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NeuroMolecular Medicine Copyright © 2007 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696/07/09:47–54/$30.00 (Online) 1559-1174 doi: 10.1385/NMM:9:1:47

ORIGINAL ARTICLE

Lack of Minocycline Efficiency in Genetic Models of Huntington’s Disease Stéphane Mievis,1 Marc Levivier,2,3 David Communi,1 Gilbert Vassart,1 Jacques Brotchi,2,3 Catherine Ledent,†,1 David Blum*,†,1,3,4,5 1

IRIBHM; 2Department of Neurosurgery; 3Laboratory of Experimental Neurosurgery ULB-Erasme, Brussels, Belgium; 4INSERM U815, IFR114, Lille, France; and 5Université Lille2, Faculté de Médicine, Lille, France Received December 29, 2005; Revised April 10, 2006; Accepted May 1, 2006

Abstract According to the recent controversy regarding the effects of minocycline in the R6/2 transgenic model of Huntington’s disease (HD), this tetracycline has been re-evaluated in another model, the N171-82Q strain. Ten miligrams per kilogram minocycline was given daily from the age of 2 mo, corresponding to an early symptomatic stage. We did not observe improvement in survival, weight loss, or motor function in treated transgenic mice. In addition, minocycline failed to mitigate the ventricle enlargement as well as the striatal and cortical atrophies induced by the transgene. Using high-performance liquid chromatography, it was observed that minocycline was similarly present in the plasma and the brain of both wild-type and N171-82Q mice following 14 daily injections. Using Western blot, we show that the increased expression of procaspase-1 induced by the transgene in the cortex was significantly reduced by the antibiotic. Combining together these data support that despite minocycline crosses blood–brain barrier in N171-82Q mice and displays an expected effect on procaspase-1 expression, it does not provide protection in this HD model. These in vivo results are in accordance with in vitro data, since minocycline failed to protect against mutated Huntingtin in an inducible PC12-clone expressing exon1 of mutated Huntingtin103Q. Altogether, the present data does not support minocycline as a beneficial drug for HD. doi: 10.1385/NMM:9:1:47 Index Entries: Huntington’s disease; minocycline; N171-82Q; transgenic mice.

Introduction

in various models of neurological diseases (Blum et al., 2004). Daily freshly prepared minocycline solution was administrated intraperitoneally (ip) at the doses of 5–10 mg/kg which was initially shown

Minocycline is an antibiotic of the tetracycline family that has displayed neuroprotective properties

*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]. †Are joint senior authors.

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48 to improve the survival and motor alterations and mitigate apoptosis-related events in the widely used R6/2 mouse model of Huntington’s disease (HD; Chen et al., 2000; Wang et al., 2003). Nonetheless, these former encouraging results could not be replicated in the same transgenic strain, following a continuous oral administration of the compound (Smith et al., 2003). Minocycline was also found ineffective (Cornet et al., 2004; Bantubungi et al., 2005) and even deleterious (Diguet et al., 2003; Diguet et al., 2004) in other nontransgenic models of HD which reliably replicates the complex II defect seen in the disease (Beal, 2005). Tolerability and safety trials have been recently realized on HD patients based on the former positive preclinical results obtained on R6/2 mice (Bonelli et al., 2003; Huntington Study Group, 2004; Thomas et al., 2004). Besides good tolerability of the compound, shortterm (2–6 mo) evaluations revealed that minocycline produced either a trend toward motor and neuropsychological improvement (Bonelli et al., 2003), no change (Thomas et al., 2004), or even slight cognitive deterioration (Huntington Study Group, 2004). Thus, from the current debate about minocycline efficacy (Hersch et al., 2003; Hockly et al., 2003; Hodl et al., 2005), it remains difficult to draw a firm conclusion about the neuroprotective effect of this molecule in HD. In this context, in order to further address the rationale of minocycline treatment in HD, the potentiality of this compound in another transgenic mouse model, the N171-82Q strain have been re-evaluated. This strain is generally considered as confirmatory for the R6/2 strain. Minocycline effects against toxicity of the N-terminal part of mutated Huntingtin were also checked in a cellular model that was previously used for drug screening assays (Aiken et al., 2004).

