,
In addition to regulation of MT synthesis, zinc also
1989; Goldblum et al., 1987; Taubeneck et al., 1995). A
plays a major role in the control of the activity of DNA
possible mechanism by which zinc may be depleted is
and RNA (Martinez-Balbas et al., 1995; Rubin & Koide,
through enhanced activities of zinc-requiring enzymes
1973; Vallee, 1983; Vallee & Falchuk, 1993). Activator
(involved in DNA/nucleic acid synthesis) of mitogen-
proteins (AP) regulating transcription and replication
or cytokine-responsive cells (Odeh, 1992).
contain zinc (Vallee & Falchuk, 1993). The genetic
Many cytokines including tumor necrosis factor-a
advantage and relative importance of incorporating
(TNF-a) and interleukin-1 (IL-1) have been implicated
zinc for folding protein domains have been reviewed
in neurological dysfunctions related to neurodegenera-
(OHalloran, 1993; Rhodes & Klug, 1993; Schwabe &
tive conditions (Griffin et al., 1989, 1995; McGeer &
Klug, 1996). Zinc is essential for binding of eukaryotic
McGeer, 1995; Stanley et al., 1994). In vivo, TNF-a
Xenopus laevis transcription factor IIIA (TFIIIA) to the
causes alterations in zinc metabolism leading to deple-
DNA site of the 5S-RNA gene (Martinez-Balbas et al.,
tion of zinc known as hypozincemia (Taubeneck et al.,
1995; Vallee & Falchuk, 1993). Zinc finger proteins
1995), while IL-1 induces not only hypozincemia, but
have high affinities for zinc (KD 5 102810212 M; Shang
also hypoferremia (iron deficiency) (Goldblum et al.,
et al., 1989). The removal of zinc by various complexing
1987).
agents, including MT, antagonizes binding to DNA
The presence of zinc has been histochemically iden-
genes by TFIIIA (Shang et al., 1989; Zeng et al., 1991a)
tified in the 7S-NGF complex (a pro-protein of which
and specificity protein type 1 (Sp1) (Zeng et al., 1991b).
NGF-b is a constituent subunit) present in nonneural
Metal abstraction, however, is reversed by zinc supple-
granule cells (Frederickson et al., 1987b). 7S-NGF has a
mentation (Zeng et al., 1991a,b). Sp1 is a zinc-
high affinity for zinc (Pattison & Dunn, 1976b), while
dependent member of the activator protein family of
murine NGF-b, a functional dimer, also contains zinc-
the transcription apparatus and the first human tran-
binding sites (Holland et al., 1994). Zinc is a potent and
scription factor identified to recognize the specific
specific inhibitor of the native 7S-NGF g-subunit con-
regulatory sequence of the guanine-cytosine box (Tjian,
taining esteropeptidase (Pattison & Dunn, 1976b). It
1995).
has been observed in vitro that chelation of zinc from
Nuclear factor-kB (NF-kB) is a zinc-dependent tran-
7S-NGF using 2,28,288-terpyridine or 8-hydroxyquino-
scription factor that translocates between cell cytoplasm
line-5-sulfonic acid activates the g-esteropeptidase en-
and nucleus (Baeuerle, 1991). NF-kB controls the expres-
zyme and liberates the a, b, and g subunits from the
sion of a variety of genes, including that of immunore-
oligomer (Pattison & Dunn, 1976a,b).
ceptors, cytokines, and several viruses (Baeuerle, 1991).
NF-kB requires zinc for its normal DNA-binding activ-
ity (Zabel et al., 1991). Gold-mediated oxidation of the
zinc-bound cysteines, as well as chelation of zinc using 2. GENERAL MECHANISMS WHICH LINK
1,10-orthophenanthroline (1 mM), can disable NF-kB ZINC TO NEURODEGENERATIVE
binding activity to DNA (Yang et al., 1995). Other DISEASES
zinc-dependent transcription factors include estrogen
receptor (EsR; Cano-Gauci & Sarkar, 1996), erythroid Oxidative Stress
receptor (Eryf-1 also known as GATA-1, NF-E1, or
GF-1; Omichinski et al., 1993), Zif268 (also known as It has been proposed that oxidative stress due to
Krox-24, NGFI-A, or Egr-1; Pavletich & Pabo, 1991), and diminished antioxidant defenses and/or increased
glucocorticoid receptor (Luisi et al., 1991), all of which production of reactive oxygen species (ROS) and free
contain the zinc finger DNA-binding motif. radicals could initiate a cascade of events leading to

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Zinc Metabolism in the Brain
neurodegeneration (Olanow, 1993). However, the pos-
sibility that free radical production is a consequence of
underlying degenerative processes, rather than a ma-
jor cause of degeneration, needs to be borne in mind
(for reviews see Coyle & Puttfarcken, 1993; Lees, 1993).
This section will provide an overview of oxidative
stress and other processes which link zinc to disease
progression. Recent reports suggest a possible involve-
ment of zinc in the regulation of oxidative stress and,
in particular, some form of association with the gluta-
thione (GSH) antioxidative system.
Energy release involves aerobic and anaerobic
mechanisms. In the aerobic process, the reduction of
molecular oxygen (O2) to water (H2O) results in the
formation of ROS (Halliwell & Gutteridge, 1984; Ola-
now, 1993). ROS and free radical species such as
superoxide radical (O22), hydrogen peroxide (H2O2),
peroxynitrite (ONOO2), nitric oxide (NO), hydroxyl
radical (OH), and hypochlorous acid (HOCl) can
cause oxidative stress and cell death when their rate of
production exceeds the cellular capacity to metabolize
them (Halliwell & Gutteridge, 1984).
The glutathione peroxidase family is an important
cellular antioxidant defense mechanism (Jenner, 1993;
Meister, 1983). Peroxidases catalyze the reduction of
H2O2 to H2O and O2. Oxidation of GSH, catalyzed by
glutathione peroxidase (GPO), results in the formation
of oxidized-glutathione (GSSG; glutathione disulfide).
Glutathione reductase (GR) catalyzes the reduction of
GSSG back to GSH. In vivo depletion of GSH shows a
marked correlation to a decrease of GR activity in rat
brains, thus decreasing the capacity to reduce GSSG
and increasing the vulnerability to oxidative stress
(Barker et al., 1996). GSH may also be converted to
mercapturate products catalyzed by glutathione S-trans-
ferase (GST) or degraded back to cysteinylglycine and
g-glutamyl amino acid by g-glutamyl-transpeptidase
(Jenner, 1993, Meister, 1983). GSH and GSH-related
enzymes are differentially distributed in the CNS
(Huang & Philbert, 1995; Kudo et al., 1990).
Disulfide products are abundant in cellular systems
and were recently found to interact with MT by
facilitating the release of zinc from its clusters (Maret,
1994, 1995). Therefore, alterations in the GSH/GSSG
ratio that result in increased concentrations of GSSG
and other biological disulfides could release and el-
evate cytoplasmic zinc levels. As a homeostatic re-
sponse, increased production of MT and/or efflux of
zinc may occur. Interestingly, hepatic GSH deficiency
results in the synthesis of MT I and II isoforms (Sato et
al., 1995). It is possible that the loss of GSH antioxidant
143
defense triggers a compensatory response by synthesiz-
ing MT to neutralize free radicals and to prevent
cytoplasmic accumulation of zinc caused by MT
disulfide interaction. Both zinc and MT have been
proposed as antioxidants against ROS and free radicals
(for reviews see Bray & Bettger, 1990 and Sato &
Bremner, 1993, respectively).
The knowledge gained involving the interactions of
zinc, MT, and the GSH antioxidant family marks an
important event that links their functions to cellular
senescence and death.
The Neurotoxicity Of Zinc
The antioxidant protective effects of zinc can be
overridden at higher levels of zinc. The importance of
increases in endogenous cellular zinc as a neuroprotec-
tive or neurotoxic mechanism (Fig. 2) is discussed in
more detail in subsequent sections. This section dis-
cusses the toxic effects of pharmacological doses of
zinc, which nevertheless correspond to levels which
may be reached in pathological states. The neurotoxic
potential of exogenous zinc in neurons has been
observed both in vivo (Chuah et al., 1995; Cuajungco &
Lees, 1996; Lees & Leong, 1994; Lees et al., 1990) and in
vitro (Choi et al., 1988; Duncan et al., 1992; Koh et al.,
1996; Yokoyama et al., 1986). It is not known, however,
if zinc-induced neurotoxicity occurs only by a necrotic
mechanism of death (cell edema and lysis). Zinc-
induced neuronal death is associated with excitotoxic
injury (see next section). Even though excitotoxic
injury typically results in necrosis, recent reports
showed that apoptosis (gene-directed cell death) may
also occur after an excitotoxic insult (Ankarcrona et al.,
1995; Bonfoco et al., 1995; Portera-Cailliau et al., 1995).
This section will concentrate on necrotic cell death,
while the association of zinc with apoptotic death will
be discussed later.
Zinc exposure at a nonphysiological dose ($600
M) for 15 min resulted in neuronal death in mixed
cultures of cortical and glial cells, while exposure for a
longer duration (1824 h) at lower concentrations
(#300 M) induces glial cell death (Choi et al., 1988;
Yokoyama et al., 1986). In vivo, intranasal irrigation of
zinc (17 mol) in mice produced axonal degeneration
of olfactory nerve bundles (Schwann cells) (Chuah et
al., 1995). Some olfactory Schwann cells were immu-
nopositive for S-100 protein and appeared to have
expanded perinuclear space (Chuah et al., 1995). An
association between S-100 proteins and neurodegenera-
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FIG. 2. A schematic summary representing several cellular protein and enzyme activities influenced by both intracellular and extracellular zinc
levels. (A) The diagram shows that an increase of extracellular levels of zinc through vesicular release and ZnT-mediated efflux may affect
several extracellular processes. Several inhibitory and excitatory receptors, neurotransmitter uptake proteins, and matrix metalloproteinases
(MMPs), enzymes that degrade basement membrane (BM) of the extracellular matrix (ECM), have been found to be modulated by the presence
of zinc. (B) The diagram illustrates several mechanisms where intracellular zinc elevation may occur. A scenario is depicted where intracellular
zinc overload may overwhelm homeostatic control mechanisms in cells, consequently producing detrimental effects on various protein and
enzyme activities necessary for normal cell functioning. It is interesting to note that zinc has also been observed to yield a positive effect on
apoptotic cell death, whereby it may inhibit endonuclease-mediated DNA cleavage or it may activate PARP DNA repair enzyme, thus,
precluding apoptosis.

