1. Modulation of zinc toxicity by tissue plasminogen activator
Mustafa M. Siddiqa,b
and Stella E. Tsirkab,*
a
Program in Molecular and Cellular Biology, University Medical Center at Stony Brook, Stony Brook, NY 11794-8651, USA
b
Department of Pharmacology, University Medical Center at Stony Brook, Stony Brook, NY 11794-8651, USA
Received 13 June 2003; revised 10 October 2003; accepted 14 October 2003
The tissue plasminogen activator (tPA)–plasmin proteolytic system
mediates excitotoxin-induced neurodegeneration in vivo and in cell
culture. tPA also confers neuroprotection from zinc toxicity in cell
culture through a proteolysis-independent mechanism. This raises two
questions: what is this non-enzymatic mechanism, and why tPA does
not synergize with zinc to promote neuronal cell death? We show here
that zinc binds to tPA and inhibits its activity in a dose-dependent
fashion, thus terminating its protease-dependent neurotoxic capacity.
We extend the previously reported culture findings to demonstrate that
elevated zinc is neurotoxic in vivo, and even more so when tPA is
absent. Thus, physiological levels of tPA confer protection from
elevated free zinc. Mechanistically, tPA promotes movement of zinc
into hippocampal neuron cells through voltage-sensitive Ca2+
channels
and Ca2+
-permeable AMPA/KA channels. Therefore, zinc and tPA
each appear to be able to limit the potential of the other to facilitate
neurodegeneration, a reciprocal set of actions that may be critical in the
hippocampus where tPA is secreted during the nonpathological
conditions of learning and memory at sites known to be repositories
of free and sequestered zinc.
D 2003 Elsevier Inc. All rights reserved.
Introduction
The secreted serine protease tissue plasminogen activator (tPA)
converts the zymogen plasminogen into the active protease plas-
min (Lijnen et al., 1994) and mediates neurotoxin-induced neuro-
nal degeneration and seizures (Tsirka et al., 1995, 1996):
intrahippocampal injection of excitotoxins into wild-type (wt) mice
results in the activation of neurodegeneration pathways and the
elimination of the pyramidal neurons in the CA1-3 hippocampal
subfields. In contrast, very limited cell death is observed in tPA-
deficient (tPAÀ/À
) or plasmin(ogen)-deficient (plgÀ/À
) mice (Tsirka
et al., 1997), result indicating that in the context of excitotoxic
injury, tPA can be neurotoxic. On the other hand, in the setting of
zinc-mediated neurotoxicity in cell cultures, the addition of tPA
confers neuroprotection (Kim et al., 1999).
Zinc is abundant in the central nervous system (CNS), playing a
role both in physiological functions (its presence is associated with
neurite outgrowth) and pathological ones (as a mediator of the
neuronal death associated with transient global ischemia and
sustained seizures) (Choi and Koh, 1998; Cole et al., 1999). Under
physiological conditions, there are high concentrations of zinc in
the hippocampus, cortex, and amygdala. Most of the zinc is bound
tightly to proteins, but a small amount exists in a chelatable (free)
state. In zinc-containing neurons, zinc localizes to vesicles where
its concentration may exceed 1 mM (Frederickson et al., 2000;
Weiss et al., 2000).
Zinc toxicity has been correlated with excitotoxicity, in which
levels of the excitatory neurotransmitter glutamate become elevat-
ed (Olney, 1986). The particular neurodegeneration pathway
involves activation of a-amino-3-hydroxy-5-methyl-4-isoxazole
propionic acid (AMPA) and kainate (KA) types of glutamate
receptors in the cortex. In contrast, toxicity induced via the N-
methyl-D-aspartate (NMDA) type of glutamate receptors is atten-
uated by zinc elevation (Weiss et al., 1993).
We propose here an explanation for this variable response to
zinc elevation by showing that tPA opposes the action of zinc and
vice versa, suggesting that where they coincide in the brain, zinc
and tPA have neuroprotective roles rather than neurotoxic ones.
Results
tPA attenuates zinc neurotoxicity
Hippocampal neuronal cultures prepared from wild-type and
tPAÀ/À
newborn mouse pups were exposed to increasing concen-
trations of ZnCl2. After 24 h, the culture medium was collected.
Neuronal cell death was analyzed by LDH release assay. Signif-
icantly increased neuronal death was detected when either wt or
tPAÀ/À
hippocampal cultures were exposed to 350 AM of zinc.
Wild-type neurons were modestly less susceptible to zinc-induced
death than tPAÀ/À
neurons at higher concentrations (350 and 455
AM zinc; Fig. 1A, P < 0.05), indicating the presence of a threshold
concentration above which zinc is toxic. Zinc toxicity was atten-
uated for both neuronal genotypes in the presence of 10 Ag/ml of
exogenously supplied tPA (Fig. 1A), in agreement with reports on
rat mixed cortical cells (Kim et al., 1999). Similar results were
observed in tPAÀ/À
cultures upon addition of 10 Ag/ml of catalyt-
ically inactive (S478A) tPA (Fig. 1D). The chloride anion did not
1044-7431/$ - see front matter D 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.mcn.2003.10.007
* Corresponding author. Department of Pharmacology, University
Medical Center at Stony Brook, BST 7-183, Stony Brook, NY 11794-
8651. Fax: +1-631-444-3218.
E-mail address: stella@pharm.sunysb.edu (S.E. Tsirka).
