Neurobiology of Disease 34 (2009) 221–229

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Neurobiology of Disease

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Accumulation of labile zinc in neurons and astrocytes in the spinal cords of G93ASOD-1 transgenic mice

Jean Kim a, Tae-Youn Kim a, Jung Jin Hwang b, Joo-Yong Lee b, Jin-Hee Shin c,Byung Joo Gwag c, Jae-Young Koh a,b,⁎a Neural Injury Res. Lab., Univ. Ulsan Col. Med., Seoul, Republic of Koreab Asan Institute for Life Science, Univ. Ulsan Col. Med., Seoul, Republic of Koreac Pharmacol., Univ. Ajou Col. Med. Suwon, Republic of Korea

⁎ Corresponding author. Neural Injury Res. Lab., Univ. Uof Korea.

E-mail address: jkko@amc.seoul.kr (J.-Y. Koh).Available online on ScienceDirect (www.scienced

0969-9961/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.nbd.2009.01.004

a b s t r a c t

a r t i c l e i n f o

Article history:

Zinc dyshomeostasis may t Received 18 September 2008Revised 7 January 2009Accepted 9 January 2009Available online 21 January 2009

Keywords:ExcitotoxicityMotoneuronsHNEZinc chelatorALS

rigger oxidative stress, which is likely the key mechanism of neuronal death inamyotrophic lateral sclerosis (ALS), including familial forms such as G93A SOD-1 ALS. Since zinc binding byG93A SOD-1 is weaker than by normal SOD-1, we assessed whether labile zinc levels are altered in the spinalcords of G93A SOD-1 transgenic (Tg) mice. Whereas no zinc-containing cells were found in wild-type (WT)mice, neurons and astrocytes with high levels of labile zinc appeared in G93A SOD-1 Tg mice, in correlationwith motoneuron degeneration. The level of HNE, an endogenous neurotoxic molecule, was increased aroundzinc-accumulating cells and mSOD-1 positive cells, suggesting a link between HNE, SOD-1 mutation and zincaccumulation. Moreover, exposure of cultured spinal neurons and astrocytes from G93A SOD-1 Tg mice toHNE increased labile zinc levels, and exposure to zinc increased 4-hydroxynonenal (HNE) levels, to a greaterdegree than in WT neurons and astrocytes. Administration of the zinc chelator TPEN extended survival inG93A SOD-1 Tg mice. These results indicate that zinc dyshomeostasis occurs in the spinal cords of Tg mice,and that this dyshomeostasis may contribute to motoneuron degeneration.

© 2009 Elsevier Inc. All rights reserved.

Introduction

Amyotrophic lateral sclerosis (ALS) is a chronic progressiveneurodegenerative disease mainly afflicting upper and lower moto-neurons in the central nervous system. Although the pathogenicmechanisms of sporadic ALS are largely unknown, oxidative stress(Robberecht, 2000; Bishop et al., 2007), mitochondrial damage(Menzies et al., 2002), abnormal protein aggregation (Bruijin et al.,1998), excitotoxicity (Choi, 1998; Rao andWeiss, 2004), and increasedlevels of HNE (Pedersen et al., 1998; Malecki et al., 2000) have beenimplicated. In contrast, the G93Amutation in the SOD-1 gene has beenshown to cause a form of familial ALS (fALS) (Gurney et al., 1994).Although this mutation was initiating thought to lead to a deficiencyin SOD-1 activity, it has been shown to cause a toxic gain of function.For example, transgenicmice containing the human G93A SOD-1 genehave sufficient SOD-1 activity, but consistently undergo motoneurondegeneration (Matyja et al., 2006; Nagai et al., 2007).

Despite the identification of thismolecular abnormality, the precisemechanism linking the G93A SOD-1mutation to selectivemotoneurondegeneration is not yet completely understood. Chemically, binding of

lsan Col. Med., Seoul, Republic

irect.com).

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zinc to G93A SOD-1 is weaker than that toWT SOD-1, whereas there isno difference in binding of copper (Lyons et al.,1996; Crowet al.,1997),suggesting that abnormal zinc homeostasis may contribute to thepathogenesis of this form of fALS. In this regard, it is intriguing thatlevels of metallothioneins, which function primarily to counter metaltoxicity, are greatly increased in G93A SOD-1 Tg mice (Puttaparthiet al., 2002; Groeneveld et al., 2003; Smith and Lee, 2007).

