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Mol. Cell. Neurosci. 25 (2004) 162–171

Modulation of zinc toxicity by tissue plasminogen activator

Mustafa M. Siddiqa,b and Stella E. Tsirkab,*

aProgram in Molecular and Cellular Biology, University Medical Center at Stony Brook, Stony Brook, NY 11794-8651, USAbDepartment 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 (tPA�/�) or plasmin(ogen)-deficient (plg�/�) 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).

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.)

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

tPA�/� 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

tPA�/� hippocampal cultures were exposed to 350 AM of zinc.

Wild-type neurons were modestly less susceptible to zinc-induced

death than tPA�/� 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 tPA�/� cultures upon addition of 10 Ag/ml of catalyt-

ically inactive (S478A) tPA (Fig. 1D). The chloride anion did not

Fig. 1. (A) Exposure to zinc is toxic to hippocampal neurons. Primary hippocampal neuronal cultures from wild-type or tPA� /� mice were exposed to

increasing concentrations of zinc for 24 h. At 350 AM zinc, tPA� /� 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 tPA� /� 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 tPA� /� 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

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, tPA�/� 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, tPA�/� animals displayed considerable amounts of

TUNEL+ neuronal death along the CA1 region with the same zinc infusion.

M.M. Siddiq, S.E. Tsirka / Mol. Cell. Neurosci. 25 (2004) 162–171164

contribute to the toxicity, because there is already 137 mM of

NaCl in the medium, and the addition of the highest dose of ZnCl2constituted 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 AMzinc + tPA).

The extent of neurodegeneration in wt and tPA�/� 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.

tPA�/� 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

Table 1

tPA�/� hippocampal cells are more sensitive to zinc toxicity

Rate of ZnCl2 (nmol) Wild-type tPA�/�

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

(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

tPA�/� 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 CaCl2did 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

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) 162–171 165

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 of65ZnCl2. Binding of radioactive zinc to tPAwas

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 65ZnCl2. 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 tPAwas

examined using 65Zn2 + in a filter-binding assay. The residual, non-filter-

bound zinc was washed away. (B) Recombinant tPA (2 and 5 Ag, lanes 1and 2) and S478A tPA (2 and 5 Ag, lanes 3 and 4) were subjected to SDS-

PAGE, transferred to PVDF membrane, and incubated with 65Zn. 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 tPA�/� 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 tPA�/� 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) tPA�/� hippocampal neurons were

pretreated with either tPA or S478A tPA for 30 min, and then 0.5 ACi 65ZnCl2was 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 tPA�/� (red symbols) hippocampal neurons were incubated in

the absence (E) or presence (n) of 10 Ag/ml tPA and 0.5 ACi 65ZnCl2 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.

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 65ZnCl2 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).

Cell. Neurosci. 25 (2004) 162–171

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. tPA�/� 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 tPA�/� 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 tPA�/� hippocampal neu-

rons were challenged with tPA in the presence of 65Zn 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 tPA�/� 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 tPA�/� 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 tPA�/� 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 tPAwere 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 tPA�/� hippocampal neurons were preincubated with tPA or

M.M. Siddiq, S.E. Tsirka / Mol.166

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 tPA�/� 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. 6. Ca2+ channel inhibitors reduce the tPA-facilitated zinc transport into hippocampal neurons. Wild-type (A, C, E) and tPA�/� (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

inhibitory effect on zinc transport in wild-type neurons, but had no

effect in tPA�/� 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

M.M. Siddiq, S.E. Tsirka / Mol. Cell. Neurosci. 25 (2004) 162–171168

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 tPA�/� 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) tPA�/� 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) 162–171 169

secreted by the neurons and found that the exogenous is about

100� 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, tPA�/�

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 tPA�/�-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 AMNimodipine 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.

tPA�/� 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 tPA�/� 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 tPA�/� 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.170

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 65ZnCl2-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 of65ZnCl2 (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.65ZnCl2-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 micromolar65ZnCl2 (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 tPA�/� mice

Adult wild-type (wt) and tPA�/� 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 �80jC. Neuronal survivalwas 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 65ZnCl2) or cold zinc. After

a further incubation for 30, 60, 120, 180, or 240 min at 37jC, zincimport 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 tPA�/� mice infused with zinc were

embedded in Tissue-Tek OCT, frozen on dry ice, and stored at

�80jC until use. Coronal sections (14 Am) were cut on a cryostat

(Leica Inc) at �20jC. 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

Neurosci. 25 (2004) 162–171

M.M. Siddiq, S.E. Tsirka / Mol. Cell. Neurosci. 25 (2004) 162–171 171

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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