Rescue from death but not from functional ... - Wiley Online Library

Journal of Neurochemistry, 2001, 77, 263±273

Rescue from death but not from functional impairment: caspase inhibition protects dopaminergic cells against 6-hydroxydopamine-induced apoptosis but not against the loss of their terminals Rainer von Coelln,* Sebastian KuÈgler,² Mathias BaÈhr,² Michael Weller,³ Johannes Dichgans*,²,³ and JoÈrg B. Schulz* *Neurodegeneration, ²Neuroregeneration and ³Neurooncology Laboratories, Department of Neurology and Medical School, TuÈbingen, Germany

Abstract Despite the identi®cation of several mutations in familial Parkinson's disease (PD), the underlying mechanisms of dopaminergic neuronal loss in idiopathic PD are still unknown. To study whether caspase-dependent apoptosis may play a role in the pathogenesis of PD, we examined 6-hydroxydopamine (6-OHDA) toxicity in dopaminergic SH-SY5Y cells and in embryonic dopaminergic mesencephalic cultures. 6-OHDA induced activation of caspases 3, 6 and 9, chromatin condensation and cell death in SH-SY5Y cells. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)¯uoromethylketone (zVAD-fmk) or adenovirally mediated ectopic expression of the X-chromosomal inhibitor of apoptosis protein (XIAP)

blocked caspase activation and prevented death of SH-SY5Y cells. Similarly, zVAD-fmk provided protection from 6-OHDAinduced loss of tyrosine hydroxylase-positive neurones in mesencephalic cultures. In contrast, zVAD-fmk failed to protect mesencephalic dopaminergic neurones from 6-OHDA-induced loss of neurites and reduction of [3H]dopamine uptake. These data suggest that, although caspase inhibition provides protection from 6-OHDA-induced death of dopaminergic neurones, the neurones may remain functionally impaired. Keywords: apoptosis, caspases, gene therapy, 6-hydroxydopamine, Parkinson's disease, X-chromosomal inhibitor of apoptosis protein. J. Neurochem. (2001) 77, 263±273.

Pathologically, the hallmark of Parkinson's disease (PD) is loss of dopaminergic neurones in the substantia nigra, leading to the major clinical and pharmacological abnormalities that characterize the disease. In recent years, mutations in four genes have been identi®ed to cause either autosomaldominant (Polymeropoulos et al. 1997; Gasser et al. 1998; Leroy et al. 1998) or autosomal-recessive PD (Kitada et al. 1998) in rare families. Although the genetic contribution to the aetiology of PD is intensively discussed, the majority of PD is sporadic, and the cause of neuronal loss in the substantia nigra is not known. Recent advances have been made in de®ning morphological and biochemical events in the pathogenesis of the disease. Inhibition of oxidative phosphorylation, excitotoxicity, and generation of reactive oxygen species (ROS) are considered important mediators of neuronal death in PD (Beal 1995; Jenner and Olanow 1998). Today, only symptomatic but no neuroprotective therapies are available for the treatment of PD.

6-Hydroxydopamine (6-OHDA) is a selective catecholaminergic neurotoxin that is widely used to study mechanisms Received September 18, 2000; revised manuscript received December 20, 2000; accepted December 27, 2000. Address correspondence and reprint requests to JoÈrg B. Schulz, MD, Department of Neurology, Hoppe-Seyler-Str. 3, D-72076 TuÈbingen, Germany. E-mail: [email protected] Abbreviations used: AdV, adenoviral vector; AdV-LacZ, adenoviral vector encoding LacZ; AdV-EGFP, adenoviral vector encoding EGFP; AdV-XIAP, adenoviral vector encoding XIAP; DEVD-amc, N-acetylAsp-Glu-Val-Asp-amino-4-methylcoumarin; DIV, day in vitro; DMSO, dimethyl sulphoxide; EGFP, enhanced green ¯uorescent protein; IAP, inhibitor of apoptosis protein; MOI, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD, optical density; 6-OHDA, 6-hydroxydopamine; PBS, phosphate-buffered saline; PD, Parkinson's disease; ROS, reactive oxygen species; SDS, sodium dodecyl sulphate; TH-ir, immunoreactive for tyrosine hydroxylase; XIAP, X-chromosomal IAP; zVAD-fmk, benzyloxycarbonylVal-Ala-Asp-(O-methyl)¯uoromethylketone.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 263±273

