www.elsevier.com/locate/ynbdi

Neurobiology of Disease 18 (2005) 206–217

Minocycline in phenotypic models of Huntington’s disease

Kadiombo Bantubungi,a Carine Jacquard,b Anita Greco,c Annita Pintor,d Abdelwahed Chtarto,e,f

Khalid Tai,e,f Marie-Christine Galas,g Liliane Tenenbaum,e,f Nicole Deglon,h Patrizia Popoli,d

Luisa Minghetti,c Emmanuel Brouillet,b Jacques Brotchi,e,i Marc Levivier,e,i

Serge N. Schiffmann,a and David Blume,f,*

aLaboratory of Neurophysiology, ULB-Erasme, Brussels, BelgiumbURA CEA-CNRS 2210, Service Hospitalier Frederic Joliot, CEA, Orsay, FrancecDepartment of Cell Biology and Neuroscience, Istituto Superiore di Sanita, Roma, ItalydDepartment of Drug Research and Evaluation, Istituto Superiore di Sanita, Roma, ItalyeLaboratory of Experimental Neurosurgery, ULB-Erasme, CP602, Bat C-6/113, 808 Route de Lennik, B-1070

Brussels, BelgiumfIRIBHM, ULB-Erasme, Brussels, BelgiumgINSERM U422, Lille, FrancehDepartment of Medical Research and ImaGene Program, CEA, Orsay, FranceiDepartment of Neurosurgery, ULB-Erasme, Brussels, Belgium

Received 20 July 2004; revised 23 September 2004; accepted 30 September 2004

Available online 2 December 2004

Minocycline has been shown to be neuroprotective in various models of

neurodegenerative diseases. However, its potential in Huntington’s

disease (HD) models characterized by calpain-dependent degeneration

and inflammation has not been investigated. Here, we have tested

minocycline in phenotypic models of HD using 3-nitropropionic acid

(3NP) intoxication and quinolinic acid (QA) injections. In the 3NP rat

model, where the development of striatal lesions involves calpain, we

found that minocycline was not protective, although it attenuated the

development of inflammation induced after the onset of striatal

degeneration. The lack of minocycline activity on calpain-dependent

cell death was also confirmed in vitro using primary striatal cells.

Conversely, we found that minocycline reduced lesions and inflamma-

tion induced by QA. In cultured cells, minocycline protected against

mutated huntingtin and staurosporine, stimulations known to promote

caspase-dependent cell death. Altogether, these data suggested that, in

HD, minocycline may counteract the development of caspase-dependent

neurodegeneration, inflammation, but not calpain-dependent neuronal

death.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Huntington’s disease; 3-Nitropropionic acid; Quinolinic acid;

Minocycline; Striatum; Cell death

0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2004.09.017

* Corresponding author. Laboratory of Experimental Neurosurgery,

Universite Libre de Bruxelles, Campus Erasme, CP602, Bat C-6/113, 808

Route de Lennik, B-1070 Brussels, Belgium. Fax: +32 2 555 46 55.

E-mail address: dablum@ulb.ac.be (D. Blum).

Available online on ScienceDirect (www.sciencedirect.com).

Introduction

Huntington’s disease (HD) is a genetic neurodegenerative

disorder characterized by motor and cognitive impairments mainly

due to neuronal degeneration within the striatum and the cerebral

cortex (Blum et al., 2003a; Brouillet et al., 1999). The mutation

involved produces a polyglutamine expansion within the N-

terminal part of the huntingtin protein (The Huntington’s Disease

Collaborative Research Group, 1993), which leads to many

neuronal alterations such as impairments in transcription (Cha,

2000; Zuccato et al., 2003), calcium signaling (Tang et al., 2003),

or axonal transport (Gunawardena et al., 2003; Szebenyi et al.,

2003) and promotes mitochondrial complex II dysfunction (Beal,

2000), mitochondrial Ca2+ defects (Panov et al., 2002), NMDA

receptor sensitization (Song et al., 2003; Zeron et al., 2002), as well

as proapoptotic and pronecrotic protease activation (Wellington et

al., 2003). However, there is no efficient treatment that slows down

or halts the evolution of HD.

Minocycline is an antibiotic of the tetracycline family that

displays beneficial activity in various models of neurodegenera-

tion (Parkinson disease, amyotrophic lateral sclerosis, spinal cord

injury, ischemia) (Brundula et al., 2002; Kriz et al., 2002; Lee et

al., 2003; Stirling et al., 2004; Tomas-Camardiel et al., 2004; Wu

et al., 2002; Yrjanheikki et al., 1999; see also Blum et al., 2004,

for review), although recent studies pointed out that it may also

be detrimental (Diguet et al., 2004a; Tsuji et al., 2004; Yang et

al., 2003; see also for review Blum et al., 2004; Diguet et al.,

2004b). Beneficial activity of minocycline has been related to its

ability to inhibit both mitochondrial mechanisms leading to cell

death and inflammatory processes (Blum et al., 2004; Scarabelli

K. Bantubungi et al. / Neurobiology of Disease 18 (2005) 206–217 207

et al., 2004; Tikka et al., 2002; Wang et al., 2003, 2004; Zhu et

al., 2002).

Minocycline is being tested in HD patients (http://www.

huntington-study-group.org), and preliminary observations are en-

couraging (Bonelli et al., 2003; Thomas et al., 2004). However, the

potential of minocycline for treating HD is far from clear (for a

review, see Blum et al., 2004). While a former study (Chen et al.,

2000) demonstrated that i.p. injections of minocycline improve

motor alterations in the R6/2 transgenic mouse model of HD—that

is characterized by very limited neuronal loss even at very late

stages (Turmaine et al., 2000)—Smith et al. (2003) failed to

reproduce this effect in the same strain following continuous oral

administration. Additionally, although the inhibitory effect of

minocycline on caspase activation has been well described (Wang

et al., 2003), it is not known whether the antibiotic would be

effective against striatal degeneration involving brain calpains.