Available online on ScienceDirect (www.sciencedirect.com.)
www.elsevier.com/locate/ymcne
Mol. Cell. Neurosci. 25 (2004) 162–171

2. Fig. 1. (A) Exposure to zinc is toxic to hippocampal neurons. Primary hippocampal neuronal cultures from wild-type or tPAÀ / À
mice were exposed to
increasing concentrations of zinc for 24 h. At 350 AM zinc, tPAÀ / À
cultures exhibited a significantly greater amount of cell death than wild-type cultures.
Addition of tPA resulted in a significant attenuation of toxicity for both wild-type and tPAÀ / À
hippocampal neurons. tPA by itself (in the absence of
excitotoxicity) did not cause significant death. (B) Representative panels from wild-type hippocampal neurons cultured in the absence or presence of 10 Ag/ml
tPA, zinc, or zinc + tPA. Neurons were stained with Neurofilament H antibody. Note the absence of degeneration of neurites in the panel where only zinc is
present. (C) Exposure (1 h) to zinc is toxic to rat hippocampal neurons. Cell death was determined 24 h after zinc exposure by LDH release assay. Addition of
tPA resulted in significant attenuation of toxicity in rat hippocampal neuronal cultures. ##P < 0.01 when compared to the cultures with the same concentration
of zinc but without tPA. (D) Addition of the catalytically inactive S478A tPA resulted in a significant attenuation of zinc toxicity for both wild-type (not shown)
and tPAÀ / À
hippocampal neurons. ##P < 0.01; #P < 0.05, when compared to cultures with the same concentration of zinc but without S478A tPA.
M.M. Siddiq, S.E. Tsirka / Mol. Cell. Neurosci. 25 (2004) 162–171 163

3. contribute to the toxicity, because there is already 137 mM of
NaCl in the medium, and the addition of the highest dose of ZnCl2
constituted increase of only 1.0 mM. Prior studies used rats as the
animal model (Kim et al., 1999); rat hippocampal neurons
exposed to 210 or 420 AM of zinc for 1 h similarly underwent
widespread cell death, with the addition of tPA being neuro-
protective (Fig. 1C).
Cell injury was confirmed using immunocytochemistry. Mouse
hippocampal neurons were stained with neurofilament antibody
(Fig. 1B). In control cells, the antibody stained both the cell body
and the neurites. No changes were observed in neurons exposed to
10 Ag/ml tPA (Fig. 1B, +tPA). The exposure of neurons for 24 h to
280 AM of zinc resulted in abnormal cell bodies and diminished
neurites (Fig. 1B, +280 AM of zinc). However, the addition of
tPA protected the neurons from zinc toxicity (Fig. 1B, +280 AM
zinc + tPA).
The extent of neurodegeneration in wt and tPAÀ/À
mice was
assessed also in vivo after intrahippocampal infusion of zinc over
the CA1 region (Fig. 2A). Using Timm staining (data not
shown), we only obtain limited diffusion of the zinc infused,
which could explain the limited area of cell death only over the
CA1 subfield.
tPAÀ/À
hippocampal neurons were more sensitive to the local
delivery of zinc compared to the wild-type ones even at concen-
trations as low as 5 nmol/h (Table 1), a result that agrees with the
culture data obtained (Figs. 1A, B). We used terminal deoxynu-
cleotidyl transferase-mediated biotinylated dUTP nick end labeling
(TUNEL) staining to assess the type of neuronal death we observe
with the delivery of zinc. As shown in Fig. 2B, TUNEL staining,
indicating apoptotic cell death, was evident in the CA1 region of
tPAÀ/À
mice in the area where zinc was infused.
tPA activity is inhibited by zinc
The fact that tPA protects neurons in culture from zinc toxicity
is striking in that tPA does not act synergistically with the zinc.
This suggested that zinc might neutralize tPA’s neurotoxic proper-
ties, which depend on its enzymatic activity. We found that
dramatic inhibition of tPA activity was observed as a function of
increasing zinc concentration (Table 2). This result was anticipated
because in early reports tPA was purified using zinc-agarose
columns (Rijken and Collen, 1981). Comparable inhibition was
observed using ZnSO4. When zinc was incubated with equal molar
concentrations of TPEN (a specific zinc chelator) before its
addition to tPA, tPA’s proteolytic activity was not inhibited (data
not shown), indicating that it is free zinc that inhibits tPA. The
inhibition was specific to zinc; increasing concentrations of CaCl2
did not significantly alter tPA’s activity (data not shown). The
inhibition of tPA activity by zinc was compared to the inhibitory
effect by plasminogen activator inhibitor-1 (PAI-1), a PA specific
endogenous inhibitor. We found that we could obtain the same
degree of inhibition when 50 ng of tPA was incubated either with
Fig. 2. Physiological concentrations of tPA are sufficient to confer neuroprotection against zinc. (A) Representative sections of wild-type mice
intrahippocampally infused with 10 nmol/h zinc for 6 days displayed no neurodegeneration. In contrast to wild-type mice, tPAÀ/À
animals displayed
considerable amounts of neuronal death along the CA1 region with the same zinc infusion. (B) Representative sections of wild-type mice intrahippocampally
infused with 10 nmol/h zinc for 6 days displayed no apoptotic, TUNEL-positive cell death. In contrast, tPAÀ/À
animals displayed considerable amounts of
TUNEL+
neuronal death along the CA1 region with the same zinc infusion.
