Death of cortical and striatal neurons induced by mitochondrial defect involves differential molecular mechanisms

www.elsevier.com/locate/ynbdi Neurobiology of Disease 15 (2004) 152 – 159

Death of cortical and striatal neurons induced by mitochondrial defect involves differential molecular mechanisms Marie-Christine Galas, a Nicolas Bizat, b Laetitia Cuvelier, a Kadiombo Bantubungi, a Emmanuel Brouillet, b Serge N. Schiffmann, a and David Blum a,* a b

Laboratoire de Neurophysiologie, ULB-Erasme, 1070 Brussels, Belgium URACEA-CNRS 2210, Service Hospitalier Fre´de´ric Joliot, DRM, CEA, 91406 Orsay Cedex, France

Received 25 July 2003; revised 24 September 2003; accepted 26 September 2003

An important aspect of Huntington’s disease (HD) pathogenesis which may have important therapeutic implications is that the cellular events leading to cell death may be different in cortical and striatal neurons. In the present study, we characterized cellular changes in cortical and striatal neurons treated with the mitochondrial toxin 3nitropropionic acid (3NP) in culture. Degeneration induced by 3NP was similar in both striatal and cortical neurons as observed using markers of cell viability and DNA fragmentation. However, in striatal neurons, 3NP produced a marked delocalization of Bad, Bax, cytochrome c and Smac while this was not observed in cortical neurons. Death of striatal neurons was preceded by activation of calpain and was blocked by calpain inhibitor I. In cortical neurons, calpain was not activated and calpain inhibitor I was without effect. In both cell types, caspase-9 and -3 were not activated by 3NP and the caspase inhibitor zVAD-fmk did not provide neuroprotective effect. Interestingly, treatment with staurosporine (STS) triggered caspase-9 and -3 in cortical and striatal cells, suggesting that the molecular machinery related to caspase-dependent apoptosis was functional in both cell types even though this machinery was not involved in 3NP toxicity. The present results clearly demonstrate that under mitochondrial inhibition, striatal and cortical neurons die through different pathways. This suggests that mitochondrial defects in HD may trigger the death of cortical and striatal neurons through different molecular events. D 2003 Elsevier Inc. All rights reserved. Keywords: Cell death; Huntington’s disease; 3-Nitropropionic acid; Apoptosis; Cortex; Striatum

Introduction Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by an abnormal expansion of a CAG repeat within the IT15 gene leading to an expanded polyglutamine stretch in the

* Corresponding author. Laboratoire de Neurophysiologie, ULBErasme, CP601, 808 route de Lennik, 1070 Brussels, Belgium. Fax: +322-555-41-21. E-mail address: [email protected] (D. Blum). Available online on ScienceDirect (www.sciencedirect.com.) 0969-9961/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2003.09.013

N-terminal part of the huntingtin protein (The Huntington’s disease research group, 1993). This mutation results in a preferential loss of the striatal GABAergic medium-sized spiny neurons (Brouillet et al., 1999; Vonsattel et al., 1985) which contrasts with the ubiquitous pattern of expression of huntingtin. However, other cerebral regions are also affected in HD patients, especially the cerebral cortex (Albin, 1995). The cause of neurodegeneration in HD is probably multifactorial (Cattaneo et al., 2001). A number of ‘‘initiator’’ mechanisms have been suggested including abnormal interactions between mutated huntingtin and neuronal proteins (Cattaneo et al., 2001; Gervais et al., 2002), transcriptional dysregulations (Cha, 2000; Zuccato et al., 2001), mitochondrial defects (Beal, 2000; Panov et al., 2002; Sawa et al., 1999) and indirect activation of the excitotoxic cascade leading to apoptosis (Ona et al., 1999; Sanchez et al., 1999; Saudou et al., 1998) or necrosis (Brouillet et al., 1999). Supporting the hypothesis that mitochondrial defects could play a role in HD pathogenesis (Beal, 2000; Blum et al., 2003b), intoxication with the succinate dehydrogenase (SDH) inhibitor 3-nitropropionic acid (3NP) in rats and nonhuman primates produces abnormal movements, cognitive deficits and striatal degeneration (Beal et al., 1993; Blum et al., 2001, 2002a,b, 2003a; Brouillet et al., 1995; El Massioui et al., 2001). Despite the preferential vulnerability of the striatum to 3NP toxicity, cell loss in certain areas of the cerebral cortex anatomically connected to the striatum has also been observed several weeks after onset of striatal degeneration (Mittoux et al., 2000, 2002). The exact mechanisms underlying striatal and cortical neurodegeneration in HD and 3NP animal models remain unknown. It is generally suggested that in both cases, the execution phase resulting in striatal and cortical neurodegeneration may involve cellular machineries that participate in apoptosis, necrosis and autophagy. However, a crucial aspect of HD pathogenesis, and possibly 3NP-induced degeneration, is that the death of striatal neurons may involve selective cascades of molecular events that may differ markedly from the cascade underlying degeneration of cortical neurons. This hypothesis has major consequences for the development of new therapies aiming to slow down neurodegeneration in HD.

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To study this important aspect of HD pathogenesis, using primary cultures, we examined whether, in presence of 3NP, the mechanisms underlying the degeneration of striatal neurons were similar to those underlying death of cortical neurons. Results show clear cut differences in the molecular machineries involved in 3NP-induced death of each cell type.