Indeed, increased calpain activity has been reported not only in the

striatum of HD patients, but also in transgenic and nontransgenic

rodent models of the disease (Bizat et al., 2003a; Gafni and Ellerby,

2002; Gafni et al., 2004). In the same way, the ability of

minocycline to modulate the development of striatal inflammation,

another important pathological feature of HD (Sapp et al., 2001), is

ill-defined. Hence, to further analyze the neuroprotective potential

of minocycline for HD, we tested whether it could attenuate striatal

neurodegeneration in phenotypic models of HD based on complex

II inhibition (Beal et al., 1993) or NMDA receptor overactivation

(excitotoxicity; Beal et al., 1986), both known to mimic numerous

behavioral, histological, neurochemical, and biochemical events

seen in HD patients (Brouillet et al., 1999).

Material and methods

Animals

We used adult male Lewis rats, 12 weeks of age, for the 3-

nitropropionic (3NP) model and adult Wistar rats (250 g) for the

quinolinic acid (QA) model and microdialysis experiments.

Animals were housed three per cage and maintained in a

temperature- and humidity-controlled room on a 12-h light/dark

cycle with food and water ad libitum. The number of animals was

kept to a minimum, and all efforts to avoid animal suffering were

made in accordance with the standards of the Institutional Ethical

Committees.

Treatments

3NP (Fluka, Belgium) was dissolved in 0.1 M PBS, pH 7.4,

adjusted to pH 7.3–7.4 with 5 N NaOH and quinolinic acid (Sigma,

Belgium) was dissolved in 2 N NaOH, the pH was adjusted to 7.4,

and the volume completed with PBS (pH 7.4) as previously

described (Bensadoun et al., 2001; Ouary et al., 2000). Minocy-

cline and doxycycline were dissolved in NaCl 0.9%. Fresh

solutions were prepared each day before injections. The volume

injected was adjusted according to the body weight of each rat (200

Al/100 g, i.p).

Chronic 3NP delivery (Fig. 1A)

Chronic treatments with 3NP (56 mg/kg/d) using 2mL1 (10 Al/h, 7 days) Alzet minipumps and neurological scoring were

performed as previously described (Blum et al., 2002a,b, 2003b;

Mittoux et al., 2002; Ouary et al., 2000). Rats were anesthetized

with a mixture containing xylazine hydrochloride (Rompun, Bayer;

4.5 mg/kg) and ketamine hydrochloride (Imalgene, Merial; 90 mg/

kg). An incision was made below the base of the neck and a 2mL1

Alzet osmotic minipump (delivering 10 Al/h for 7 days; IFFA

Credo, Belgium) containing 3NP (Fluka) was positioned under the

skin. The final concentration of 3NP in the pump was adjusted to

the weight of the rats on the day of implantation in order to exactly

deliver 56 mg/kg/d. Sham rats and animals treated with pharmaco-

logical compounds alone underwent all the surgical procedures

(without minipump implantation). Thirty-nine rats (sham/vehicle,

n = 5; 3NP/vehicle, n = 8; sham/minocycline 10 mg/kg, n = 5;

3NP/minocycline 10 mg/kg, n = 8; sham/minocycline 50 mg/kg,

n = 5; 3NP/minocycline 50 mg/kg, n = 8) were challenged for the

neuroprotective ability of minocycline. Animals were injected with

vehicle or minocycline just before minipump implantation and

each day until sacrifice. Controls and 3NP-treated animals were

evaluated every day for motor impairments. Briefly, behavioral

abnormalities were determined according to the presence and

severity of motor symptoms consisting of dystonia, gait abnormal-

ities, recumbency and also grasping and the ability to remain on a

small platform for N10 s. The final neurological score was assessed

as described (Mittoux et al., 2002; Ouary et al., 2000; minimum =

0, normal animal; score = 8, animal showing near-death

recumbency). All rats were killed after 5 days of 3NP subcutaneous

infusion (10 h after the last minocycline injection) according to the

known kinetics of striatal lesion occurrence in this model as we

previously reported (Bizat et al., 2003a; Blum et al., 2001, 2002a,b,

2003b; Ouary et al., 2000).

Chronic 3NP delivery and minipump removal (Fig. 1B)

We developed another 3NP protocol in which inflammation

was induced in addition to the striatal lesion (see also the Results

section) and determined the effect of minocycline on its develop-

ment. In this model, minipumps were positioned under the skin as

indicated above and removed under light anesthesia with halothane

5 days after implantation. Then, animals were injected daily with

vehicle or 10 mg/kg minocycline until the sacrifice for 5 days.

Thirty-two rats were used: sham/vehicle, n = 6; 3NP/vehicle, n =

10; sham/minocycline 10 mg/kg, n = 6; 3NP/minocycline 10 mg/

kg, n = 10. All rats were killed 5 days after minipump removal.

Quinolinic acid-induced striatal lesions (Fig. 1C)

Animals were anesthetized with xylazine/ketamine mixture and

received an intrastriatal stereotaxic injection of QA (1 Al; 180 nmol)

using the following coordinates: 1.0 rostral to bregma, 3.5 mm

lateral to midline and 5 mm ventral from the dural surface. The

toxin was injected over 4 min, and the needle was left in place for an

additional 2 min. Eighteen rats were used: QA/vehicle, n = 6; QA/

minocycline 10 mg/kg, n = 6; QA/doxycycline 10 mg/kg, n = 6. All

rats were killed 7 days after surgery.