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of cell death in dopaminergic neurones (Ungerstedt 1968; Angeletti and Levi-Montalcini 1970) and in animal models of PD (Sauer and Oertel 1994; Przedborski et al. 1995). In the latter, 6-OHDA selectively eliminates dopaminergic terminals in the striatum and axons within a circumscribed area of the injection site, followed by a degeneration of substantia nigra neurones. The toxicity of 6-OHDA is thought to be mediated by selective uptake through the dopamine transporter (Shimada et al. 1991; Usdin et al. 1991). Under physiological conditions 6-OHDA is rapidly and nonenzymatically oxidized by molecular oxygen to form hydrogen peroxide (H2O2) and the corresponding p-quinone. Downstream various mechanisms for its toxicity have been put forward, including the production of ROS, the interaction of one or more of its oxidation products (quinones) with nucleophilic groups of several intraneuronal proteins, or with peptides like glutathione (Graham et al. 1978; Gee and Davison 1989) and inhibition of mitochondrial complexes I and IV (Glinka and Youdim 1995). In addition, evidence is accumulating that 6-OHDA leads to the activation of caspases and induces apoptosis in vitro in PC12 and MN9D cells, in primary cerebellar granule neurones and mesencephalic cultures (Walkinshaw and Waters 1994; Wu et al. 1996; Ochu et al. 1998; Takai et al. 1998; Choi et al. 1999; Dodel et al. 1999; Lotharius et al. 1999) and in vivo (Offen et al. 1998; Jeon et al. 1999; He et al. 2000). Because 6-OHDA was detected in human brain (Curtius et al. 1974) and urine of PD patients (Andrew et al. 1993), 6-OHDA may not only serve to model dopaminergic cell death experimentally, but has been proposed as a putative endogenous neurotoxic factor in the pathogenesis of PD (Jellinger et al. 1995). Caspases are the major mammalian cell death effector proteins during apoptosis (Nicholson 1999). They execute cell death but may also be linked to the initiation of chronic neurodegenerative diseases (Schulz et al. 1999). Therefore, the inhibition of caspases by peptide inhibitors or by members of the inhibitor of apoptosis protein (IAP) family may offer a therapeutic opportunity to prevent neuronal death (Robertson et al. 2000). IAPs are an evolutionarily conserved family of proteins which regulate apoptosis in diverse species ranging from insects to humans (Deveraux and Reed 1999). Human IAP family members are potent caspase inhibitors (Deveraux et al. 1997; Roy et al. 1997). The human neuroblastoma cell line SH-SY5Y possesses many of the qualities of human dopaminergic neurones (Takahashi et al. 1994) and is therefore widely used to study dopaminergic cell death (Sheehan et al. 1997). To more clearly de®ne the role of caspase activation and apoptosis in 6-OHDA-induced toxicity to dopaminergic cells, we studied the activation of caspases and the protective effects of caspase inhibitors in the human neuroblastoma cell line SH-SY5Y and in primary dopaminergic neurones. We show that 6-OHDA-induced cell death is mediated by caspases.