144
Zinc Metabolism in the Brain
tion is discussed elsewhere in this review. While the
above in vitro and in vivo observations clearly show
that zinc is neurotoxic, due to diffusion and dilution by
the extracellular fluid (in vivo) or intracellular fluid (in
vitro), the final zinc levels reached in cells after expo-
sure to zinc is difficult to determine. In addition, the
minimum concentration required to induce neuronal
death in vivo is unknown (Lees et al., 1990).
Blocking the Toxic Effects of Zinc
Our endeavor to prevent zinc neurotoxicity in vivo
led us to look for complexing agents that would inhibit
the effects of exogenous zinc-induced neuronal death
(Cuajungco & Lees, 1996). This rationale is analogous
to a report that chelating calcium in vitro and in vivo
protects neurons from damage due to prolonged hippo-
campal tissue stimulation (Scharfman & Schwartz-
kroin, 1989) or focal cortical ischemia (Tymianski et al.,
1993), respectively. The protective effect of the calcium
chelator, however, was evident only in cells that do not
contain CBPs (Scharfman & Schwartzkroin, 1989).
CBPs are known to buffer Ca21 and contribute to
intracellular calcium concentration ([Ca21]i) homeosta-
sis (Heizmann & Braun, 1995). Using metal complex-
ing agents, it was shown that membrane permeable
chelators N,N,N8,N8-tetrakis(2-pyridylmethyl)ethylene-
diamine (TPEN) (Cuajungco & Lees, 1996) and pyrithi-
one (Cuajungco & Lees, manuscript submitted) attenu-
ate zinc neurotoxicity in vivo, while the membrane
impermeant chelator ethylenediamine tetraacetic acid
(EDTA) reduces zinc toxicity in vitro (Koh et al., 1996).
Note that neither TPEN nor pyrithione showed inher-
ent toxicity in the absence of exogenous metal ions in
the rat hippocampus. The implications of chelating
intracellular zinc using TPEN both in vivo and in vitro
are discussed in the forthcoming section on apoptosis.
Putative Mechanisms of Zinc-Induced Neuronal
Cell Death
The presence of zinc-binding proteins (Ebadi, 1991;
Ebadi & Hama, 1986; Itoh et al., 1983) and possibly an
efflux protein transporter similar to ZnT-1 (Palmiter &
Findley, 1995) serves as homeostatic control mecha-
nisms. However, aberrations in the metabolism and
buffering of zinc resulting in an excess of [Zn21]i and
its subsequent extrusion resulting in elevated [Zn21]e
could be detrimental to cells. The exact mechanism of
zinc-induced cell death is currently unknown. There
are several ways zinc may augment or directly trigger
145
neuronal death by interactions with various cell recep-
tors, enzymes, and/or other metal ions present in the
cellular environment (Fig. 2).
a. Receptor Interactions
The ultrastructural localization of several neuro-
transmitters present in the hippocampal mossy fiber
terminals, especially glutamate (Beaulieu et al., 1992;
Bramham et al., 1990; Kesslak et al., 1987; Stengaard-
Pedersen et al., 1983; Storm-Mathisen & Ottersen,
1990), coincides with the location of ZENs (Frederick-
son et al., 1982, 1983; Haug, 1967; Haug et al., 1971;
Perez-Clausell & Danscher, 1985). Modification of the
toxicity of excitatory amino acids (EAAs) is thus a
possible mechanism by which zinc may induce neuro-
nal death. For example, zinc exacerbates the toxicity of
a-amino-3-hydroxy-5-methyl-4-isoxazole propionic
acid (AMPA) (Freund & Reddig, 1994; Koh & Choi,
1988; Koh et al., 1996; Weiss et al., 1993) and KA (Nave
& Connor, 1993; Shiraishi et al., 1993; Yin & Weiss,
1995), but reduces N-methyl-D-aspartate (NMDA) tox-
icity (Koh & Choi, 1988; Peters et al., 1987; Xie et al.,
1993). The alteration of EAA neurotoxicity is due to an
interaction of zinc with both ionotropic and metabo-
tropic glutamate receptors (Bresink et al., 1996; Dreixler
& Leonard, 1994; Harrison & Gibbons, 1994; Koh &
Choi, 1994; Smart et al., 1994; Xie et al., 1993).
In addition, zinc binds to the g-aminobutyric acid
(GABA) receptors and noncompetitively inhibits
GABA-mediated responses (Kumamoto & Murata,
1995; Kume et al., 1994; Mayer & Vyklicky, 1989;
Ricciardi & Malouf, 1995; Smart, 1992; Smart et al.,
1994; Turgeon & Albin, 1992; Wang et al., 1995; White &
Gurley, 1995). The neuronal death in cerebral ischemia
is due in part to the ratios of glutamate, glycine, and
GABA released extracellularly (the excitotoxic index)
(Globus et al., 1991). An interaction of zinc with these
receptors could thus modify the excitotoxic index and
be region specific, due to the localization of neurons
containing vesicular zinc. Interestingly, zinc modulates
both glutamate (Dreixler & Leonard, 1994) and GABA
(Wang et al., 1995; White & Gurley, 1995) responses of
neurons that contain only specific splice-variant recep-
tor subunits of NMDA and GABAA, respectively. The
dissociation constants (KD) for zinc antagonism of
NMDA (Mayer et al., 1989) and GABA (Mayer &
Vyklicky, 1989) responses are approximately 13 and 11
M, respectively. These are well within the concentra-
tions released during synaptic activity, which implies
an association of zinc with the regulation of neurotrans-
mission. Likewise, zinc can modulate the ATP P2X-
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146
purinergic (Cloues, 1995; Cloues et al., 1993; Koizumi et
al., 1995), opiate (Connor & Chavkin, 1992), and gly-
cine (Kumamoto & Murata, 1996; Laube et al., 1995)
receptors, as well as the dopamine transporter com-
plex (Richfield, 1993). Recently, zinc was observed to
inhibit the actions of a competitive NMDA receptor
antagonist, CGP-39653, on glutamate and glycine bind-
ing on NMDA receptors (Terse & Komiskey, 1997). This
finding has implications regarding the efficacy of
competitive NMDA receptor antagonists for use as
neuroprotectants in CNS injuries caused by cerebral
ischemia or epilepsy.
b. Interactions with the Activities of Critical
Enzymes and Transport Proteins
Zinc can inhibit the cell respiratory chain (Ki 5 1027
M) by blocking the initial step of respiration of electron
transfer between ubiquinone (coenzyme Q) and cyto-
chrome b of the bc1 complex (complex III) (Hunter &
Ford, 1955; Kleiner & von Jagow, 1972; Link & von
Jagow, 1995). At higher concentrations (1023 M), zinc
may further inhibit at the levels of flavoprotein 1 or 2
(complex I or II) and cytochrome c oxidase (complex
IV) activities (Skulachev et al., 1967).
Zinc also inhibits the ion transporter enzymes such
as Na1/K1-ATPase both in vitro and in vivo (ID50 5 20
M, Donaldson et al., 1971; Hexum, 1974; Mustafa et
al., 1971) and calcium-ATPase in vitro (IC50 5 50100
M; Brewer, 1980; Brewer et al., 1979; Zhang et al.,
1990). The Na1/K1-ATPase enzyme activity has been
found to be highly concentrated in the hippocampus
and hypothalamus (Donaldson et al., 1971). Inhibition
of Na1/K1-ATPase is known to cause neuronal death
(e.g., see Lees, 1991; Lees & Leong, 1994, 1995).
Zinc inhibits a number of enzymes critical for
metabolism of neurotransmitters (Dreosti, 1989; Freder-
ickson, 1989). It inhibits uptake transporters for both
glutamate and GABA (Km 5 50 M) in synaptosomal
fractions of mice (Gabrielsson et al., 1986). Inhibition of
the glutamate uptake mechanism produces progres-
sive extracellular glutamate accumulation and subse-
quent death of cortical neurons in vitro (Velasco et al.,
1996). However, the ability of physiological zinc concen-
trations to depress glutamate/aspartate uptake trans-
porter activity was recently disputed (Killinger et al.,
1995).
c. Interactions with Structural Proteins
Zinc has been shown to influence both assembly and
disassembly of tubulin (KD 5 10251027 M; Eagle et al.,
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Cuajungco and Lees
1983) and a number of microtubule-associated proteins
in vitro (Banerjee et al., 1982; Gaskin & Kress, 1977;
Gaskin et al., 1978; Kress et al., 1981). An excess zinc
concentration in nervous tissues could interfere with
the microtubule structure by disrupting the normal
functions of the cytoskeleton (Kress et al., 1981). The
metal chelator, TPEN, prevented protein kinase C
(PKC)-mediated actin cytoskeletal disruption induced
by phorbol ester in cultured C6 rat glioma cells, while
the addition of zinc reversed the protective effects of
TPEN (Hedberg et al., 1994).
d. Perturbation of Intracellular Calcium and
Interactions with Calcium-Binding Proteins
It has been observed that neuronal swelling caused
by zinc is dependent on the presence of sodium in vitro
(Choi et al., 1988). Koh and Choi (1994) proposed that
zinc influx via the NMDA-receptor channel may be a
mechanism of zinc-induced neurotoxicity. Recently,
however, Koh et al. (1996) showed that activation of
AMPA receptors but not NMDA receptors mediated
zinc toxicity in cultured neurons. Zinc influx may
depolarize the cell and consequently trigger a rise in
intracellular calcium concentration ([Ca21]i). It is also
conceivable that Zn21 influx may perturb buffering of
[Ca21]i by interacting with CBPs, thus exacerbating the
condition. A rise in [Zn21]i could result in the activa-
tion of second-messenger systems via PKC-mediated
(Csermely et al., 1988; Murakami et al., 1987) phosphor-
ylation of receptor ion channels and/or voltage-
dependent gene expression (Atar et al., 1995; Rubin &
Koide, 1973) which could initiate cellular death path-
ways. NMDA receptor-dependent Ca21 influx and
overload are known to result in excitotoxic damage
(Mody & MacDonald, 1995). In contrast, it has been
reported that Zn21 attenuates NMDA receptor-acti-
vated channel currents by binding to two different
receptor sites in vitro (Mayer et al., 1989), which
suggests some protective role for zinc. Therefore, it
remains to be delineated if zinc toxicity is caused by an
excitotoxic mechanism mediated by overaccumulation
of [Ca21]i through activation of either NMDA- or
non-NMDA-gated ion channels or whether it acts via
direct inhibition of processes critical for cell survival or
both.