Material and Methods Animals Transgenic N171-82Q mice were obtained from Jackson Laboratories (Jaxmice, USA) and maintained on a B6C3F1 background as described in Schilling et al., (1999). The offspring was genotyped using a polymerase chain reaction (PCR) assay on tail DNA. Mice were housed 10/cage with free access to food and water, under standard


Mievis et al. conditions with a 12-h light/dark cycle. Experiments were carried out in accordance with Enzyme Commission Regulations for animals use in research (CEE no. 86/609).

Survival and Clinical Assessments To evaluate the effects of minocycline, N171-82Q mice (HD) received either ip injections of the antibiotic (n = 38 including 20 males and 18 females; 10 mg/kg/d freshly prepared in 0.9% NaCl at 1 mg/ mL; Sigma, Belgium) or vehicle (n = 36 including 21 males and 15 females; 0.9% NaCl) every day from 8 wk of age. Littermate controls (WT) were treated in a similar way (n = 40 per groups). The modalities of minocycline treatment (dose, injection way) were chosen according to the previous beneficial effects found in the R6/2 strain following ip and not oral administration (Chen et al., 2000; Smith et al., 2003; Wang et al., 2003). Survival, bodyweight and motor performances were monitored throughout the study. Death of the animal was checked daily. Weight was measured weekly. Evaluation of motor coordination was performed using a rotarod apparatus (Ugobasile, Italy) maintained at 28 rpm. To acclimate the mice training sessions were realized the week before treatment onset. Animals were tested by three successive trials of 60s and quoted on 180s. Locomotor activity was assessed at constant light (20 lux) by testing the animals in 40 × 40 cm2 open-field chambers in a quiet room separated from the colony area. Activity was measured as the total distanced moved over a 5-min period by an automatic video tracking system (Noldus, Netherlands). Motor parameters were determined at 8 wk of age to establish the baseline performances (Table 1) and again at 12, 16, and 20 wk of age.

Histological and Biochemical Evaluations Additional N171-82Q or littermate wild-type mice were submitted to a similar treatment and sacrificed at 16.5 wk of age (WT, n = 6; WT + minocycline, n = 5; HD, n = 10; HD + minocycline, n = 6). All animals were killed by decapitation. Brains were quickly removed and frozen in 2-methylbutane, which was cooled by dry ice at –80°C. Right hemisphere of each animal was used to measure morphological changes and left hemispheres for biochemistry.

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Table 1 Basal Characteristics of Each Group at the Time of Treatment Onset Group WT WT+Mino HD HD+Mino

Age (d) 56.6 ± 0.7 56.6 ± 0.9 56.3 ± 0.7 55.1 ± 0.8

Weight (g) 22.1 ± 0.4 21.6 ± 0.5 20.0 ± 0.5b 20.0 ± 0.5b

Latency (s) 163.6 ± 4.5 157.9 ± 5.8 163 ± 4.4 159 ± 6.7

Distance (cm) 1111 ± 132 1111 ± 133 797 ± 72a 841 ± 67a

Both groups of N171-82Q (HD) mice were matched for basal parameters. ap < 0.05. bp < 0.01 vs respective wild-type (WT) littermates control using LSD fisher posthoc test.

For histological evaluations, half right brains were cut at a thickness of 20 µm using a cryostat (Leitz, Germany) and serial coronal sections were mounted onto gelatine-coated slides and stored at –20°C until use. The surface of ventricle and striatum as well as the thickness of the cerebral cortex were determined following succinate dehydrogenase histochemistry as previously described in Bantubungi et al. (2005). Surfaces of the lateral ventricle and striata were measured for every 300 µm on five coronal planes along the antero-posterior axis of the striatum (bregma +1.2 to 0 mm). The thickness of the cerebral cortex was calculated as the averaged value for these five coronal planes. For Western blotting experiments, striata and surrounding cortices were dissected out from three frozen left hemispheres per group and homogenized in lysis buffer (M-PER, Pierce, UK), containing a protease inhibitor mixture (Complete, Roche Molecular Biochemicals, Belgium). Protein concentrations were determined using MicroBCA protein assay (Pierce, UK). Equal amounts of proteins (20 µg) were denaturated in 5X Laemmli buffer at 100°C for 5 min and then separatedon 12% sodium dodecyl sulfate (SDS)polyacrylamide gels and transferred to nitrocellulose. Membranes were blocked at 30 min with 5% milk in Tris-buffer saline (TBS) buffer and then incubated overnight at 4°C with polyclonal anticaspase-1 (sc-514; Santa Cruz, USA) diluted in TBS at 1/200 containing 0.1% Tween-20 and 5% milk powder. After washing in 0.1% Tween-20/TBS buffer, membranes were blocked again and then incubated with an antirabbit horseradish peroxidase (HRP)-labeled antiboby (enhanced chemiluminescence [ECL] Antirabbit-HRP, 1/2000 in 0.1% Tween-20/TBS; Amersham, Belgium) for 2 h at room temperature.


