partial resistance to malonate‐induced striatal cell death in transgenic mouse models of...

Journal of Neurochemistry, 2001, 78, 694±703

Partial resistance to malonate-induced striatal cell death in

transgenic mouse models of Huntington's disease is dependent on

age and CAG repeat length

Oskar Hansson,* Roger F. Castilho,² Laura Korhonen,³ Dan Lindholm,³ Gillian P. Bates§ andPatrik Brundin*

*Section for Neuronal Survival, Wallenberg Neuroscience Center, Department of Physiological Sciences, Lund University, Lund,

Sweden

²Department of Clinical Pathology, School of Medical Sciences, State University of Campinas, Campinas, Brazil

³Department of Neuroscience, Neurobiology, Uppsala University, Uppsala, Sweden

§Medical and Molecular Genetics, GKT School of Medicine, London, UK

Abstract

Transgenic Huntington's disease (HD) mice, expressing exon

1 of the HD gene with an expanded CAG repeat, are totally

resistant to striatal lesion induced by excessive NMDA

receptor activation. We now show that striatal lesions induced

by the mitochondrial toxin malonate are reduced by 70±80%

in transgenic HD mice compared with wild-type littermate

controls. This occurred in 6- and 12-week-old HD mice with

150 CAG repeats (line R6/2) and in 18-week-old, but not

6-week-old, HD mice with 115 CAG repeats (line R6/1).

Therefore, we show for the ®rst time that the resistance to

neurotoxin in transgenic HD mice is dependent on both the

CAG repeat length and the age of the mice. Importantly, most

HD patients develop symptoms in adulthood and exhibit an

inverse relationship between CAG repeat length and age of

onset. Transgenic mice expressing a normal CAG repeat (18

CAG) were not resistant to malonate. Although endogenous

glutamate release has been implicated in malonate-induced

cell death, glutamate release from striatal synaptosomes was

not decreased in HD mice. Malonate-induced striatal cell

death was reduced by 50±60% in wild-type mice when they

were treated with either the NMDA receptor antagonist

MK-801 or the caspase inhibitor zVAD-fmk. These two

compounds did not reduce lesion size in transgenic R6/1

mice. This might suggest that NMDA receptor- and caspase-

mediated cell death pathways are inhibited and that the limited

malonate-induced cell death still occurring in HD mice is

independent of these pathways. There were no changes in

striatal levels of the two anti cell death proteins Bcl-XL and

X-linked inhibitor of apoptosis protein (XIAP), before or after

the lesion in transgenic HD mice. We propose that mutant

huntingtin causes a sublethal grade of metabolic stress which

is CAG repeat length-dependent and results in up-regulation

over time of cellular defense mechanisms against impaired

energy metabolism and excitotoxicity.

Keywords: cell death, excitotoxicity, Huntington's disease,

malonate, N-methyl-D-aspartate, transgenic mouse.

J. Neurochem. (2001) 78, 694±703.

Huntington's disease (HD) is a devastating autosomal

dominantly inherited neurodegenerative disorder manifested

by chorea, personality changes and dementia. Typically, HD

starts in mid-life and progress to death within 10±20 years

(Harper 1996). The pathology is focused on the brain, with

selective neuronal loss occurring preferentially in the

striatum and to a lesser extent in the cortex (Vonsattel and

DiFiglia 1998). The mutation causing the disease is an

unstable expansion of a CAG trinucleotide repeat encoding a

polyglutamine stretch near the N-terminus of the huntingtin

protein (The Huntington's Disease Collaborative Research

694 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 694±703

Received December 5, 2000; revised manuscript received March 17,

2001; accepted March 17, 2001.

Address correspondence and reprint requests to Oskar Hansson,

Section for Neuronal Survival, Wallenberg Neuroscience Center, Lund

University, BMC A10, 221 84 Lund, Sweden.

E-mail: [email protected]

Abbreviations used: BSA, bovine serum albumin; DMSO, dimethyl

sulfoxide; HD, Huntington's disease; MK-801, (5R,10S)-(1)-5-methyl-

10,11-dihydro[a,d ]cyclohepten-5,10-imine hydrogen maleate; 3-NP,

3-nitropropionic acid; SDH, succinate dehydrogenase; PB, phosphate

buffer; PBS, phosphate-buffered saline; SDS, sodium dodecyl

sulfate; XIAP, X-linked inhibitor of apoptosis protein; zVAD-fmk,

N-benzyloxycarbonyl-Val-Ala-Asp-¯uoromethylketone.

Group 1993). Patients exhibit in excess of 36 CAG repeats

and there is an inverse correlation between the length of the

CAG repeat expansion and the age of onset of the disease

(Duyao et al. 1993; MacDonald and Gusella 1996). The

molecular pathways mediating the neuropathology of HD

are still poorly understood (Reddy et al. 1999). Both wild-

type and mutant huntingtin are widely expressed in the CNS

as well as in non-neuronal tissues (Trottier et al. 1995;

DiFiglia et al. 1997). Therefore, the cause of the selective

neuronal death is truly enigmatic.

