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Experimental Neurology 162, 27–36 (2000)doi:10.1006/exnr.2000.7323, available online at http://www.idealibrary.com on

Metallothionein Expression Is Altered in a Transgenic Murine Modelof Familial Amyotrophic Lateral Sclerosis

Yun Hua Gong and Jeffrey L. Elliott1

Department of Neurology, University of Texas, Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9036

Received March 11, 1999; accepted November 17, 1999

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Missense mutations in the gene encoding copperinc superoxide dismutase (SOD1) have been found toause one form of familial amyotrophic lateral sclero-is (FALS). Although the exact mechanism of disease isnknown, abnormalities in the ability of mutant SOD1o bind zinc or copper ions may be crucial in theathogenesis of disease. Because members of the metal-

othionein (MT) family of zinc and copper bindingroteins function as important cellular regulators ofetal ion bioavailability in the central nervous sys-

em, we used in situ hybridization and immunohisto-hemistry to study the expression pattern of theseolecules in a transgenic mouse model of familial ALS.

n adult wild-type mouse spinal cord, expression ofT-I and MT-II is restricted to ependymal cells and a

ubset of astrocytes located in white matter tracts,hile MT-III synthesis is limited to neurons withinray matter. Compared to wild-type littermates, trans-enic mice carrying the G93A SOD1 mutation demon-trate markedly increased expression of MT-I andT-II within astrocytes in both white and gray matter

s weakness develops. MT-III synthesis in neurons islso greatly upregulated as G93A SOD1 animals age,ith glial cell expression of MT-III evident by later

tages of the disease. Changes in MT expression occurefore the onset of motor deficits or significant motoreuron pathology in G93A SOD1 mice and remarkablyxtend beyond ventral horn populations of neuronsnd glia. These results are consistent with the hypoth-sis that metallothioneins may serve an early andmportant protective function in FALS. r 2000 Academic Press

Key Words: superoxide dismutase; zinc; motor neu-on; astrocyte; ALS.

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is characterizedy the progressive degeneration of motor neurons lead-ng to profound weakness and death in affected individu-ls. The etiology of ALS is currently unknown although

1

CTo whom correspondence should be addressed.

27

isense mutations in the gene encoding Cu/Zn superox-de dismutase (SOD1) have been found to cause oneamilial form of the disease (FALS) linked to chromo-ome 21q (27). Genetic manipulations of mice either byargeted deletion of SOD1 alleles or by overexpressionf mutant SOD1 have demonstrated a toxic gain ofunction for the altered SOD1 protein, but exactly howutant SOD1 activity leads to motor neuron death is

nclear (12, 24, 25, 34). SOD1 is a cytosolic metalloen-yme with a zinc and copper binding site, active as aomodimer. Virtually all of the SOD1 mutations linkedo FALS are located outside the active site of thenzyme in regions that would influence the stableonformation of the molecule, particularly with regardo heavy metal binding (13). Recently, Beckman andolleagues have reported that mutant SOD1 possesses5- to 30-fold less affinity for zinc ion binding than doesild-type SOD1, suggesting that abnormalities in zincomeostasis may be observed in FALS (7).Within the CNS, zinc is normally distributed among

hree pools including a small intracellular free ioniceservoir, a vesicular compartment localized in theynaptic vesicles of nerve terminals, and a membrane-r protein-bound component (8). Included within thisast compartment is the presence of low-molecular-eight metal binding proteins known as metallothio-eins (MTs), three of which, MT-I, MT-II, and MT-III,re found in the murine nervous system. These bindingroteins have complementary expression patternsithin the CNS in that MT I and MT-II immunoreactiv-

ty has been localized in astrocytes and ependymal cellsut not oligodendrocytes or microglia (3, 21, 33), whilexpression of MT-III is restricted largely to neurons19, 23). Although the precise function of MTs in vivo isnclear (22), MTs are likely important in the regulationf zinc bioavailability within cells, by acting as chaper-nes during the synthesis of metalloenzymes and byuffering cells against essential metal toxicity or oxida-ive injury (8, 15). MT expression within cells is par-ially dependent on zinc via a zinc-mediated activationf the MT transcription factor (MTF-1). Because ofltered zinc binding by mutant SOD1, we asked whether

NS regions particularly affected by FALS, such as the

0014-4886/00 $35.00Copyright r 2000 by Academic Press

All rights of reproduction in any form reserved.