High levels of endogenous zinc may act as a potential neurotoxin(Choi et al., 1988). Accumulation of labile zinc in degenerating cellbodies has been observed in animal models of brain ischemia,seizures, brain trauma, and hypoglycemia (Frederickson et al., 1989;Koh et al., 1996; Choi and Koh, 1998; Suh et al., 2000; Suh et al., 2004).In these models, measures that reduce zinc accumulation areneuroprotective, indicating that zinc dyshomeostasis contributes toneuronal death (Koh et al., 1996; Suh et al., 2000; Lee et al., 2000; Leeet al., 2002; Suh et al., 2004). In neuronal cultures, increases in zinclevels trigger oxidative stress via protein kinase C (PKC) and NADPHoxidase (Noh and Koh, 2000). Oxidative stress itself, however, mayrelease zinc from zinc-binding proteins such as metallothioneins(Hidalgo et al., 2001; Lee et al., 2003), which may play a crucial role incausing neuronal death (Sillevis Smitt et al., 1994; Gong and Elliott,2000). Hence, oxidative stress and increases in intracellular labile zincmay constitute a vicious cycle.

Since the binding of zinc by G93A SOD-1 is weaker than by normalSOD-1 and since zinc dyshomeostasis may trigger oxidative stress,

Fig.1. Zinc accumulation in degenerating cells in the spinal cords of G93A SOD-1 Tgmice.Horizontal sections of the lumbar spinal cord were stained with TFL-Zn (A, C), andsubsequentlywith acid fuchsin (B, D). No zinc-containing or acidophilic cells were notedin the spinal cords of wild-type (WT)mice (A, B), whereas Tgmice contained a few zinc-containing cells (C), which were also stained with acid fuchsin (D). Scale bar, 50 μm.

222 J. Kim et al. / Neurobiology of Disease 34 (2009) 221–229

which is likely the key mechanism of neuronal death in ALS, weassessed whether zinc dyshomeostasis occurs in the spinal cords ofG93A SOD-1 Tg mice.

Materials and methods

Transgenic mice

The animal experiment protocol was approved by Internal ReviewBoard for Animal Experiments of Asan Life Science Institute, Universityof Ulsan College of Medicine (Seoul, Korea). G93A transgenic mice[high copy number, B6SJL-Tg (SOD1-G93A)1Gur/J] (Gurney et al.,1994) were obtained from The Jackson Laboratories (Bar Harbor, ME,USA). Mice were distinguished using the following scoring system:normalwithno signofmotordysfunction (4 points); hind limb tremorsevident when suspended by the tail (3 points); gait abnormalitiespresent (2 points); dragging of at least one hind limb (1 point); andinability to right itself within 30 s (0 point) (Weydt et al., 2003).

Tissue preparation

G93A transgenic mice and non-transgenic littermates were deeplyanesthetized with zoletil and sacrificed at points 4 (four Tg, four NTg;8 weeks), points 3 (three Tg; 14–15 weeks), points 2 (three Tg; 15–16 weeks), point 1 (four Tg;16–17 weeks ), and point 0 (three Tg; 17–19weeks). Their spinal cordswereharvested and immediately frozen indry ice and stored at−70 °C. Lumbar spinal cords (L1–L5, 10 μm)werecut by cryostat and mounted onto poly-L-lysine coated glass slides.

Immunohistochemistry and cytochemistry

Spinal cord sections and cells were fixedwith 4% paraformaldehyde(Sigma, St. Louis, MO), and incubated in blocking solution containing1% bovine serum albumin (BSA, Sigma) and 0.2% Triton X-100 for30min at room temperature. The sections were incubated overnight at4 °C with antibodies against the motor neuron marker SMI32 (1:1000,Sternberger Monoclonal Inc., Lutherville, MD), the astrocyte markerGFAP (1:1000, Chemicon, Temecula, CA), the microglial marker CD11(1:100, Serotec, Oxford, UK), HNE (1:250, Alpha Diagnostic, SanAntonio, TX), and anti-SOD1 (1:100, Biovision, Mountain View, CA),each in PBS containing 0.5% sodium azide. Tissue samples were rinsedin PBS and incubated for 1 hwith secondary antibody (Alex fluorescentTRITC or FITC, Invitrogen, Carlsbad, CA) at room temperature. Thestained tissues were photographed using a CCD camera (DP70;Olympus, Tokyo, Japan), and the stained cells were photographed byconfocal microscope (TCS-SP2; Leica, Nussloch, Germany).