Tissue postprocessing

All animals were killed by decapitation, and their brains were

quickly removed and frozen in 2-methylbutane cooled by dry ice

(�408C). The tissue was cut at 20-Am thickness on a cryostat

(Leitz), and serial coronal sections were mounted onto poly-l-

lysine and stored at �208C until use. In 3NP-treated rats, since

lesions are bilateral with similar extents (Mittoux et al., 2002;

Brouillet et al., unpublished results), hemispheres were separated at

K. Bantubungi et al. / Neurobiology of Disease 18 (2005) 206–217208

the time of sacrifice. One was used for histology and the other for

prostaglandin E2 (PGE2) dosage. Hematoxylin staining or succi-

nate dehydrogenase (SDH) histochemistry was used to reveal

striatal lesions. The latter was measured every 300 Am along the

anteroposterior axis of the striatum (bregma +1.6 to �0.9 mm), and

the lesional volume was calculated for each animal using

Cavalieri’s method as described (Coggeshall, 1992).

Semiquantitative measurement of succinate dehydrogenase activity

Measurement of SDH activity in control and 3NP-treated rats

was performed as described previously (Brouillet et al., 1998).

Frozen sections mounted on poly-l-lysine-coated slides were air

dried and then incubated for 15 min in 0.1 M PBS (pH 7.4, 0.9%

NaCl) at 378C followed by incubation in 0.3 mM nitroblue

tetrazolium (Sigma), 0.05 M sodium succinate (Sigma), and 0.05

M phosphate buffer, pH 7.6, for 30 min at 378C. Finally, sectionswere rinsed successively for 5 min in cold PBS and deionized

water and dried at room temperature. The image of each section

was acquired, and the quantification was performed as described

previously (Brouillet et al., 1998) using NIH image software.

In situ hybridization

The hybridization technique was adapted from previous reports

(Blum et al., 2002a,b). The sections mounted on RNAse-free poly-

l-lysine-coated slides were fixed in 4% freshly prepared parafor-

maldehyde for 30 min and rinsed in PBS 0.1 M. All sections were

dehydrated and dipped for 3 min in chloroform. After air drying, the

sections were incubated overnight at 428C with 0.35� 106 cpm per

section of 35S-labeled probes diluted in hybridization buffer, which

consisted of 50% formamide, 4� SSC (1� SSC: 0.15 m NaCl,

0.015 M sodium citrate, pH 7.4), 1� Denhardt’s solution (0.02%

each of polyvinylpyrrolidone, bovine serum albumin, Ficoll), 1%

sarcosyl, 0.02 M sodium phosphate at pH 7.4, 10% dextran sulfate,

yeast tRNA at 500 Ag/ml, salmon sperm DNA at 100 Ag/ml, and 60

mM dithiothreitol. Compounds were provided by Sigma. After

hybridization, the sections were rinsed for 4� 15 min in 1� SSC at

558C, dehydrated, and covered with Hyperfilm-gmax film (Amer-

sham, Belgium) for 2 or 3 weeks. The oligonucleotide probes were

synthesized by Eurogentech with the following sequences: GFAP

5V-CAGCTCCCGAAGTTCTGCCTGGTAAACGTCAGCCAG-TTTGGTGGG-3V and CD11b/MAC-1 5V-GACCTGGGGAG-GATCCCATATGGTCACCTTGTTGATCTGG-3V. They were

labeled with a-35S dATP (DuPont-NEN, Belgium) at it the 3V endby terminal DNA deoxynucleotidylexotransferase (Gibco, Bel-

gium) and purified with a G50 column (Pharmacia) according to the

manufacturer’s instructions. Digitalized images with 256 gray

levels were generated from the autoradiograms with the public

domain NIH image 1.61 program (National Institute of Health,

USA), a Power Macintosh G3, and a CCD video camera (Dage-

MTI, IN, USA) with fixed gain and black level as previously

described (Blum et al., 2002a,b). On each section, an averaged

optical density of the background level was subtracted from that of

the measured areas to obtain corrected values.

CD11b immunohistochemistry

Immunohistochemistry was performed on paraffin sections as

we previously described (Blum et al., 2002a). We used a

monoclonal mouse antirat CD11b antibody from Serotec (MCA

275R; France) at 1/100 dilution. For revelation, we used

biotinylated donkey antimouse secondary antibody (1/200 in 1%

normal horse serum; Jackson Immuno Research, USA) and the

ABC method (Vector Laboratories, Belgium) and diaminobenzi-

dine (Dako, Belgium).

Prostaglandin E2 (PGE2) dosage

PGE2 dosage was performed as previously described (Minghetti

et al., 2000). Striata were dissected out from frozen hemispheres of

3NP-treated animals, weighted, and homogenized in 50 mM Tris

buffer (pH 7.5) containing 10 Ag/ml indomethacin (Sigma) and 10

AM butylated hydroxytoluene (Sigma) to avoid ex vivo generation

of prostanoids. Homogenates were vigorously vortexed and

incubated for 5 min on ice, before centrifugation at 14,000 rpm

for 45 min at 48C. The recovery of the extraction procedure,

assessed by adding 5000 cpm of tritium-labeled PGE2 to the

homogenate and measuring the recovered radioactivity, was N70%.

Supernatants were collected and stored at �808C until use. PGE2

content was assessed using a specific high-sensitivity enzyme-

immunoassay (detection limit: 4 pg/ml; Assay Design, Ann Arbor,

MI, USA). Results were expressed as pg PGE2/mg of wet tissue.