Experimental procedures Cell culture Primary striatal and cortical neurons were obtained from 17to 18-day-old Wistar rat embryos and prepared as followed. Briefly, brain and meninges were removed. Caudate-putamen or cortex was carefully dissected out and mechanically dissociated in culture medium by trituration with a polished Pasteur pipette. Once dissociated and after blue trypan counting, cells were plated in 24-well plates or 25-cm2 flasks at a density of 900 cells/mm2. For dissociation, plating and maintenance, we used Neurobasal medium supplemented with 1% B27 containing 200 mM glutamine and 1% antibiotic – antimycotic agent (Gibco, Belgium). Cells were then treated with 3NP or staurosporine (STS) at 7 DIV. Stock 3NP solution (100 mM; Fluka, Belgium) was dissolved in 0.1 M PBS (pH 7.4) and brought to pH 7.3 – 7.4 with 5 N NaOH. Stock solution of staurosporine (STS; 1 mM; Sigma, Bornem, Belgium) was prepared in DMSO. Viability assay Cell viability was assessed 72 h after 3NP treatment by colorimetric measurement of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT; Sigma) reduction as we previously described (Blum et al., 2002b, 2003a). 3NP (100 AM) was added directly to the culture medium 8 – 10 days after plating. When appropriate, neurons were treated with either calpain inhibitor I (CI-1; Biomol, Belgium), pan-caspase inhibitor zVAD-fmk (Biomol) or both of them concomitantly with 3NP. Morphologic assessment of cell death TUNEL staining was performed using an in situ cell death detection kit (Roche Molecular Biochemicals, Indianapolis, IN, USA) with a protocol previously described (Koshimizu et al., 2002). At the end of staining, nuclei were counterstained with ethidium bromide (5 Ag/ml in presence of 0.5 mg/ml RNAse for 5 min in PBS at 37jC). Observations were made using a confocal microscope (Bio-Rad, CA) fitted on an Axiovert 100 inverted microscope (Zeiss, United Kingdom). Whole cell lysates Briefly, cells were lysed in a solubilization buffer (M-PER, Pierce, Rockfold, IL, USA) containing a protease inhibitor cocktail (Complete, Roche Molecular Biochemicals, Mannheim, Germany) as described by the manufacturer (6  106 cells in 500 Al). Samples were stored at 20jC until analyzed. Protein concentration was determined using MicroBCA Protein Assay (Pierce).

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Cell permeabilization Selective plasma membrane permeabilization was performed using a modified digitonin method previously described by Leist et al. (1998). After treatment, cells (6  106 cells in 5 ml) were washed with PBS (10 mM) at 37jC. After 5 min, PBS was exchanged for permeabilization buffer (210 mM D-mannitol, 70 mM sucrose, 10 mM HEPES pH 7.2, 5 mM succinate, 0.2 mM EGTA and 80 Ag/ml digitonin; Sigma) and cell plates were gently shaken for 4 min at 4jC (6  106 cells in 1 ml). Buffer was collected and centrifuged for 10 min at 15000  g. Cytosolic proteins from the supernatant were precipitated with 5% trichloracetic acid on ice for 1 h. After centrifugation at 15000  g for 10 min at 4jC, the pellet (cytosolic fraction) was solubilized into sample buffer and the pH neutralized with 10 N NaOH. The permeabilized cells were washed in cold PBS, scraped into 1 ml PBS at 4jC and centrifuged at 5000  g for 10 min at 4jC. The cell pellet (mitochondria-enriched fraction) was solubilized into sample buffer. As a control for the latter conditions, untreated striatal neurons were submitted to the permeabilization protocol. Using Western blotting (see below) and, as expected, cytochrome c and Bad stainings were essentially observed in mitochondria-enriched and cytosolic fractions, respectively, corresponding to their normal subcellular localization (not shown). Electrophoresis and immunoblotting Electrophoresis was performed as previously described (Galas et al., 2000). Equal amounts of proteins (10 Ag) or equal volumes of cytosolic or mitochondria-enriched fractions were denaturated in Laemmli buffer at 100jC, loaded on 15% SDSpolyacrylamide gels and transferred to nitrocellulose. Primary antibodies used were anti-cytochrome c (556433, 1/5000; BD Biosciences, Germany), anti-Bad (B36420, 1/500; BD Biosciences), anti-Smac or DIABLO (SA-219, 1/1000; BIOMOL Inc, PA), anti-Bax (sc-493, 1/5000; Santa Cruz Biotechnology Inc., CA) and anti-actin (A-2066, 1/2000; Sigma). Membranes were incubated with HRP-labeled secondary antibody (goat anti-rabbit or anti-mouse IgGs, 1/10000; NEN, MA) and bands were visualized by chemiluminescent ECL reagent (Amersham Pharmacia Biotech., UK). Actin was used as an internal loading control. We previously verified that actin was not cleaved under the different treatments. Signals were acquired using a CDD camera with fixed gain and black level and analyzed using NIH Image software. Measurement of succinate dehydrogenase (SDH) activity Succinate dehydrogenase activity in control and 3NP-treated neurons was measured by adapting the histochemical method used by Brouillet et al. (1998) as followed. Succinate was used as the specific substrate and MTT was used as an artificial electron acceptor, which is eventually oxidized in formazan. After a 2-h treatment with 3NP, cell plates were frozen and kept at 20jC until measurement. Cells were thawed for 15 min in 0.1 M PBS (0.9% NaCl) at 37jC followed by incubation in 0.1 mg/ml MTT (Sigma), various concentrations of sodium succinate (Sigma) and 0.05 M phosphate buffer pH 7.6 for 30 min at 37jC. Incubation medium was carefully removed and formazan crystals dissolved in DMSO. The