Table 1
tPAÀ/À
hippocampal cells are more sensitive to zinc toxicity
Rate of ZnCl2 (nmol) Wild-type tPAÀ/À
Delivery per hour % Neurodegeneration
F SD
% Neurodegeneration
F SD
2.5 No death (n = 4) No death (n = 4)
5 No death (n = 3) 31.0 F 5.2 (n = 4)
10 3.5 F 3.4 (n = 3) 36.0 F 3.0 (n = 3)
25 47.7 F 1.3 (n = 2) Not tested
Table 2
Zinc inhibits the proteolytic activity of tPA
Concentration of
ZnCl2 (AM)
% tPA activity
(DA405nm)
0 100
8.75 52 F 2
17.5 48 F 2
35 31 F 1
105 23 F 2
175 19 F 1
350 8 F 4
M.M. Siddiq, S.E. Tsirka / Mol. Cell. Neurosci. 25 (2004) 162–171164

4. 175 AM zinc (approximately 80% inhibition of tPA activity) or 25
units of PAI-1 (78% inhibition).
Zinc binds to tPA
The inhibition of tPA’s proteolytic activity by zinc suggested
that there may be a physical interaction between zinc and tPA. To
evaluate whether tPA can directly bind zinc, we used two
approaches, the first of which involved a solid phase binding
assay: 1 Ag of tPA was incubated with varying concentrations of
ZnCl2 and 2 ACi of 65
ZnCl2. Binding of radioactive zinc to tPA was
observed and was specifically competed away as the concentration
of cold Zn increased (Fig. 3A).
The second approach detected zinc binding on proteins after
their electrophoretic separation by SDS-PAGE. Different amounts
of tPA, S478A (catalytically inactive) tPA, and control proteins
were subjected to electrophoresis, transferred onto a PVDF mem-
brane, and incubated with 65
ZnCl2. Strong and dose-dependent
binding was detected both for wild-type tPA (Fig. 3B, lanes 1 and
2) and for S478A tPA (Fig. 3B, lanes 3 and 4). The relative
efficiency of binding was examined by inclusion of two known
zinc-binding proteins (BSA and collagenase), which yielded pos-
itive signals (Fig. 3B, lane 5, data not shown for collagenase),
whereas nonspecific binding was controlled by inclusion of a
protein (cytochrome c) known not to bind zinc (Fig. 3B, lanes 6
and 7). The results confirm that tPA not only binds zinc but does
not need to be proteolytically active to do so.
Fig. 3. Zinc binds tPA. (A) The direct interaction between zinc and tPA was
examined using 65
Zn2 +
in a filter-binding assay. The residual, non-filter-
bound zinc was washed away. (B) Recombinant tPA (2 and 5 Ag, lanes 1
and 2) and S478A tPA (2 and 5 Ag, lanes 3 and 4) were subjected to SDS-
PAGE, transferred to PVDF membrane, and incubated with 65
Zn. BSA
(10Ag, lane 5) and cytochrome c (10 and 20 Ag, lanes 6 and 7) were used as
positive and negative controls, respectively. The protein concentration was
quantified using a Bradford assay.
Fig. 4. (A) tPA decreases free zinc levels in culture medium and facilitates
zinc import into hippocampal neurons independently of its proteolytic
function. Wild-type and tPAÀ/À
neuronal culture medium was collected after
exposure of the cells to zinc with or without tPA for 24 h, and TSQ
fluorescence was quantified. In the presence of tPA alone (no exogenously
added zinc), there was significant decrease in TSQ fluorescence in the culture
medium from both wild-type and tPAÀ/À
neurons ( P < 0.05). In the presence
of tPA and with increasing concentrations of zinc, for all concentrations
tested, there was a very significant decrease ( P < 0.01) in TSQ fluorescence
for both genotypes of neurons. (B) tPAÀ/À
hippocampal neurons were
pretreated with either tPA or S478A tPA for 30 min, and then 0.5 ACi 65
ZnCl2
was added to all wells. Later (1 h) cells were lysed and samples were read on a
gamma counter and analyzed for total protein content. (C) Wild-type (black
symbols) and tPAÀ/À
(red symbols) hippocampal neurons were incubated in
the absence (E) or presence (n) of 10 Ag/ml tPA and 0.5 ACi 65
ZnCl2 in
Locke’s buffer and the indicated concentrations of cold zinc. Zinc import and
total protein content was quantified 2 h later. n = 9, *P < 0.05 for both
genotypes in the presence of tPA.