A number of EF-hand CBPs have been implicated in
neurodegenerative disorders (Heizmann & Braun,
1995). Some of these proteins contain distinct zinc-
binding sites such as S-100b, S-100A1/S-100a, S-100A3/
S-100E, S-100A6/calcyclin, calmodulin (CaM), and
calgranulin C (for a review, see Schafer & Heizmann,
Zinc Metabolism in the Brain
1996). Zinc was observed to bind CaM and induces a
slight conformational change (Baudier et al., 1983).
However, calcium has a greater affinity for CaM
(KD 5 10281026 M) and that zinc binding was sug-
gested to be nonspecific (Baudier et al., 1983). Zinc has
been found to inhibit the CaM-complexed Ca21-
ATPase of erythrocyte membranes (I-310) (Brewer,
1980; Brewer et al., 1979). Calcyclin, homologous to
S-100b, is mainly present in neurons and is not found
in glial cells (Filipek et al., 1993). Its function is
currently unknown, but has a specific binding site for
zinc (Filipek et al., 1990) with a half-maximal affinity
(Ks) of 2 mM (Pedrocchi et al., 1994). Generally, the
affinity of most CBPs for zinc is very low, such that it is
unlikely to affect them under normal physiological
zinc concentrations (typically in the nanomolar range).
Zinc Deficiency and Neurological Dysfunction
The consequential effects of nutritional zinc defi-
ciency in biological systems have been documented.
Lack of zinc in animals results in teratogenic and CNS
abnormalities in vivo (Dreosti, 1989; McNall et al., 1995;
Taubeneck et al., 1995). For example, chronic zinc
deficiency has been observed to affect neuronal func-
tions in the hippocampal mossy fibers (Hesse, 1979;
Wensink et al., 1987). Likewise, dietary zinc deficiency
for 3 months in rats showed a 30% maximum reduc-
tion of mossy fiber zinc content in the hippocampus
(Wensink et al., 1987). Further, Hesse (1979) found that
zinc-deficient rats showed abnormal hippocampal
mossy fiber synaptic responses during low-frequency
stimulation. Recently, it was found that functional
NMDA receptors in cortical synaptic membranes in
vitro were decreased due to low zinc bioavailability
(Browning & ODell, 1995). Low zinc status also
activates brain MT-III expression, which results in cell
growth retardation (Palmiter, 1995). Finally, it was
observed that maternal zinc deficiency ($4 days) in
rats produces excessive embryonic apoptotic cell death
especially within the neural crest (Rogers et al., 1995). It
is interesting to note that in several human neurodegen-
erative diseases, zinc is specifically depleted in several
brain regions (see Part 3).
Zinc and Apoptosis
This section discusses in detail the involvement of
zinc in apoptosis occurring in cells from nonnervous
tissues (Sunderman, 1995) as it provides a basis for
147
comparative studies for the role of zinc in apoptotic
death in the CNS. Apoptosis (or programmed cell
death) was first described by Kerr et al. (1972) from
their observation of distinct morphological changes
occurring in cells in vitro. Apoptosis and necrosis are
two distinct ways a cell can die. The characteristic
features of apoptosis are membrane blebbing, chroma-
tin condensation, DNA fragmentation, organellar pack-
aging, and cell shrinkage (Wyllie et al., 1973). Necrosis,
however, is characterized by cell swelling and lysis, is
accompanied by inflammation, and is thus a rather
violent way for a cell to die (Wyllie et al., 1973).
Neuronal apoptosis has been postulated to be a
mechanism typically occurring in neurodegenerative
disorders (Thompson, 1995, for a review). Evidence
comes from in vitro and in vivo models of brain injury
caused by applications of excitotoxins (Ankarcrona et
al., 1995; Bonfoco et al., 1995; Portera-Cailliau et al.,
1995) or periods of hypoxiaischemia (Beilharz et al.,
1995; Nitatori et al., 1995), or paroxysmal activities
(Charriaut-Marlangue et al., 1996; Pollard et al., 1994a,b;
Simonian et al., 1996). There is also some evidence that
apoptosis occurs in the striatum and temporal lobes of
Huntingtons disease and Alzheimers disease pa-
tients, respectively (Dragunow et al., 1995; Su et al.,
1994). Recently, evidence of apoptotic cell death in the
substantia nigra of Parkinsons disease brains was also
confirmed (Mochizuki et al., 1996), although this is
contradictory to the report of Dragunow et al. (1995).
Free radicals have been proposed to play a major role
in the initiation of the apoptotic process (Bonfoco et al.,
1995; Greenlund et al., 1995; Ratan et al., 1994).
Surprisingly, the involvement of zinc in neuronal
apoptosis is just beginning to be explored. Zinc may be
very relevant in neuronal apoptosis as cells require
many zinc-dependent transcription factors in normal
functioning (OHalloran, 1993; Rhodes & Klug, 1993;
Schwabe & Klug, 1996; Vallee et al., 1991) and during
induction of apoptosis (Xu et al., 1996). There is
currently no information on the relevance of zinc in
neuronal apoptosis, and thus, we discuss correlative
evidence that zinc plays a role in neuronal apoptotic
death.
a. Accumulation of Intracellular Zinc
in Apoptotic Cells
Cultured lymphoblasts undergoing early events of
apoptosis have an increased intracellular zinc content
(Zalewski et al., 1994). This was demonstrated by the
use of a zinc-specific fluorophore, Zinquin. The investi-
gators emphasized that the observed enhanced intracel-
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148
lular zinc fluorescence was not due to zinc uptake from
the medium since the latter was zinc-free and occurred
before cell membrane disruption (see Zalewski et al.,
1994 for discussion).
While the mechanism for [Zn21]i mobilization is not
known, enhanced zinc dissociation from metallopro-
teins in vitro can be produced by free radicals and ROS
such as HOCl, H2O2, and O22 (Fliss & Menard, 1991,
1992; Fliss et al., 1991), EAAs such as L-glutamate,
L-aspartate, and L-cysteic acid (Baba et al., 1989), and
biological disulfides (Maret, 1994, 1995). These observa-
tions could provide a pivotal role for dissociated mechanism. In contrast, the rise of zinc in ischemic
Zn21 to reduce or exacerbate pathological conditions in
certain disease states, depending on the levels of [Zn21]i.
After a period of cerebral ischemia (Koh et al., 1996;
Tonder et al., 1990) or seizure activity (Frederickson et
al., 1989), degenerating neurons stain positively for
zinc. These neurons appear to be dying by an apoptotic
process (Beilharz et al., 1995; Charriaut-Marlangue et
al., 1996; Nitatori et al., 1995; Pollard et al., 1994a,b;
Simonian et al., 1996). The increase in cellular zinc
occurs only after a delay of 324 h following an insult,
but precedes other markers for cellular damage such
as cellular staining with acid fuchsin. The marked
decrease in neuronal death brought about by EDTA
implies that cellular uptake of zinc is critical for
neuronal death caused by a period of ischemia (EDTA
is not taken up by cells) (Koh et al., 1996). Translocation
from presynaptic ZEN terminals to postsynaptic neuro-
nal perikarya appears to be the mechanism (Frederick-
son et al., 1989; Koh et al., 1996; Tonder et al., 1990).
Hence, both increased uptake and intracellular mobili-
zation are possible sources for the increased intracellu-
lar zinc following an insult.
b. Induction and Prevention of Apoptosis by
Manipulating Intracellular Zinc
i. Modulation of induction of apoptosis by the
extracellular concentration. Exogenously added zinc
can have biphasic effects on nonneuronal cells by
either initiating or preventing apoptosis (Cohen &
Duke, 1984; Provinciali et al., 1995; Sunderman, 1995;
Telford & Fraker, 1995; Treves et al., 1994). There is,
however, a variable range of concentrations where zinc
may induce or preclude apoptotic death. For example,
cultured mouse thymocytes incubated with zinc at
concentrations #15 M undergo apoptosis (Provinciali
et al., 1995), while another study found induction of
apoptotic death when extracellular zinc concentrations
were 80200 M (Telford & Fraker, 1995). On the other
hand, zinc concentrations from 75 M and up to 5000
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Cuajungco and Lees
M have been shown to prevent DNA fragmentation
and apoptotic death in a variety of nonneural cells
induced by either serum/growth factor deprivation or
exposure to toxic/chelating agents (Cohen & Duke,
1984; Jiang et al., 1995; McCabe et al., 1993; Provincialli
et al., 1995; Shimizu et al., 1990; Telford & Fraker, 1995;
Treves et al., 1994; Zalewski et al., 1993, 1994). These
results contrast markedly with induction of c-myc-
dependent apoptosis by zinc (37.5 M). Thus, the
increased intracellular zinc in apoptotic lymphoblasts
reported by Zalewski et al. (1994) may be a protective
neurons would appear to be neurotoxic as a chelating
agent reduced neuronal death (Fig. 4) (Koh et al., 1996).
ii. Activation of apoptosis by chelation of intracel-
lular zinc. Apoptosis with or without elevation of
[Ca21]i, in vitro can be triggered by chelating [Zn21]i
using TPEN in thymocytes, lymphocytes, and spleno-
cytes (Jiang et al., 1995; Treves et al., 1994; Zalewski et
al., 1993). TPEN is a cell-permeant complexing agent
with a high affinity for zinc but low affinity for calcium
(Anderegg et al., 1977; Arslan et al., 1985). These results
are comparable to recent findings in which apoptosis
was induced by exposure to low levels of Zn21 (Provin-
cialli et al., 1995; Telford & Fraker, 1995). Note that zinc
chelation-mediated death is not only independent of,
but also additive to, apoptosis induced by exogenous
addition of Ca21 (Jiang et al., 1995). These observations
clearly indicate that Zn21 is important in at least some
apoptotic cell death pathways.
iii. Hippocampal zinc chelation in vivo. We found
that intrahippocampal injections of TPEN (10 nmol)
alone were not toxic to hippocampal neurons (Cuajungco
& Lees, 1996) in spite of complexing most of, if not all,
chelatable mossy fiber zinc for up to 8 h (Cuajungco &
Lees, unpublished observation). This result is in direct
contradiction with in vitro observations that nonneuro-
nal cells loaded with TPEN undergo apoptosis (Jiang et
al., 1995; McCabe et al., 1993; Treves et al., 1994).