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Results 3NP-induced inhibition of neuronal SDH activity We first characterized the primary effect of 3NP (i.e., inhibition of SDH) in striatal and cortical cells. We determined for neurons similar apparent Km of SDH for sodium succinate (0.79 F 0.12 and 1.01 F 0.25 mM for untreated striatal and cortical neurons, respectively), consistent with the previously reported values in peripheral cells (Ackrell et al., 1978) and brain (Brouillet et al., 1998). We compared the ratio of the apparent Vmax of 3NP-treated vs. control (Fig. 1) and found that SDH was inhibited by 3NP to the same extent in both cell types, that is, striatal culture, 79.1

Fig. 1. 3NP-induced SDH inhibition in striatal and cortical neurons. SDH activity (OD/30 min) was measured at varying concentrations of sodium succinate in striatal (A) or cortical (B) neurons untreated (square) or treated with 100 AM 3NP for 2 h (triangle). These data are representative of three independent experiments.

optical density from 3  105 cells was measured at a wavelength of 540 nm on a Titertek Multiskan MCC/340 (ICN Biomedicals, CA). Proteolytic activity assay using fluorogenic substrate for caspases and calpain Fluorescent assays for calpain and caspase activities are based on a previously described protocol (Bizat et al., 2003a). Calpain activity was determined using N-succinyl-Leu-Tyr-(Nsuccinyl-LY)-AMC, a substrate preferentially cleaved by A/m calpain. Caspase-3 and -9 activities were tested on peptidic substrates (Biomol) using respectively N-acetyl-Asp-Glu-ValAsp-AFC (DEVD-AFC) and N-acetyl-Leu-Glu-His-Asp-AFC (LEHD-AFC). Enzyme activity was calculated using standard curves of AFC or AMC and expressed as pmol AFC-AMC released per min/mg of protein. Analysis and statistics Results were expressed as means F SEM of at least three independent experiments. Statistics were performed by one way ANOVA followed by post hoc Newman – Keuls.

Fig. 2. Effect of 3NP on striatal and cortical cell viability. (A) 3NP induces similar dose-dependent decrease of cell viability in primary striatal (S) and cortical (C) cultures. Striatal and cortical neurons were treated for 72 h with 100 AM 3NP and cell viability was assayed using MTT test. Data shown are the mean F SEM of eight independent experiments. (B) Treatment of both neuronal types with 3NP (100 AM, 72 h) leads to the appearance of DNA alterations such as condensation or marginalization and fragmentation detected using ethidium bromide (EthB) and TUNEL method. (C) The percentage of TUNEL-positive cells is similar for both neuronal types following a 72-h treatment with 100 AM 3NP. ***P < 0.001 vs. corresponding untreated cells.

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F 3.5% and cortical culture, 76.4 F 1.1% of inhibition vs. respective untreated controls. This maximal inhibition was reached with similar time course in both neuronal types (data not shown). These data indicate that, in our conditions, 3NP induced a similar SDH inhibition in both striatal and cortical neurons.

ginalization or condensation and fragmentation (Fig. 2B). In accordance to what has been found using MTT assay, the percentage of TUNEL-positive cells was similar in both neuronal cultures (Fig. 2C), suggesting that 3NP, in vitro, has no preferential toxic effect regardless of cell type.

Effect of 3NP on neuronal cell death

3NP-induced redistribution of proapoptotic proteins

3NP treatment of either striatal or cortical cultures resulted in a similar dose-dependent neurotoxic effect as assessed by MTT assay (Fig. 2A). Seventy-two hours after treatment, 100 AM of 3NP induced a maximal toxic effect as compared to control cells (striatal culture: 48.3 F 5.9%; cortical culture: 46.1 F 3.4% of cell death). Similar results were found using the TUNEL method which detects in situ nuclei with fragmented DNA. Indeed, 72 h after treatment, a substantial number of TUNEL-positive cells were seen in both striatal and cortical cultures as compared to respective untreated cultures (Figs. 2B and C). TUNEL-positive nuclei presented abnormal morphologies such as chromatin mar-

According to the maximal toxic effect previously observed, cells were continuously exposed to 100 AM 3NP and we analyzed, at various time points from 2 to 72 h, the level of cytochrome c, Smac, Bad and Bax by Western blot in cytosolic or mitochondria-enriched fractions prepared from striatal or cortical cultures. We observed that 3NP produced a differential protein redistribution in striatal neurons as compared to cortical cells (Fig. 3). In cortical cultures, there was no significant redistribution of Bax, Bad and Smac. Only a slight but significant release of cytochrome c was detected 24 h after 3NP exposure (+25 F 1.9%, P < 0.05; Fig. 3A).