Counting

Serial transverse sections of spinal cords (10 mm, L1–L5) wereprepared using cryocut. Twenty sections, each 100 mm apart fromadjacent ones (∼2 mm total length) were used for the assessment ofcell numbers. These sections were stained with 0.5% cresyl violet and/or TFL-Zn and then were photographed using a CCD camera under alight microscope (200× magnification). TFL-Zn positive signals(N15 mm2) and motor neurons were counted in the anterior horn(800×650 mm2) of 20 sections using an image analysis software(ImagePro, Media cybernetics, Silver Spring, Maryland). The countingof motor neurons was according to the following formula:

P= AT M= L+Mð Þð Þ

“P is the average number of nuclear points per section, A is thecrude count of number of nuclei seen in the section, M is the thickness(in μ) of the section, and L the average length (in μ) of the nuclei.”(Abercrombie, 1946)

Treatment with TPEN or vehicle

To study the survival, G93A SOD-1 Tg male mice, at 14 weeks ofage, were divided into two groups, and injected intraperitoneally withTPEN (20 mg/kg/day, n=7) (N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine, Sigma, dissolved in 1:4 DMSO:water) or vehicle(n=12) once a day for 7 days. The identity of treatments was blindedto experimenters until the completion of analysis.

To test whether TPEN reaches the central nervous system whengivenas above, levels of labile zinc inhippocampusweremeasured (Fig.S1). Frozen sections of hippocampus were obtained from G93A SOD-1NTg male mice (10–12 weeks of age, n=6/each group) that hadreceived TPEN (20 mg/kg/day, i.p.) or vehicle for 7 days. TFL-Znfluorescence intensity in the dentate gyrus andCA3wasmeasuredwiththe fluorescence microscope using an image analysis program (Image-Pro). To determine specific zinc fluorescence, background fluorescencein hippocampus was subtracted. Specific fluorescence values werenormalized to the mean specific value of vehicle group (=100%).

Astrocyte cultures

Spinal cord astrocytes were prepared from postnatal (P7) G93ASOD-1 Tg and NTg mice. Spinal cords were digested with 0.025%trypsin in Hank's Balanced Salt Solution (HBSS), and isolated spinalcord cells were plated onto poly-L-lysine coated cover glasses inplatingmedium.All cultureswereused atDIV 14,with themedium(5%FBS, 5% HS) changed every 3 days until the cells became confluent. Tgoffspring were genotyped by PCR assay of DNA obtained from the tail.

Spinal cord slice cultures

Organotypic slice cultures were prepared from G93A SOD-1 Tgmice (postnatal days 7–8) as described (Stoppini et al., 1991). Briefly,mice were decapitated and their spinal cords were removed rapidly inice-cold dissecting HBSS. Spinal cord slices (300 mm) were cut with aMcIlwein tissue chopper (Mickle Laboratory Engineering Co. Ltd.,Gomshall, Surrey, UK) and plated onto poly-L-lysine coated glasscoverslips containing culture medium (50% MEM, 25% HBSS, 25% HS,6.5 mg/ml glucose, 2 mM glutamine, pH 7.2), with culture mediumchanged twice weekly. The slices were used at DIV 12–16.

Confocal live cell images and ROI active analysis

Astrocytes and neurons were stained with 5 μM FluoZin-3-AM(Molecular Probes, Eugene, OR) in Minimal Essential Medium (MEM)

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for 30 min in a CO2 incubator and transferred to HBSS. Live cellconfocal images were obtained using an Ultra View Confocal Live CellImaging System (Perkin Elmer, Waltham, MA) with a Nikon ECLIPSETE2000 microscope (Nikon, Melville, NY). We calculated the intensityof intracellular zinc level in each region for 1 h using software for acircular region of interest (ROI).

Statistical analysis

All data are presented as mean±SEM. Comparisons for cellnumbers in multiple groups were assessed using one way ANOVAwith post-hoc Student–Newman–Keuls test, and zinc levels in live cellswere statistically compared using repeated-measures ANOVA with

Fig. 2. Correlation between zinc accumulation and disease progression. Lumbar spinal cordsJ). Bars denote the number of TFL-Zn (+) cells (K) and of motoneurons (L) at indicated stagKeuls test). Scale bar, 100 μm.

post hoc Student–Newman–Keuls test. The survival datawere assessedby theKaplan–Meier survival analysis andMantel–Cox log-rank test. Inall cases, p valueb0.05 was considered statistically significant.