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28 GONG AND ELLIOTT

pinal cord, exhibited changes in metallothionein ex-ression and if so, what would be the temporal patternf that change.Although human necropsy material is useful for

tudying pathological changes in MT expression occur-ing in later stages of ALS, its value is extremelyimited for assessing potential alterations in very earlyr presymptomatic phases of the disease when autopsyissue is not available. Transgenic mice expressingutant SOD1 develop progressive motor weakness

oncomitant with pathologic accumulation of neurofila-ent and ubiquitin-positive inclusions in a pattern

esembling human disease and consequently representn excellent animal model of human FALS (5, 12,2, 34). Consequently, we used these mice to study MTxpression during various stages of disease using bothn situ hybridization and immunohistochemicalethods.

METHODS

eneration of G93A SOD1 and Wild-Type Mice

Transgenic mice expressing the human G93A SOD1utation (B6SJL-TgNSOD1-G93A; JR2300) were ob-

ained from The Jackson Laboratory (Bar Harbor, ME).ounders were bred with CF1 wild-type mice andffspring were genotyped using PCR on genomic DNAsolated from tail digested overnight with proteinase Kt 50°C. Primers for PCR were used according to theecommendations of The Jackson Laboratory using theorward primer 58-CAT CAG CCC TAA TCC ATCGA-38 and the reverse primer 58-CGC GAC TAA CAACA AAG TGA-38 for 30 cycles at 94°C for 1 min, 55°C

or 45 s, and 72°C for 1 min 20 s. These primers willmplify a 236-bp fragment from mice carrying theuman G93A SOD1 transgenic construct. Transgenic93A SOD1 mice and wild-type littermates were then

acrificed at various time points for study. This line of93A SOD1 mice typically develops clinical signs ofotor weakness at 6.5 to 7 months of age and will die of

espiratory failure due to motor neuron loss at 8onths of age (mean 244 6 4 days). Before 5.5 to 6onths of age, G93A SOD1 mice appear normal both to

linical examination and to motor stride length testingJ. L. Elliott, unpublished observations). Three to fournimals per group were used in the experimentalrocedures given below. All procedures performed onice were approved by the animal research committee

t the university and conform to NIH guidelines.

n Situ Hybridization for Detection of MT-1, MT-II,and MT-III Transcripts

Mice were overdosed with sodium pentobarbital (150g/kg), and the brains and spinal cords were quickly

emoved, frozen on dry ice, and stored at 280°C. In situ i

ybridization was performed according to methodsescribed previously (9). In brief, 12-µm sections ofhoracolumbar spinal cord were cut, thaw mounted,nd fixed in 4% paraformaldehyde, followed by washesn phosphate-buffered saline (PBS) and PBS/glycine,ehydration through graded alcohols, and delipidationith chloroform.Antisense deoxynucleotide probes 58-CAC AGC ACG

GC ACT TGT CCG CGG CGC CTT TGC AGA CACGC C-38 for MT-I, 58-GCA CAG CAG CTG CAC TTGCG GAA GCC TCT TTG CAG ATG CAG CC-38 forT-II, and 58-CTG GCA GCA GCT GCA TTT CTC GGCTC TGC CTT GGC CCC CTC TTC ACC-38 for MT-IIIere obtained from a commercial source (Oligos Etc.,ilsonville, OR). Oligonucleotides (3 pmol) were 38

nd-labeled with [a-33P]dATP (2000 Ci mmol21 (NENife Science) using terminal deoxynucleotidyl transfer-se (Boehringer Mannheim). Hybridization was per-ormed overnight at 39°C following the methods oflliott and Snider (9). After posthybridization washes

n 13 SSC, slides were dipped in Kodak NBT-2 emul-ion and stored in a light-tight container at 4°C for 2eeks. Slides were developed using Kodak D19 andxed before counterstaining with hematoxylin andosin. Specimens were viewed with a Nikon ES800icroscope, and photographic images (35-mm slides)ere obtained. These photographic slides were then

canned into a power PC (Macintosh) using a Polaroidprint scan and Adobe PhotoShop(4.0) software. Senseligonucleotides for the MTs were also used and gave noignal when hybridized to spinal cord sections. Onlylides from the same hybridization experiments weresed for comparisons between groups.