Fig. 3. Proapoptotic protein redistribution in 3NP-treated striatal and cortical neurons. Striatal (S) or cortical (C) neuronal cultures were exposed for various times to 100 AM 3NP. Cytosolic and mitochondria-enriched fractions were prepared from permeabilized neurons as described in Experimental procedures. Representative Western blots are presented for cytosolic cytochrome c (cyto. C) and Smac or DIABLO (A) and membrane-bound Bad and Bax (B). Proteinloading controls are shown for actin. Graphics represent the corresponding densitometric analysis of three pooled independent experiments. #P < 0.05 vs. untreated cortical cells. *P < 0.05 vs. untreated striatal cells.

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Involvement of caspase and calpain We then examined the activity of downstream cell death proteases in cortical and striatal neurons treated with 3NP. Regardless of the cell type considered, exposure to 3NP did not lead to increased caspase-9- or -3-related activities. The latter was rather slightly reduced in striatal cells (Fig. 4). No significant change in calpain activity was detected in cortical cells treated with 3NP. In contrast, in striatal neurons, 3NP produced a strong and sustained elevation of calpain proteolytic activity (Fig. 4). We then studied the ability of the caspase inhibitor zVAD-fmk and the calpain inhibitor I (CI-1) to protect striatal and cortical cells against 3NP-induced toxicity (Fig. 5). The pan-caspase inhibitor zVAD-fmk did not reduce cell death in striatal and cortical cultures exposed to 3NP, consistent with the lack of activation of caspase-9 and -3 in these cells. In contrast, the calpain inhibitor CI-1 significantly prevented 3NP-induced death of striatal neurons, demonstrating that calpain activation in these cells was instrumental. CI-1 alone or in combination with zVADfmk did not protect cortical cells against 3NP (Fig. 5). These data thus demonstrate that cell death proteases involved in 3NPinduced degeneration are differentially implicated depending on the neuronal type considered. Cell death induced by staurosporine

Fig. 4. Protease activation in 3NP-treated neurons. Striatal (S) and cortical (C) cultures were subjected to 100 AM 3NP for different times. Cells were then lysed and calpain (A), caspase-9 (B) and caspase-3 (C) related activities measured. Data represent the mean F SEM of two experiments performed in quadruplicate. *P < 0.05, **P < 0.01 vs. respective untreated control.

To determine whether the molecular machinery leading to caspase activation was functional in striatal and cortical cells, both cultures were treated with staurosporine (STS), a broad protein kinase inhibitor, used as a control for apoptotic induction. Twenty-four hours post-treatment, staurosporine (500 nM) induced a significant decrease of cell viability reaching 45.8 F 6.6% and 70.4 F 6.7% (n = 8; P < 0.05) for striatal and cortical cells, respectively, and the appearance of numerous cells presenting chromatin condensation and fragmentation as well as TUNEL-positive nuclei (data not shown). At the same time, we

Conversely, in striatal cultures treated with 3NP, both Smac and cytochrome c were strongly released into the cytoplasm (Fig. 3A). Noteworthy, using immunocytochemistry, we did not observe nuclear translocation of AIF, another mitochondrial apoptogenic factor acting directly within the nucleus to induce DNA degradation (Susin et al., 1999), in 3NP-treated cortical or striatal neurons (data not shown). Furthermore, 3NP produced a Bad translocation detected after 7 h and reaching a plateau at 48 h (Fig. 3B). Interestingly, this translocation was associated with an increased expression of Bad during 3NP exposure (6 h: +74 F 25.1%, ns; 24 h: +116 F 11.5%, ns; 48 h: +248 F 36.1%, P < 0.05; 72 h: +168 F 46.7%, P < 0.05 vs. untreated control). In cortical neurons, 3NP produced only a slight and transient increase in Bad expression at 48 h (+46.5 F 7.2%, P < 0.05 vs. untreated control). In striatal cells, a delayed release of Bax was also observed (Fig. 3B). However, on whole cell lysates, the levels of cytochrome c, Smac and Bax remained unchanged during 3NP exposure (data not shown), suggesting that 3NP produced no major modification of expression of these proteins in striatal and cortical neurons. Altogether, our observations showed that, in striatal cells, 3NP produced a redistribution of pro-apoptotic proteins and a modification of Bad expression, whereas in cortical neurons, no or only minor changes were detected.

Fig. 5. Effect of a pharmacological inhibition of calpain and caspase against 3NP-induced cell death. Striatal (S) and cortical (C) neurons were treated with 1 AM calpain inhibitor (CI-1), 10 AM zVAD-FMK or combination of both inhibitors concomitantly with 100 AM 3NP. Viability was determined 72 h following treatment. Data represent the mean F SEM of three experiments performed in quadruplicate. ###P < 0.001 and ***P < 0.001 vs. respective untreated control. jjjP < 0.001 vs. 3NP-treated striatal neurons. No difference was noticed between cortical neurons treated with 3NP and those treated with 3NP and protease inhibitors.

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Fig. 6. Protease activation in staurosporine-treated neurons. Striatal (S) and cortical (C) cultures were subjected to 500 nM staurosporine for different times. Cells were then lysed and calpain (A), caspase-9 (B) and caspase-3 (C) activities measured. *P < 0.01 vs. respective untreated control.

were able to observe, in both cell types, a significant cytosolic release of cytochrome c (+79 F 12%, P < 0.001 and +89 F 25%, P < 0.01 in cortical and striatal cells, respectively) and Smac (+68 F 7.5%, P < 0.001 and +118,0 F 5%, P < 0.01 in cortical and striatal cells, respectively). As shown in Fig. 6, STS did not induce calpain activity in striatal or in cortical neurons. Conversely, we found a prominent increase in caspase-9- and -3related proteolytic activities 7 and 24 h after treatment in both cell types.