M.M. Siddiq, S.E. Tsirka / Mol. Cell. Neurosci. 25 (2004) 162–171 165

5. tPA decreases extracellular free zinc levels and facilitates the
transport of zinc into neurons
How might tPA counter zinc toxicity? Although zinc interacts
with tPA physically, the concentration of zinc in the cultures (300
AM) was far higher than the concentration of tPA (approximately
170 nM). Hence, although zinc could be physically inhibiting tPA,
tPA could not be sequestering zinc to any significant extent. We
examined whether tPA might have an indirect effect on free zinc
concentrations. tPAÀ/À
hippocampal cultures were exposed to zinc
for 24 h, with or without tPA (Fig. 4A). Culture supernatants were
collected and the free zinc fluorescent indicator, TSQ, was added.
The amount of free zinc detected at each concentration decreased
in the presence of tPA. Furthermore, the detection of free zinc in
culture was specific, because the addition of TPEN (a chelating
agent specific for zinc) to the collected supernatants before adding
TSQ eliminated detectable fluorescence (data not shown). Wild-
type hippocampal cultures exhibited lower levels in free zinc
compared to tPAÀ/À
cultures, presumably because wild-type cells
generate and secrete endogenous tPA. Similar results were ob-
served with another fluorescent indicator for free zinc, Newport
Green (Molecular Probes, data not shown).
These results suggested that the cells might be importing the
zinc into storage vesicles in response to tPA stimulation. To
examine this possibility, wild-type and tPAÀ/À
hippocampal neu-
rons were challenged with tPA in the presence of 65
Zn and the
amount of zinc that became cell-associated was determined. An
increase in the amount of cell-associated zinc was observed in the
presence of tPA (Fig. 4B). S478A tPA was equally effective,
indicating that the proteolytic activity of tPA was not required to
promote the import event.
Significant increases in the amount of cell-associated zinc were
also evident when wild-type and tPAÀ/À
hippocampal neurons
were incubated with tPA and radioactive zinc progressively com-
peted by increasing concentrations of cold zinc (Figs. 4C and 5A).
To determine whether the cell-associated increase in zinc reflected
import into cells rather than cell-surface binding, wild-type neurons
were treated with pronase to eliminate all cell surface receptors (but
without interfering with cell integrity; (Carroll et al., 1993). The
amount of cell-associated zinc was not altered by the pronase
treatment, indicating that the radioactive zinc detected was intra-
cellular and hence reflected import (not shown). At baseline
conditions (in the presence only of trace amounts of radiolabeled
zinc), zinc import was still evident and occurred at higher levels for
wild-type hippocampal neurons (3.19 + 0.48 cpm/Ag protein) in
comparison to tPAÀ/À
cells (2.22 + 0.27 cpm/Ag protein, P <
0.00005, n = 9). We suggest that this difference is evident because
only exogenous tPA is present in tPAÀ/À
neurons, as opposed to
the presence of both endogenously secreted tPA from the wild-type
neurons and exogenously added tPA. Similar increases in intracel-
lular zinc in the presence of tPA were observed for rat hippocampal
and cortical neuronal cultures (Figs. 5B, C), indicating that
although there are differences in the degree of susceptibility to
zinc-induced cell death between the two species, import takes place
similarly as a consequence of the presence of extracellular tPA.
Because a significant component of zinc trafficking into neu-
rons is thought to be mediated by the voltage-sensitive calcium
channels (VSCC), the Ca2+
-permeable AMPA/KA channels, and
NMDA receptors, we used specific inhibitors for each of these
channels to determine which route(s) was being used. Wild-type
and tPAÀ/À
hippocampal neurons were preincubated with tPA or
NAS (specific inhibitor for Ca2+
permeable AMPA/KA channels)
(Figs. 6A, B). NAS significantly reduced the amount of zinc
imported into neurons in the presence of tPA for both wild-type
and tPAÀ/À
cultures, indicating that these channels constitute one
way via which tPA facilitates the import of zinc into the cells.
Nimodipine (5 AM), a specific inhibitor of the L-type VSCC, was
used next (Figs. 6C, D). Nimodipine elicited only a minimal
Fig. 5. tPA facilitates zinc import in rat hippocampal and cortical neuronal
cultures. (A) Wild-type mouse hippocampal, (B) rat hippocampal, and (C)
cortical neuronal cultures were pretreated with (.) or without (n) tPA for
30 min, and then 0.5 ACi 65
ZnCl2 was added to all wells along with the
indicated concentrations of cold zinc. Zinc import was quantified 2 h later
(*P < 0.05 and **P < 0.01).
M.M. Siddiq, S.E. Tsirka / Mol. Cell. Neurosci. 25 (2004) 162–171166

6. inhibitory effect on zinc transport in wild-type neurons, but had no
effect in tPAÀ/À
neurons, suggesting that the primary route of tPA-
mediated zinc entry into neurons would be via the A/K Ca channels.
However, exposure to both NAS and nimodipine resulted in an
additive effect, diminishing the levels of import lower than that of
cells incubated without added tPA (Fig. 7A). The effect of inhibitors
on tPA-mediated zinc import is small, and the question then emerges
whether such small changes in zinc uptake could mediate the tPA
protective effect. We evaluated the extent of cell death after exposure
of wild-type neurons to zinc in the presence or absence of tPA or
NAS. As shown in Fig. 7B, the presence of NAS could reverse the
neuroprotective effect of tPA against zinc exposure. NAS by itself
had no effect on neurons. Similar results were obtained when
nimodipine was used instead of NAS (data not shown).