Morphological evidence of apoptosis was not apparent
4 to 14 days after TPEN administration neurons (Cu-
ajungco & Lees, 1996). However, molecular techniques
are necessary to corroborate whether in vivo exposure
of neurons to TPEN did not induce apoptotic death.
These results imply that different classes of cells may
have different intracellular zinc requirements for the
initiation of apoptosis.
c. Mechanisms by Which Zinc May Modify
Apoptotic Pathways
i. Interactions between zinc and immediate early
genes in apoptosis. Immediate early genes (IEGs)
Zinc Metabolism in the Brain
and their products, known as inducible transcription
factors (ITFs), are known to play a role in the cascade
of events leading to apoptosis (for a review, see
Dragunow & Preston, 1995). Several ITFs such as c-Jun,
JunB, JunD, c-Fos, Fos-B, and the zinc-dependent ITF
Krox-24 show basal, but diverse expression in the
mammalian CNS (Herdegen et al., 1995). Both neural
and nonneural cells undergoing apoptotic death have
been found to express IEGs such as c-fos, c-jun, and
hsp-70 (Dragunow et al., 1990; Estus et al., 1994).
Induction of either c-fos or c-jun in neurons is highly
correlated with apoptotic death (Dragunow et al., 1990;
Estus et al., 1994). In contrast, c-fos in nonneural murine
cell cultures was recently demonstrated to be unneces-
sary in cell suicide, as cells with a disrupted c-fos2/2
gene (spleen and thymus) underwent spontaneous
and etoposide-induced apoptosis (Gajate et al., 1996).
Zinc is able to induce c-jun and c-fos expressions in
hepatocyte (H-7) cells (Xu et al., 1996). However, no
further data are available to indicate whether zinc
produces a transient or prolonged expression of c-fos
and c-jun or whether zinc-induced expression of the
two IEGs is directly associated with the induction of
cell death. A more direct involvement of zinc in IEG
regulation of apoptosis was shown when zinc (37.5
M) facilitated the c-myc-dependent apoptosis of cul-
tured H-7 cells (Xu et al., 1996). Cells exposed to either
copper or cadmium did not produce any effects,
suggesting a specific zinc-dependent step for c-myc-
induced apoptosis (Xu et al., 1996). Apoptotic death via
c-myc occurs without DNA fragmentation (Xu et al.,
1996).
ii. Inhibition of endonuclease. DNA fragmenta-
tion is one of the hallmarks of apoptosis which is
closely associated with key morphological changes
(Wyllie, 1980), but may not be essential. Glucocorticoid-
induced thymocyte apoptosis results in the activation
of the calcium/magnesium (Ca21/Mg21)-dependent
endonuclease in which DNA linker regions are cleaved
into around 200-basepair fragments (Wyllie, 1980).
Hence, one hypothesis is that zinc halts apoptosis in
both cell cultures and cell-free isolated nuclei via
competitive inhibition of endonuclease activity (Cohen
& Duke, 1984). This proposal was challenged when
Barbieri et al. (1992) found that zinc inhibition (15
mM) of DNA fragmentation did not protect rat thymo-
cytes from spontaneous or dexamethasone-induced
apoptosis. Furthermore, it has been reported that the
main morphological features of apoptosis may occur
without DNA cleavage, thus suggesting that DNA
fragmentation is not a critical event in the death
process (Barbieri et al., 1992; Cohen et al., 1992; Xu et al.,
149
1996). Other recent studies have challenged the idea
that the Ca21/Mg21-dependent endonuclease activity
is controlled solely by [Ca21]i (e.g., see Jiang et al., 1995;
Treves et al., 1994). It is interesting to note that an
increase in [Ca21]i induces apoptosis in immature
thymocytes (Cohen & Duke, 1984), while mature
thymocytes could resist apoptosis even with an ele-
vated [Ca21]i (McCabe et al., 1993). This is further
evidence supporting differential induction of apopto-
sis among various cell types.
iii. Regulation of growth cycle. A rather interest-
ing observation was noted in mouse thymocytes in
which preferential inhibition or induction of apoptosis
occurs during the G0/G1 phase (Provinciali et al., 1995;
Telford & Fraker, 1995). This could be explained by
previous findings that cultured fibroblasts required
zinc-dependent processes (i.e., activation of thymidine
kinase and possibly DNA histone H3) (Chesters et al.,
1993) in order for them to progress from G1 to S phase
(Chesters et al., 1989). According to Willson (1989) cells
may not divide during low intracellular zinc status at
critical sites, as zinc is thought to protect cells from
iron-catalyzed thiol oxidation during mitosis, thus
preventing intramolecular disulfide formation.
iv. DNA strand breakage repair enzyme. DNA
strand breakage results in the activation of poly-
[adenosine diphosphate(ADP)-ribose] synthetase
(PARS) and poly-[ADP-ribose] polymerase (PARP),
which recognize and repair strand damage (de Murcia
& de Murcia, 1994; Szabo et al., 1996). PARP is a zinc
finger protein whose activity depends on the presence
of zinc (Zahradka & Ebisuzaki, 1984). PARP catalyzes
the attachment of poly(ADP-ribose) from the coen-
zyme nicotinamide adenine dinucleotide (NAD1) (de
Murcia & de Murcia, 1994). Activation of PARP/PARS
significantly depletes NAD1 (de Murcia & de Murcia,
1994; Szabo et al., 1996). The process of replenishing the
diminished NAD1 is postulated to consume too much
energy, consequently exhausting the cellular ATP stores,
which then ultimately leads to the cells demise. While
zinc (1 mM) inhibits DNA fragmentation and cell
death, it does not prevent the N-methyl-N8-nitro-N-
nitrosoguanidine-mediated increase of PARP synthesis
(Shimizu et al., 1990). Thus, the protective effects of
zinc on apoptosis do not appear to be due to a direct
inhibition of PARP activity.
v. Protein kinase regulation. PKC is an enzyme
activated by the second messengers Ca21 and diacyl-
glycerol that then exerts its effects at certain cytoplas-
mic locations (Huang, 1989). PKC contains zinc finger
domains and has a high-affinity binding site for zinc
(Hubbard et al., 1991; Murakami et al., 1987; Schwabe &
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150
Klug, 1996). Zinc has biphasic effects on PKC activity,
with stimulation at low concentrations (,300400 M)
and inhibition at high concentrations (.400 M), both
in the presence of calcium (Murakami et al., 1987).
Binding of zinc can result in the translocation of PKC
to the membrane and augment its activity on the
membrane cytoskeleton (Baba et al., 1991; Csermely et
al., 1988; Zalewski et al., 1990, 1991). Downregulation
of PKC activity is highly correlated with the induction
of apoptosis in vitro (Lin et al., 1995; Rinaudo et al.,
1995). Taken together, these results indicate that zinc
may have an intrinsic role in PKC activity in terms of
either initiating or preventing apoptotic cell death.
The above observations demonstrate an important
role for zinc in the complex processes of apoptotic cell
death and provide further evidence that diverse and
complex mechanisms of cell suicide exist among differ-
ent cell types.
3. POSSIBLE INVOLVEMENT
OF ZINC IN SELECTED
NEUROLOGICAL DISORDERS
In any discussion of causation in neurological dis-
eases, correlations can often be made between pathol-
ogy and abnormal concentrations or metabolism of
many factors. Which events are primary and which are
secondary is often unclear, and in many cases changes
may be downstream events not involved in pathology.
In the following discussion on the role of zinc in CNS
dysfunctions, aberrations in zinc metabolism are high-
lighted, with emphasis on those processes which are
likely to be involved in the causation of the disorder. In
a number of cases, the changes observed are mutually
inconsistent. However, all possibilities are listed, in the
hope of stimulating future research to determine which
changes in zinc metabolism are most important. Fu-
ture research will inevitably find that in some, if not
most, of these diseases, abnormal zinc metabolism is a
secondary event that may be critical in the common
pathways by which neurons die (e.g., the function of
zinc in cell suicide) rather than for the initiation of the
disease process.
Hypoxia-Ischemia
The role of zinc in neuronal death caused by cerebral
ischemia was first investigated by Tonder and col-
leagues (1990). Recent confirmation by Chois group
indicates that zinc is in fact involved in the pathology
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Cuajungco and Lees
resulting from transient global cerebral ischemia (Koh
et al., 1996). A growing amount of evidence suggests
that selective delayed neuronal death in cerebral isch-
emia occurs via apoptosis (Ankarcrona et al., 1995;
Beilharz et al., 1995; Charriaut-Marlangue et al., 1996).
Thus, it is reasonable to postulate that zinc status may
be involved in ischemia-induced apoptosis.
The apparent similarity between neurodegeneration
following ischemia and kainic acid-induced toxicity
(an experimental model of epilepsy) led Tonder and
colleagues (1990) to propose that zinc has an important
function in cell death. Transient cerebral ischemia in
rats produced a zinc-dependent increase in the fluores-
cence of N-(6-methoxy-8-quinolyl)-p-toluene sulfon-
amide quinoline (TSQ) in the dentate hilus with a
reduction of TSQ fluorescence in the mossy fiber
terminals (see Fig. 3) (Koh et al., 1996; Tonder et al.,
1990). Neither neuronal perikarya nor glial cell bodies
normally exhibit positive Zn21 fluorescence (Koh et al.,
1996). Prior chelation of Zn21 by dithizone abrogated
Zn21 fluorescence within degenerating neurons (Koh
et al., 1996). TSQ-positive neurons in the hilus (Koh et
al., 1996; Tonder et al., 1990) and pyramidal CA1 cells
(Koh et al., 1996) were in fact degenerating, as evi-
denced by positive staining with acid fuchsin, a marker
for damaged neurons (Fig. 3). Furthermore, other brain
areas such as the neocortical laminae (II/III/IV), tha-
lamic reticular nucleus, amygdala, and striatum, known
to selectively degenerate after transient global isch-
emia, also showed positive TSQ fluorescence within
neuronal perikarya (Koh et al., 1996). Interestingly,
Chois group found that Zn21 accumulation preceded
neuronal death and that chelation of Zn21 via intraven-
tricular injection of calcium-EDTA (Ca-EDTA) 30 min
prior to insult decreased ischemic-related neurodegen-
eration by up to 70% (Fig. 4) (Koh et al., 1996). Hence,
zinc is one of many factors involved in ischemia-
induced neuronal death. It was suggested that zinc
cytotoxicity and other excitotoxic insults may be code-
pendent processes required to induce lethal injury
after global cerebral ischemia (Koh et al., 1996). There is
a need, however, to define the mechanisms involved in
zinc neurotoxicity, in order to devise an efficacious
counteractive measure to minimize neuropathological
sequelae following brain trauma.