Microdialysis experiments

Microdialysis and determination of glutamate concentrations by

high-performance liquid chromatography (HPLC) (electrochemical

detection) were performed as we previously reported (Blum et al.,

2003b; Popoli et al., 2002). Under Equithesin anesthesia (3 ml/kg

i.p.), Wistar rats were placed in a stereotaxic frame and implanted

with a concentric dialysis probe (mod CMA/12, 3-mm length,

Carnegie Medicine, Sweden) into the striatum. Stereotaxic

coordinates were as follows A = + 1; L = +3; V = �6.5 (anterior

striatum) according to the atlas of Paxinos and Watson. Twenty-

four hours later, the probe was perfused at a rate of 2 Al/min with a

Ringer solution (NaCl 147, CaCl2 2.3, and KCl 4.0 mM). After a

washout period of at least 90 min, samples were collected every 5

min into a refrigerated fraction collector (mod CMA/170) and then

frozen until assay. Glutamate outflow was induced by quinolinic

acid (QA) as previously shown (Popoli et al., 2002). Since the

intracerebral injection of QA induces tremors and convulsions in

rodents, these experiments were performed under general anes-

thesia (Equithesin). Results were expressed as percentage changes

of extracellular glutamate levels induced by probe perfusion with

QA (5 mM over 30 min) with respect to basal (predrug) values

(mean of three to four samples collected after the induction of

general anesthesia). Minocycline (10 mg/kg, i.p.) was given daily

for 3 or 7 days with the last injection 20 min before QA perfusion.

At the end of the experiments, each rat was sacrificed with an

overdose of Equithesin, and the brain was cut to verify the probe

location. The glutamate content of all samples was measured by

reverse-phase high-performance liquid chromatography coupled to

a fluorometric detector (Perkin Elmer LC240 at wavelength of 335

nm and emission cutoff filter of 425 nm), using a 15-min gradient

elution program (methanol from 20% to 80% with 50 mM

NaH2PO4 and CH3COONa) and automatic precolumn derivatiza-

tion with o-phthalaldehyde and h-mercaptoethanol. Cysteic acid

was used as internal standard. The concentration of the standard

was linear (r2 = 0.99) between 0.2 to 25 ng/10 Al. Basal glutamate

levels were calculated by comparison of sample peak height with

external standard peak height, both corrected for the internal

Fig. 1. Schematic drawing representing the experimental protocols used for

chronic intoxication with 3NP (protocols A and B), QA-induced lesions

(protocol C), and minocycline treatments.

K. Bantubungi et al. / Neurobiology of Disease 18 (2005) 206–217 209

standard peak height and expressed as ng/10 Al without probe

recovery correction.

Primary striatal cultures

Primary cultures of striatal neurons were obtained from 17- to

18-day-old Wistar rat embryos as described (Blum et al., 2002b,

2003b; Galas et al., 2004). Cells were exposed for 3 days to 100

AM 3NP with or without a daily treatment with 1–50 AMminocycline or doxycycline (Sigma; 10 mM stock in ethanol

50%) in 24-well plates. Cell viability was assessed by MTS assay

(Promega, Belgium) according to the manufacturer’s instructions.

The optical density was measured on a Titertek Multiskan MCC/

340 (ICN Biomedicals, Costa Mesa, CA).

Staurosporine treatment and transfection of HEK 293T cells

The HEK 293T immortalized human embryonic kidney cell line

was purchased from Q-One Biotech (Glasgow, UK). Cells were

cultured in Dulbecco’s modified Eagle medium (DMEM; Life

Technologies) supplemented with 10% FCS (Sigma), penicillin-

streptomycin, l-glutamine, nonessential amino acids, and sodium

pyruvate. Cells were seeded at 100,000 293T cells in 12-well tissue-

culture plates (Nunc). For staurosporine experiments, cells were

treated three times with minocycline (25–100 AM): 12 h after

seeding, the day after, and 2 h before staurosporine treatment (1 AMfrom a 1 mM stock solution in DMSO). MTS viability assay was

performed 24 h later. For huntingtin transfection experiments, we

used SIN-PGK-HD plasmids previously described (de Almeida et

al., 2002) encoding for 171-aa N-terminal fragments of huntingtin

carrying 19 or 82 glutamines. Twenty-four hours after seeding, the

medium was replaced by 500 Al DMEM 1% heated FCS containing

the transfection mixture which consisted DNA (1 Ag), 5 Alpolyethyleneimine (10 mM; 25 kDa, Aldrich, cat n8 4087270),

and 25 Al OptiMEM (Life Technologies) incubated 30 min at room

temperature. Twenty hours later, 500 Al DMEMmedium containing

heated 5% FCS (Sigma) was added to the wells. Cell viability was

analyzed 72 h later. Minocycline (100 AM) treatment was started 12

h after transfection and continued for the 2 following days.

Proteolytic activity assay using fluorogenic substrate for caspase-3

and calpain

Fluorescent assays for proteolytic activity of calpain and

caspase were performed according to previously described

methods (Bizat et al., 2003a). Calpain activity was determined

using N-succinyl-Leu-Tyr-(N-succinyl-LY)-AMC, a substrate pref-

erentially cleaved by A/m calpains. Caspase-3 activity was

determined using the substrate N-acetyl-Asp-Glu-Val-Asp-AFC

(DEVD-AFC) (Biomol). Enzyme activity was calculated using

standard curves of AFC or AMC and expressed as pmol AFC-

AMC released per min/mg of protein. It is noteworthy that

presence of minocycline in protein extracts led to apparent

decrease in calpain and caspase-3 activity. This occurred also with

recombinant caspase-3 and purified A-calpain. We found that this

apparent inhibition was due to the fact that minocycline produced

quenching of the fluorescence of AMC and AFC (concentrations

leading to 50% fluorescence quenching, 300 AM for AMC, and 1

mM for AFC). Thus, protease activity values determined in brain

homogenates from minocycline-treated animals could not reliably

be interpreted and were not included in the present results.