An important advance in HD research was the develop-

ment of a transgenic mouse model that expresses exon 1 of a

human HD gene with an expanded number of CAG

trinucleotide repeats (Mangiarini et al. 1996). Two different

lines, R6/1 (CAG)115 and R6/2 (CAG)150 display a

progressive neurological phenotype. Importantly, striatal

neurons of both affected transgenic lines exhibit some key

neuropathological features of HD, including intranuclear

inclusions that contain huntingtin and reduced levels of

dopamine receptors prior to onset of overt symptoms

(Davies et al. 1997; DiFiglia et al. 1997; Cha et al. 1998).

In addition, recent reports provide evidence for morpho-

logical changes, suggestive of degeneration, in striatal

neurons of both R6/1 and R6/2 mice (Turmaine et al.

2000; Iannicola et al. 2000).

Impaired energy metabolism has been hypothesized to

play a central role in the pathogenesis of HD by causing

neurodegeneration via secondary excitotoxicity (Albin and

Greenamyre 1992; Beal 1992, 1995). In line with this, there

is evidence for decreased glucose utilization, increased

levels of lactate and signs of impaired mitochondrial

function, including decreased succinate dehydrogenase

(SDH, complex II) activity, in the striatum of HD patients

(Jenkins et al. 1993; Gu et al. 1996; Browne et al. 1997;

Koroshetz et al. 1997). In addition, treatment of animals

with the SDH inhibitors 3-nitropropionic acid (3-NP,

systemic injection) and malonate (intrastriatal injection)

mimic some of the features of the striatal neuropathology of

HD (Beal et al. 1993a,b; Brouillet et al. 1995). Interestingly,

Bogdanov et al. (1998) found that transgenic R6/2 mice (12

weeks old) are more susceptible to systemic injections of

3-NP. While this provided tentative support for excito-

toxicity in this model of HD, we found that both R6/1 (18

weeks old) and R6/2 (6 weeks old) are totally resistant to

intrastriatal injections of the endogenous NMDA-receptor

agonist quinolinic acid (Hansson et al. 1999). The cellular

mechanisms underlying this resistance remain unknown. In

a similar vein, Hickey and Morton (2000) recently found

that fewer R6/2 (7±10 weeks old) than wild-type mice

exhibit striatal lesions after systemic injections of 3-NP.

However, they were not able to de®nitely ascertain whether

the R6/2 striatal neurons are resistant to mitochondrial toxin

because the sizes of the striatal 3-NP lesions (when they

occurred) were not signi®cantly different between R6/2 and

wild-type mice. Furthermore, because Bogdanov et al.

(1998) studied 12-week-old R6/2 mice, Hickey and Morton

(2000) speculated whether R6/2 mice older than 10 weeks

may be more susceptible to mitochondrial toxin.

We wanted to examine whether striatal neurons in mice

expressing exon 1 of the HD gene really are resistant to

damage following inhibition of mitochondrial function, and

if this is an age-dependent phenomenon. Striatal lesions

caused by systemic 3-NP injections in mice tend to be very

variable. We therefore injected malonate into the striatum of

transgenic mice expressing exon 1 of the HD gene with 18

(HDex6), 115 (R6/1) or 150 (R6/2) CAG repeats. Mice of

different ages were studied. As malonate-induced striatal

lesions are believed to depend upon an intact corticostriatal

pathway (Henshaw et al. 1994), we studied the capacity of

synaptosomes prepared from corticostriatal terminals from

transgenic mice to release glutamate in vitro. We also

examined the involvement of malonate-induced NMDA

receptor- and caspase-mediated cell death pathways in vivo

in wild-type and transgenic HD mice. Finally, we also

measured levels of the two anti cell death proteins Bcl-XL

and X-linked inhibitor of apoptosis protein (XIAP) in the

intact and malonate-injected striatum, to determine whether

changes in these proteins could underlie toxin-resistance in

the transgenic HD mice.

Materials and methods

Animals and malonate lesion

Heterozygous transgenic R6/1 and R6/2 males of CBA � C57BL/6

strain were purchased from Jackson Laboratories (Bar Harbor, ME,

USA) and maintained by back-crossing carrier males with

CBA � C57BL/6 F1 females. All mice used in this study were

taken from the 12±18th generations of back-crossing. HDex6 mice

were obtained from the breeding colony established at Medical and

Molecular Genetics, GKT School of Medicine, London, UK. The

total numbers of mice used in this study were: 260 R6/1 and wild-

type littermate mice; 134 R6/2 and wild-type littermate mice; and

16 HDex6 and age-matched wild-type mice. The exact number of

mice used in each experiment is presented in the ®gure legends.