Discussion Our results demonstrate that a metabolic compromise induces the death of primary striatal and cortical neurons to the same extent but through different molecular mechanisms. Whereas cell death induced by staurosporine is mediated by cytochrome c release and caspase activation in both neuronal types, striatal and cortical neurons develop different molecular pathways under 3NP treatment. In striatal neurons, we observed Bad and Bax membrane translocation and cytochrome c and Smac cytosolic release as well as calpain activation. On the other hand, response of cortical neurons to 3NP involved neither Bad and Bax and cytochrome c and Smac relocalization nor substantial caspase and calpain activation. Such differences were not related

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to a differential effect of 3NP on SDH activity because the latter was similarly inhibited by 3NP in both neuronal types. To our knowledge, this is the first demonstration of the existence, in cortical and striatal neurons, of an in vitro cell type and biochemical cascade specificity in neuronal death mechanisms triggered by mitochondrial impairment. Unlike STS, 3NP-induced striatal cell death was not associated with detectable caspase-9 and -3 activation despite cytochrome c and Smac cytosolic release that are classical apoptotic events. We observed instead a marked activation of the Ca2+activated protease calpains. Accordingly, in striatal neurons, whereas the broad caspase inhibitor zVAD-fmk did not display any significant effect against 3NP, the calpain inhibitor CI-1 was neuroprotective. Conversely, in cortical neurons, only a small cytochrome c release was evidenced, but this was followed neither by caspase nor calpain activation. The results found in striatal cells reinforce recent studies supporting an instrumental role of calpains in striatal degeneration observed both in the caudate nucleus of HD patients (Gafni and Ellerby, 2002) and the striatum of rats chronically treated with 3NP (Bizat et al., 2003a). The reasons for the specific activation of calpains observed in striatal cultures chronically treated with 3NP are not totally understood but are consistent with observations recently made in vivo in the rat striatum (Bizat et al., 2003a). As discussed previously (Bizat et al., 2003a), it remains possible that the intrinsic ability of striatal neurons to regulate cytosolic Ca2+ increases is lower as compared to cortical neurons. This hypothesis is supported by a recent study which demonstrates that striatal mitochondria are more vulnerable to Ca2+-induced permeability transition than cortical mitochondria (Brustovetsky et al., 2003). Thus, it is possible that such a vulnerability of striatal mitochondria to Ca2+ could facilitate cytosolic Ca2+ rises and a preferential activation of calpain within striatal neurons exposed to 3NP. In addition, it remains possible that systems regulating calpain activity such as the endogenous calpain inhibitor calpastatin may be less efficient in striatal cells as compared to cortical cells. The lack of caspase-3 activation in striatal cells treated with 3NP is also surprising considering that the biochemical changes observed (i.e. Bax, Bad, cytochrome c, Smac redistribution) are reminiscent of apoptosis and generally lead to caspase activation. Since staurosporine is able to induce caspase activation in our culture conditions, the lack of caspase3 activation in striatal cells exposed to 3NP is not due to a constitutive defect in the molecular pathways leading to the processing and the activation of caspases. The absence of caspase-9 and -3 activation in striatal neurons may be related to calpain activation. Indeed, recent data support the ability of calpain to inactivate the caspase-9 and -3 pathway in excitotoxic cell death (Lankiewicz et al., 2000) and to degrade caspase-9 and -3 during 3NP-induced degeneration in vivo (Bizat et al., 2003b). The lack of caspase and calpain activation in cortical neurons treated with 3NP despite obvious signs of degeneration (including DNA fragmentation) is very intriguing, and the mechanisms leading to their death remain speculative. The data we obtained using staurosporine demonstrate that, in our experimental conditions, caspase activation is possible in primary cortical cells in culture. Additionally, 3NP can induce calpain activation in cortical neurons when applied at very high concentrations (5 mM) (Goffredo et al., 2002). Thus, cortical neurons possess the molecular machineries involved in apoptosis and necrosis-related

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cell death, but neither caspases nor calpain, classically involved in these death processes, are activated by 3NP treatment. It remains possible that ROS-mediated pathway or other mitochondrial apoptogenic factors acting directly within the nucleus such as endonuclease G or Par-4 might be involved (Duan et al., 2000; van Loo et al., 2002). Alternatively, it is possible that other death effector proteases such as cathepsins (Leist and Jaattela, 2001) may be implicated. Finally, our data suggest that for a given chronic and partial mitochondrial defect, each neuronal type triggers differential death pathways. Such events could explain the differential reactivity of cortical and striatal neurons overexpressing mutated huntingtin to apoptotic stressors or excitotoxins as described and discussed recently (Snider et al., 2003 and references therein). For now, the reasons for explaining these differential mechanisms remain obscure. However, it is now admitted that, in HD pathogenesis, cortical dysfunctions are as much important as striatal dysfunctions. Cortical metabolic modifications (Sax et al., 1996) and alterations along the corticostriatal pathways appear at early stages of the disease. Such changes may be important for the development of motor symptoms and striatal cell death (Cepeda et al., 2003; Laforet et al., 2001) through, for example, the lack of trophic support afforded by the cortical synthesis of BDNF (Duan et al., 2003; Laforet et al., 2001). Therefore, therapeutically, it is crucial to promote the function and the survival of both cortical and striatal neurons. Biochemical and NMR spectroscopy studies have previously suggested a defect in oxidative energy metabolism in the HD striatum and cortex (Browne et al., 1997; Gu et al., 1996; Jenkins et al., 1993, 1998; Tabrizi et al., 1999) that could be involved in striatal and cortical degeneration and facilitate certain complex molecular pathways leading to cell demise. Our results suggest that, depending on the neuronal type considered, such pathways may differ substantially. This may have important implications for designing new efficient therapies. Particularly, ‘‘cocktail’’ pharmacological therapies or neurotrophic factors acting on various cellular signaling pathways may be more efficient than strategies with a single cellular target. In conclusion, our data support that different effector systems are involved in striatal and cortical cells submitted to a similar mitochondrial perturbation. Such intrinsic differences are of particular interest to further understand HD pathogenesis and design future neuroprotective therapies.