Caspase inhibition by benzyloxycarbonyl-Val-Ala-Asp-(Omethyl)¯uoromethylketone (zVAD-fmk) or by adenovirusmediated overexpression of the X-chromosomal inhibitor of apoptosis protein (XIAP) signi®cantly decreases the amount of cell death. However, in spite of improved survival, dopaminergic neurones appear to be functionally impaired due to the toxin-induced loss of their processes. Materials and methods Materials If not stated otherwise, chemicals were purchased from SigmaAldrich (Deisenhofen, Germany) and cell culture medium ingredients from Gibco (Karlsruhe, Germany). SH-SY5Y neuroblastoma cell cultures SH-SY5Y cells were maintained in Dulbecco's modi®ed Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 IU/mL penicillin and 100 mg/mL streptomycin and incubated at 378C in a humidi®ed atmosphere of 95% air and 5% CO2. Cells were split (1 : 10) every 5 days. For immunoblot analysis, cells were seeded on 6 cm-dishes at a density of 1.2 106 cells/dish. For all other experimental procedures, cells were seeded in 96-well microtitre plates (20 000 cells/well). After 24 h culture medium was replaced by medium containing zVAD-fmk (Bachem, Heidelberg, Germany) dissolved in dimethyl sulphoxide (DMSO) or DMSO as vehicle alone. Two hours later, 6-OHDA (dissolved at 20-fold concentration in ice-cold 0.01 m HCl containing 4 mm ascorbic acid) was added to the culture medium. After 12 h cells were processed for assessment of viability or immunoblotting. Determination of viability in SH-SY5Y neuroblastoma cells 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was dissolved in phosphate-buffered saline (PBS) at a concentration of 5 mg/mL and added 1 : 10 to the culture medium. After 2 h, medium was removed and the remaining MTT crystals were dissolved in 100 mL DMSO. Optical density was assessed using a plate photometer (MRX Dynatech Laboratories, Chantilly, VA, USA). The MTT reduction assay was validated as a tool to determine cell viability by comparing the results of this assay with the trypan blue exclusion assay (Soldner et al. 1999). Application of both assays resulted in equal percentages of surviving cells after treatment with different concentrations of 6-OHDA as compared with respective control values (data not shown). Mesencephalic neuronal cultures Primary neuronal cultures were prepared from the ventral mesencephalon which was dissected from E14 rat embryos (Charles River, Sulzfeld, Germany) (von Coelln et al. 1995). In brief, tissue pieces were dissociated enzymatically in 0.25% trypsin and mechanically by trituration using ®re-polished glass pipettes, then washed with DMEM/F12, 1 : 1 mixture (BioWhittaker, Verviers, Belgium). Complete medium (DMEM/F12, containing 0.25% BSA, N1 supplements, 50 IU/mL penicillin, 50 mg/mL streptomycin and 33 mm glucose) was used for single cell suspension. Cells were seeded at a density of 1.5 105/cm2 on 10 mm-glass coverslips


Caspase inhibition in 6-OHDA toxicity 265

coated with polyornithine and laminin. For transmitter uptake experiments, neurones were seeded in polyornithine-laminin-coated 24-well plates at the same density. At DIV4 two-thirds (500 mL) of the culture medium were replaced and zVAD-fmk (dissolved in DMSO) was added to the culture medium at ®nal concentrations ranging from 0 mm (vehicle alone) to 200 mm. 6-Hydroxydopamine (dissolved as described above) was added 2 h later. After 24 h of incubation with the toxin, cultures were processed for immunocytochemistry or transmitter uptake analysis.

EDTA, 0.1% Nonidet P-40, 10% sucrose, 3 mm 1,4-dithio-dlthreitol, pH 7.5) containing 10 mm DEVD-amc. Fluorescence was measured every 10 min at excitation 360 nm, emission 460 nm, using a CytoFluor ¯uorescent plate reader (CytoFluor II, PE Biosystems, Weiterstadt, Germany). Analysis of the time course of ¯uorescence revealed a proportional increase of optical density (od) between 20 and 40 min corresponding to a stable enzymatic hydrolysis in this time range. Dod/min was determined as a measure of enzyme activity of caspase 3.