Immunoreactive bandswere visualized using chemiluminescent reagent (Western Lightning, Perkin Elmer, Belgium). Brain sections and western blots were digitalized using a CCD video camera (DageMTI, Indiana, USA) and analyzed using the public domain ImageJ software (NIH, USA).

High-Performance Liquid Chromatography Experiments For the detection of minocycline levels within the plasma and brains we use WT and HD animals daily treated with 10 mg/kg ip minocycline (WT, n = 9; HD, n = 8) or with saline (WT, n = 10; HD, n = 10) for 14 d. Minocycline was assayed using a reversephase high-performance liquid chromatography (HPLC) method based on the protocol used by Smith et al. (2003). After 1 h, following the last ip injection, blood was collected into tubes containing EDTA (Sarstedt, Nümbrecht). Animals were killed by decapitation and the brains were removed quickly and frozen immediately in liquid nitrogen. Plasma was prepared by centrifugating blood samples at 3000g for 5 min. It was then immediately frozen in liquid nitrogen and stored at –80°C until it is required. Brains were homogenized in 10 volumes of homogenization buffer (2 M urea, 50 mM Tris-HCl, 17 mM NaCl, 2.68 mM EDTA, 0.1% Brij35 [Sigma], final pH 7.6), using porcelain homogenizers and centrifuged at 4°C for 1 h at 10,000g. Supernatants were stored at −80°C until it is required for HPLC. Samples (plasma and supernatants from homogenates) were denaturated in 1% v/v phosphoric acid, centrifuged and the supernatants were loaded onto a 1-mL OASIS HLB cartridges (Waters, UK) preconditioned with 1-mL methanol, followed by

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Fig. 1. Analysis of minocycline effects on N171-82Q HD mice survival, body weight, and motor performances. HD and WT mice were treated from 8 wk of age by daily intraperitoneal (ip) injections of 10 mg/kg minocycline. (A) Kaplan-Meier analysis of survival. (B) Effect of ip minocycline treatment on body weight of HD and WT littermate mice. (C–D) Effects of ip minocycline treatment on rotarod performances (C) and locomotion (D). ,,p < 0.05; ,,p < 0.001 vs respective WT control using LSD Fisher posthoc test. ∇, WT-NaCl; , WT-Minocycline; , HD-NaCl; , HD-Minocycline.

1-mL 5% methanol in water. Absorbent was washed with three volumes of 5% methanol in water and sample eluted with 1-mL 70% acetonitrile in water. Reverse-phase HPLC was performed using a Waters 600 solvent delivery module and Waters 490E UV detector (Milford, MA). Samples were separated on a Hypersil H5ODS column (15 cm, 4.6 mm; Hichrom) by isocratic elution with 1 to 1 water to acetonitrile, 1.5% Trifluoroacetic acid, at flow rate of 1 mL/min, with absorbance detector at 350 nm. Spike samples of all tissue with known concentrations of minocycline (added before purifica-


tion on OASIS) were run as standards. No peak was resolved in saline-injected animals.

In Vitro Assays PC12 cell-clone expressing exon1 of mutated Huntingtin (PC12-Ecd-Htt103Q) fused to green fluorescent protein (GFP) under the control of an ecdysoneinducible promoter was used to determine the effect of minocycline in the in vitro condition (Aiken et al., 2004; generous gift from Erik Schweitzer, UCLA, USA). Cells were maintained in Dubelcco’s Modified

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Eagle’s Medium (DMEM) containing 25 mM 4-(2Hydroxyethyl)piperazine-1-ethanesulfonic acid N-(2-Hydroxyethyl)piperazine-Nα(2-ethanesulfonic acid) (HEPES) (Gibco, Belgium), 10% fetal calf serum (Gibco), 5% horse serum (Gibco), penicillin, and streptomycin at 37°C, 5% CO2. Stocks were maintained in the same medium with 0.5 mg/mL G418 (Invitrogen, Belgium). Gene induction was realized by adding 1 µM Tebufenozide (Tebu; gift from Fred Gage, Salk Institute) according to previous description (Aiken et al., 2004). Minocycline was added at the time of gene induction at various concentrations. Percentage of aggregates was assessed under a fluorescence microscope. Cell viability was measured through lactate dehydrogenase (LDH) release within the culture medium and the metabolic activity of the cells was determined by using MTS (3-[4,5-dimethylthiazol-2yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]2H-tetrazolium) assay as previously described (Aiken et al., 2004; Bantubungi et al., 2005).