Acknowledgments This work was supported by the FMRE (Belgium, Neurobiology 99-01 and 02-04), FRSM (Belgium, 3.4551.98/ 3.4507.02), Action de Recherche Concerte´e and the A. & D. Van Buuren Foundation. M.C.G. is a researcher of the CNRS (France). K.B. is supported by a Televie grant and D.B. and M.C.G by FNRS (Belgium) post-doctoral fellowships. We thank Dr. Raphae¨l Hourez for comments.

References Ackrell, B.A., Kearney, E.B., Singer, T.P., 1978. Mammalian succinate dehydrogenase. Methods Enzymol. 53, 466 – 483. Albin, R.L., 1995. Selective neurodegeneration in Huntington’s disease. Ann. Neurol. 38, 835 – 836.

Beal, M.F., 2000. Energetics in the pathogenesis of neurodegenerative diseases. Trends Neurosci. 23, 298 – 304. Beal, M.F., Brouillet, E., Jenkins, B.G., Ferrante, R.J., Kowall, N.W., Miller, J.M., Storey, E., Srivastava, R., Rosen, B.R., Hyman, B.T., 1993. Neurochemical and histologic characterization of striatal excitotoxic lesions produced by the mitochondrial toxin 3-nitropropionic acid. J. Neurosci. 13, 4181 – 4192. Bizat, N., Hermel, J.M., Boyer, F., Jacquard, C., Creminon, C., Ouary, S., Escartin, C., Hantraye, P., Kajewski, S., Brouillet, E., 2003a. Calpain is a major cell death effector in selective striatal degeneration induced in vivo by 3-nitropropionate: implications for Huntington’s disease. J. Neurosci. 23, 5020 – 5030. Bizat, N., Hermel, J.M., Humbert, S., Jacquard, C., Creminon, C., Escartin, C., Saudou, F., Kajewski, S., Hantraye, P., Brouillet, E., 2003b. In Vivo Calpain/Caspase Cross-talk during 3-Nitropropionic Acid-induced Striatal Degeneration. J. Biol. Chem. 278, 43245 – 43253. Blum, D., Gall, D., Cuvelier, L., Schiffmann, S.N., 2001. Topological analysis of striatal lesions induced by 3-nitropropionic acid in the Lewis rat. NeuroReport 12, 1769 – 1772. Blum, D., Galas, M.C., Gall, D., Cuvelier, L., Schiffmann, S.N., 2002a. Striatal and cortical neurochemical changes induced by chronic metabolic compromise in the 3-nitropropionic model of Huntington’s disease. Neurobiol. Dis. 10, 410 – 426. Blum, D., Gall, D., Galas, M.C., d’Alcantara, P., Bantubungi, K., Schiffmann, S.N., 2002b. The adenosine A1 receptor agonist adenosine amine congener exerts a neuroprotective effect against the development of striatal lesions and motor impairments in the 3-nitropropionic acid model of neurotoxicity. J. Neurosci. 22, 9122 – 9133. Blum, D., Galas, M.C., Pintor, A., Brouillet, E., Ledent, C., Muller, C.E., Bantubungi, K., Galluzzo, M., Gall, D., Cuvelier, L., Rolland, A.S., Popoli, P., Schiffmann, S.N., 2003a. A dual role of adenosine A2A receptors in 3-nitropropionic acid-induced striatal lesions: implications for the neuroprotective potential of A2A antagonists. J. Neurosci. 23, 5361 – 5369. Blum, D., Hourez, R., Galas, M.C., Popoli, P., Schiffmann, S.N., 2003b. Adenosine receptors and Huntington’s disease: implications for pathogenesis and therapeutics. Lancet Neurol. 2, 366 – 374. Brouillet, E., Hantraye, P., Ferrante, R.J., Dolan, R., Leroy-Willig, A., Kowall, N.W., Beal, M.F., 1995. Chronic mitochondrial energy impairment produces selective striatal degeneration and abnormal choreiform movements in primates. Proc. Natl. Acad. Sci. U. S. A. 92, 7105 – 7109. Brouillet, E., Guyot, M.C., Mittoux, V., Altairac, S., Conde, F., Palfi, S., Hantraye, P., 1998. Partial inhibition of brain succinate dehydrogenase by 3-nitropropionic acid is sufficient to initiate striatal degeneration in rat. J. Neurochem. 70, 794 – 805. Brouillet, E., Conde, F., Beal, M.F., Hantraye, P., 1999. Replicating Huntington’s disease phenotype in experimental animals. Prog. Neurobiol. 59, 427 – 468. Browne, S.E., Bowling, A.C., MacGarvey, U., Baik, M.J., Berger, S.C., Muqit, M.M., Bird, E.D., Beal, M.F., 1997. Oxidative damage and metabolic dysfunction in Huntington’s disease: selective vulnerability of the basal ganglia. Ann. Neurol. 41, 646 – 653. Brustovetsky, N., Brustovetsky, T., Purl, K.J., Capano, M., Crompton, M., Dubinsky, J.M., 2003. Increased susceptibility of striatal mitochondria to calcium-induced permeability transition. J. Neurosci. 23, 4858 – 4867. Cattaneo, E., Rigamonti, D., Goffredo, D., Zuccato, C., Squitieri, F., Sipione, S., 2001. Loss of normal huntingtin function: new developments in Huntington’s disease research. Trends Neurosci. 24, 182 – 188. Cepeda, C., Hurst, R.S., Calvert, C.R., Hernandez-Echeagaray, E., Nguyen, O.K., Jocoy, E., Christian, L.J., Ariano, M.A., Levine, M.S., 2003. Transient and progressive electrophysiological alterations in the corticostriatal pathway in a mouse model of Huntington’s disease. J. Neurosci. 23, 961 – 969. Cha, J.H., 2000. Transcriptional dysregulation in Huntington’s disease. Trends Neurosci. 23, 387 – 392.