Analysis and statistics

Results were expressed as means F standard error. Depending

on the parameter studied, comparisons among groups were made

using Student t test or one-way analysis of variance (ANOVA)

followed by a LSD Fisher or Kruskal-Wallis post hoc tests using

GraphPad Instat and Statistica softwares.

Results

Minocycline is not neuroprotective against 3NP toxicity in vivo

We determined whether minocycline given daily at 10 and 50

mg/kg could protect striatal neurons from degeneration induced by

the chronic delivery of the complex II inhibitor 3NP in rats

(protocol A, Fig. 1). We first evaluated the evolution of motor

symptoms following 3NP treatment in animals receiving or not

minocycline. As shown on Fig. 2A, motor disabilities were not

significantly improved by minocycline. At the time of sacrifice,

that is, 5 days after the onset of intoxication, all animals displayed

gait abnormalities and dystonia. The neurological scores were

similar in all groups. Accordingly, using hematoxylin staining, we

found that the volume of the striatal lesions was similar between

groups despite a trend to a decrease in the 3NP/minocycline 10 mg/

kg group as compared to the 3NP/vehicle group (Fig. 2B). The lack

of significant effect was further assessed using quantitative

measurement of the activity of the complex II enzyme succinate

dehydrogenase (SDH), which active site is selectively inhibited by

3NP. The treatment with 3NP produces a loss of SDH activity in

the striatum that results from both 3NP-induced inactivation of the

enzyme at the catalytic site and degradation of the enzyme

Fig. 2. Minocycline is not protective against chronic intoxication with 3NP in vivo and in vitro. (A) Evolution of neurological score in rats treated with 3NP

(protocol A) with or without minocycline 10 or 50 mg/kg. (B) Histological evaluation of minocycline effect against 3NP-induced striatal lesions. Upper panel

shows typical hematoxylin staining for rats treated with 3NP and injected with vehicle or minocycline. Lower panel shows quantitative measurement of the

striatal lesion volume. (C) Determination of SDH activity in the striatum and the cortex of 3NP-treated rats receiving vehicle or minocycline. Upper panel:

representative SDH histochemistry in sham/vehicle or 3NP/vehicle rats. White lines represent regions of interest used for OD analysis. Lower panel represents

the OD analysis of SDH activity for all groups. ***P b 0.001, ###P b 0.001 using LSD post hoc test versus sham/vehicle rats. (D) Weight loss in sham rats

treated with vehicle, minocycline 10 or 50 mg/kg. Note that at the highest dose of minocycline, rats failed to gain body weight [F(5,45) = 22,58, P b 0,0001 vs.

vehicle]. (E) Effect of various concentrations of minocycline against the deleterious effect of 3NP (100 AM) on striatal cells in vitro. Data were obtained in

quadruplicate and were reproduced in two separate experiments. ***P b 0.001 using LSD post hoc test versus untreated controls. Note that minocycline alone

did not influence cell viability.

K. Bantubungi et al. / Neurobiology of Disease 18 (2005) 206–217210

associated with ongoing degeneration (Brouillet et al., 1998). In the

present study, the level of SDH activity in the cerebral cortex that is

only due to 3NP-induced inactivation of the catalytic site of the

enzyme (Blum et al., 2002a; Brouillet et al., 1998) remained

similar in animals treated or not with minocycline (Fig. 2C),

suggesting that minocycline did not interact directly with 3NP at

the level of SDH. In the striatum, the neurotoxin produced a major

loss of SDH activity consistent with severe ongoing degeneration.

This SDH loss was not modified by minocycline treatment (Fig.

2C), further indicating that minocycline was unable to block

striatal degeneration induced by 3NP. Interestingly, body weight

evaluation of non-3NP-lesioned rats during the 5-day antibiotic

treatment suggested that 50 mg/kg minocycline may be toxic (Fig.

2D): animals treated with 10 mg/kg minocycline gained weight

similarly to control animals, although rats treated with 50 mg/kg

minocycline significantly failed to gain weight.

Fig. 3. Effect of minocycline on staurosporine (STS) and mutated

huntingtin-induced cell death. Viability was determined 24 h after treatment

of HEK293T cells with either STS (1 AM; A; ***P b 0.001 using LSD post

hoc test vs. untreated controls, ###P b 0.001 LSD post hoc test vs. STS-

treated cells) or 72 h following transfection with a plasmid encoding for N-

terminal part of huntingtin carrying 19 or 82 glutamines repetitions (B;

***P b 0.001 using LSD post hoc test vs. controls wells transfected with

the plasmid carrying 19 repetitions). Experiments were performed in

sixplicate for at least four times.