Immunocytochemical analysis Cells were ®xed with 4% paraformaldehyde (10 min, 208C), permeabilized with acetone (10 min, 2 208C) and blocked with H2O2 (10 min, 208C) and 10% horse serum (10 min, 378C). All steps were separated by washing three times with PBS. Incubation with mouse monoclonal antibody to rat tyrosine hydroxylase (TH, 1 : 200 with 5% horse serum, 1 h at 378C, Boehringer, Mannheim, Germany) and biotinylated horse anti-mouse IgG antibody (1 : 200 with 5% horse serum, 15 min, 378C; Vector Laboratories, Burlingame, CA, USA) was followed by the staining procedure using Vectastain ABC kit (15 min, 378C, Vector Laboratories) in combination with 3,3 0 -diaminobenzidene (DAB) reagents (5 min, 208C). Alternatively, a Cy3-labelled goat anti-mouse IgG antibody (1 : 400 with 5% goat serum, 15 min, 378C; Rockland, Gilbertsville, PA, USA) was used as second antibody for ¯uorescence labelling.

Immunoblot analysis Blotting was performed essentially as described previously (Schulz et al. 1996). Neuronal cells seeded on 6-cm dishes were washed once with cold PBS and then lysed using ice-cold lysis buffer [1% Triton X-100 and 0.1% sodium dodecyl sulphate (SDS) with 10 mg/mL leupeptin and aprotinin]. Cell debris was removed by high-speed centrifugation at 48C. Samples containing 20 mg of protein were boiled in 1% SDS and 5% b-mercaptoethanol for 10 min, separated by 10±15% SDS±polyacrylamide gel electrophoresis and electroblotted to nitrocellulose. Filters were blocked for 1 h in blocking solution (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% Tween 20, 5% skimmed milk, and 2% BSA), followed by incubation with primary antibodies directed to the large (p17/20) subunit of caspase 3 (MF 393, D.W. Nicholson, Merck Frosst, Pointe-Claire-Dorval, Kirkland, Quebec, Canada), the large subunit of caspase 6 (MF 473, D.W. Nicholson, Merck Frosst; Zheng et al. 2000), the `pro-large' subunit of caspase 9 (Kevin Tomaselli, IDUN Pharmaceuticals, San Diego, CA, USA), and to XIAP (Liston et al. 1996) overnight at 48C and anti-rabbit IgG horseradish peroxidaselinked antibody (1 h, 208C). Bound antibody was visualized using enhanced chemiluminescence (ECL, Amsterdam, Netherlands).

Determination of viability of mesencephalic dopaminergic neurones Using an Aristoplan microscope (Leica, Bensheim, Germany) with a counting grid enclosed in the ocular, TH-immunoreactive (TH-ir) neurones with intact perikarya were counted at a 100-fold magni®cation. Moving optically across one diameter of the coverslip, all TH-ir neurones inside the grid were counted. Thereby, the analysed portion of the coverslip comprised an area of 8 mm2 (equivalent to 10% of the whole culture). For further morphometric analysis, the number of TH-ir neurones carrying processes was determined. The total length of the cellular processes was measured using an image analysis system (MCID-IV, Imaging Research, St. Catharines, Ontario, Canada). Only the ®rst branch of each main neurite was measured, as second and third branches were inconsistently stained. Chromatin staining (Hoechst dye 33258) SH-SY5Y neuroblastoma cells were ®xed with 4% paraformaldehyde (dissolved in PBS) for 10 min, rinsed twice with PBS and then incubated with 1 mg/mL Hoechst dye 33258. Stained chromatin was visualized using a DM IRBE ¯uorescence microscope (Leica, Bensheim, Germany). N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage analysis Cleavage of the caspase substrate DEVD-amc was determined as described previously (Armstrong et al. 1997). Brie¯y, cells cultured in 96-well microtitre plates were lysed in 50 mL of buffer A (10 min, 378C; 10 mm HEPES, pH 7.4, 42 mm KCl, 5 mm MgCl2, 1 mm phenylmethylsulphonyl ¯uoride, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm 1,4-dithio-dl-threitol, 1 mg/mL pepstatin A, 1 mg/mL leupeptin, 5 mg/mL aprotinin, 0.5% Nonidet P-40). Subsequently, cells were incubated for 40 min in buffer B (25 mm HEPES, 1 mm