To examine the third potential pathway for zinc import,
NMDA receptors (NMDAR), we used the NMDAR specific
Fig. 6. Ca2+
channel inhibitors reduce the tPA-facilitated zinc transport into hippocampal neurons. Wild-type (A, C, E) and tPAÀ/À
(B, D, F) hippocampal
neurons were exposed to 200 AM NAS (A, B), 5 AM nimodipine (C, D), and 50 AM MK-801 (E, F) in the presence of zinc F tPA as in Figs. 5 and 6. *P <
0.05, **P < 0.01 for comparing no exogenous tPA to added tPA; #P < 0.05, ##P < 0.01 for comparing the addition of tPA only to co-administration of
inhibitor and tPA.
M.M. Siddiq, S.E. Tsirka / Mol. Cell. Neurosci. 25 (2004) 162–171 167

7. inhibitor, MK-801. MK-801 (50 AM) had no effect on zinc import
(Figs. 6E, F), indicating that this route of zinc entry is not
facilitated by tPA. This result agrees with previous findings
(Canzoniero et al., 1999).
It is possible that the observed increase in zinc import via the
VSCC and Ca A/K pathways was due to the fact that these
experiments were performed in the absence of the natural ion
permeating these channels, calcium. To address this possibility, we
co-administered Ca2+
with zinc or tPA. There was still a significant
increase in the amount of zinc imported into the cells under all
conditions tested (Fig. 7C), indicating that zinc enters neurons via
VSCC and Ca A/K under conditions of physiological levels of
calcium.
Discussion
It was previously reported that the Zn2+
toxicity observed for rat
cortical cells could be countered by the addition of wild-type tPA, or
tPA that had been preincubated with plasminogen activator inhib-
itor-1 (a specific tPA inhibitor, Kim et al., 1999). We show here that
tPA can regulate the concentration of extracellular zinc both by
binding to it (which becomes important at very low concentrations
of zinc) and by facilitating its import into neuronal cells. Converse-
ly, tPA’s proteolytic activity is inhibited by zinc. In addition to its
effects on tPA that we report here, zinc has been shown to inhibit
cysteine proteases and the HIV protease by binding to their active
sites (Katz et al., 1998). We observed that the sensitivity of primary
hippocampal neurons to zinc toxicity is heightened if the neurons
lack the capacity to express endogenous tPA, suggesting that the
sequestration and neuroprotection mediated by endogenous levels
of tPA may have physiological significance.
In vivo, wild-type mice are more resistant to zinc toxicity
compared to tPAÀ/À
mice (Table 1), suggesting that under normal
physiological conditions, tPA may be functioning to alleviate some
of the toxic effects risked by an overload of synaptically released
zinc.
Zinc has been shown to bind to several proteins expressed in
the CNS. Metallothioneins are vital zinc-binding proteins
expressed in astrocytes (MT-I and -II) and in zinc-containing
dentate granule neurons (MT-III, Aschner, 1996). Overexpression
or elimination of MT-III has dramatic consequences on CNS
homeostasis. Increased neuronal damage and frequency and sever-
ity of epileptic events occur in mice lacking MT-III (Erickson et al.,
1997) or a combination of MT-I and -II (Carrasco et al., 2000).
Conversely, overexpression of MT-III protects neurons from exci-
totoxic and radiation damage (Cai et al., 2000; Erickson et al.,
1997). tPA could function to some extent as a native chelator of
zinc in a mechanism analogous to or in combination with that of
MT proteins (Cole et al., 2000).
Our data concur with the previous report (Kim et al., 1999) for a
potential neuroprotective role for tPA. Endogenous neuronal tPA
does not seem to be strongly protective against zinc toxicity in our
culture assays. We think that this is due to the lower tPA
concentration secreted by neurons compared to the exogenous
tPA added in the assay (when we measured the amount of tPA
Fig. 7. tPA facilitates zinc import through the Ca2+
permeable channels in
the presence of Ca2+
. (A) The presence of exogenous tPA resulted in a
significant increase in the amount of zinc imported into wild-type
hippocampal neurons. The combination of NAS and nimodipine decreased
the amount of zinc imported into neurons below the level that each inhibitor
alone did, suggesting that they may act synergistically. *P < 0.05; **P <
0.01; ##P < 0.01. (B) Addition of zinc import inhibitors results in the
reversal of tPA’s neuroprotective effect. The presence of NAS blocked the
protection conferred by tPA against the toxicity of 280 AM zinc. Wild-type
hippocampal neurons were pretreated for 30 min with tPA or NAS. Cell
death was determined 24 h after zinc exposure by LDH release assay.