etallothionein, Glial Fibrillary Acidic Protein,and Lectin Immunohistochemistry

For immunohistochemistry, mice were overdosed withodium pentobarbital (150 mg/kg) and perfused intra-ardiac with 4% paraformaldehyde (pH 7.4). Brain andpinal cord were dissected out, embedded in paraffin,ectioned (4 µm), and then deparaffinized throughylene and graded alcohols. Sections were then washedn 0.05 Tris (pH 7.6)/1.5% sodium chloride, treated with.3% hydrogen peroxide for 30 min, and then washedgain. Immunohistochemistry was performed using anmmunoperoxidase reagent kit, the One Kit (Stern-erger Monoclonals, Inc.), following the instructions ofhe manufacturer. Primary antibodies used were (1)T-E9, a mouse monoclonal directed against MT-I andT-II (Dako Corp.) at 1:50 dilution, and (2) a rabbit

olyclonal antibody from Dako directed against glialbrillary acidic protein (GFAP) at 1:250 dilution. Slidesere counterstained in 0.25% cresyl violet, followed byehydration through graded alcohols, and then cover-lipped with Permount. For dual MT-I/II and GFAP

mmunofluorescence, deparaffinated sections were

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29METALLOTHIONEIN EXPRESSION IN G93A SOD1 MICE

ashed in TBS, blocked, and incubated overnight withnti-MT-I/II and then anti-GFAP antibodies as above.ections were then washed in TBS, followed by incuba-ions with a Cy-3-conjugated goat anti-rabbit and aITC-conjugated goat anti-mouse secondary antibody

Jackson ImmunoResearch). Sections were washed inBS, mounted with Vectashield aqueous mount (Vectororp.), and viewed under fluorescence with a NikonS800 microscope.For dual immuno- and lectin fluorescence, 4-µm

epariffinized sections were washed with TBS andncubated overnight with the mouse monoclonal di-ected against MT-I and MT-II as above. Sections wereashed in TBS and then incubated with a secondaryoat anti-mouse IgG conjugated with Cy-3 (JacksonmmunoResearch). Sections were washed in TBS fol-owed by overnight incubation with a tomato (L. escul-

FIG. 1. Metallothionein mRNA synthesis in wild-type and G9-month-old wild-type and G93A spinal cords, respectively. (C) MT-IIentral spinal cord in 5-month-old G93A SOD1 mice. (E) MT-II exprexpression in 8-month wild-type spinal cord. (G and H) MT-III exespectively. *Medial ventral horn. Arrowheads indicate small focal

003 for D and 503 for the remaining photos.

ntum) lectin (Sigma) conjugated to fluorescein. Thisectin recognizes poly-N-acetyl lactosamine residues on

icroglia but does not bind to neurons, astrocytes, orligodendroglia (1). Sections were washed in TBS,ounted with Vectashield aqueous mount (Vectororp.), and viewed under fluorescence with a NikonS800 microscope.

RESULTS

T-I and MT-II Expression in Spinal Cordsfrom G93A SOD1 and Wild-Type Mice

In the normal adult murine spinal cord, MT-I andT-II mRNA expression is largely restricted to whiteatter tracts with only scattered expression foundithin gray matter (Figs. 1A and 1C). Within the white

SOD1 mice. Dark-field microscopy. (A and B) MT-I expression inression in 8-month-old wild-type spinal cord. (D) MT-II expression inn in spinal cord from an 8-month-old G93A SOD1 mouse. (F) MT-IIIssion in spinal cords from 5- and 8-month-old G93A SOD1 mice,ression of MT-II in white matter astrocytes. Original magnification