M.-C. Galas et al. / Neurobiology of Disease 15 (2004) 152–159 Duan, W., Guo, Z., Mattson, M.P., 2000. Participation of par-4 in the degeneration of striatal neurons induced by metabolic compromise with 3-nitropropionic acid. Exp. Neurol. 165, 1 – 11. Duan, W., Guo, Z., Jiang, H., Ware, M., Li, X.J., Mattson, M.P., 2003. Dietary restriction normalizes glucose metabolism and BDNF levels, slows disease progression, and increases survival in huntingtin mutant mice. Proc. Natl. Acad. Sci. U. S. A. 100, 2911 – 2916. El Massioui, N., Ouary, S., Cheruel, F., Hantraye, P., Brouillet, E., 2001. Perseverative behavior underlying attentional set-shifting deficits in rats chronically treated with the neurotoxin 3-nitropropionic acid. Exp. Neurol. 172, 172 – 181. Gafni, J., Ellerby, L.M., 2002. Calpain activation in Huntington’s disease. J. Neurosci. 22, 4842 – 4849. Galas, M.C., Chasserot-Golaz, S., Dirrig-Grosch, S., Bader, M.F., 2000. Presence of dynamin – syntaxin complexes associated with secretory granules in adrenal chromaffin cells. J. Neurochem. 75, 1511 – 1519. Gervais, F.G., Singaraja, R., Xanthoudakis, S., Gutekunst, C.A., Leavitt, B.R., Metzler, M., Hackam, A.S., Tam, J., Vaillancourt, J.P., Houtzager, V., Rasper, D.M., Roy, S., Hayden, M.R., Nicholson, D.W., 2002. Recruitment and activation of caspase-8 by the Huntingtin-interacting protein Hip-1 and a novel partner Hippi. Nat. Cell Biol. 4, 95 – 105. Goffredo, D., Rigamonti, D., Tartari, M., De Micheli, A., Verderio, C., Matteoli, M., Zuccato, C., Cattaneo, E., 2002. Calcium-dependent cleavage of endogenous wild-type huntingtin in primary cortical neurons. J. Biol. Chem. 277, 39594 – 39598. Gu, M., Gash, M.T., Mann, V.M., Javoy-Agid, F., Cooper, J.M., Schapira, A.H., 1996. Mitochondrial defect in Huntington’s disease caudate nucleus. Ann. Neurol. 39, 385 – 389. Jenkins, B.G., Koroshetz, W.J., Beal, M.F., Rosen, B.R., 1993. Evidence for impairment of energy metabolism in vivo in Huntington’s disease using localized 1H NMR spectroscopy. Neurology 43, 2689 – 2695. Jenkins, B.G., Rosas, H.D., Chen, Y.C., Makabe, T., Myers, R., MacDonald, M., Rosen, B.R., Beal, M.F., Koroshetz, W.J., 1998. 1H NMR spectroscopy studies of Huntington’s disease: correlations with CAG repeat numbers. Neurology 50, 1357 – 1365. Koshimizu, H., Araki, T., Takai, S., Yokomaku, D., Ishikawa, Y., Kubota, M., Sano, S., Hatanaka, H., Yamada, M., 2002. Expression of CD47/ integrin-associated protein induces death of cultured cerebral cortical neurons. J. Neurochem. 82, 249 – 257. Laforet, G.A., Sapp, E., Chase, K., McIntyre, C., Boyce, F.M., Campbell, M., Cadigan, B.A., Warzecki, L., Tagle, D.A., Reddy, P.H., Cepeda, C., Calvert, C.R., Jokel, E.S., Klapstein, G.J., Ariano, M.A., Levine, M.S., Difiglia, M., Aronin, N., 2001. Changes in cortical and striatal neurons predict behavioral and electrophysiological abnormalities in a transgenic murine model of Huntington’s disease. J. Neurosci. 21, 9112 – 9123. Lankiewicz, S., Marc, L.C., Truc, B.N., Krohn, A.J., Poppe, M., Cole, G.M., Saido, T.C., Prehn, J.H., 2000. Activation of calpain I converts excitotoxic neuron death into a caspase-independent cell death. J. Biol. Chem. 275, 17064 – 17071. Leist, M., Jaattela, M., 2001. Triggering of apoptosis by cathepsins. Cell Death Differ. 8, 324 – 326. Leist, M., Volbracht, C., Fava, E., Nicotera, P., 1998. 1-Methyl-4-phenylpyridinium induces autocrine excitotoxicity, protease activation, and neuronal apoptosis. Mol. Pharmacol. 54, 789 – 801. Mittoux, V., Joseph, J.M., Conde, F., Palfi, S., Dautry, C., Poyot, T., Bloch, J., Deglon, N., Ouary, S., Nimchinsky, E.A., Brouillet, E., Hof, P.R., Peschanski, M., Aebischer, P., Hantraye, P., 2000. Restoration of cog-