The experimental procedure was approved by the ethical committee

at Lund University, and the animals were handled according to the

animal protection act of the Swedish Government. Mice were

genotyped using a polymerase chain reaction assay as previously

described (Mangiarini et al. 1996). Malonate (Sigma, Stockholm,

Sweden) was dissolved in 0.1 m phosphate buffered-saline (pH 7.4).

Under halothane anesthesia, the mice received intrastriatal injections

of 1 mmol (1 mL) malonate, using a 2-mL Hamilton microsyringe

®tted with a glass micropipette (outer diameter 60±80 mm), at the

following stereotaxic coordinates: 0.9 mm rostral to bregma, 2.0 mm

lateral to midline, 3.2 mm ventral from the bone surface, with the

tooth-bar set at zero. The toxin was injected over 5 min, and

thereafter the cannula was left in place for an additional 5 min to

minimize the risk of the retrograde leakage of toxin. Body

temperature was controlled using a heating pad set at 378C.

Resistance to malonate in transgenic HD mice 695

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 694±703

In a pharmacological protection study, wild-type and R6/1

transgenic mice were treated with NMDA antagonist MK-801 or

the caspase inhibitor zVAD-fmk. Mice were injected intraperi-

toneally with MK-801 (5 mg/kg) 30 min before intrastriatal

injection of malonate, or alternatively, zVAD-fmk [1 mg dissolved

in 1 mL of 1% dimethyl sulfoxide (DMSO); Enzyme Systems,

Livermore, CA, USA] was injected into the striatum 3 h after the

intrastriatal injection of malonate. These treatment regimens have

been shown to ef®ciently reduce malonate-induced lesion size in

rats (Schulz et al. 1998). Control mice received injections of both

saline intraperitoneally 30 min before and 1 mL of 1% DMSO into

the striatum 3 h after injection of malonate.

Perfusion

Either 2 (for Fluoro-Jade staining) or 4 days (for DARPP-32

immunostaining) after malonate injection, mice were deeply

anesthetized with pentobarbitone (200 mg/kg, i.p.) and trans-

cardially perfused with 50 mL of isotonic saline followed by

150 mL of 4% paraformaldehyde in 0.1 m phosphate buffer (PB;

pH 7.4). The brains were removed, placed in 4% paraformaldehyde

for 4 h at 48C and then dehydrated in 20% sucrose/0.1 m phosphate

buffer. Coronal sections were cut (30 mm) and collected in

phosphate-buffered saline (PBS).

Fluoro-Jade

Fluoro-Jade staining was performed as described by Schmued et al.

(1997). Brie¯y, perfusion ®xed brain sections were mounted, dried

and immersed in 100% ethanol, followed by 70% ethanol. Sections

were then treated with 0.06% potassium permanganate for 15 min.

After rinsing, sections were immersed in Fluoro-Jade (0.001%

Fluoro-Jade/0.1% acetic acid; Histo-Chem Inc., Jefferson, AR,

USA) for 30 min, followed by a 5-min incubation with Hoechst

33342 (2 mg/mL) in order to label the nuclei of all cells.

DARPP-32 immunohistochemistry

Free ¯oating sections were processed for DARPP-32 (dopamine-

and cyclic AMP-regulated phosphoprotein of a molecular weight of

32 kDa) immunohistochemistry. Free-¯oating sections were incu-

bated in 10% horse serum/0.2% Triton-X-100/0.1 m PBS for 1 h at

room temperature, followed by a reaction with an antibody against

DARPP-32 (1 : 20 000; donated by Drs P. Greengard and

H. Hemmings, Weill Medical College, New York, NY, USA) for

48 h at 48C. Sections were incubated with a biotinylated secondary

antibody (1 : 200) for 1 h, and bound antibody was visualized

using the ABC system (Vectastain ABC Kit, Vector Laboratories,

Burlingame, CA, USA), with 3,3 0-diaminobenzidine as chromogen.

Fig. 1 Photomicrographs of striatal sections prepared from brains

after intrastriatal malonate injection, labelled with the cell death

marker Fluoro-Jade (a, b, e and f ) or processed for cresyl violet

staining (c and g) or for DARPP-32 immunohistochemistry (d and h).

Fluoro-Jade staining and DARPP-32 immunohistochemistry was per-

formed on striatal sections from brains perfused at 48 h and 96 h

after malonate injection, respectively. Sections from wild-type mice

(all panels except e) contain numerous Fluoro-Jade-stained cells (a

and b) and show massive loss of cresyl violet- (c) and DARPP-32-

(d) stained cells, when compared with the contralateral non-lesioned

side (f, g and h). In sections from transgenic R6/2 HD mice (e) cells

are labelled with Fluoro-Jade only close to the injection site.

[Bar � 700 mm (a and e), 20 mm (b and f ) and 40 mm (c, d, g

and h)].