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30 GONG AND ELLIOTT

atter, MT-I mRNA expression is limited to a smallopulation of glial cells which on H and E staining havearger and oblong nuclei, while the majority of glialells in white matter containing smaller round andark nuclei do not express MT-I (Fig. 2B). The punctateT-II mRNA expression observed in the white matter

racts of the spinal cord is also, like MT-I, limited to glia

FIG. 2. Metallothionein mRNA expression in wild-type and G93dult wild-type ventral horn (A) and spinal cord white matter (B), 5-orn (D). MT-II expression in 5-month G93A SOD1 ventral horn (E). M

OD1 ventral horn (G) and spinal cord white matter (H). Original magni

ith large and oblong nuclei. Within the gray matter, initu hybridization does indicate low levels of MT-Iignal within scattered nonneuronal cells (Figs. 1A andA), while MT-II mRNA expression could not be de-ected within gray matter. Ependymal cells lining theentral canal demonstrate robust MT-I expression withore limited MT-II mRNA synthesis. In contrast, most

OD1 spinal cord under bright-field microscopy. MT-I expression innth G93A SOD1 ventral horn (C), and 8-month G93A SOD1 ventralIII expression in adult wild-type ventral horn (F) and 8-month G93A

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31METALLOTHIONEIN EXPRESSION IN G93A SOD1 MICE

eurons and, particularly, motor neurons do not exhibitetectable levels of signal for MT-I and II mRNA (Figs.C and 2A). The overall pattern and relative intensityf MT-I and MT-II mRNA expression within the spinalord does not appear to significantly change as theild-type animal matures from 5 to 8 months. Immuno-istochemical study demonstrates that MT-I and MT-IIeactivity is again confined to stellate-appearing cellsn white matter tracts and ependymal cells lining theentral canal (Fig. 3A). MT-I or MT-II immunoreactiv-ty is not observed in the ventral horn of the cord eithern neurons or in glial cells. In particular, spinal motoreurons do not appear to express MT-I or MT-II reactiv-

ty in normal adult mice, at least during the periodnder study from 5 to 8 months of age.The pattern of MT-I and MT-II expression in the

pinal cords of G93A SOD1 transgenic mice demon-trate significant differences from wild-type mice event 5 months of age. This time point represents alinically normal period in which G93A SOD1 exhibitotor skills comparable to and are clinically indistin-

uishable from wild-type littermates. As in wild-typeice, MT-I and MT-II mRNA expression in G93A SOD1ice is found in astrocytes within white matter tracts

f the spinal cord as well as ependymal cells lining theentral canal. However, by this age, glia surrounding amall number of ventral horn motor neurons now begino demonstrate increased MT-I and MT-II mRNA syn-hesis (Figs. 1D, 2C, and 2E). Similarly, MT-I and MT-IImmunoreactivity is now visible within certain glia inhe ventral horn surrounding a subset of motor neuronsFig. 3B) and colocalizes well with GFAP immunoreac-ivity (Fig. 3E). At this time point, expression of MT-Ind MT-II is limited to gray matter astrocytes withinhe ventral horn and does not extend to cells in thentermediate lateral or dorsal zones of the cord. These

ore dorsal areas of spinal cord are devoid of MTmmunoreactivity, resembling the pattern found inild-type mice. Motor neurons in the anterior horn doot express MT-I or MT-II immunoreactivity at thisime point in G93A SOD1 mice.

As G93A SOD1 mice mature and develop overt signsf weakness, MT-I and MT-II expression increases inhe spinal cord. By 7 months of age, increased intensityf expression is observed in astrocytes within the whiteatter tracts of G93A SOD1 mice (Fig. 3F). In addition,larger number of astrocytes within the ventral horn

nd extending into the lateral horns now express MT-Ind MT-II protein (Fig. 3C). The intensity of positiveT-I and MT-II reactivity in astrocytes steadily in-

reases as the mice age and develop more profoundeakness (from 7 to 8 months), and the distribution ofositive astrocytes also appears to significantly change,xtending throughout the gray matter of the cord bothaterally and dorsally. Within the spinal cord gray

atter, there is a marked increase in both MT-I and b

T-II mRNA expression (Figs. 1B and 1E). High-powerright-field imaging demonstrates that the increasedRNAexpression is nonneuronal (Fig. 2D). In 8-month-