K. Bantubungi et al. / Neurobiology of Disease 18 (2005) 206–217 211

Minocycline is not protective against 3NP in vitro

As shown in Fig. 2E, 3NP significantly decreased neuronal

viability, an effect which was not prevented by 1 to 50 AMminocycline. In line with this, 3NP toxicity was not modified by

minocycline in primary cortical neurons (data not shown). We

previously demonstrated that chronic 3NP intoxication in Lewis

rats and in primary striatal neurons produced calpain-dependent

striatal cell death (Bizat et al., 2003a,b; Galas et al., 2004). In line

with our previous data, we found that on the fourth day of the

3NP treatment, activity of calpain was increased by 2.8-fold as

compared with sham (3NP animals: 91.78 F 4.56; sham animals:

32.25 F 4.67 pmol AMC released/min/mg of proteins; P b

0.0001), although caspase-3 was not activated (not shown). In

vitro, we observed that 48 h after 3NP treatment, calpain activity

was significantly increased by 1.5-fold (3NP-treated neurons:

40 F 1.82; control neurons, 60.22 F 4.12 pmol AMC released/

min/mg of proteins; P b 0.0001). Similarly to in vivo

observations, this effect was not accompanied by caspase-3

activation (not shown). The lack of protection we found with

minocycline against 3NP toxicity in vivo and in vitro thus

suggested that the antibiotic may not be protective against

calpain-dependent degeneration.

However, as previously suggested (Wang et al., 2003; Zhu et

al., 2002), we found that minocycline protected HEK 293T cells

from conditions (staurosporine and expression of an N-terminal

fragment of mutated huntingtin) known to promote caspase-

dependent cell death (Galas et al., 2004; Yu et al., 2003). In the

present studies, we also found that a 6-h staurosporine treatment

led to a significant 1.6-fold increase in caspase-3 activity (control:

30.47 F 0.86; staurosporine: 47.01 F 3.71 pmol AFC released/

min/mg of proteins; P b 0.0016). Calpain activity remained

unchanged during staurosporine treatment (data not shown). In

agreement with this, a 24-h treatment with 1 AM staurosporine

significantly decreased 293T cell viability, an effect dose-depend-

ently inhibited by minocycline (Fig. 3A). At a 100-AMconcentration, the antibiotic produced a complete protection

against staurosporine toxicity. We also found that this optimal

concentration of antibiotic significantly protected 293T cells from

death induced by ectopic expression of an N-terminal fragment of

mutated huntingtin carrying 82 glutamines (Fig. 3B). Similar

findings were observed using doxycycline, another tetracycline

analogue (data not shown).

Effect of minocycline on 3NP-induced striatal inflammation

Previous works have shown that minocycline can promote

neuroprotection through inhibition of inflammation and particu-

larly of microglial activation (Brundula et al., 2002; Tikka et al.,

2002; Tomas-Camardiel et al., 2004; Wu et al., 2002). It was also

recently reported that the antibiotic was able to reduce PGE2

produced by microglial cells (Kim et al., 2004).

We previously reported using MAC-1/CD11b immunohisto-

chemistry that on the fifth day of chronic 3NP treatment, no sign of

striatal inflammation was detected (Blum et al., 2003b). In the

present study, analysis of MAC-1 mRNA expression and PGE2

concentrations, two indices of inflammation, confirmed this

finding: none of these markers increased following 3NP treatment

(Figs. 4A and E), and 3NP treatment rather decreased both

markers, suggesting an inhibitory effect of the toxin on microglial

cells. A similar decrease was observed in rats cotreated with

minocycline (data not shown). In addition, on the fifth day of the

3NP treatment, the increase in GFAP mRNA expression seen

around the lesion and the cortex was not modified by the antibiotic

(Fig. 4B).

Given the lack of microgliosis in our chronic model and the

inhibitory effect exerted by 3NP on microglial cells (Ryu et al.,

2003), we performed an experiment where osmotic minipumps

were removed on the fifth day of 3NP treatment, and animals

were allowed to survive for 5 additional days, thus permitting

striatal inflammation to occur in absence of 3NP. In a pilot

experiment, we previously observed, using CD11b immunohis-

tochemistry, that such a protocol led to microglial activation

within the lesion core (Fig. 4C). We thus performed a trial in

which animals were treated with 10 mg/kg minocycline once

the minipumps were removed (protocol B, Fig. 1). Using such

a protocol, we did not observe a change in the volume of

striatal lesions (22.9 F 4.7 mm3 for the 3NP/vehicle group and

18.8 F 1.9 mm3 for the 3NP/minocycline 10 mg/kg group, ns

using Student t test). However, 5 days after removal of osmotic

pumps, striatal MAC-1 mRNA expression and PGE2 levels

were significantly increased, suggesting inflammation in the

striatum (Figs. 4D and E). In 3NP-treated animals receiving

Fig. 4. Effect of minocycline against inflammation induced by minipump removal following chronic 3NP intoxication. (A) Representative MAC-1 mRNA

expression in the cortex and the striatum of rats chronically treated for 5 days with 3NP (protocol A). Note that 3NP does not induce increase of MAC-1

expression and rather decrease it in the lesion area (star). (B) GFAP mRNA expression in the cortex and the striatum of rats chronically treated for 5 days with

3NP (protocol A). Upper panel: representative in situ hybridization signals. Lower panel: densitometric quantification of the signal within the area of interest,

indicated as a square in the upper panel, in sham or 3NP rats treated with vehicle or minocycline (**P b 0.01 using LSD post hoc test vs. sham/vehicle rats). (C)

CD11b/MAC-1 immunoreactivity in the lateral striatum of a sham rat (left) or a rat submitted to a 5-day 3NP intoxication in which the minipump has been

removed for additional 5 days (pilot experiment; protocol B). Note the increased number and size of positive microglial cells. (D) Quantification of MAC-1

mRNA expression in the striatum of rats chronically treated for 5 days with 3NP in which minipumps have been removed for additional 5 days and then injected

with vehicle or 10 mg/kg minocycline (protocol 1B). *P b 0.05 using LSD post hoc test versus sham/vehicle rats. Note that in the presence of minocycline,

MAC-1 mRNA expression decreased and remained nonsignificantly different from the controls. (E) Quantification of PGE2 levels in the striatum of rats

submitted to protocols A and B. ***P b 0.001 using LSD post hoc test versus sham/vehicle rats, #P b 0.05 using LSD post hoc test versus 3NP/vehicle rats.