Results

Zinc accumulation in degenerating cells

Horizontal frozen sections of the lumbar spinal cords of 8–18week-old WT and G93A SOD-1 Tg mice were stained with TFL-Zn andobserved under a fluorescencemicroscope (Figs.1A, C). InWTmice, nolabile zinc-containing cell bodies were noted. In contrast, several cellscontaining dense zinc fluorescence were noted in Tg mice. To

frommice at different disease stages were stained with TFL-Zn (A–E) or cresyl violet (F–es (##pb0.001, #pb0.005, ⁎pb0.05, one-way ANOVAwith post hoc Student–Newman–

Fig. 3. Accumulation of zinc and mSOD-1 in spinal cord motor neurons and astrocytes. Lumbar spinal cords of 18 week-old G93A SOD-1 Tg mice were stained with TFL-Zn (A, D, G)and then with antibody to SMI32 (B), GFAP (E) or human SOD-1 (H). Merged images show that some zinc accumulating cells were SMI32 (+) motoneurons (C, arrow), GFAP (+)astrocytes (F, arrowheads, yellow arrowhead; inset) or human SOD-1 (I, arrow; inset). Scale bar, 50 μm.

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determine if these zinc-accumulating cells were degenerating, thesesections were subsequently fixed and stained with acid-fuchsin (Figs.1B, D).Whereas no acidophilic cellswere identified inWTspinal cords,some acidophilic cells were found inTg spinal cords. There appeared tobe a close correlation between zinc fluorescence and acidophilicchanges, indicating that, in the spinal cords of Tg mice, zincaccumulation occurred mainly in degenerating cells.

Correlation between zinc accumulation and disease progression inTg mice

We next examined whether the appearance of zinc accumulationcorrelated with disease progression. Motor symptoms of Tg mice werescored as described in Methods. In Tg mice with a score of 4 (normal,presymptomatic), no zinc accumulation in cells was noted. In contrast,Tgmicewith score of 0 (fullymanifested), numerous zinc accumulating

Fig. 4. Correlation between HNE accumulation and zinc accumulation. Spinal cords of 8 (A–Cantibody to HNE. Whereas no zinc-accumulating cells were noted in presymptomatic 8 weemice (D). The level of HNE immunostaining was also significantly greater in 18 week-old (E)zinc (+) cells has increased levels of HNE (F, arrows, yellow arrow; inset). Scale bar, 50 μm

cell bodies were found. Spinal cords of Tg mice with scores of 1–3showed score-dependent appearance of zinc-accumulating cells (Figs.2A–E, K). To determine if zinc accumulation correlated with moto-neuronal loss,motoneuronswere counted in the spinal cords of Tgmiceat different stages (Figs. 2F–J, L). We found that the number ofmotoneurons decreased in a stage-dependent manner, indicating aninverse correlation between motoneuronal loss and the appearance ofzinc-accumulating cells.

Accumulation of zinc and mSOD-1 in spinal cord motor neuronsand astrocytes

Although the degree of spinal motoneuronal loss correlated wellwith the number of zinc-accumulating cells, it was unclear whethermotoneurons belong to the zinc accumulating cell population. Spinalcord sectionswere therefore double stainedwith TFL-Zn and for SMI32

) and 18 (D–F) week-old G93A SOD-1 Tg mice were stained with TFL-Zn and then withk-old Tg mice (A), many zinc (+) cells were found in fully symptomatic 18 week-old Tgthan in 8 week-old Tg mice (B). Merged images (C, F) show that the cytoplasm of some.

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to identify spinal motoneurons. In some sections, zinc-accumulatingcells were also positively stained for SMI32 (Figs. 3A–C), indicatingthat at least a fraction of zinc-accumulating cells are likely motoneur-ons. Other sections were doubly stained with TFL-Zn and for GFAP.Although GFAP staining was more diffuse and hence difficult tocolocalize, some cells were clearly double stained (Figs. 3D–F). Thesefindings indicate that both astrocytes and motoneurons may undergozinc dyshomeostasis. In contrast, we were unable to find zincaccumulation in microglial cells positively stained for CD11 (data notshown).