ld G93A SOD1 mice, MT-I and MT-II immunoreactiv-ty is now present in stellate glial cells not only in theentral horn but also extending throughout the entirepinal cord gray matter (Fig. 3D).To ascertain which population of glial cells in the

pinal cord have begun to express MT-I and MT-II inhese animals, we colocalized MT-I/II reactivity witharkers for astrocytes and microglia. Astrocytes were

dentified by GFAP immunoreactivity and microglialells were recognized by positive lectin L. esculentuminding. Colocalization of MT-I/II and GFAP immunoflu-rescence on spinal cord sections from 8-month G93AOD1 mice confirmed that MT-I/II-positive cells arestrocytes (Figs. 4Aand 4B), while the lack of colocaliza-ion between MT-I/II-positive cells and those stainingositive with L. esculentum lectin demonstrated thaticroglial cells do not express metallothionein (Figs.

C and 4D).Thus in 8-month-old G93A SOD1 mice, MT-I andT-II expression is now present in astrocytes through-

ut the entire gray matter of spinal cord, even inocations not in proximity to degenerating motor neu-ons. Interestingly, in clinically weak G93A SOD1 mice,ray matter astrocytic expression of MT-I and MT-IIecomes evident even in areas of the CNS that areemote from neuronal populations susceptible to mu-ant SOD1 toxicity (i.e., motor neurons) as illustratedn the rostral pons (Figs. 3G and 3H). At all time pointsxamined, MT-I and MT-II synthesis appears restrictedo astrocytes, with motor neurons not expressing MT-Ir MT-II even at the end stages of the disease. Theemporal and spatial pattern of MT expression withinhe spinal cord is summarized in Table 1.

T-III Expression in Spinal Cords from G93A SOD1and Wild-Type Mice

Because no antibody is readily available to studyT-III protein expression, we relied on in situ hybrid-

zation to investigate MT-III mRNA expression in wild-ype and G93A SOD1 mice. In the spinal cords of adult,ild-type mice, MT-III mRNA expression is limited toray matter (Fig. 1F). High-power bright-field micro-raphs confirm that MT-III expression within grayatter is neuronal, with moderate levels of expression

n motor neurons (Fig. 2F). At 5 months of age, bothild-type and G93A SOD1 mice demonstrate a similarattern of MT-III mRNA expression within the spinalord, although the intensity of expression appearsildly increased in 5-month-old G93A SOD1 mice

Fig. 1G). Interestingly, that increased expression is notestricted to ventral horn motor neurons but appears to

e upregulated in neurons throughout the gray matter

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FIG. 3. MT-I/II and GFAP immunoreactivity in wild-type and G9ild-type mouse. (B–D) MT-I/II reactivity in the ventral horn of

mmunoreactivity in ventral horn from 5-month G93A SOD1 mouse-month G93A SOD1 mouse. (G and H) MT-I/II reactivity in the poeruleus. Original magnification 2003 for A–E, 6003 for F, and 403 f

3SOD1 mice. (A) MT-I/II reactivity in the ventral horn from an 8-month5-, 7-, and 8-month-old G93A SOD1 mice, respectively. (E) GFAP(serial section to B). (F) MT-I/II reactivity in white matter tract from

ns of an 8-month wild-type (G) and G93A SOD1 mouse (H). LC, locusor G and H.

32

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33METALLOTHIONEIN EXPRESSION IN G93A SOD1 MICE

ncluding those located more dorsally and laterally.gain at this age, MT-III expression is restrictedrimarily to neurons within the cord of G93A SOD1ice. However, at 8 months of age, G93A SOD1 mice

emonstrate significantly increased MT-III mRNA ex-ression in gray matter not only in neurons but also inlial cells (Figs. 1H and 2G). In addition, at 8 months of

FIG. 4. (A and B) Dual immunofluorescence for GFAP (A) and MTOD1 mouse. (C and D) Dual MT-I/II immuno- (C) and L. esculentum93A SOD1 mouse. Arrows and arrowheads label identical landmark

TAB

Relative Expression of Metallothio

Line

Gliawhite matter

Gliaventral gray matte

MT-I MT-II MT-III MT-I MT-II MT

ild type 111 1 2 1/2 2 2-month G93A SOD1 111 1 2 11 11 2-month G93A SOD1 1111 111 11 1111 1111 11

ntense expression.

ge, G93A SOD1 mice also express MT-III MRNA in aubset of glial cells within white matter tracts (Fig.H). Based on morphological appearance and pattern ofistribution these MT-III-expressing cells in the whiteatter of 8-month G93A SOD1 likely represent astro-

ytes, but whether some microglia also begin to expressT-III mRNA cannot be excluded.