Statistical analysis (Student’s t test) comparing the values obtained for
neuronal death of cells exposed to zinc versus those exposed to zinc + tPA,
or zinc + tPA compared to the treatments with NAS revealed that the
differences were significant ( P < 0.01). (C) tPAÀ/À
hippocampal neurons
were treated with varying concentrations of Ca2+
in Locke’s buffer for a 2-h
import assay. In the absence of Ca2+
, there was a very significant increase in
the amount of zinc imported in the presence of 280 AM zinc. However, zinc
import was still observed in the presence of Ca2+
(0.1 and 0.5 mM, **P <
0.01; 2 mM, *P < 0.05).
M.M. Siddiq, S.E. Tsirka / Mol. Cell. Neurosci. 25 (2004) 162–171168

8. secreted by the neurons and found that the exogenous is about
100Â in excess than what the neurons normally secrete). However,
this is a non-indicative measurement, because in the mouse brain, it
is also microglial cells that secrete tPA, especially after injury (such
as zinc toxicity). The tPA contribution of microglial is quite
significant (Tsirka et al., 1995); when we perform the zinc toxicity
assays in mixed cortical cultures (which include both neurons and
microglial cells), the neuroprotective effect of endogenous tPA is
quite prominent, even at lower concentrations of exogenous tPA.
Furthermore, even in neuronal cells, we cannot be sure of what the
localized concentration of tPA might be in the perisynaptic space.
However, adding to the importance of endogenous tPA, tPAÀ/À
pyramidal hippocampal neurons show increased susceptibility to
zinc toxicity compared to wild-type ones (Fig. 2A). As we show,
tPA facilitates the transport of zinc into neurons independently of
its proteolytic activity. The tPA-mediated accumulation of intra-
cellular zinc combined with decreased levels of cell death suggest
that tPA contributes to the sequestration of free zinc, either by
mediating its entry into vesicles or by up-regulating the expression
of zinc-chelating proteins such as the MTs.
We think that it is unlikely that the import of zinc into neurons
is due to formation or endocytosis of a Zn–tPA complex for the
following reasons:
1. We inhibited the low-density lipoprotein receptor-related
protein (LRP), a known receptor for tPA, which is expressed
in neurons and has been shown to mediate the endocytosis and
clearance of tPA (Zhuo et al., 2000). We used 500 nM of
receptor-associated protein (RAP, a specific inhibitor for LRP)
in the import assay. No inhibition of zinc import into wild-type
hippocampal neurons was observed (data not shown), indicating
that LRP-mediated specific tPA endocytosis is not the
mechanism through which zinc enters.
2. Generic endocytosis was inhibited in cultured hippocampal
neurons by decreasing the temperature of the import assays to
20jC. No change in the amount of zinc entering the neurons in
the presence of tPA was observed.
The attenuation of zinc toxicity by tPA and the facilitation of
zinc import into hippocampal neurons were reproduced using
mixed cortical cultures (data not shown). Zinc toxicity on mixed
cortical cultures was attenuated with the addition of tPA or
S478A tPA. Furthermore, there was a significant increase in
the amount of intracellular zinc when tPAÀ/À
-mixed cortical
cultures were exposed to tPA. Both the zinc import and cell
death results for mixed cortical cells were carried out in DMEM
with 1% FBS, indicating that the attenuation of zinc toxicity and
increased import of zinc were due to tPA specifically, not to the
absence of other CNS cell types, and not localized solely to the
hippocampus.
Using rat cortical and hippocampal neuronal cultures, we
confirmed the protection against zinc toxicity conferred to these
neurons in the presence of tPA. We found that rat neuronal cells are
more sensitive to zinc compared to mice, which is consistent with
previous reports that mouse cortical neurons require 2–7-fold
higher concentrations of zinc to exhibit similar neuronal death
(Yokoyama et al., 1986). Rat hippocampal neurons exposed to 210
or 420 AM zinc alone for 1 h resulted in widespread cell death (in
agreement with previous reports, Chen and Liao, 2003), whereas
only minimal cell death is detected in mouse hippocampal cells
exposed to similar concentrations of zinc for the same time (data
not shown). Zinc import was observed at concentrations below
those that induced cell death, suggesting that tPA facilitates zinc
import in physiological concentrations of zinc.
In PC12 cells, blockade of the L-type voltage-gated Ca2+
channels with 1 AM Nimodipine markedly attenuated Zn2+
-in-
duced neurotoxicity (Kim et al., 2000). Under normal ionic
conditions encountered in the brain, cortical cultures exhibit a
small, non-inactivating, voltage-gated inward current that is sensi-
tive to L- and N-type high voltage-activated Ca2+
channel inhib-
itors (Kerchner et al., 2000). Similarly in hippocampal neurons,
tPA movement of zinc into neurons is dependent on the VSCC, as
determined by the modest inhibition of zinc transport with the
addition of 5 AM Nimodipine in wild-type cultures. Taken together,
our data suggest that the mechanism through which tPA opposes
zinc neurotoxicity involves direct physical interaction and indirect
cellular responses.
The role of neuronal zinc in the brain has not been well defined.