Epilepsy
a. The Role of Zinc in Modulating Seizure Activity
Ever since the discovery that zinc induces seizures in
rats, it has been used as an experimental model of
Zinc Metabolism in the Brain 151

epilepsy (Pei & Koyama, 1986; Pei et al., 1983). This

finding has also resulted in the suggestion that the
release of cellular zinc plays a major role in the
generation and propagation of epileptic activity (Assaf
& Chung, 1984; Frederickson et al., 1988, 1989; Pei &
Koyama, 1986; Pei et al., 1983; Sloviter, 1985). However,

FIG. 4. CaEDTA protects hippocampal hilar and CA1 neurons

FIG. 3. TSQ staining and neuronal degeneration in the hippocam-
pus. (A) Fluorescent photomicrograph of normal rat hippocampus
after staining with TSQ, showing dense fluorescence in the hilus (H)
and the stratum lucidum (SL) of CA3. In addition, TSQ staining is
seen in the stratum radiatum (SR) and the stratum oriens (SO) of
CA1 (DG, dentate gyrus). No Zn21 fluorescence is seen in the
stratum pyramidale (SP) or alveus (alv.) of CA1 (dark bands). (B)
Twenty-four hours after a 10-min period of forebrain ischemia, TSQ
fluorescence is reduced in presynaptic terminals and is newly
apparent in the cell bodies of some hilar neurons. (C) The same
hippocampal section as in (B), with subsequent acid fuchsin stain-
ing. All the TSQ-fluorescent neurons in (C) exhibited ischemic
acidophilic changes (pink cytoplasm, arrows). (D) Seventy-two
hours after a 10-min period of ischemia, dense TSQ staining
appeared in degenerating CA1 pyramidal neurons. (E) The same
section as in (D), with acid fuchsin staining. All the CA1 neurons
with Zn21 fluorescence showed acidophilic changes. Bars, 800 m
(A); 200 m (B and C); 100 m (D and E). Reprinted with permission
from Koh et al. (1996) Science 272, 10131016. Copyright 1996
American Association for the Advancement of Science.
against ischemic injury. (A) Hilar neuron counts (mean 1 SEM) in
normal rats (n 5 4) or in saline-injected controls (n 5 20), Ca-EDTA-
treated rats (n 5 12), and Zn-EDTA-treated rats (n 5 7), 72 hours
after a 10-min period of ischemia. (B) CA1 pyramidal neuron counts
for the same rats. Three rats injected with Ca-EDTA were studied
after 14 days. Asterisks denote difference from controls ( p , 0.05
two-tailed t test with Bonferroni correction for multiple compari-
sons). Reprinted from Koh et al. (1996) Science 272, 10131016 with
permission.
opposing lines of evidence suggest that endogenous
zinc may have an anticonvulsant effect. These contrast-
ing observations are reviewed below.
Zinc in synaptic vesicles of the hippocampal mossy
fibers (Danscher, 1981; Haug, 1967; Perez-Clausell &
Danscher, 1985, 1986) is proposed to be co-localized
with glutamate (Beaulieu et al., 1992; Frederickson,
1989). Glutamate is believed to be the major EAA
neurotransmitter in excitatory hippocampal pathways
(Bramham et al., 1990; Storm-Mathisen & Ottersen,

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152
1990). The suggestion that glutamate and zinc are
co-localized within neurons comes from observations
that: (i) ZENs in the hippocampus release the cation in
a calcium-dependent manner during synaptic activity
(Aniksztejn et al., 1987; Assaf & Chung, 1984; Charton
et al., 1985; Howell et al., 1984); and (ii) the release of
zinc is abolished following chemical destruction of the
hippocampal mossy fibers (Aniksztejn et al., 1987). It is
noteworthy that not all glutamatergic neurons contain
zinc (Beaulieu et al., 1992; Frederickson, 1989).
There are conflicting results regarding the role of
endogenous zinc in the propagation of seizures
(Mitchell & Barnes, 1993; Mitchell et al., 1990; Morton et
al., 1990; Theoret et al., 1988). For example, Mitchell
and Barnes (1993) observed that intraperitoneal injec-
tion (ip) of diethyldithiocarbamate (DEDTC, .50 mg/
kg) chelates endogenous zinc and lowers the threshold
for convulsions when the rat hippocampal perforant
path was stimulated, indicating a protective role for
the release of zinc. Furthermore, DEDTC and dithi-
zone were found to augment the severity of KA-
induced seizures (Mitchell et al., 1990). However,
dithizone (100 mg/kg) and DEDTC (1000 mg/kg)
administered ip 30 min prior to intrahippocampal (ih)
injection of zinc (10 nmol) did not protect hippocampal
neurons from zinc-induced neurotoxicity (Lees, Cu-
ajungco, & Leong, unpublished observation). Like-
wise, co-injection (ih) of DEDTC (100 nmol) with zinc
(10 nmol) had no significant effect on zinc neurotoxic-
ity (Cuajungco & Lees, unpublished observation).
Thus, it is uncertain whether the proconvulsant effect
of DEDTC and dithizone reported by other groups is
due to chelation of zinc or a side effect of the chelating
drugs. Morton et al. (1990) found that subcutaneous
preadministration of zinc (100 mg/kg) significantly
decreased the frequency of audiogenic seizures in
mice, but did not significantly attenuate KA-induced
seizures. There is, however, a considerable doubt as to
whether this effect is due to a central action of zinc. The
amount of zinc crossing the bloodbrain barrier within
minutes of injection is likely to be insufficient to
modify seizures. This is because previous studies have
shown that peripheral zinc is only slowly taken up into
the cerebrospinal fluid (Blair-West et al., 1990) and into
the brain (Kasarskis, 1984), where it is rapidly bound to
plasma proteins and nonneural tissues (Kasarskis,
1984). Although Morton et al. (1990) estimated that a
minimum of 2 M zinc (i.e., 0.2% of 100 mg/kg
dosage) would have reached the extracellular compart-
ments of the CNS via facilitated diffusion of the
microligand-bound fraction, it can be argued that the
brain has high-affinity zinc-binding proteins to buffer
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Cuajungco and Lees
any excess extracellular zinc (Ebadi, 1991; Ebadi &
Hama, 1986; Itoh et al., 1983).
A very recent study using resected hippocampal
tissue from medial temporal lobe sclerosis patients has
reported that exogenously applied zinc (300 M)
attenuated the dentate granule cell hyperexcitability,
thus limiting seizure activity in situ (Williamson &
Spencer, 1995). From this finding, the authors pro-
posed that zinc may act as inhibitory neuromodulator
in pathological situations, as zinc is only normally
released during repetitive stimulation (Williamson
& Spencer, 1995). More recently, however, Buhl and
co-workers (1996) have suggested that zinc released
from aberrantly sprouted mossy fiber terminals in the
human temporal lobe epilepsy (HTLE) kindling
model could cause a direct collapse of an intensified
autoinhibition by GABA through attenuation of the
GABA response. This is evidenced by the observation
that zinc (200 M) significantly blocked the inhibitory
postsynaptic currents in kindled dentate granule cells
of hippocampal slices, whereas similar concentrations
of zinc did not produce any effects on control neurons
(Buhl et al., 1996). Whether zinc is important for the
neuronal death resulting from sustained seizure activ-
ity is a separate question from its involvement in
seizure activity per se. Nonetheless, the association of
zinc in epilepsy needs confirmation.
b. Zinc and Neuronal Death in Epilepsy
From the clear involvement of zinc in neurotransmis-
sion and neuromodulation, it was proposed that synap-
tic zinc mobilization during paroxysmal activity could
be involved in the consequent neuropathology of
epileptic activity (Assaf & Chung, 1984; Frederickson
et al., 1988, 1989; Fukahori et al., 1988; Sloviter, 1985).
Some researchers have observed an elevated level of
zinc in the brains of rats and mice following epileptic
seizures (Chung & Johnson, 1983; Mody & Miller,
1985), while another study reported otherwise (Fuka-
hori et al., 1988). These variations could be due to
differences between the types of seizure model used
and the time between seizure and sacrifice. Mody and
Miller (1985) used a rat commissural-kindling model,
Chung and Johnson (1983) used the audiogenic seizure-
prone DBA/2J mice strain, while Fukahori et al. (1988)
used E1 mice, a strain that is seizure-prone to postural
stimulations or paraboloid movements. Mody and
Miller (1985) sacrificed and measured hippocampal
zinc levels of the rats 24 h after the last stimulation,
whereas Fukahori et al. (1988) sacrificed the E1 mice at
7 days after the last induced seizure. Chung and
Zinc Metabolism in the Brain
Johnson (1983) did not specify the time at which
hippocampal zinc levels of the DBA/2J mice were
measured. It is possible then that the observed differ-
ences in hippocampal zinc concentration after a sei-
zure could be due to variations in the lag time before
zinc levels were measured. Moreover, the severity and
latency of paroxysmal activities may also explain the
anomalous findings of the above studies.
A consistent observation is that zinc is translocated
from presynaptic boutons to postsynaptic neurons as
revealed by TSQ fluorescence 12 h after KA-induced
seizures (Frederickson et al., 1988, 1989) or by Timms
stain 24 h following perforant path stimulation-
induced seizures (Sloviter, 1985). Experimental models
of epilepsy have provided increasing evidence that
neurons severely affected by seizures die by an apop-
totic process (Charriaut-Marlangue et al., 1996; Pollard
et al., 1994a,b; Simonian et al., 1996). While the rise in
[Ca21]i resulting from the activation of glutamate
receptors is one likely cause of cell death (Choi, 1988,
1992), zinc may also be involved in the pathology.