K. Bantubungi et al. / Neurobiology of Disease 18 (2005) 206–217212

minocycline, striatal MAC-1 mRNA expression was similar to

control rats, suggesting an inhibitory effect of the antibiotic on

the microglial activation (Fig. 4D). In line with this, minocy-

cline significantly reduced striatal PGE2 levels in the 3NP/

minocycline group as compared to 3NP/vehicle animals (Fig.

4E). Conversely, minocycline did not reduce astrogliosis as

K. Bantubungi et al. / Neurobiology of Disease 18 (2005) 206–217 213

assessed by GFAP mRNA in situ hybridization (data not

shown).

Altogether, our results suggested that minocycline could not

counteract calpain-dependent degeneration but reduced the

development of inflammation in a lesioned striatum. To

Fig. 5. Minocycline is protective against QA-induced striatal lesions in vivo. (A) E

kg minocycline or doxycycline (protocol C). **P b 0.001 using LSD post hoc t

striatum in QA rats treated with 10 mg/kg minocycline or doxycycline. **P b 0.001

treated rats treated with vehicle, minocycline or doxycycline. (D) MAC-1 mR

minocycline or doxycycline. Upper panel: representative in situ hybridization sign

0.001 using LSD post hoc test versus QA/vehicle rats. (E) GFAP mRNA express

Lower panel: quantification of signal shown on the upper panel.

determine whether this property could be associated with

neuroprotection, we tested minocycline in the QA model of

HD in which inflammation is known to occur and contribute to

the development of the lesion (Dihne et al., 2001; Topper et al.,

1993).

valuation of the size of the striatal lesion core of QA rats treated with 10 mg/

est versus QA/vehicle rats. (B) Evaluation of the percentage of the spared

using Kruskal-Wallis test versus QA/vehicle rats (C) Weight curves in QA-

NA expression in the striatum of QA-injected rats treated with vehicle,

als. Lower panel: quantification of signal shown on the upper panel. **P b

ion in the striatum of QA-injected rats treated with vehicle or minocycline.

Fig. 6. Minocycline does not affect the basal and QA-evoked striatal

glutamate release. Evaluation of the influence of minocycline on the basal

and QA-evoked glutamate outflow in the striatum of rats chronically treated

for 3 (A) or 7 (B) days with vehicle or 10 mg/kg minocycline. Each

experimental group was made of three animals. The bar indicates the period

of QA perfusion through the probe.

K. Bantubungi et al. / Neurobiology of Disease 18 (2005) 206–217214

Minocycline is protective against QA-induced striatal lesions in

vivo

Animals unilaterally injected with QAwere treated with 10 mg/

kg minocycline for 7 days (protocol C, Fig. 1) and the size of the

striatal lesion core was then measured using SDH histochemistry.

We found that the volume of the lesion core was significantly

reduced in minocycline-treated animals (Fig. 5A). The percentage

of the spared striatum was then significantly increased in the

minocycline-treated animals (Fig. 5B). Noteworthy, at the dose

tested, minocycline did not alter body weight gain (Fig. 5C),

although higher dose of the antibiotic (50 mg/kg) prevented weight

gain of QA-treated animals and impaired the protective activity

found with the 10 mg/kg dose (not shown). Microglial activation

was assessed using MAC-1 mRNA in situ hybridization at the end

of the experiment. As compared to the unlesioned side, we found

that striatal MAC-1 mRNA expression was strongly increased in

animals treated by quinolinic acid (Fig. 5D). The MAC-1 mRNA

level was significantly decreased in the lesioned striatum of

minocycline-treated animals compared with the lesion striatum of

vehicle-treated rats. In contrast, minocycline did not exert an effect

on the increase of GFAP expression induced by QA (Fig. 5E). The

present results thus suggested that minocycline is beneficial in a

phenotypic model of HD in which striatal lesion involves

inflammatory processes. Noteworthy, the beneficial effects of

minocycline were not observed in animals injected with a similar

and nontoxic dose of doxycycline, another tetracycline analogue

(Figs. 5A–D).

Of interest, it was previously found that glutamate or glutamate

receptor agonists can induce microglial activation (Tikka and

Koistinaho, 2001). Given that QA neurotoxicity is mediated at least

in part by an increased glutamate release (Blum et al., 2003b; Popoli

et al., 2002), we determined whether the protective and anti-

inflammatory activity of minocycline could be related to a reduction

of evoked glutamate outflow. Using microdialysis, we found that

basal and QA-induced glutamate release remained unchanged in

animals chronically treated with minocycline at a protective dose

(Fig. 6). This thus suggested that the observed protective effect of

the antibiotic was not related to a presynaptic effect but rather to the

inhibition of events downstream glutamate release.

Discussion

In the present study, we assessed the effects of minocycline in

phenotypic models of HD. Our results showed that minocycline is

not beneficial against calpain-dependent striatal degeneration but is

able to counteract the development of striatal inflammation.

In a first attempt, we found that minocycline was not protective

against chronic 3NP intoxication both in vitro and in vivo. This

absence of protection was unlikely related to a lack of minocycline

activity which could arise from an insufficient dosage or to a

chemical inactivation of the molecule since minocycline was

prepared fresh before each injection, and the molecule displayed

anti-inflammatory and protective activities in the other models we

used. We previously showed that in both models, striatal cell death

was mediated through calpain but not caspase activation (Bizat et

al., 2003a,b; Galas et al., 2004), reproducing an important feature of

the human pathology (Gafni and Ellerby, 2002; Gafni et al., 2004).