We have previously reported that tPA plays a physiological role in
neurite outgrowth in the hippocampal mossy fiber formation,
which is a region of intense zinc concentration. tPA functions in
this setting through both enzymatic and non-enzymatic pathways.
tPAÀ/À
mice have distorted and punctuated mossy fibers, with less
zinc accumulation (Wu et al., 2000). If tPA is required for the
movement of zinc into the neurons, then it is not hard to envision
how tPAÀ/À
mice have abrogated mossy fiber sprouting after
kainate-induced seizures. It is tempting to speculate that zinc
may act in this setting to regulate tPA’s downstream enzymatic
pathway, and possibly even its non-enzymatic pathway, if binding
of zinc to tPA at the noncatalytic sites interferes with tPA’s
stimulation of microglial activation. Accordingly, zinc and tPA
may act to fine-tune or place constraints on promotion of mossy
fiber outgrowth. It is also interesting to speculate that tPA may
function to sequester free zinc and make it cryptic, so that it can do
no harm to the neurons.
Experimental methods
Hippocampal neurons
Hippocampal cultures were prepared from C57BL/6 (wild-type,
wt) and tPAÀ/À
mice (Rogove and Tsirka, 1998). Hippocampi were
dissected out from newborn brains and trypsinized. Trypsinization
was terminated by the addition of soybean trypsin inhibitor. The
tissue was washed in Neurobasal medium supplemented with B27,
25 AM L-glutamate, 0.5 mM L-glutamine, and 40 mg/l gentamycin
sulfate (G3 medium). The cells were separated by trituration,
seeded onto poly-L-lysine and laminin-coated 96-well plates, and
maintained at 37jC in 5% CO2.
Embryonic day 18 rat cortices and hippocampi were purchased
from BrainBits (Southern Illinois University), and primary neuro-
nal cultures were prepared according to the provider’s protocol.
The cells were switched at day 3 to G3 medium without L-
glutamate (G2 medium) and used 4–7 days later for experiments.
The primary mouse hippocampal neurons (6–9 days in culture)
were exposed to zinc and tPA in Neurobasal medium supplemented
with 14.5 mM glucose and L-glutamine in the presence of 1.36 mM
Ca2+
. The primary rat cultures were exposed to zinc and tPA for 1
h in HEPES-buffered saline solution (HSS). Primary rat hippo-
campal neuronal cultures were first washed three times with HSS
(120 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 20 mM HEPES, pH
M.M. Siddiq, S.E. Tsirka / Mol. Cell. Neurosci. 25 (2004) 162–171 169

9. 7.4, 15 mM Glucose, and 1.8 mM CaCl2) and then exposed to 210
or 420 AM zinc for 1 h in HSS, and then subsequently changed to
G2 media.
Lactate dehydrogenase (LDH) release assay
Overall cell injury in rat or mouse neuronal cells was quantified
by release of LDH into the medium after 24 h of exposure to zinc
or tPA. LDH release was determined using a commercial kit
(Roche, Inc). To quantify percent toxicity, LDH values were
determined for control cells killed by lysis using 1% Triton-X or
by exposure to 100 AM NMDA. These values indicated the amount
of LDH that would be released if 100% toxicity occurred, and
experimental values were normalized to them.
Immunohistochemistry
Wild-type hippocampal neurons were fixed on coverslips with
4% paraformaldehyde–4% sucrose and permeabilized using 0.1%
Triton X-100. After blocking with goat serum (10% in PBS), the
mouse anti-Neurofilament antibody (Sternberger Monoclonals Inc)
was used at a 1:1000 dilution followed by incubation with goat
anti-mouse IgG (Alexa 488) (Molecular Probes, Inc). The cover-
slips were mounted with Vectashield medium (Vector Labs) and
the cells imaged.
Amidolytic assay
tPA’s proteolytic activity was measured by a colorometric
assay (Gualandris et al., 1996). Zinc and tPA were incubated at
RT for 15 min in chelex-treated PBS (PBS was treated with the
weak cation-chelating resin Chelex 100 to remove traces of
divalent cations). The samples were incubated in 0.1 M Tris–
HCl, pH 8.1, 0.1% (v/v) Tween 80, and 0.3 mM of the plasmin
substrate S-2251 (DiaPharma, Inc) at 25jC. The change in
absorbance at 405 nm was measured against blanks that lacked
tPA. The data presented are the average of three independent
experiments.
Binding of zinc to tPA
The 65
ZnCl2-binding assay was performed as described (Stradal
et al., 2000). tPA (1 Ag) was diluted into 100 Al of chelex PBS and
incubated with varying concentrations of ZnCl2 and 2 ACi of
65
ZnCl2 (NEN). The samples were spotted onto glass microfiber
filters (Whatman) and placed on a vacuum apparatus. The filters
were washed three times with 10% trichloroacetic acid and then
twice with 100% ethanol. The dried filters were counted in a gamma
counter to quantify the amount of zinc bound to tPA.
65
ZnCl2-overlay experiments were performed as described
(Serrano et al., 1988) with minor modifications. tPA (or
S478A tPA) was analyzed by SDS-PAGE and transferred onto
a PVDF membrane. The PVDF membrane was soaked in 0.05%
Tween 20 in PBS for 3 h at RT, followed by a 2-h incubation in
10 mM Na 1,4-piperazinediethanesulfonic acid, pH 6.9, 50 mM
NaCl, 0.5 mM MnCl2, and 5 mM dithiothreitol. Five micromolar
65
ZnCl2 (1 ACi/ml) was added and the incubation continued
overnight. The membrane was washed once for 1 min in the
above buffer without zinc, twice more for 30 s with distilled
water, and then dried on filter paper and exposed for 2–4 h on
Kodak XAR-5 film.