However, it remains to be determined whether the
toxic and convulsant effects of pharmacological doses
of zinc occur in vivo during release of endogenous zinc
from neurons.
c. Zinc, Cytokines, and Neuronal Reorganization
Following Seizure Activity
Dense sprouting and distinctive reorganization of
the hippocampal mossy fibers are commonly found in
HTLE tissue and experimental brain models (Babb et
al., 1991; Cronin & Dudek, 1988; Sundstrom et al., 1993;
Sutula et al., 1989). Sundstrom et al. (1993) found an
increased staining of ZEN terminal fibers of the den-
tate granule cell layer (inner molecular) after KA
lesioning of the CA3 pyramidal cells and CA4 commis-
sural association fibers. The group inferred that the
presence of suprapyramidal band stain provides a
direct link between aberrant sprouting and propaga-
tion of epileptiform activity. A zinc-containing growth
factor-like protein has been suggested to be the cause
of sympathetic sprouting in deafferented septo-
hippocampal projections (Kesslak et al., 1987; Frederick-
son et al., 1983; Stewart et al., 1984; Yu & Crutcher,
1995). The zinc-containing NGF-b is one such growth
factor which promotes highly specific sprouting of
dennervated septo-hippocampal (cholinergic) connec-
tions (Kesslak et al., 1987; Frederickson et al., 1983;
Stewart et al., 1984; Yu & Crutcher, 1995).
Another important finding is the significant and
specific up-regulation of S-100b expression, but not
153
GFAP in HTLE resected tissues, possibly signifying a
physiological role for this protein in aberrant axonal
sprouting seen in epilepsy (Griffin et al., 1995). It was
suggested that overexpression of S-100b could be a
factor in excessive formation of dystrophic neurites in
HTLE, which are also typically found in neuritic
plaques in Alzheimers disease (Griffin et al., 1995;
Ihara, 1988; Marshak et al., 1991). S-100b has a higher
affinity for zinc than for calcium (Baudier & Gerard,
1983), induces calcium influx which elevates [Ca21]i in
glial and neuronal cells (Barger & Van Eldik, 1992), and
promotes neurite outgrowth (Kligman & Marshak,
1985), as well as axonal and glial cell proliferation
(Reeves et al., 1994; Selinfreund et al., 1991). These
factors may be substantial enough to associate interac-
tions of zinc with neurotrophins which promote aber-
rant sproutings seen in HTLE hippocampus. Studies to
show whether zinc chelation affects seizure-induced
sprouting may prove useful.
Alzheimers Disease
Senile dementia exists in many forms. Alzheimers
disease (AD) is a commonly observed type of demen-
tia which afflicts millions worldwide. The disease is
progressive, exhibiting a widespread deterioration of
memory, intellect, will, self-care, and social functions
(Drachman & Leavitt, 1974; Rubin, 1990). Cortical
atrophy in the frontal and temporal cortices (especially
in the hippocampus) is the neuropathological hall-
mark of the disease (Ball, 1988). Furthermore, selective
loss of central cholinergic neurons in the brains of AD
patients is also observed (Davies & Maloney, 1976;
Whitemore et al., 1982; Wurtman et al., 1990). In the
basal nucleus system, the cholinergic receptor activity
is of the muscarinic type, is believed to be important in
memory, and is markedly affected in AD (Drachman &
Leavitt, 1974). Other findings in AD brains include a
significant accumulation of senile and diffuse plaques
and neurofilament plaques (paired helical filament)
(Brun et al., 1990; Mattson, 1995a,b; Selkoe et al., 1990).
Insoluble amyloid-b protein (bA) is the main constitu-
ent of senile plaques and vascular tissue amyloid; it
typically has 3943 amino acid residues with a molecu-
lar mass of around 4 kDa (Mattson, 1995a; Selkoe et al.,
1990). It originates from cleavage of the amyloid
precursor protein (APP) by the putative proteinase
enzymes designated a, b, and g secretases (Checler,
1995; Mattson, 1995b). Several genetic mutations iden-
tified from familial AD have been identified and
characterized (Levy-Lahad et al., 1995a,b; St. George-
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154
Hyslop et al., 1987; Sherrington et al., 1995; Tanzi et al.,
1987).
a. Zinc Interactions with Amyloid Precursor
Protein Gene Expression and Metabolism
The genetic mutation that links bA and the causation
of one form of familial AD has been identified in
chromosome 21 (St. George-Hyslop et al., 1987; Tanzi et
al., 1987). It is believed that possible overexpression of
the APP gene has etiologic consequences for the AD
and AD-like neuropathology seen in Downs syn-
drome (trisomy 21) (for a review, see Ashall & Goate,
1994, and Hendriks & Van Broeckhoven, 1996). The
function of zinc in regulating the synthesis of APP is
thus of critical importance.
Zinc is important in gene transcription as evidenced
by its diverse presence in many transcription factors
(OHalloran, 1993; Rhodes & Klug, 1993; Schwabe &
Klug, 1996). Of these, APP synthesis is regulated by
zinc-containing transcription factors NF-kB and Sp1.
The APP promoter contains two NF-kB/Re1 (desig-
nated APBa/APBb) binding sites in its proximal (58-
regulatory) region, which, upon activation, increases
gene transcription by several-fold (Grilli et al., 1995;
Quitschke, 1994). Furthermore, Sp1 has been found to
bind to the positive regulatory region of the mouse
APP gene promoter which is highly homologous to the
human APP gene (Izumi et al., 1992). Zinc is easily
abstracted from NF-kB and Sp1, but is essential for
their activity (Yang et al., 1995; Zabel et al., 1991; Zeng et
al., 1991a,b). Whether their activity in vivo is regulated
by zinc availability is hence relevant to the synthesis of
APP.
APP has a ligand-binding site for zinc (764 nM), and
zinc (#50 M) inhibits degradation of APP and proteo-
lytic cleavage of bA at its a secretase site (Bush et al.,
1993, 1994b). While this concentration is high, it is
nevertheless well within the range of concentrations
which may occur under pathological conditions (dis-
cussed in previous sections). Thus, zinc may be a factor
in both stimulating the synthesis of APP and inhibiting
its subsequent degradation. The regulation by zinc of
the activities of NF-kB and Sp1 and their subsequent
effects on APP expression should be fruitful areas of
research.
b. Zinc Interactions with Basement Membrane and
Matrix Metalloproteinase
Basement membranes (BMs) are distinct types of
extracellular matrices (ECMs) that support cells, pro-
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Cuajungco and Lees
vide filtration, and sequester growth factors (Yur-
chenco & Schittny, 1990). BMs have been implicated in
the pathogenesis of AD as APP isoforms were found to
interfere with the normal BM proteinprotein interac-
tions in vitro (Narindrasorasak et al., 1995). Several BM
proteins have been found to contain zinc-binding sites
(Reinhardt et al., 1993), as well as a zinc finger-like
structural motif (Narindrasorasak et al., 1992). Zinc (10
M) can stimulate the binding of laminin to several
APP isoforms in vitro (Narindrasorasak et al., 1992).
Likewise, zinc (1 M) enhances binding of APP to
heparin side chains of proteoglycans in vitro (Mul-
thaup et al., 1994). Recently, zinc (15 M) was observed
to effectively augment both lamininentactin and lam-
inincollagen type IV associations (Ancsin & Kisi-
levsky, 1996). This is the first report of a function for
zinc as a cofactor/modulator of BM assembly and the
first identification of high-affinity proteinprotein inter-
actions involving an extracellular zinc finger motif
(Ancsin & Kisilevsky, 1996).
Matrix metalloproteinases (MMPs) degrade and re-
structure the ECM (Baramova & Foidart, 1995;
Gottschall & Yu, 1995). MMPs are a family of enzymes
(collagenases and gelatinases) whose activities are
dependent on the presence of zinc in their catalytic
sites (Baramova & Foidart, 1995; Gottschall & Yu,
1995). In vitro, zinc has been shown to completely
inhibit two types of MMPs (MMP-2 and MMP-9)
found in the CNS (Backstrom et al., 1992). However,
the potency of zinc as an inhibitor is unknown (only
one high concentration of zinc was tested). MMP-2
degrades soluble forms of bA140/bA142, but not in-
soluble aggregates of bA140/bA142 (Roher et al., 1994).
MMP-9 is also capable of degrading synthetic bA140
peptides (Backstrom et al., 1996). Thus, regulation of
MMP activity by zinc could also affect the degradation
of bA, a possible initiator of neuronal death.
Further investigations are required in order to deter-
mine whether zinc plays a major role in the changes
that occur between APP metabolism and its interac-
tions with BM proteins or with the metabolism of BMs,
and any association of these activities with amyloid
deposition.
c. Zinc, b-Amyloid, and Oxidative Stress
The aggregated form of bA is cytotoxic (Iversen et
al., 1995) and can induce apoptosis both in vivo (LaFerla
et al., 1995) and in vitro, although this action is depen-
dent on the type of cultured cells used (Gschwind &
Huber, 1995). bA toxicity can trigger activation of the
stress-related transcription factor NF-kB (Behl et al.,
Zinc Metabolism in the Brain
1994). Recent studies have implicated oxidative injury
to mitochondrial DNA damage and malfunctions in
the mitochondrial respiration chains as key players in
the pathogenesis of AD (Hutchin & Cortopassi, 1995;
Mecocci et al., 1994; Mutisya et al., 1994), as well as to
the mediation of bA toxicity in vitro (Behl et al., 1994).
The relevance of oxidative stress to the release of zinc
from MT has been discussed previously.
Several metal ions such as iron and aluminum
(Mantyh et al., 1993), as well as zinc (Bush et al.,
1994a,b, 1995, 1996; Clements et al., 1996; Esler et al.,
1996; Fuson et al., 1996; Mantyh et al., 1993), induce bA
protein aggregation. Of these metal ions, it is claimed
that only zinc can induce bA precipitates at low,
physiological concentrations #1026 M (Bush et al.,
1994a,b, 1995, 1996; Fuson et al., 1996). However, other
recent studies do not support the initial report of Bush
and colleagues (e.g., see Clements et al., 1996; Esler et
al., 1996), and the subject is rather controversial (e.g.,
see Kaiser, 1994). It is interesting to note that zinc-
mediated bA140 precipitation per se is not sufficient for
neurodegeneration as co-addition of zinc in rat hippo-
campal primary cultures specifically and in a dose-
dependent manner reduced bA140 neurotoxicity (Fu-
son et al., 1996).