I reactivity (B) on a single spinal cord section from an 8-month G93Actin (D) fluorescence on a single spinal cord section from an 8-monththe section. Original magnification 4003.

1

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Gliadorsal gray matter

Neuronsventral gray matter

Neuronsdorsal gray matter

MT-I MT-II MT-III MT-I MT-II MT-III MT-I MT-II MT-III

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DISCUSSION

In this paper, we examined the expression pattern ofembers of the metallothionein family of heavy metal

inding proteins in the spinal cords of wild-type and93A SOD1 mice, an animal model of 21q-linked FALS.e show that synthesis of MT-I and MT-II within

pinal cord astrocytes, particularly in the gray matter,rom G93A SOD1 mice is upregulated in the earlyymptomatic stages of disease and continues to in-rease as motor weakness progresses. Expression ofT-III, which in wild-type animals is neuronal, is not

nly upregulated in neurons as the G93A SOD1 miceevelop weakness, but also begins to be expressed bypinal cord astrocytes both in gray matter and in whiteatter tracts. Although it is unclear whether similar

hanges occur in patients with 21q-linked FALS, ele-ated levels of MT-I/MT-II expression in astrocytesave previously been found in the spinal cords ofnd-stage sporadic ALS patients (4, 31). The currentnvestigation is the first to show that a similar processccurs in an animal model of ALS that has a knowntiology. Previous studies of MT expression in humanave also been complicated by the fact that humanissue in early or presymptomatic stages of the diseases not available for study, and consequently observa-ions concerning temporal and spatial changes in MTxpression during disease progression cannot be made.These results indicate that changes in MT expression

re not manifestations of agonal events in the degenera-ive process. Altered MT expression both in glia and ineurons has already begun in G93A SOD1 mice by 5onths of age, at a time point when they do not yet

xhibit clinical motor deficits or obvious motor neuronoss. Interestingly, for both MT-I and MT-II, increasedxpression begins focally in glia surrounding a smallubset of motor neurons in the ventral horn. Thisocalization is logical given that motor neurons are theusceptible neuronal population in the disease. How-ver, it is not clear why astrocytes distant from motoreuron pools begin to express MT-I and MT-II as theisease progresses. One possibility is that other neuro-al populations are also affected by the mutant SOD1ctivity (although to a lesser extent than motor neu-ons) and only later begin to manifest abnormalitieshat then trigger neighboring astrocytic responses. Butecause mutant SOD1 expression in these mice isnder the control of the native SOD1 promoter whichrives expression in neurons and astrocytes, it is alsoossible that mutant SOD1 may be affecting astrocytesirectly (5, 28). Expression of mutant SOD1 in astro-ytes might perturb astrocytic function in some wayhat eventually results in subsequent expression ofTs. The issue of primary or secondary astrocytic

ysfunction and its relationship to eventual neuronal

egeneration in ALS is of critical importance, since w

ecent evidence has suggested that astrocytes may benvolved in the pathogenesis of the disease (see below).

Changes in expression of the neuronal MT-III inome ways resemble those of the astrocytic MTs. MT-IIIRNA expression in neurons is similarly increased

elatively early in the disease course, before clinicalotor dysfunction or motor neuron loss occurs. How-

ver, even at 5 months, the subtle increase in MT-IIIxpression is widespread and not limited to motoreurons. By 8 months, levels of neuronal MT-III synthe-is are even more significantly elevated in G93A SOD1urine spinal cord, but likewise not restricted to motor

euron pools. This result again suggests that manyiffering neuronal populations are in some way affectedy mutant SOD1 activity and as part of that neuronalesponse increase expression of MT-III. Unlike MT-Ind MT-II expression which remains restricted to a cellopulation which normally expresses the protein (i.e.,strocytes), MT-III synthesis in older G93A SOD1 mices not limited to neurons but is observed in glial cells,ikely astrocytes. Using both in situ and knock-inechnology, Palmiter and colleagues were unable toetect glial MT-III expression in normal mice in vivo,ut could detect astrocytic MT-III expression in vitronder certain experimental conditions, including se-um deprivation (19). The authors then speculated thatT-III might be induced in glia in vivo particularly

nder stressful conditions. Mutant SOD1 toxicity inater stages of the disease does appear capable ofnducing glial expression of MT-III.