In vivo zinc toxicity in wild-type and tPAÀ/À
mice
Adult wild-type (wt) and tPAÀ/À
mice (approximately 25 g)
were injected intraperitoneally with atropine (0.6 mg/kg body
weight) and then deeply anesthetized with 2.5% avertin (0.02 ml/
g body weight). The intrahippocampal infusion coordinates were:
bregma, 2.5 mm; medial–lateral, 1.7 mm; and dorsoventral, 1.6
mm. Zinc was infused at 2.5, 5, 10 and 25 nmol/h over 6 days. The
mice were then perfused with 0.2% Na2S in 0.15 M SØrensen
phosphate buffer, pH 7.4 (Holm and Geneser, 1991). The brains
were removed and embedded in OCT at À80jC. Neuronal survival
was determined by cresyl violet staining. The stained sections were
photographed (Nikon CoolPix 990) on a Nikon Eclipse TS100
microscope under brightfield optics. The degree of neurodegener-
ation was measured using the NIH1.62f Image software package.
The length of pyramidal neuronal loss was traced and measured as
arbitrary units. The entire length of the hippocampal pyramidal
layer was also measured, and the percent loss of neurons on the
infused side calculated. Five sections in minimum were quantified
for each zinc dosage per genotype.
TSQ detection of free zinc in culture medium
The levels of free zinc were measured with TSQ in culture
medium from hippocampal neurons exposed to zinc, with or
without tPA. An equal volume of TSQ solution (10 AM in 280
mM Na barbital, 280 mM Na acetate, pH 10) to culture medium
was added and the fluorescence was read on a microplate reader
(excitation: 355 nm, emission: 460 nm). A standard curve of
known concentrations of zinc in culture medium was generated
to ensure linear detection of zinc in the cultures.
Import assay
The zinc import assay was carried out as described (Colvin et
al., 2000), with modifications. Hippocampal neurons were washed
three times with Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 5.0
mM HEPES, pH 7.4, and 10 mM Glucose). tPA or inhibitors
[nimodipine and 1-Naphtyl-acetyl spermine trihydrochloride
(NAS) (Sigma)] were added in Locke’s buffer for 30 min, followed
by the addition of radioactive (0.5 ACi 65
ZnCl2) or cold zinc. After
a further incubation for 30, 60, 120, 180, or 240 min at 37jC, zinc
import was terminated by washing the cells twice (5 min each
wash) with ice-cold Locke’s buffer with 1 mM EGTA. Cells were
washed one more time with ice-cold Locke’s buffer, lysed with 0.5
N NaOH, then collected and counted on the gamma counter. The
results are presented as counts of zinc over the total protein content
(DC Protein Assay, Bio-Rad). All data represented were carried out
in triplicate or quadruplicate.
Terminal deoxynucleotidyl transferase-mediated biotinylated
dUTP nick end labeling (TUNEL) reactivity
Frozen sections were used for TUNEL assay (In Situ Cell Death
Detection Kit, POD-conjugated, Boehringer Mannheim). Hippo-
campi from wild-type or tPAÀ/À
mice infused with zinc were
embedded in Tissue-Tek OCT, frozen on dry ice, and stored at
À80jC until use. Coronal sections (14 Am) were cut on a cryostat
(Leica Inc) at À20jC. Then sections were fixed in 4% parafor-
maldehyde and permeabilized with 0.1% Triton X-100 in 0.1%
sodium citrate. Terminal deoxynucleotidyl transferase (TdT) and
M.M. Siddiq, S.E. Tsirka / Mol. Cell. Neurosci. 25 (2004) 162–171170

10. fluorescein-dUTP were then added to cover the sections and
incubated in a humidified chamber for 60 min at 37jC in the
dark. The reaction was terminated by washing with PBS. Then the
slides were covered with antifade and analyzed using a confocal
microscope (PCM2000, Nikon, Inc).
Hippocampal neurons from both genotypes of mice incubated
with or without zinc or with zinc and tPA were also fixed as above
and subjected to the TUNEL staining procedure.
Statistical analysis
Data are presented as mean F standard deviation. The signif-
icance of the difference between the mean was calculated by
unpaired Student’s t test, as appropriate. Numbers of individual
experiments are indicated by n. Probability values of P < 0.05 were
considered to represent significant differences, and P < 0.01 were
considered to represent very significant differences.
Acknowledgments
We would like to thank Dr. Chia-Jen Siao for instruction
regarding preparing the primary cultures, Drs. Dan Bogenhagen
and Sanford Simon for reagents, and members of the Tsirka lab and
Dr. M. Frohman for critical comments on the manuscript. We are
also grateful to Genentech Inc for providing recombinant human
tPA and S478A tPA. This work was supported by NIH and
Klingenstein Foundation grants to SET.
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