Nonetheless, in line with recent studies (Scarabelli et al., 2004;

Wang et al., 2003; Zhu et al., 2002), we found that minocycline

protects HEK 293T cells against experimental conditions known to

promote caspase induction. These results indicate that minocycline

would not be protective against calpain-dependent degeneration. At

present, it is not clear which proteases are directly responsible for

neuronal degeneration in HD. Although caspases participate to the

pathological development in HD through, particularly, the cleavage

of mutated full-length huntingtin into shorter fragments (Goldberg

et al., 1996; Hermel et al., 2004; Kiechle et al., 2002; Ona et al.,

1999; Wellington et al., 2003), their role may not be directly related

to the death of striatal neurons. Indeed, it has been recently shown

that R6/2 mice displayed many features of mitochondrial-depend-

ent and independent apoptosis (Wang et al., 2003; Zhang et al.,

2003), despite lacking massive neuronal cell loss (Turmaine et al.,

2000) in contrast to what occurs in HD patients (Vonsattel et al.,

1985). Alternatively, calpains may play an important role as

suggested by recent studies performed on postmortem HD brains

and animal models of the disorder (Bizat et al., 2003a,b; Gafni and

Ellerby, 2002; Gafni et al., 2004). Then, benefits afforded by

minocycline in HD patients might depend on the cell death

processes involved. Furthermore, given that striatal and cortical

degeneration may involve differential mechanisms (Galas et al.,

2004), minocycline efficiency may be anatomically determined.

Using a paradigm of 3NP intoxication allowing the development

of a secondary microgliosis, we found that minocycline reduced

striatal inflammation. This finding was further confirmed using the

K. Bantubungi et al. / Neurobiology of Disease 18 (2005) 206–217 215

quinolinic acid model in which inflammation is concomitant to

degeneration (Dihne et al., 2001; Topper et al., 1993). In this model,

we found that minocycline reduced not only the MAC-1 mRNA

expression, but also the size of the lesion core produced by the

NMDA agonist. Conversely, minocycline did not act on the

increased GFAP mRNA expression associated with the lesion,

consistent with previous works demonstrating that minocycline does

not modify astrogliosis (Diguet et al., 2004a; Kriz et al., 2002;

Yrjanheikki et al., 1998, 1999). It remains also possible that the

neuroprotective activity ofminocycline against QA involves caspase

inhibition despite that its contribution has been poorly characterized

in this model (Nakai et al., 2000; Qin et al., 2000). However, our

results are in accordance with the known anti-inflammatory activity

of minocycline found in other models of degenerative diseases and

suggested in the R6/2 HD model (Amin et al., 1996; Chen et al.,

2000; Dommergues et al., 2003; He et al., 2001; Kriz et al., 2002;

Tikka and Koistinaho, 2001; Tomas-Camardiel et al., 2004). Given

that the presence of microglial activation in HD brains evenly

correlated with the neuropathological grade (McGeer et al., 1988;

Sapp et al., 2001), the ability of minocycline to inhibit striatal

inflammation could lead to clinical benefit in HD patients.

Previous observations reported that high doses of both

minocycline and doxycycline provided protection against cerebral

ischemia (Yrjanheikki et al., 1998). However, in vivo, we found that

at low doses (10 mg/kg/d), only minocycline displayed protective

potential, although in vitro, doxycycline displayed a similar

protective activity than minocycline. This difference may be due

to the lowest ability of doxycycline to cross the blood–brain barrier

as compared to minocycline (Smith et al., 2003). This suggests that,

clinically, minocycline may be more appropriated than doxycycline.

It is also important to note that, as reviewed by Diguet et al.

(2004b) and Blum et al. (2004), positive effects of minocycline

formerly found in models of Parkinson disease, HD, or ischemia

(see Blum et al., 2004; for review) have been recently questioned

regardless the mode of administration or the species involved

(Diguet et al., 2004a; Smith et al., 2003; Tsuji et al., 2004; Yang et

al., 2003). Additionally, repeated i.p. delivery of minocycline at

doses N45 mg/kg/d may even lead to unintended morbidity (Fagan

et al., 2004). In accordance, we found that high doses of

minocycline (or doxycycline, data not shown) impaired body

weight gain in rats as previously reported (Smith et al., 2003; Yang

et al., 2003). Together, this shows that minocycline may have

variable and contradictory effects and that neuroprotective mech-

anisms remain to be further clarified. Although it remains difficult to

extrapolate to humans observations made in models, these

controversies should encourage a careful evaluation of potential

minocycline toxicity at the peripheral (liver, kidney. . .) or immuno-

logical levels (Gottlieb, 1997; Knowles et al., 1996) in patients

enrolled in clinical trials (Blum et al., 2004; Diguet et al., 2004b).

In conclusion, our data suggest that minocycline, at low dose,

may be beneficial to HD patients but that its clinical efficiency will

depend on the relative contribution of caspase- and calpain-

dependent mechanisms and of inflammation in the development of

the pathology and neurodegeneration.

Acknowledgments

This work was supported by fundings from the Hereditary

Disease Foundation (DB), FNRS (DB/LT/JB, SNS), Region-

Bruxelles Capitale (JB/LT), Fondation Medicale Reine Elisabeth

(SNS), Action de Recherche Concertee (SNS), and Fondation

Universitaire David and Alice Van Buuren (DB/JB, SNS). KB and

AC hold grants from Televie. DB is bCharge de RecherchesQ of theFNRS.

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