Data Analysis Data were expressed as the means ± SEMs. Data were compared using the ANOVA followed by LSD Fisher posthoc test or, when appropriate, using unpaired Student’s t-test. Survival data were analyzed using Kaplan-Meier survival curves and the Mantel-Cox log-rank test.

Results and Discussion At the time of treatment onset, N171-82Q HD mice were matched for age, weight, rotarod score, and open-field score (Table 1). Although motor coordination was not already affected, HD mice had a lower spontaneous locomotion as well as a slightly lower body weight than littermate controls. Thus, when minocycline treatment was initiated, animals were in a very early symptomatic stage. As shown in Fig. 1A, minocycline did not improve survival of transgenic mice. The averaged survival of vehicle- and minocycline-treated HD animals was, 119.6 ± 2.7 and 121.2 ± 2.9 d, respectively. As shown in Fig. 1B, minocycline did not improve the failure to gain weight observed in vehicle-treated HD mice and also did not affect the gain of bodyweight of littermate WT controls. Moreover, minocycline did not mitigate the time-dependent decrease of rotarod and


Fig. 2. Morphological alterations in HD and WT animals treated daily by ip injections of either NaCl or 10 mg/kg minocycline. Animals treated with minocycline were killed at 16.5 wk of age. The ventricle enlargement (A) and the striatal atrophy (B) were estimated every 300 µM along the rostro-caudal axis of the striatum from planes +1.2–0 mm from bregma. ∇, WT-NaCl; , WT-Minocycline; , HD-NaCl; , HD-Minocycline. Averaged cortical thickness (C) was calculated from these planes (open bars: NaCl, black bars: minocycline). ,p < 0.05; p < 0.01; ,p < 0.001 vs WT controls using LSD Fisher Post hoc test.

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Fig. 3. Procaspase-1 expression in the cortex (A) and the striatum (B) of HD and WT animals treated daily by ip injections of either NaCl or 10 mg/kg minocycline and sacrificed at 16.5 wk. Open bars: NaCl, black bars: minocycline p < 0.05 vs WT controls using LSD Fisher posthoc test. Note that for quantifications, WT value was set at 1.

open-field scores (Fig. 1C,D). Analyzing data independently for male and female subgroups demonstrated that lack of minocycline beneficial effects was similar in both the sexes (data not shown). A similar experiment was performed to monitor whether minocycline affects histological changes in N171-82Q mice at 16.5 wk of age. Ventricle and striata surface along the rostro-caudal axis of the striatum as well as the averaged cortical thickness were monitored. As shown in Fig. 2, we observed that ventricle enlargement (2A), striatal atrophy (2B) as well as a decrease in cortical thickness (2C) of HD mice were not mitigated by the minocycline treatment. The presence of minocycline in plasma and brain of wild-type and transgenic animals following 14 d of daily injection with the antibiotic was confirmed by HPLC determinations on samples processed 1 h after the last injection (plasmatic concentration: WT, 2.26 ± 0.27 µM; HD, 2.39 ± 0.24 µM; brain concentration: WT, 0.65 ± 0.02 µM; HD, 0.86 ± 0.16 µM). Interestingly, such amount of minocycline within brain was able to reduce induced procaspase-1 expression, in accordance with previous reports (Chen et al., 2000; Du et al., 2001). Indeed, it was found that procaspase-1 expression was significantly increased in the cortex, but not in the striatum, of HD