696 O. Hansson et al.


Morphological analyses

The numbers of cells in the striatum stained with Fluoro-Jade or

DARPP-32 immunohistochemistry were estimated by using the

stereology optical fractionater, as previously described (Hansson

et al. 2000). This sampling technique is not affected by tissue

volume changes and does not require reference volume determi-

nation (West et al. 1991). Cell loss was examined in every ®fth

section (120 mm apart) in a region starting caudally at the level of

the ventral hippocampal commissure and reaching up to the level of

the genu of the corpus callosum rostrally. This encompasses a

major portion of the head and part of the tail of the caudate-

putamen and typically 10 sections were examined per brain.

Sampling was done using the CAST-Grid system (Olympus

Denmark A/S, Albertslund, Denmark). It is composed of an

Olympus BX50 microscope, an X-Y motor stage run by a

computer, and a microcater (Heidenhain, ND 281) connected to

the stage. The CAST-Grid software (version 1.10) was used to

delineate the striatum at 4 � objective and generate counting areas

of 150 � 150 mm. Using a 40 � oil objective, the counting frame

(3980 mm2) was randomly placed on the ®rst counting area and

systemically moved through all counting areas until the entire

delineated area was sampled. The sampling frequency was chosen

so that about 300 cells were counted in each mouse. An estimate of

the number of cells was calculated according to the optical

fractionater formula (for more details, see West et al. 1991).

Succinate dehydrogenase activity

Brains were frozen with liquid nitrogen and striata were dissected

out at 2178C and stored at 2808C until analysis. Striata were

homogenized in a buffer containing 300 mm sucrose, 30 mm

HEPES and proteinase inhibitors. Equal amounts of protein

(200 mg/mL) were used for measurement of SDH activity. The

activity of SDH was quanti®ed spectrophotometrically at 600 nm in

a reaction medium containing 0.3 mm PB, pH 7.5, 1 mm antimycin

A, 0.1% Triton X-100, 2 mm succinate, 2 mm phenazine metho-

sulfate and 80 mm 2,6-dichlorophenolindophenol (Singer 1974).

The assay is based on the reduction of phenazine methosulfate by

SDH. Reduced phenazine methosulfate is immediately reoxidized

by 2,6-dichlorophenolindophenol resulting in a decrease of the

absorbance of the latter dye (Singer 1974).

Synaptosomal preparation and glutamate release

The striata from 18-week-old wild-type and R6/1 mice were

dissected bilaterally and homogenized in buffer containing 320 mm

sucrose, 1 mm EGTA, 20 mm HEPES, pH 7.4, 1.2 mm MgCl2,

10 mm glucose, proteinase inhibitors and 1 mg/mL bovine serum

albumin (BSA). The homogenate was ®lter through 2 layers of

nylon mesh (pore size 100 mm) and centrifuged at 800 g for 5 min

at 48C. The supernatant was divided into four tubes, centrifuged

at 12 000 g for 10 min, and pellets were stored at 48C. The

synaptosomal pellet was resuspended in 2 mL of incubation buffer

consisting of 140 mm NaCl, 5 mm KCl, 5 mm NaHCO3, 1.2 mm

NaH2PO4, 1.2 mm MgCl2, 10 mm glucose and 20 mm HEPES

(pH 7.4). After 4 min of preincubation at 308C, 1 mm NADP1,

100 U glutamate dehydrogenase, and 1.3 mm CaCl2 or 1 mm

Fig. 2 Quanti®cation of the number of striatal Fluoro-Jade and

DARPP-32 positive cells after intrastriatal malonate injection in wild-

type control, R6/1 and R6/2 HD transgenic mice at different ages

(wk � weeks of age). Fluoro-Jade staining and DARPP-32 immuno-

histochemistry were performed on striatal sections from brains per-

fused at 48 h and 96 h after malonate injection, respectively. The

number of Fluoro-Jade positive cells is expressed as total number of

positive cells in the striatum. The number of DARPP-32 positive

neurons is expressed as percentage of the contralateral intact side.

Data are mean ^SD (n � 12 in each group for R6/1 and n � 15 in

each group for R6/2). *Signi®cantly different from wild-type littermate

mice, p , 0.001.

Fig. 3 Quanti®cation of the number of striatal Fluoro-Jade and

DARPP-32 positive cells after intrastriatal malonate injection in

18-week-old HDex 6 mice expressing 18 CAG repeats. Fluoro-Jade

staining and DARPP-32 immunohistochemistry were performed on

striatal sections from brains perfused at 48 h and 96 h after malo-

nate injection, respectively. The number of cells were quanti®ed and

expressed as described in the legend of Fig. 2 (n � 4 in each

group).



EGTA were added. Glutamate release was determined by a

continuous ¯uorometric assay as described previously (Nicholls

et al. 1987), at excitation and emission wavelengths of 340 nm and

460 nm, respectively.