Mutant SOD-1 aggregates and accumulates in various cell types inG93A SOD-1 Tgmice (Raoul et al., 2002; Pramatarova et al., 2001; Linoet al., 2002; Gong, 2000). To examine the possible associationbetween zinc and mutant SOD-1, we double stained spinal cordsections with anti-SOD-1 antibody andwith TFL-Zn. In G93A SOD-1 Tg

Fig. 5. HNE-induced zinc increases in astrocytes and neurons from G93A SOD-1Tg and WT40 min to 300 μM HNE. To visualize zinc release, astrocytes were loaded with FluoZin-3-AMbetweenWT (A) and G93A SOD-1 astrocytes (D), the latter exhibited greater increases in labfluorescencemeasured in each cell with an image analyzer confirmed the difference (G). SimiG93A SOD-1 Tg (K–M)micewere exposed for 30min to 300 μMHNE. Data denote cellular zin##pb0.001, repeated-measures ANOVA). Scale bar, 20 μm.

mice, zinc accumulation occurred in cells exhibiting increased levelsof SOD-1 in Tg mice (Figs. 3G–I), whereas neither zinc accumulationnor SOD-1 accumulation was noted in NTg mice (data not shown).

Correlation between HNE accumulation and zinc accumulation

HNE is a potent endogenous neurotoxin, which has been reportedto increase in the spinal cord of ALS patients (Simpson et al., 2004;Vigh et al., 2005). Since zinc and HNE can increase the level of theother in cultured cortical neurons (Hwang et al., 2008), we examinedwhether zinc accumulation correlates with HNE accumulation in Tgmice. In presymptomatic spinal cords, there were no zinc accumulat-ing cells, while the level of HNE was very low (Figs. 4A, B). In contrast,the spinal cords of fully symptomatic mice (18 weeks of age)contained numerous zinc-accumulating cells, with these cells also

mice. Astrocytes cultured from spinal cords of WT and mSOD-1 mice were exposed forand observed under a confocal microscope. Whereas baseline zinc levels did not differile zinc levels (E, F) thanWT cells (B, C) upon 10–40 min exposure to HNE. Cellular zinclar findings were observedwhen spinal cords slice cultures obtained fromWT (H–J) andc fluorescence in each cell asmeasuredwith an image analyzer (N) (⁎pb0.05, #pb0.005,

Fig. 6. Zinc-induced increase in HNE levels in astrocytes and neurons from G93A SOD-1Tg andWTmice. Astrocytes cultured from spinal cords of WT (A, B) and G93A SOD-1 Tg(C, D) mice were stained with anti-HNE antibody before (A, C) or 1 h after exposure to400 μM zinc (B, D). Whereas baseline HNE levels did not differ, G93A SOD-1 astrocytesexhibited greater increases in HNE levels (D) upon exposure to zinc thanWT astrocytes(B). Spinal cord slice cultures of WT and G93A SOD-1 Tg mice were exposed for 1 h to400 μM zinc. Zinc exposure increased HNE in G93A SOD-1 neurons (I–L) more than inWT neurons (E–H). Scale bar, 20 μm.

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showing high levels of HNE (Figs. 4D, E). Overlay of the two imagesshowed that the cytoplasm of some zinc-accumulating cells exhibitedincreased HNE levels (Figs. 4C, F).

Increased zinc release by HNE in mSOD-1 astrocytes and slice cultures

Since exposure to HNE increases labile zinc levels in culturedcortical neurons, an increase that may be critical for HNE toxicity(Baldwin et al., 1998; Malecki et al., 2000; Zarkovic, 2003), weexamined whether G93A SOD-1-expressing primary cultured astro-cytes and slice cultured neurons were more prone to zinc dysho-meostasis than WT cells. Exposure of WT astrocytes and neurons to300 mMHNE slightly increased intracellular zinc levels (Figs. 5A–C, G,H–J, N), whereas mSOD-1 Tg astrocytes and neurons showed greaterincreases in intracellular zinc levels upon exposure to HNE (Figs. 5D–F,G, K–M, N), suggesting that mSOD-1 astrocytes and motor neuronsmay be more prone to oxidative stress-triggered zinc dyshomeostasis.

Increased HNE levels by zinc in mSOD-1 astrocytes and slice cultures

While HNE increases labile zinc levels, zinc increases HNE levels incultured cortical neurons, thus likely constituting a vicious cycle(Hwang et al., 2008). We found that exposure of astrocytes andneurons to zinc increased HNE levels to a greater degree in mSOD-1astrocytes and neurons (Figs. 6C, D, I–L) than in WT astrocytes andneurons (Figs. 6A, B, E–H).