Postmortem studies report that zinc is significantly
reduced in the temporal lobes including the hippocam-
pus, as well as in the inferior parietal and primary
visual cortices of AD brain (Andrasi et al., 1995;
Constantinidis, 1990, 1991; Corrigan et al., 1993; Deng
et al., 1994; Ward & Mason, 1987; Wenstrup et al., 1990).
On the other hand, other investigations report no
change (Basun et al., 1991; Wenstrup et al., 1990) or an
increase in specific brain areas (Danscher et al., per-
sonal communication; Deibel et al., 1996; Samudralwar
et al., 1995; Thompson et al., 1988). Thus, the specificity
and location in brain regions/neurons of any changes
in zinc need further work. It is not known if zinc
depletion is simply due to a reduction in number of
zinc-containing neurons (i.e., a later-stage event) or
whether it is the cause of neuronal death found at
specific areas of AD brains. Observations indicating a
decrease in brain zinc concentration contrast with the
postulated role of zinc in APP metabolism, bA aggrega-
tion, and abnormal BM metabolism. This paradox
could be resolved if the observed zinc reduction in AD
brain is a secondary effect of neurodegenerative pro-
cesses (Cuajungco & Lees, 1997). There is also a
controversy regarding the observed levels of MT-III in
AD brains (e.g., see Tsuji et al., 1992, and Uchida et al.,
1991, versus Erickson et al., 1994).
155
d. Zinc and EF-Hand S-100 Protein
S-100b protein is an isoform localized in the brain
(Baudier & Gerard, 1983; Baudier et al., 1983; Kligman
& Marshak 1985). S-100b has a greater affinity for zinc
than calcium, and zinc increases its affinity for calcium
by one order of magnitude (Baudier & Gerard, 1983).
The physical binding of zinc or calcium produces
distinct conformational changes in the proteins struc-
ture (Baudier & Gerard, 1983; Calissano et al., 1974). It
is not known whether the conformational changes
induced by either ion are necessary for the normal
functions of the protein. S-100b is mainly observed in
glial cells and is believed to regulate their proliferation
(Reeves et al., 1994; Selinfreund et al., 1991). However,
it was also suggested to be involved in neuronal
differentiation and maturation (Azmitia et al., 1990;
Van Eldik et al., 1991; Yang et al., 1996).
Several lines of evidence have implicated S-100b in
the pathogenesis of neurological dysfunctions in AD
and Downs syndrome (DS) (Allore et al., 1988; Griffin
et al., 1989; Marshak et al., 1991; Sheng et al., 1996; Van
Eldik & Griffin, 1994). S-100b, interleukin-1 (IL-1), and
glial fibrillary acidic protein (GFAP) are increased in
the temporal lobes of AD and DS brains where neuritic
plaques are densely located (Marshak et al., 1991;
Griffin et al., 1989). The observed elevation of S-100b
content in the brain of rats (Kato et al., 1990) and
humans (Sheng et al., 1996) is closely correlated with
aging and age-dependent changes in the CNS, lending
support for a role of S-100b in AD neuropathology. On
the contrary, elevation of S-100b has no effect on APP
mRNA induction nor does it promote bA deposits in
the brains of aging transgenic mice (Yao et al., 1995).
e. AD and Protein Kinases
The role of zinc in regulating the activity of PKC and
the relationship between down-regulation of PKC and
apoptosis has already been described. PKC activation
has been found to modulate APP degradation, thus
inhibiting bA protein formation (Buxbaum et al., 1993;
Gabuzda et al., 1993; Hung et al., 1993). The regulation
of APP secretion by PKC activity was found to be
defective in cultured neurons (Wang et al., 1994) and
fibroblasts (Bergamaschi et al., 1995) obtained from AD
patients. Both cytosolic and membrane-associated PKC
responses were attenuated in frontal and temporal
cortices, including the hippocampi of AD brains (Wang
et al., 1994). Further evidence comes from a recent
report that both Ca21-dependent and Ca21-indepen-
dent PKC activities in cytosolic and membranous
fractions obtained from AD specimens were signifi-
Copyright r 1997 by Academic Press
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156
cantly decreased (Matsushima et al., 1996). The tyro-
sine kinase trk proteins are another group of protein
kinases which are significantly reduced in AD hippo-
campi (Vener et al., 1993). Zinc (0.5 mM, or magnesium
at 10 mM) added to membrane fractions of the AD
hippocampus activates the phosphorylation of trk
proteins (Vener et al., 1993). It is conceivable then that a
decreased zinc content in the AD hippocampus (if
confirmed) could produce defective PKC and/or pro-
tein trk activities (Vener et al., 1993). A future study
would be to determine if addition of zinc to membrane
fractions taken from specific areas of AD brain would
lead to a recovery of PKC activity.
Amyotrophic Lateral Sclerosis
Amyotrophic lateral sclerosis (ALS) is a neurodegen-
erative disorder that affects motor neurons in the
spinal cord, brain stem, and cerebral (motor) cortex
(Olanow, 1993). Growing evidence suggests that ALS
pathology involves free radical toxicity (Olanow, 1993;
Przedborski et al., 1996).
Even though the etiology of sporadic ALS is still
unknown, there are approximately 510% reported
familial cases of this disease (FALS). A genetic defect in
the superoxide dismutase type 1 (SOD1) gene of FALS
was discovered in chromosome q21 which codes for
Cu,Zn-SOD (Rosen et al., 1993). It is not known,
however, how this particular mutation may lead to
FALS symptomatology and pathology. Recent studies
suggest that FALS may not be a result of decreased
enzymic function owing to mutation in the SOD1 gene,
but due to a gain-of-function by the enzyme (Rabiza- The observations presented here suggest some associa-
deh et al., 1995; Yim et al., 1996). In vitro studies showed
that the SOD1 mutation is proapoptotic in both neu-
rons (Rabizadeh et al., 1995) and immune cells (Peled-
Kamar et al., 1995).
Cu,Zn-SOD is a cytosolic antioxidant that catalyzes
the dismutation of O22 to H2O2 and O2, and it repre-
sents the first line of defense against oxidative toxicity
(Olanow, 1993). There are two zinc atoms and two
copper atoms in the enzyme, and all four ions are
necessary for optimal SOD activity (Willson, 1989).
Zinc is believed to have a structural function, while
copper is involved in the catalytic sites of dismutation
(Willson, 1989). SOD is structurally very stable and
remains active even at high (8 M) urea concentrations
(Willson, 1989). SOD has a relatively low affinity for
zinc (KD 5 1025 M) (Fabris & Mocchegiani, 1995).
Nonetheless, the metal chelators 1,10-phenanthroline
D 5 10
(KZn 217 M, pH 5 5.5) and EDTA (KZn 5 10216 M,
D
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All rights of reproduction in any form reserved.
Cuajungco and Lees
pH 7.4), chelate only 13 and 10% of Zn21 in SOD,
respectively (Willson, 1989), which indicates that most
of the structural zinc is inaccessible to zinc chelators.
Very recently, the effect of the mutation on the binding
of the metal constituents of the enzyme was reported
(Lyons et al., 1996). It was found that SOD1 mutation
affects the native zinc-binding sites of the enzyme,
which may possibly result in an unstable protein
structure.
MMP-2 and MMP-9 activities were recently reported
to be significantly different in brain and spinal cord of
ALS patients compared with controls (Lim et al., 1996).
MMP-9 activity was markedly elevated in the frontal
and occipital cortices, particularly in the motor cortex,
and in the thoracic and lumbar regions of the spinal
cord of ALS sufferers (Lim et al., 1996). On the other
hand, MMP-2 activity in the motor cortex was signifi-
cantly reduced. As zinc may inhibit both MMP-2 and
MMP-9 activities in vitro (Backstrom et al., 1992),
evidence for an abnormal zinc metabolism in ALS
would not explain these observed changes.
PKC activity in ALS spinal cord is significantly
increased, possibly attributable to abnormal Ca21 buff-
ering (for a recent review, see Krieger et al., 1996) or
aberrant zinc levels which could influence its activity
(Csermely et al., 1988; Murakami et al., 1987; Zalewski
et al., 1990, 1991). Note that MT expression is signifi-
cantly increased in the spinal cord gray matter of
protoplasmic astrocytes (Sillevis-Smitt et al., 1992a), as
well as in liver and kidney (Sillevis-Smitt et al., 1992b)
of ALS patients. These reports could mean that either
metal ion levels are elevated or an increased oxidative
stress is present (reviewed in Sillevis-Smitt et al., 1994).
tion of metal cations and oxidative injury in the
pathogenesis of ALS. Hence, investigations need to be
done to determine whether abnormal zinc or copper
function in mutant SOD has any relevance to disease
progression.
Guam ALS-Parkinsonism Dementia
Guamanian ALS-PD is a subtype of ALS. The pres-
ence of cycad (genus Cycas) in food or medicine has
been implicated in the pathogenesis of ALS-PD of
Guam (Duncan et al., 1992). It was found that cycad
flour samples produce a significant but dose-depen-
dent cytotoxicity to mesencephalic and cerebellar gran-
ule cell cultures (Duncan et al., 1992). Zinc was found
to be the neurotoxic constituent which contaminates
cycad flour, and similar zinc levels (100 M) calculated
to be present in the flour samples also produced a
Zinc Metabolism in the Brain
concentration-dependent toxicity on cultured mesence-
phalic and cerebellar granule cells. Addition of EDTA
(200 M) in the culture medium conferred protection
against zinc neurotoxicity. Meanwhile, zinc levels in
the gray matter of the frontal cortex of Guam ALS-PD
brains were reported to be markedly reduced com-
pared with normal controls (Yasui et al., 1993). Further
investigations are necessary in order to understand the
relationship between alterations in zinc metabolism
and exacerbation of the disease.
Parkinsons Disease
As with ALS, oxidative stress may be an etiologic
factor in Parkinsons disease (PD) (Jenner et al., 1992).