Adenoviral vector construction and virus puri®cation Adenoviral vectors coding for XIAP (AdV-XIAP), b-galactosidase (AdV-LacZ) and EGFP (AdV-EGFP) were constructed as described previously (KuÈgler et al. 1999). High titre stocks were raised in 293 suspension cells and puri®ed twice by CsCl-density gradient centrifugation. Viral titres were determined by plaque assay. Adenoviral infection of cultured neuronal cells SH-SY5Y neuroblastoma cells were seeded in 96-well microtitre plates or on 6-cm dishes as described above. After 12 h, the volume of culture medium was reduced to one-half and recombinant adenovirus was added at a multiplicity of infection (MOI) of 10. After an incubation period of 6 h, medium was replenished. Another 6 h later, 6-OHDA was added to the culture medium (dissolved as described above) if applicable. After a further 12 h incubation, cells were processed for assessment of viability or immunoblotting. For adenoviral infection of mesencephalic neuronal cultures, mesencephalic neurones were seeded on coated coverslips as described above. For immunoblotting experiments, cells were seeded on polyornithine-laminin-coated 6-cm dishes. At DIV1, culture medium was removed and the culture surface was carefully covered with medium containing recombinant adenovirus at 200 MOI. After an incubation period of 12 h, medium was replenished. Cell cultures were incubated with 6-OHDA for 24 h starting at DIV4 and then processed for immunocytochemistry or immunoblot analysis as described above.



Detection of b-galactosidase-positive cells and EGFP-positive cells b-Galactosidase activity was measured as described previously (Simons et al. 1999). Cells were ®xed in 4% paraformaldehyde (10 min, 208C) and washed twice with PBS. Cells were then incubated for 12 h in X-gal stain [2 mm MgCl2, 5 mm K3Fe(CN)6, 5 mm K4Fe(CN)6 and 1 mg/mL X-gal (5-bromo-4-chloro-3-indolyl b-d-galactoside)]. Mesencephalic neurones expressing EGFP after adenoviral transduction were visualized and locally correlated to TH-ir (Cy3-positive) neurones using a LSM 510 laser scanning microscope (Zeiss, Jena, Germany). Transmitter uptake analysis High af®nity uptake of [3H]dopamine was carried out as described previously (Krieglstein and Unsicker 1997). At DIV5, cells were washed three times with the incubation solution (5 mm glucose, 1 mm ascorbic acid in PBS, pH 7.4) and incubated (15 min, 378C) in this solution, before adding 50 nm [3H]dopamine (15 min, 378C, Amersham). Uptake was stopped by removal of the incubation mixture, followed by three rapid washes with ice-cold PBS. After removal of PBS, 300 mL of distilled water were added, cultures frozen (2 h, 2 808C), thawed, and cells were scraped twice with an additional volume of 200 mL of distilled water. Radioactivitycontaining water was collected in vials and extracted radioactivity was measured by liquid scintillation counting after addition of 10 mL of scintillation cocktail per vial.

Fig. 2 SH-SY5Y neuroblastoma cells undergoing 6-OHDA-induced cell death show morphological features of apoptosis. After exposure to vehicle (a) or 40 mM of 6-OHDA for 6 h (b), cells were stained with the ¯uorescent chromatin dye Hoechst 33258. Vehicle-treated cultures showed high cell density and no condensed chromatin, whereas in 6-OHDA-treated cultures cell density was markedly decreased and many of the remaining cells exhibited chromatin condensation and nuclear fragmentation.

Statistical analysis Data are expressed as mean ^ SEM. Statistical signi®cance was assessed by one-way anova followed by Student±Newman±Keuls' multiple comparisons test. If not stated otherwise, all experiments reported represent at least three independent replications performed in triplicate.