159

nitive and motor functions by ciliary neurotrophic factor in a primate model of Huntington’s disease. Hum. Gene Ther. 11, 1177 – 1187. Mittoux, V., Ouary, S., Monville, C., Lisovoski, F., Poyot, T., Conde, F., Escartin, C., Robichon, R., Brouillet, E., Peschanski, M., Hantraye, P., 2002. Corticostriatopallidal neuroprotection by adenovirus-mediated ciliary neurotrophic factor gene transfer in a rat model of progressive striatal degeneration. J. Neurosci. 22, 4478 – 4486. Ona, V.O., Li, M., Vonsattel, J.P., Andrews, L.J., Khan, S.Q., Chung, W.M., Frey, A.S., Menon, A.S., Li, X.J., Stieg, P.E., Yuan, J., Penney, J.B., Young, A.B., Cha, J.H., Friedlander, R.M., 1999. Inhibition of caspase1 slows disease progression in a mouse model of Huntington’s disease. Nature 399, 263 – 267. Panov, A.V., Gutekunst, C.A., Leavitt, B.R., Hayden, M.R., Burke, J.R., Strittmatter, W.J., Greenamyre, J.T., 2002. Early mitochondrial calcium defects in Huntington’s disease are a direct effect of polyglutamines. Nat. Neurosci. 5, 731 – 736. Sanchez, I., Xu, C.J., Juo, P., Kakizaka, A., Blenis, J., Yuan, J., 1999. Caspase-8 is required for cell death induced by expanded polyglutamine repeats. Neuron 22, 623 – 633. Saudou, F., Finkbeiner, S., Devys, D., Greenberg, M.E., 1998. Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell 95, 55 – 66. Sawa, A., Wiegand, G.W., Cooper, J., Margolis, R.L., Sharp, A.H., Lawler Jr., J.F., Greenamyre, J.T., Snyder, S.H., Ross, C.A., 1999. Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization. Nat. Med. 5, 1194 – 1198. Sax, D.S., Powsner, R., Kim, A., Tilak, S., Bhatia, R., Cupples, L.A., Myers, R.H., 1996. Evidence of cortical metabolic dysfunction in early Huntington’s disease by single-photon-emission computed tomography. Mov. Disord. 11, 671 – 677. Snider, B.J., Moss, J.L., Revilla, F.J., Lee, C.S., Wheeler, V.C., Macdonald, M.E., Choi, D.W., 2003. Neocortical neurons cultured from mice with expanded CAG repeats in the huntingtin gene: unaltered vulnerability to excitotoxins and other insults. Neuroscience 120, 617 – 625. Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo, I., Snow, B.E., Brothers, G.M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., Larochette, N., Goodlett, D.R., Aebersold, R., Siderovski, D.P., Penninger, J.M., Kroemer, G., 1999. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441 – 446. Tabrizi, S.J., Cleeter, M.W., Xuereb, J., Taanman, J.W., Cooper, J.M., Schapira, A.H., 1999. Biochemical abnormalities and excitotoxicity in Huntington’s disease brain. Ann. Neurol. 45, 25 – 32. The Huntington’s disease research group, 1993. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. The Huntington’s Disease Collaborative Research Group. Cell 72, 971 – 983. van Loo, G., Saelens, X., van Gurp, M., MacFarlane, M., Martin, S.J., Vandenabeele, P., 2002. The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet. Cell Death Differ. 9, 1031 – 1042. Vonsattel, J.P., Myers, R.H., Stevens, T.J., Ferrante, R.J., Bird, E.D., Richardson Jr., E.P., 1985. Neuropathological classification of Huntington’s disease. J. Neuropathol. Exp. Neurol. 44, 559 – 577. Zuccato, C., Ciammola, A., Rigamonti, D., Leavitt, B.R., Goffredo, D., Conti, L., MacDonald, M.E., Friedlander, R.M., Silani, V., Hayden, M.R., Timmusk, T., Sipione, S., Cattaneo, E., 2001. Loss of huntingtinmediated BDNF gene transcription in Huntington’s disease. Science 293, 493 – 498.