According to Dexter et al. (1989a), postmortem studies
revealed that polyunsaturated fatty acid levels in the
substantia nigra (SN) of patients with PD were re-
duced in comparison with other brain regions and that
of control (normal) tissue. Moreover, malondialdehyde
levels were elevated in the SN, suggesting occurrence
of lipid peroxidation (Dexter et al., 1989a). Zinc and
both cytoplasmic and mitochondrial SOD activities in
SN of patients with PD were also markedly increased
(Dexter et al., 1989b, 1991, 1992; Jenner et al., 1992),
while GSH levels were reduced in both early- and
late-stage PD (Jenner et al., 1992; Sofic et al., 1992). In
contrast, there were no significant alterations in the
activity of H2O2-degrading enzymes (catalase and
GPO) or in the concentrations of free radical scaven-
gers (ascorbic acid and a-tocopherol) (Jenner et al.,
1992), as well as GSSG (Sofic et al., 1992). The SN
contains a collection of neurons that utilize dopamine
as their major transmitter and which are selectively
lost in PD. These dopaminergic neurons also contain
melanin, an autoxidation by-product of dopamine
metabolism. Nonmelanized neurons are believed to be
less susceptible to oxidative stress than melanized
cells, since they are not likely to experience iron
melanin interactions that could produce excessive free
radicals (Ben-Shachar et al., 1991; Hirsch, 1992). Metabo-
lism of catecholamines such as DA autoxidation pro-
duces ROS and free radicals, thus making nigral cells
more susceptible to oxidative stress (Ben-Shachar et al.,
1991; Hirsch, 1992). Intracerebral injection of 6-hy-
droxydopamine in rats, a drug known to produce free
radicals, resulted in a reduction of zinc and MT
concentrations in the striatum, but not in any other
brain areas tested (Shiraga et al., 1993). Shiraga et al.
(1993) postulated that MT may reduce the effects of
generating ROS and free radicals by releasing zinc to
157
neuronal membrane and/or receptors, suggesting an
antioxidant role for both zinc and MT (Bray & Bettger,
1990; Sato & Bremner, 1993). Further, zinc may prevent
excessive NO production as it inhibits nNOS activity
(one Zn21 per NOS molecule) (Persechini et al., 1995).
One possible source of zinc is its release from MT, since
cells have been estimated to contain approximately 13
M zinc-bound MT in vitro (Krezoski et al., 1988).
HIV Neuropathy/AIDS Dementia Complex
The mechanisms of human immunodeficiency virus
(HIV) neuropathy and acquired immunodeficiency
syndrome (AIDS) dementia complex (ADC) are not
fully understood. ADC is progressive and is character-
ized by memory impairment, motor deficits, organic
psychosis, and other behavioral dysfunctions (Navia et
al., 1986). There is a great deal of neuropathological
variability in patients with CNS HIV-1 infection and
ADC (Price et al., 1988). The major brain areas that
show abnormalities are central white matter and deep
gray matter, including the basal ganglia, thalamus,
brain stem, and spinal cord, with the exception of
relative sparing of the cerebral cortex (Navia et al.,
1986). The potential involvement of zinc-dependent
processes on HIV neuropathy and ADC is on three
fronts: (i) zinc-dependent synthesis of, or binding to,
cytokines; (ii) oxidative stress-mediated neuronal death;
and (iii) regulation of viral replication.
An abundance of reactive microglial cells is com-
monly found in HIV-infected brain, as well as intense
IL-1a immunoreactivity (Stanley et al., 1994). Reactive
astroglial cells with up-regulated S-100b expression
are also observed (Stanley et al., 1994). Generally, glial
changes are associated with an elevated b-APP expres-
sion in neurons and overgrown neurites, including
detection of tau-2 immunoreactivity among NFT-like
formations (Stanley et al., 1994). Not surprisingly, the
histopathological detection of NFTs and reactive glial
cells, as well as marked elevations of IL-1 and S-100b
in ADC suggest a close association with the correspond-
ing neuropathological changes in both AD and DS
pathologies (Griffin et al., 1989). Although the above
observations suggest a direct relationship between
enhanced expression of cytokines and neurodegenera-
tion, it remains to be proven whether these entities
provide a primary involvement in the pathogenesis of
neurological dysfunctions. The physiological role of
zinc in AIDS and its importance in the immune
function have been reviewed (Odeh, 1992; Ripa &
Ripa, 1995). There is a significant reduction in the
Copyright r 1997 by Academic Press
All rights of reproduction in any form reserved.
158
plasma zinc level among asymptomatic HIV patients,
which suggests that deficiencies in trace metals and
antioxidants are not secondary effects of malnutrition
commonly found in the late AIDS stage (Favier et al.,
1994). Zinc deficiency, on the other hand, could also be
due to an intense immune response against HIV,
because endotoxin- and viral-induced immune/inflam-
matory reactions result in low serum zinc (Bremner &
May, 1989; Goldblum et al., 1987; Taubeneck et al., 1995)
and iron (Goldblum et al., 1987). Zinc deficiency may
also be due to utilization by HIV during replication
(see below). In contrast, a previous study did not show
any association between HIV infection and zinc status
(Walter et al., 1990).
HIV type 1 (HIV-1) infection has been linked with
oxidative stress-mediated cell death (McCord & Flores,
1994). Evidence comes from findings that GSH levels
(Staal et al., 1992) and other antioxidants (Favier et al.,
1994) were significantly decreased in tissues of in-
fected patients. Such condition may increase the intra-
cellular zinc pool as disulfideMT interactions are
known to liberate zinc from MT (Maret, 1994, 1995).
In regards to the involvement of zinc in the replica-
tion of the HIV virus, zinc (10 M) was recently found
to stimulate the Mg21-dependent 38-processing activ-
ity of HIV-1 integrase in vitro (Lee & Han, 1996). Note
also that zinc is an essential component of retroviral
nucleocapsid protein which is present in viral RNA
genomes (Green & Berg, 1990). Zinc is also important
for the activity of gag polyprotein, which serves to
identify and form viral RNA genome complexes (Sum-
mers et al., 1990). The final mature viral product is
HIV-1 nucleocapsid protein type 7 (NCp7), a zinc-
dependent protein (Mely et al., 1996). The importance
of zinc in HIV replication was demonstrated when
HIV activity and infectivity in vitro were inhibited by
ejecting zinc from intact HIV-1 virions and NCp7,
using disulfide benzamides and C-nitroso compounds
(Rice et al., 1993; Tummino et al., 1996). Furthermore,
zinc is an essential component of NF-kB, which regu-
lates the transcription of immuno-receptors, cytokines,
and viruses (Baeuerle, 1991). The zinc-dependent,
oxidoreductive activation of NF-kB protein stimulates
viral replication by binding to HIV long terminal
repeats (LTRs) (Baeuerle, 1991), although it is not
essential as other transcription promoters are involved
(Leonard et al., 1989). Deletion of two NF-kB-binding
sites and the three Sp1 sites, or mutation of the
tat-responsive region, completely abolished viral repli-
cation (Leonard et al., 1989). TNF-a has been found to
independently instigate HIV-1 transcription, and it
acts in synergy with the presence of mitogens in vitro,
Copyright r 1997 by Academic Press
All rights of reproduction in any form reserved.
Cuajungco and Lees
via the enhancer element of the HIV-LTR NF-kB-
binding region (Israel et al., 1989). Further, TNF-a was
shown to induce zinc deficiency and teratogenicity in
developing mice (Taubeneck et al., 1995).
The mechanism of damage in cerebral ischemia is
also closely associated with the possible pathogenesis
of HIV neuropathy and ADC. The glycoprotein coat of
HIV-1 (gp120) was found to increase [Ca21]i in astro-
cytes (Codazzi et al., 1996) and increase NO levels
among cortical neurons in vitro (Dawson et al., 1993).
HIV-1 gp120 induces apoptosis in hippocampal neu-
rons and interneurons in vitro (Aggoun-Zouaoui et al.,
1996) and in cortical neurons in vivo (Bagetta et al.,
1996; Charriaut-Marlangue et al., 1996). NO-mediated
toxicity may be one of the underlying mechanisms of
death in both HIV neuropathy and ADC neuropathol-
ogy, since inhibition of NOS abolished the cytotoxic
effects of gp120 (Dawson et al., 1993). Likewise, inhibi-
tion of NOS by L-NAME reduces the extracellular
glutamate concentration in the rat hippocampus
(Rigaud-Monnet et al., 1995). Thus, zinc depletion
could be detrimental as it is a potent inhibitor of nNOS
activity (Persechini et al., 1995).
Provided that the chelation of zinc is not too toxic for
human cells (e.g., due to inhibition of nNOS activity),
the above results indicate that chelation of zinc from
HIV proteins could be effective in reducing the infectiv-
ity of the virus.
CONCLUDING REMARKS
Zinc is critical for the growth and survival of cells.
However, an abnormal metabolism of zinc in cells can
have deleterious effects. The evidence presented here
indicates that while the importance of changes in zinc
metabolism in causing neurological dysfunctions can
be debated, zinc does seem to play a role in ischemic-
related neuropathology. Although there is a high asso-
ciation with zinc and epilepsy, some contentious issues
need to be resolved. Studies have shown that zinc can
induce cytotoxicity in nervous and nonnervous tissue.
Zinc was also shown to play a role in apoptotic death
of nonneuronal cells. Whether zinc also serves any
function in neuronal apoptosis needs to be elucidated.
The involvement of zinc in Alzheimers and other
neurodegenerative diseases is far from being demon-
strated, but is currently the subject of thorough investi-
gation. Whether zinc is directly responsible in the
etiologic processes of these disorders or is a secondary
consequence of the disease process remains to be
proven. Therefore, further investigations are needed in
Zinc Metabolism in the Brain
order to determine if there is any role for zinc in
dysfunctions of the central nervous system. The knowl-
edge gained from investigations of the mechanism of
zinc-induced cell death may enable researchers to
develop therapeutic interventions that would reduce
or perhaps prevent further destructive processes occur-
ring in the brain.
ACKNOWLEDGMENTS
We thank the NZ Lotteries Grant Office, the University of
Auckland Staff Research Fund, and the Health Research Council of
NZ for providing financial support to G.J.L. M.P.C. is a recipient of
the Miller scholarship from the NZ Neurological Foundation. We are
grateful to Professor Richard Palmiter and Professor Gorm Danscher
for providing results prior to publication, and we thank the Ameri-
can Association for the Advancement of Science for allowing us to
reproduce published materials.
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