Fig. 1 Effects of zVAD-fmk on 6-OHDA-induced cell death of SH-SY5Y neuroblastoma cells. After pretreatment with different concentrations of zVAD-fmk for 2 h, cells were exposed to 6-OHDA for 12 h in the absence or presence of the caspase inhibitor. Viability was assessed at 12 h using the MTT assay and photometric evaluation. Data are mean ^ SEM, n 4. **p , 0.01; ***p , 0.001 compared with the respective toxin-treated control values (corresponding white bar), ANOVA followed by Student±Newman±Keuls' multiple comparisons test.

Fig. 3 6-OHDA-induced activation of caspases 3, 6 and 9 is blocked by zVAD-fmk and adenovirus-mediated XIAP expression. Lysates of SH-SY5Y cells were harvested after 12 h of 6-OHDA treatment (lanes 1 and 2). Alternatively, cells were pretreated with zVAD-fmk for 2 h (lanes 3 and 4) or were infected with 10 MOI of AdV-LacZ (lane 5) or AdV-XIAP (lane 6) 12 h prior to 6-OHDA exposure. Twenty micrograms of protein were subjected to SDS gel analysis. The full-length proforms and 6-OHDA-induced cleaved fragments of caspase 3 (top), caspase 6 (middle) and caspase 9 (bottom) were detected by speci®c antibodies.



In the absence of 6-OHDA, caspase inhibitors did not signi®cantly affect survival. SH-SY5Y neuroblastoma cells undergoing 6-OHDA-induced cell death show morphological features of apoptosis The morphological features of SH-SY5Y-neuroblastoma cells undergoing 6-OHDA-induced cell death were examined using the ¯uorescent chromatin dye, Hoechst 33258. At 6 h of toxin exposure cell density decreased rapidly due to the detachment of damaged cells from the surface of the cell culture well. A high percentage of the remaining cells showed both chromatin condensation and nuclear fragmentation (Fig. 2).

Fig. 4 Time course of 6-OHDA-induced activation of caspase 3 in SH-SY5Y cells as measured by DEVD-amc hydrolysis. Cell extracts were prepared after treatment with 40 mM of 6-OHDA for 1, 3, 6, 9 and 12 h and were evaluated for DEVD-amc cleavage. Vehicle-treated cultures did not show any change of DEVD-amc cleavage over time. Data are mean ^ SEM, n 5.

Results The caspase inhibitor zVAD-fmk inhibits 6-OHDA-induced cell death of SH-SY5Y neuroblastoma cells The toxicity of 6-OHDA on SH-SY5Y neuroblastoma cells was tested at different concentrations by measuring viability at 12 h of toxin exposure. 6-OHDA concentration-dependently induced cell death (Fig. 1). Next, the protective effects of 100 mm and 200 mm of zVAD-fmk on 6-OHDAinduced toxicity were examined. Concomitant incubation with zVAD-fmk signi®cantly protected SH-SY5Y cells from death induced by 20 mm or 40 mm of 6-OHDA (Fig. 1). Both concentrations of zVAD-fmk were equally effective.

6-OHDA induces activation of caspases 3, 6 and 9 in SH-SY5Y neuroblastoma cells Using antibodies against speci®c caspases, we sought to identify the caspases that are activated in the process of 6-OHDA-induced cell death. Cleaved fragments, indicating the activation of the respective caspases, were detected with speci®c antibodies against caspases 3, 6 and 9 in lysates of SH-SY5Y cells treated with 6-OHDA for 12 h (Fig. 3, lane 2). The antibodies against caspases 3 and 6 detected the large p17 or p20 subunit, whereas the caspase 9 antibody detected an intermediary fragment (p37/39). To investigate whether treatment with caspase inhibitors interferes with the activation of caspases, SH-SY5Y cells were incubated for 2 h with 100 mm or 200 mm of zVAD-fmk prior to exposure to 6-OHDA. Caspase activation at 12 h was completely blocked by zVAD-fmk (Fig. 3, lanes 3 and 4) Time course of 6-OHDA-induced activation of caspase 3 in SH-SY5Y neuroblastoma cells Activation of the effector caspase 3 has been described as one key event of the apoptotic cascade in many cell culture models of apoptotic cell death. Proteolytic cleavage of procaspase 3 occurs within 12 h after exposure of SH-SY5Y cells to 6-OHDA (Fig. 3). To further characterize the time course of its activation, DEVD-amc cleavage was measured