Glucose tolerance test

Blood samples for determination of glucose concentration were

obtained from the tail and analyzed with a Glucometer II (Modell

5529; Bayer Diagnostics, Gothenburg, Sweden). Transgenic R6/1

(18-week-old) and R6/2 (6- and 12-week-old) mice and wild-type

littermates were food deprived for 8±10 h and the basal levels of

glucose were measured. Thereafter the mice were given a bolus

injection of glucose (2 g/kg) intraperitoneally and blood levels of

glucose were measured after 40 min.

Western blotting

Striata from wild-type and 18-week-old R6/1 mice were dissected

bilaterally, as described above, at 6 or 18 h after unilateral

instrastriatal injection of malonate. Tissue was homogenized and

lysed in an ice-cold RIPA-buffer [150 mm NaCl, 1% Triton-X,

0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS),

50 mm Tris-HCl (pH 8.0)] supplemented with protease inhibitor

cocktail (Roche, Mannheim, Germany). Equal amounts of proteins

(15 mg/lane) were loaded on a 12% SDS-gel. After separation,

proteins were transferred onto a polyvinylidene di¯uoride-

membrane (Pall Gell Laboratories, Lund, Sweden), which was

blocked with 5% skimmed milk in TBS-0.5% Tween-20 (TBS-T).

Following washing with TBS-T, the membrane was incubated with

anti-XIAP (1 : 5000, Transduction Laboratories, Lexington, KY,

USA), anti-Bcl-XL (1 : 5000, Transduction Laboratories) or

b-tubulin (1 : 5000, BioSite, Taby, Sweden) followed by an

incubation with secondary horseradish peroxidase-coupled anti-

bodies (1 : 5000, Dako, Glostrup, Denmark). Signals were detected

with the enhanced chemiluminescence method and protein sizes

analysed using prestained protein markers obtained from BioRad.

Statistical analysis

Statistical comparisons of the dependent factors (genotype and age/

treatment) were undertaken by two-factor analyses of variance

using the Statview 5.4 package (Abacus Concepts, Berkley, CA,

USA). When the main effect was signi®cant, Bonferroni±Dunn's

post hoc test was used to correct for multiple analyses. Data are

presented as means ^ SD.

Results

In wild-type control mice (irrespective of age, between 6

and 18 weeks), intrastriatal injections of malonate caused

large lesions encompassing a major part of the head of the

caudate-putamen and even including parts of the tail of the

caudate-putamen. In Fluoro-Jade stained sections, obtained

2 days after injection, there were numerous positively

stained cells (Figs 1a and b). On average, each mouse

contained around 400,000 Fluoro-Jade labelled striatal cells

(Fig. 2), which is equivalent to approximately 50% of the

number of cells (both neurons and glia) in the whole

striatum (Hansson et al. 1999). Four days after injection of

malonate the majority of the cells were lost in the striatum,

including approximately 75% of the DARPP-32 positive

Fig. 4 Measurement of succinate dehydrogenase (SDH) activity in

striatal tissue homogenates from wild-type control and transgenic

R6/1 HD mice at 18 weeks of age. SDH activity was measured

under basal conditions or in the presence of different concentrations

of malonate (0.3 mM and 3.0 mM) (n � 4 for each group).

Fig. 5 Measurement of glutamate release from striatal synapto-

somes obtained from wild-type control and transgenic HD R6/1 mice

at 18 weeks of age. Glutamate release from synaptosomes was fol-

lowed in basal conditions in reaction medium containing 1.3 mM

Ca21 except when EGTA (1 mM) was present. After 5 min of incuba-

tion, KCl (30 mM) or ionomycin (10 mM) was added to evoke the

release of glutamate from synaptosomes. Triton X-100 (0.25%) was

added at the end of each experiment to determine the total content

of glutamate. (a) Representative trace of release of glutamate from

striatal synaptosomes in a reaction medium containing 1.3 mM Ca21

(TX � Triton X-100) (n � 5 in each group).



striatal medium spiny projection neurons (Figs 1d and h and

Fig. 2). In cresyl violet stained sections, the core of the

lesion exhibited an almost complete loss of cells (Figs 1c

and g), which was surrounded by a region of neuronal loss

and gliosis. In the lesion core, there were groups of

surviving cells which displayed an elongated shape and

tended to be located adjacent or close to blood vessels

(Fig. 1c).

When compared with littermate control mice, R6/1 and

R/2 mice exhibited partial resistance to malonate-induced

striatal damage (Figs 1e and Fig. 2). Thus, in 6- and

12-week-old R6/2 mice (150 CAG repeats), and 18-week-

old R6/1 mice (115 CAG repeats), both the numbers of

Fluoro-Jade stained cells (at 2 days) and the loss of

DARPP-32 stained cells (at 4 days) were signi®cantly

reduced to 20±30% of that observed in wild-type mice

(Figs 1a and e and Fig. 2). Interestingly, this partial

resistance was not observed in younger, 6-week-old, R6/1

mice with the shorter 115 CAG repeat length. Instead, they

displayed lesion sizes comparable to control mice (Fig. 2).