MT expression in astrocytes is clearly upregulated in93A SOD1 mice. Localization to this cell type mayave importance given that recent evidence has sug-ested a potential role for astrocytes in the pathogen-sis of ALS. Astrocytes from the brains and spinal cordsf ALS patients manifest selective decreases in gliallutamate transporter (GLT-1) protein expression andunction likely mediated by aberrant mRNA processing17, 29). Such changes in GLT-1 expression/functionxplain the elevated CSF glutamate levels observed inLS patients and have provided support for a gluta-ate-based model of motor neuron degeneration. Simi-

ar alterations in GLT-1 expression and function haveeen found in mutant SOD1 transgenic mice. In addi-ion, pathologic changes in astrocytes are among thearliest abnormalities observed in G86R SOD1 mice5). These pieces of evidence suggest that changesbserved in astrocytes may not just represent benigneaction to neuronal abnormalities, but that astrocyticysfunction itself may contribute to the disease pro-ess.What role, if any, MTs play in the degenerative

rocess in FALS is unclear. Part of this uncertaintyrises from the fact that MT function during normal oristurbed cellular homeostasis also remains elusive in

ild-type animals. For example, MT expression within

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35METALLOTHIONEIN EXPRESSION IN G93A SOD1 MICE

he nervous system may be upregulated in response toertain excitotoxic or cytokine-mediated insults, buthe function of MTs during the injury response remainsomewhat unclear (2, 14). Several experiments utiliz-ng genetically altered mice either with MT overexpres-ion or with targeted MT deletions have suggested thathese proteins may be protective under particularnjurious conditions. Mice deficient for MT-I and MT-IIre viable but demonstrate a markedly increased sensi-ivity to heavy metal toxicity (20). Astrocytes fromhese mice exhibit significantly enhanced vulnerabilityn vitro to zinc toxicity and ischemia (18). MT-IIInockout mice exhibit greater susceptibility to excito-oxic neuronal injury (10). Overexpression of MTs alsoonfers resistance to oxidative injury and decreasesensitivity to heavy metal toxicity in vitro (26). Thesexperiments overall suggest a protective role for MTsarticularly against oxidative injury or heavy metaloxicity from copper or zinc. Zinc itself may potentiallyontribute to neuronal degeneration as accumulation ofntracellular zinc has been implicated in cell deathollowing excitotoxic and ischemic insults possibly viariggering increased mitochondrial production of reac-ive oxygen species (11, 16, 30).

However, the significance of elevated MT expressionn astrocytes and neurons in this genetic animal modelf FALS is unclear. It is possible that increased MTxpression may be a protective cellular response to thencreased oxidative or toxic insults mediated by mutantOD1, thus delaying onset of dysfunction. In addition,utant SOD1 possesses significantly diminished affin-

ty for zinc ion binding, potentially allowing for alter-tions in zinc homeostasis. Because MT expressionithin cells is partially dependent on zinc levels via

inc-mediated activation of MTF-1, it is also possiblehat changes in zinc levels may drive increased MTynthesis. However, this mechanism is more likely toccount for changes in glial MT-I and MT-II expressionather than for neuronal MT-III expression based onecent findings of decreased zinc responsiveness withinetal regulatory elements of the MT-III promoter (6).f course, changes in MT expression may be nonspe-

ific, reflecting only reactive changes in astrocytes andeurons rather than significant indicators of diseaseechanisms. Future experiments using mice with ge-etically altered levels of MTs should be able to distin-uish between these two possibilities.

ACKNOWLEDGMENT

This work is supported in part by grants from the NINDSNS01853) and the ALS association.

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