The cell-permeant zinc chelator TPEN protects against neuronal death inALS Tg mice

If labile zinc accumulation in motoneurons plays a role in neuronaldeath in ALS Tg mice, measures that reduce zinc accumulation shouldbe protective. To test this possibility in vivo, we treated ALS Tg micewith TPEN, a potent cell-permanent zinc chelator, or vehicle, for 7 daysbeginning at 14 weeks of age, shortly before the symptom onset. Noobvious toxic effects were noted in the TPEN group.

To determine whether TPEN penetrates the central nervoussystem, we examined levels of labile zinc in hippocampus beforeand after TPEN treatment (20 mg/kg/day, i.p.). TPEN administrationfor 7 days significantly reduced levels of labile zinc (by about 20%) inmossy fiber terminals, supporting that TPEN thus given is effective inreducing labile zinc levels in the CNS (Fig. S1).

Although 40–50% of vehicle-treated mice had died at 121.7±1.2 days of age, 80% of TPEN-treated mice survived, even at 129±2.5 days (Fig. 7A). The difference in survival time was between 5 and10 days. In addition, TPEN treated mice exhibited fewer zinc-accumulating cells and more surviving motoneurons, as comparedwith vehicle treated control mice (Figs. 7B–G).

Discussion

The central finding of the present study is that certain spinal cordmotoneurons and astrocytes accumulate labile zinc in symptomaticG93A SOD-1 Tg mice. The onset of the appearance of zinc-accumulat-ing cells coincided with symptom onset, and the number of zinc-accumulating cells correlated well with disease progression. Further-more, most zinc-accumulating cells exhibited acidophilic changesindicative of cell degeneration, strongly suggesting that zinc accumu-lation may be directly linked to cell degeneration. Although metalshave been suspected of playing a role in ALS, this study is the first toshow that accumulation of labile zinc occurs in individual cells thatdegenerate in the spinal cords of G93A SOD-1 Tg mice.

While the precise mechanism of ALS has yet to be determined,excitotoxicity and oxidative injury may be important contributors. InALS, the glutamate transporter (glutamate transporter-1 or excitatoryamino acid transporter-2) is downregulated, which should increase

extracellular glutamate levels (Rothstein et al., 1995; Vanoni et al.,2004). Whereas NMDA receptors play a dominant role in acuteexcitotoxic injury, AMPA/kainite receptors may be important inchronic degenerative conditions such as ALS. It is therefore intriguingthat motoneurons express calcium-permeable AMPA/kainite (CAK)channels, which are also permeable to zinc (Jia et al., 2002; Rao andWeiss, 2004; Kwak andWeiss, 2006; Tortarolo et al., 2006). Hence, themechanism underlying zinc accumulation in degenerating spinalneurons and astrocytes of mSOD-1 Tgmicemay involve increased zincinflux through CAK channels.

Alternatively, zinc accumulation may be a predominantly intra-cellular event triggered by oxidative stress (Frederickson et al., 2005).

Fig. 7. TPEN protects against motoneuronal degeneration in G93A SOD-1 Tg mice. G93ASOD-1 Tg mice were treated with TPEN (20 mg/kg/day) or vehicle from 14 to 15 weeksof age. (A) Life span was significantly longer in TPEN-treated (n=7) than in vehicle-treated mice (n=12) (pb0.01, Mantel–Cox log-rank test). Lumbar spinal cords frommice at TPEN treated group and vehicle group were stained with TFL-Zn (B, C) or cresylviolet (D, E). Bars denote the number of TFL-Zn (+) cells (F) and of motoneurons (G) atindicated stage 2 points (#pb0.005, ##pb0.001; one-way ANOVA with post hocStudent–Newman–Keuls test). Scale bar, 100 μm.