Therefore, the resistance to malonate appears to be

dependent on both the CAG repeat length and the age of

the transgenic mice. Importantly, transgenic mice expressing

a normal CAG repeat length (18 CAG; HDex6) were not

resistant to malonate-induced lesions at 18 weeks of age

(Fig. 3). This indicates that the resistance to neurotoxin is

not related to the expression of a transgene coding for exon

1 HD gene per se. Instead, in analogy to development of the

clinical disorder in humans, the length of the CAG repeat in

the HD gene must exceed a crucial threshold for neurotoxin

resistance to develop.

Possibly, the resistance to malonate-induced lesions could

be due to increased basal levels of SDH activity or lack of

sensitivity of this enzyme to malonate in the HD mice.

Therefore, we measured SDH activity in striatal homo-

genates of 18-week-old R6/1 mice and littermate control

mice (Fig. 4). The basal activity of SDH was found to be

similar in HD and wild-type control mice. In addition,

Fig. 8 Glucose measurements in blood samples of wild-type con-

trol, R6/1 and R6/2 HD transgenic mice at different ages (wk �weeks of age). Blood glucose levels were determined in fasting

conditions and after 40 min of glucose administration (2 g/kg, i.p.).

(n � 7 in each group).

Fig. 7 Western blots showing levels of XIAP and Bcl-XL in striatum

from HD transgenic and wild-type mice. Intact control striatal tissue

(C), striatal tissue 18 h after malonate lesion (L) and non-lesioned

tissue from the contralateral striatum (NL). Tissues from wild-type

(wt) and 18-week-old R6/1 (HD) mice are compared. No differences

were found in Bcl-XL and XIAP expression levels. However, an addi-

tional XIAP immunoreactive band was present in malonate lesioned

tissue from wt striatum. b-Tubulin was used as a loading control

(n � 3 in each group).

Fig. 6 Effect of the NMDA antagonist MK-801 or the caspase inhibi-

tor zVAD-fmk on malonate-induced striatal lesion in 18-week-old

wild-type control and R6/1 HD transgenic mice. The mice received

injections of the NMDA antagonist MK-801 (5 mg/kg, i.p.) 30 min

before malonate or intrastriatal injections of the caspase inhibitor

zVAD-fmk (1 mg) 3 h after malonate. Fluoro-Jade staining and

DARPP-32 immunohistochemistry were performed on striatal sec-

tions from brains perfused at 48 h and 96 h after malonate injection,

respectively. The number of cells was quanti®ed and expressed as

described in the legend of Fig. 2. (n � 10 in each group). *Signi®-

cantly different from vehicle-treated mice of same genotype,

p , 0.005.



malonate inhibited SDH to the same extent in HD mice as

compared with control mice.

Previous studies have shown that striatal lesions induced

by malonate or 3-NP partially depend upon glutamate

released from the corticostriatal pathway (Beal et al. 1993a;

Henshaw et al. 1994). Therefore we assessed basal gluta-

mate release from striatal synaptosomes in vitro, and evoked

glutamate release after membrane depolarization and calcium

in¯ux (Fig. 5). We found no difference between the glutamate

release from synapatosomes prepared from transgenic R6/1

(18-week-old) and wild-type striatal tissue, under basal or

evoked conditions (Fig. 5). This indicates that the resistance to

striatal damage in HD mice is not due to a decrease in the

release of glutamate from the corticostriatal pathway.

Next we sought to clarify which intracellular cell death

pathways are activated in striatal neurons in wild-type and

transgenic R6/1 mice (18-week-old) following intrastriatal

injections of malonate. The mice received intraperitoneal

injections of the NMDA antagonist MK-801 30 min before

malonate or intrastriatal injections of the caspase inhibitor

zVAD-fmk 3 h after malonate. In wild-type mice, both

MK-801 and zVAD-fmk reduced the number of dying cells

by around 50±60% (Fig. 6). However, neither one of the

drugs reduced lesion size in the transgenic R6/1 mice

(Fig. 6). This indicates that the NMDA receptor- and

caspase-mediated cell death pathways, which are normally

activated by malonate in striatal neurons (Schulz et al.

1998), are somehow inhibited in R6/1 mice and that the

residual cell death seen after intrastriatal malonate injection

in HD mice is independent of these pathways.

The two anti cell death proteins of Bcl-XL and XIAP are

able to inhibit caspase activation through different mechan-

isms (Boise et al. 1993; Liston et al. 1996). However,

western blotting revealed no differences between transgenic

HD and wild-type mice regarding levels of Bcl-XL and

XIAP in intact striatum (Fig. 7), at 6 h (data not shown) and

at 18 h (Fig. 7) after malonate lesion. At 18 h after the

malonate lesion there was a second band immunoreactive

for XIAP in tissue from wild-type mice, indicating

degradation of XIAP. This band was not as clearly apparent

in tissue from R6/1 mice. The degradation of XIAP has

recently been shown to occur in hippocampal neurons

following excitotoxic injury induced by kainic acid

(Korhonen et al. 2001).