Fig. 5 Adenovirus-mediated expression of LacZ and XIAP in SH-SY5Y cells. (a) At DIVI SH-SY5Y neuroblastoma cells were infected with 10 MOI of AdV-LacZ. Staining of cells expressing b-galactosidase was performed 12 h later using X-gal stain. (b) SH-SY5Y cells remained uninfected (lanes 1 and 2) or were infected with AdV-LacZ (lane 3) or AdV-XIAP (lane 4) followed by an incubation with 40 mM of 6-OHDA for 12 h. Cell lysates were prepared and subjected to SDS-polyacrylamide gel electrophoresis. XIAP was detected by a speci®c antibody.



Fig. 6 Adenovirus-mediated expression of XIAP protects SH-SY5Y neuroblastoma cells from 6-OHDA toxicity. Twelve hours after infection with 10 MOI of AdV-XIAP, 10, 20 or 40 mM of 6-OHDA were added to the culture medium. Viability was assessed at 12 h using the MTT assay and photometric evaluation. Data are mean ^ SEM, n 4. *p , 0.05; ***p , 0.001 compared with the respective toxintreated control values (corresponding white bar), ANOVA followed by Student±Newman±Keuls' multiple comparisons test.

at 1 h, 3 h, 6 h, 9 h and 12 h after addition of 6-OHDA to the cell cultures. After 3 h of exposure of SH-SY5Y neuroblastoma cells to 40 mm of 6-OHDA, there was a steep increase of caspase 3-like enzymatic activity with a peak after 6 h and a subsequent gradual decrease (Fig. 4).

Adenovirus-mediated expression of XIAP protects SH-SY5Y neuroblastoma cells against 6-OHDA toxicity To investigate the feasibility of gene transfer by adenoviral vectors in this cell culture system, we used an adenoviral construct encoding LacZ (AdV-LacZ). At DIV1, SH-SY5Y neuroblastoma cells were infected with 10 MOI of AdVLacZ. Cells were stained for b-galactosidase 12 h after infection. While there was no endogenous b-galactosidase activity in control cultures, virtually 100% of cells in AdVLacZ-treated cultures were b-galactosidase-positive (Fig. 5a). In another experiment, cells were either infected with AdVXIAP or AdV-LacZ, or not infected at all. Cultures were subsequently exposed to 40 mm of 6-OHDA for another 12 h. In cultures infected with AdV-XIAP, strong transgene expression was detected by western blot (Fig. 5b). Without transfection and in AdV-LacZ-treated cultures there was a weak endogenous expression of XIAP that was not regulated after 6-OHDA treatment. Next, we tested the ef®cacy of adenovirus-mediated gene transfer of XIAP to protect SH-SY5Y neuroblastoma cells from 6-OHDA-induced cell death. The infection procedure was the same as described above. At 12 h after infection, 6-OHDA was added to the culture medium at 10 mm, 20 mm or 40 mm. Cell viability was assessed at 12 h of treatment with the toxin. In the absence of 6-OHDA, adenovirally infected cultures showed slightly decreased cell survival compared with control cultures not exposed to viral vectors

Fig. 7 The caspase inhibitor zVADfmk inhibits 6-OHDA-induced cell death of cultured mesencephalic dopaminergic neurones. (a) Cultures were treated with different concentrations of 6-OHDA for a period of 24 h beginning at DIV4. (b) After a 2-h incubation with zVAD-fmk, 40 mM of 6-OHDA were added to the culture medium for 24 h starting at DIV4. After immunocytochemical staining TH-ir cells were counted across one diameter of the coverslip, comprising an area of 8 mm2. Data are mean ^ SEM, n 4. *p , 0.05; **p , 0.01; ***p , 0.001; ANOVA followed by Student± Newman±Keuls' multiple comparisons test.