Mievis et al. mice as compared with WT, an effect mitigated in animals treated with minocycline (Fig. 3). Altogether, these data support that despite active levels of minocycline are present in the brain of N171-82Q mice, the antibiotic fails to promote any neuroprotective effect in this strain. Such lack of minocycline is in accordance with in vitro assays. As expected (Aiken et al., 2004), the expression of mutated Huntingtin103Q in the PC12-clone significantly increased LDH release within the culture medium 24 and 48 h following the gene induction with Tebufenozide (+Tebu; Fig. 4A). Increasing concentrations of minocycline applied at the time of gene induction did not attenuate cell death at any times. To rule out the possibility that the lack of beneficial effect was seen at 48 h postinduction could be related to the instability of the molecule, the cells were treated twice with minocycline (at time of gene induction and 24 h later). Similar results were found (not shown). As measured using MTS assay, transgene expression also affected the metabolic activity of the cells (Fig. 4B). At the highest tested concentration, minocycline increased cell metabolism both in the induced and noninduced cells supporting a possible mitochondrial target. Finally, minocycline did not prevent the formation of aggregates 24 and 48 h following transgene induction (Fig. 4C). The present observations are in line with the work from Smith et al. (2003) supporting the lack of minocycline beneficial effects in the R6/2 strain but are in marked contrast with the initial positive findings obtained in the same model (Chen et al., 2000). Such inability of minocycline to counteract toxic effects of mutated Huntingtin in our experiments were not related to a lack of active compound within the brain of treated animals. Indeed, HPLC measurements demonstrate that 10 mg/kg ip administration in N171-82Q mice leads to plasma and brain concentrations very similar to the result obtained following 5 mg/kg ip injections in R6/2 mice (Smith et al., 2003), a condition previously shown to be neuroprotective in this strain (Chen et al., 2000; Stack et al., 2006). In addition, as expected (Chen et al., 2000; Du et al., 2001) minocycline can reduce induced procaspase-1 expression. In line with survival and motor observations minocycline had no effect against mutated Huntingtin toxicity in vitro. It is noteworthy to observe that the lack of minocycline effects on cell viability was measured by LDH release and was not related with the ability of the tetracycline to increase

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Fig. 4. Effect of minocycline in vitro. Cells were incubated in the presence (gray bars) or absence (black bars) of the Huntingtin103Q inducer tebufenozide (Tebu) and minocycline 24 h after plating. After 24 (upper panel) or 48 h (lower panel), cell viability was determined (A; LDH assay), metabolic activity of the cells (B; MTS assay) or counted the percentage of cells presenting aggregates (C). p < 0.05, p < 0.001 vs respective noninduced (–Tebu) control using LSD Fisher posthoc test. , p < 0.001 vs respective untreated (w/o Minocycline) control using LSD Fisher posthoc test.

metabolic activity of the cells. Thus, it remains possible that previous experiments, including ours, using metabolic assays as an index of cell survival and supporting a beneficial effect of minocycline against mutated Huntingtin in vitro may have been misinterpreted (Wang et al., 2003; Bantubungi et al., 2005). Finally, the lack of minocycline effect on aggregation observed in the inducible PC12 clonel is in accordance with previous similar experiments (Apostol et al., 2003). This is consistent with observations made on R6/2 mice, even when the antibiotic displays neuroprotective properties (Chen et al., 2000; Smith et al., 2003; Stack et al., 2006). Despite a very similar treatment paradigm and similar brain concentrations, the reasons for the


different clinical outcomes observed between N17182Q and R6/2 mice are unclear. Nevertheless, a recent comparison between these two models have been established that the respective N-terminal parts of mutated Huntingtin overexpressed in these strains mediate different forms of neurodegeneration dependent on the protein fragments used (Yu et al., 2003). Particularly, the magnitude of DNA fragmentation, caspase-3 activation, and mitochondrial degeneration was obviously different between the two models. As supported from studies using phenotypic models (Bantubungi et al., 2005), the relative contributions of different cell-death events produced by mutated Huntingtin expression may be an important feature that substantially influences the clinical outcome of

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54 minocycline treatment. Thus, the present report supports the view that replication of preclinical trials in other transgenic—or even phenotypic—models than the popular R6/2 strain is a crucial prerequisite to establish the rationale before translating preclinical trials to patients (Hersch and Ferrante, 2004). This is important seeing that it remains finally unclear to what extent compounds that displayed positive effects in R6/2 mice can benefit patients.

Acknowledgments The present work was supported by Hereditary Disease Foundation (USA), FNRS (Belgium), Région Bruxelloise (Belgium) and Van Buuren Foundation (Belgium). SM is a FRIA recipient. CL and DC are “Chercheur Qualifié,” of the FNRS. DB, formerly “Chargé de Recherches” of the FNRS, is an INSERM investigator. We thank Dr. Raphal Hourez for the critical reading of the manuscript and Laura Nebreda for technical assistance.

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