Furthermore, because R6/2 mice have been shown to

develop diabetes (Hurlbert et al. 1999), and high blood

levels of glucose could effect lesions induced by malonate

by providing additional substrate for glycolysis, we

performed glucose tolerance tests on the mice. We found

that three out of seven of the 12-week-old R6/2 mice had

reduced glucose tolerance. However, 6-week-old R6/2 and

18-week-old R6/1 mice were not diabetic (Fig. 8), excluding

the possibility that the resistance to malonate-induced lesion

is mediated by increased levels of glucose in the blood.

Discussion

We demonstrate that transgenic mice expressing exon 1 of

the HD gene with an expanded CAG trinucleotide repeat

exhibit partial resistance to neurodegeneration caused by

malonate-induced local energy depletion in the striatum.

The results highlight two novel important principles. First,

the resistance to malonate develops with time, as 18-week-

old but not 6-week-old R6/1 mice displayed resistance to

malonate-induced neurodegeneration. This is analogous to

the human disorder where the onset of symptoms most often

is in adulthood. Second, there is a relationship between CAG

repeat length and the onset of resistance to malonate because

6-week-old R6/2 (CAG 150), but not R6/1 (CAG 115), mice

were resistant to malonate. Interestingly, in patients with HD

there is an inverse correlation between the number of CAG

repeats and age of onset of symptoms (Duyao et al. 1993;

MacDonald and Gusella 1996). Malonate inhibited striatal

succinate dehydrogenase activity with equal ef®cacy in both

R6/1 and wild-type mice. Moreover, the release of glut-

amate from striatal synaptosomes in vitro was not altered in

HD mice. In addition, we provide tentative evidence that

NMDA- and caspase-mediated cell death pathways are

inhibited in the HD mice and those cells that die following

malonate-injections in HD mice undergo a form of death

that is independent of these pathways.

The present results are in good agreement with our earlier

observations that R6/1 and R6/2 mice are totally resistant to

quinolinic acid-induced striatal toxicity (Hansson et al.

1999). A recent report suggested reduced striatal sensitivity

to 3-NP in 7±10-week-old R6/2 mice, because fewer R6/2

mice than wild-type controls exhibited lesions after systemic

3-NP treatment (Hickey and Morton 2000). However, the

study did not de®nitely establish whether R6/2 striatal

neurons are resistant to 3-NP treatment as when striatal

lesions occurred they did not differ in size between R6/2

and wild-type mice. In contrast, Bogdanov et al. (1998)

previously reported an increased susceptibility to systemic

3-NP in 12-week-old R6/2 mice. Their data are dif®cult to

reconcile with our ®ndings of resistance to malonate-

induced damage in 6±12-week-old R6/2 mice and with the

study with 3-NP lesions in 7±10-week-old R6/2 mice

reported by Hickey and Morton (2000). Hickey and Morton

(2000) speculated that R6/2 mice may become more

sensitive to 3-NP beyond the age of 10 weeks. However,

we have also examined the effects of systemic 3-NP

treatment on 12-week-old R6/2 mice and littermate controls

(similar treatment regimen as Bogdanov et al. 1998). We

found that three out of ®ve wild-type mice exhibited striatal

lesions but none of the R6/2 mice showed any striatal

damage, indicating that in our hands 12-week-old R6/2 mice

are also resistant to systemic 3-NP (O. Hansson, unpub-

lished result). Taken together with the current ®ndings of

reduced striatal neurodegeneration following malonate



injections, we propose that the R6 lines of transgenic

HD mice actually develop resistance, not increased

susceptibility, to neurotoxins with age.

Malonate-induced striatal lesions are partially dependent

on NMDA receptor activation (Greene and Greenamyre

1995; Greene et al. 1993; Henshaw et al. 1994; Schulz et al.

1998 and Fig. 6 in this study). Malonate-induced energy

de®ciency leads to partial membrane depolarization, which

in turn is believed to generate secondary excitotoxicity by

causing a removal of the voltage-dependent magnesium

block of the NMDA receptor-gated calcium channel (Beal

1992, 1995). Under these circumstances, glutamate released

from the corticostriatal pathway is important for the

induction of striatal neuronal death (Greene et al. 1993;

Henshaw et al. 1994; Greene and Greenamyre 1995).

Therefore, a reduced release of glutamate in the striatum

of HD mice could contribute to the resistance. However, the

total striatal tissue levels of glutamate are not changed in

R6/2 mice (Reynolds et al. 1999) and as we show in this

study the evoked release of glutamate from striatal synapto-

somes in vitro is not decreased in 18-week-old R6/1 mice.