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G93A SOD-1 binds zinc less avidly than does WT SOD-1, whereas bothbind copper equally (Crow et al., 1997). Since oxidative stress releaseszinc from certain oxidoreduction-sensitive zinc-binding sites, theincreased oxidative stress in G93A SOD-1 Tg mice may trigger zincrelease from metallothioneins and perhaps from G93A SOD-1 itself.HNE-induced increases in labile zinc levels were more pronounced inG93A SOD-1 than in WT neurons and astrocytes. These increases inlabile zinc levels may further increase oxidative stress via variouspathways including protein kinase C and NADPH oxidase activation(Noh and Koh, 2000). We also found that exposure to exogenouslyapplied zinc increased HNE levels to a greater degree in mSOD-1 thanin WT neurons and astrocytes. Thus, once oxidative stress reaches a

certain level, a vicious cycle involving HNE and zinc may be set inmotion (Hwang et al., 2008).

Although the involvement of calcium dyshomeostasis in neuronaldeath has been investigated extensively, study of the involvement ofzinc dyshomeostasis has begun recently. Labile zinc accumulationoccurs in neuronal death following ischemia, seizures, trauma, andhypoglycemia (Koh et al., 1996; Lee et al., 2000; Lee et al., 2003; Suhet al., 2004; Suh et al., 2006; Suh et al., 2007b). Both zinc influx fromoutside and zinc release from inside may contribute to zincaccumulation in acute brain injury. Increased cellular levels of zincmay trigger various cytotoxic cascades, such as NADPH oxidase andPARP activation, which preferentially lead to necrosis, and p75NTR

activation, which leads to apoptosis (Noh and Koh, 2000; Park et al.,2000; Kim and Koh, 2002; Suh et al., 2007a, b). Although zinc is clearlyinvolved in neuronal death occurring during acute brain injury, therole of zinc in chronic neurodegenerative conditions has been largelyunclear (Lee et al., 2002; Mocchegiani and Malavolta, 2007). In thisregard, it may be noteworthy that zinc dyshomeostasis or labile zincaccumulation occurs in degenerating spinal motoneurons and astro-cytes, and thus may contribute to motoneuronal death in ALS.Supporting this possibility, HNE levels were increased in and aroundzinc-accumulating cells in the spinal cord of mSOD-1 Tg mice. Otherpossible mechanisms linking zinc dyshomeostasis to motoneurondegeneration in ALS, may include the inhibition of BDNF action andthe activation of microglia by zinc (Post et al., 2008, Kauppinen et al.,2008).

At face value, the idea of zinc dyshomeostasis may appear inconflict with the previous report showing decreased zinc, butincreased copper levels in the same mice (Tokuda et al., 2007).However, whereas ICP-MS was used to measure total levels of metals,bound and unbound, in that study, in the present study, fluorescencestaining was used to measure levels of labile (loosely-bound) zinc inindividual cells, which may be more relevant to neuronal death asdiscussed above. At any rate, our results do not rule out the possibilitythat copper dyshomeostasis also plays a role in motoneurondegeneration in these mice.

ALS is a relentlessly progressive neurodegenerative diseaseprimarily affecting upper and lower motoneurons. Although riluzolehas been approved by the FDA for the treatment of ALS, it onlymodestly prolongs the life of ALS patients. Hence, more effectivetherapy is desperately needed. If zinc dyshomeostasis contributes tomotoneuron death in ALS, as suggested by our results, agents thatreduce zinc toxicity may prove effective in ALS. For example,pyruvate, which blocks zinc-triggered toxicity in vitro and in vivo(Lee et al., 2001; Chang et al., 2003; Kelland et al., 2004; Yoo et al.,2004; Kim et al., 2007), has been shown effective in G93A SOD-1 Tgmice (Park et al., 2007). In addition, the metal chelators DP-109 andDP-460 were effective in prolonging the survival of G93A SOD-1 Tgmice (Petri et al., 2007). We have shown here that injection ofTPEN, a cell membrane-permeant zinc chelator, for 1 week prior todisease onset significantly prolonged survival by 5–10 days. Theseresults suggest that labile zinc accumulation may be a target forfuture drug development in ALS. Further studies are warranted todetermine the precise role and mechanism of zinc dyshomeostasisin ALS.

Acknowledgments

This work was supported by the Korea Science and EngineeringFoundation through the National Research Laboratory Program(M10600000181-06J0000-18110), by the Brain Research Center of21st Century Frontier Research Program (M103KV010020-06K2201-02010) funded by Korean Ministry of Science and Technology, and bythe Korea Research Foundation (Grant MOEHRD, KRF-2005-084-C00026) funded by the Korean Ministry of Education and HumanResources Development.

228 J. Kim et al. / Neurobiology of Disease 34 (2009) 221–229

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.nbd.2009.01.004.

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