Moreover, the release of glutamate from the corticostriatal

pathway has been found to be increased following

depolarization in 16-week-old R6/1 mice using intrastriatal

microdialysis (NicNiocaill et al. 2001). Taken together, it

seems unlikely that changes in corticostriatal release of

glutamate contribute to the resistance phenomenon in R6/1

and R6/2 HD mice. In addition to glutamate, it is believed

that dopamine may contribute to cell death after adminis-

tration of mitochondrial inhibitors such as malonate and

3-NP (Reynolds et al. 1998; Ferger et al. 1999). Malonate

causes an acute and massive ef¯ux of dopamine from

nigrostriatal terminals in the striatum, which could be toxic

(Ferger et al. 1999). Interestingly, 6-week-old R6/2 mice

display defective striatal dopamine signalling and down-

regulation of striatal dopamine receptors (Cha et al. 1998;

Bibb et al. 2000). However, dopamine receptor antagonists

at most reduce excitotoxic lesions by around 30% (Bakker

and Foster 1991; Filloux and Wamsley. 1991; Wahl et al.

1993), which is not enough to explain the extent of

resistance observed in malonate- and quinolinate-induced

toxicity in R6/1 and R6/2 mice (Hansson et al. 1999). In

addition, levels of striatal dopamine have been reported to

be normal in 4- and 8-week-old R6/2 mice, while at 12

weeks of age the striatal dopamine level is reduced by

50% compared with wild-type control (Reynolds et al.

1999). In the present study, we observed protection

against malonate lesions in the R6/2 mice already at 6

weeks of age, when they still have normal striatal dopamine

tissue levels.

It is likely that the resistance phenomenon in R6/1 and

R6/2 mice is due to intrinsic changes in the striatal neurons

themselves. Our earlier ®ndings indicate that the resistance

phenomenon in R6/1 mice is not due to changes in the

striatal levels of NR1 subunit of the NMDA receptor, Bcl-2,

heat shock protein 70, calbindin or activity of superoxide

dismutase (Hansson et al. 1999). In this study, we observed

that there were also no changes in the anti cell death proteins

Bcl-XL (Boise et al. 1993) and XIAP (Liston et al. 1996).

Malonate-induced striatal cell death was reduced by

50±60% in wild-type mice by treatment with either the

NMDA antagonist MK-801 or the caspase inhibitor

zVAD-fmk. However, these two compounds did not reduce

lesion size in transgenic HD mice, suggesting that NMDA

receptor- and caspase-mediated cell death pathways may be

inhibited in HD mice. Interestingly, the conductance of the

NMDA receptor is unchanged in medium spiny striatal

neurons of R6/2 mice (6±12-week-old) (E. Guatteo, O.

Hansson, P. Brundin and N. Mercuri, unpublished obser-

vation) implying that the resistance mechanism is down-

stream to NMDA receptor activation. It is possible that

compensatory mechanisms are induced in the striatal

neurons of R6/1 and R6/2 mice during the disease

progression. When subjected to sublethal grades of different

types of metabolic or excitotoxic stress, e.g. transient

ischaemia or seizures, neurons can adapt and become

resistant to subsequent, normally lethal, exposures to

hypoxia, ischemia or excitotoxicity (for reviews, see Marini

and Paul 1992; Chen and Simon 1997; Ishida et al. 1997).

Therefore, we propose that HD neurons might be subjected

to a protracted, sublethal grade of metabolic stress caused

by mutant huntingtin, which results in an adaptation of

the neurons with up-regulation of cellular defence

mechanisms against impaired energy metabolism and

excitotoxicity. Interestingly, evidence for metabolic dys-

function, including mitochondrial defects, has been

reported in R6/2 mice (Tabrizi et al. 2000; Jenkins et al.

2000). In spite of this, there is only limited neuronal death in

R6/2 mice and it does not occur until at 14±16 weeks of

age (Turmaine et al. 2000), even though the mice show

motor and cognitive dysfunction already at 3.5±5 weeks of

age (Carter et al. 1999; Lione et al. 1999). These results

indicate that compensatory mechanisms in striatal

neurons prevent them from dying even though they are

subjected to metabolic and excitotoxic stress. If the same

mechanisms are present in the striatum of HD patients, it

could explain why it usually takes several years or

decades before the striatal neurons actually die in HD.

Interestingly, the observations of neurotoxin-resistance in

transgenic HD mice exhibit parallels to the disease

process in HD patients because they both display a delay

before onset and an inverse correlation between CAG

repeat number and age at onset. Future studies will

determine to what extent the resistance is correlated to the

appearance of other phenotypic features in R6 mice, e.g.

intranuclear inclusions and behavioural changes, and focus

on the cellular mechanisms that underlie the resistance

phenomenon.



Acknowledgements

We acknowledge the excellent technical assistance of Birgit

Haraldsson and Britt Lindberg. This study was supported by

grants from the Swedish Medical Research Council, the

Swedish Association of the Neurologically Disabled, the

Swedish Society for Medical Research, the Swedish Cancer

Foundation and the Wellcome Trust. OH and LK are supported

by the National Network in Neuroscience. RFC is partially

supported by grants from CNPq and FAPESP.

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