Fig. 8 zVAD-fmk does not provide protection against 6-OHDAinduced loss of neurites and dopamine uptake sites in mesencephalic dopaminergic neurones. After a 2-h incubation with zVAD-fmk, 40 mM of 6-OHDA were added to the culture medium. TH-ir neurones were visualized by immunocytochemical staining and further analysed at 250-fold magni®cation. (a±c) TH-ir mesencephalic dopaminergic neurones at 24 h after treatment with (a) vehicle alone (b) 40 mM of 6-OHDA, and (c) 200 mM of zVAD-fmk for 2 h

followed by addition of 40 mM of 6-OHDA. (d) The length of the cell processes of TH-ir neurones was determined by morphometric analysis. Data are mean ^ SEM, n 200. (e) The percentage of TH-ir neurones with cell processes shorter than 10 mm was determined counting 100 TH-ir cells. Data are mean ^ SEM, n 3. (f) At 24 h cell cultures were incubated with [3H]dopamine for 15 min and subsequently processed for liquid scintillation counting. Data are mean ^ SEM, n 4.

(Fig. 6). However, the extent of 6-OHDA-induced cell death was not affected by adenovirus-mediated expression of LacZ. In contrast, ectopic expression of XIAP signi®cantly inhibited 6-OHDA toxicity. The activation of caspases 3, 6 and 9 was blocked by adenovirus-mediated ectopic expression of XIAP, while transfection with AdV-LacZ did not inhibit cleavage of procaspases (Fig. 3, lanes 5 and 6).

immunocytochemical staining for TH-ir neurones. 6-OHDA induced cell death of dopaminergic mesencephalic neurones (Fig. 7a) in a dose-dependent manner. Incubation with zVAD-fmk starting 2 h before the exposure to 6-OHDA concentration-dependently protected TH-ir neurones from 6-OHDA-induced cell death (Fig. 7b). A higher concentration of zVAD-fmk (400 mm) resulted in toxicity to mesencephalic dopaminergic neurones and did not provide better protection against toxin-induced cell death (data not shown). Morphologically, treatment of mesencephalic cultures with 6-OHDA resulted not only in cell death but also in a marked loss of cell processes of the remaining TH-ir neurones (Figs 8a and b). In spite of the increased number of surviving TH-ir neurones, the addition of zVAD-fmk did not provide protection against the loss of neurites after 6-OHDA-treatment (Fig. 8c). To quantify this effect, we determined the mean length of TH-ir cell processes (Fig. 8d) as well as the number of TH-ir neurones with only rudimentary cell processes (length , 10 mm) (Fig. 8e) under the different culture conditions. zVAD-fmk was not effective to block the toxic effects of 6-OHDA on neurites. The same result was found using the caspase inhibitor at a concentration of 400 mm (data not shown).

The caspase inhibitor zVAD-fmk inhibits 6-OHDA-induced cell death of mesencephalic dopaminergic neurones but does not attenuate the disruption of neuronal processes Next, we investigated the ef®cacy of these anti-apoptotic strategies in differentiated, nonmitotic dopaminergic cells using a cell culture model of rat embryonic mesencephalic dopaminergic neurones. As previously described, under the conditions used here these cultures contain approximately 98% neuronal cells and virtually no cells expressing the glial ®brillary acidic protein (GFAP) (Krieglstein et al. 1995; Krieglstein and Unsicker 1997). The percentage of TH-positive cells amounts 8±15%. At DIV4, the mesencephalic cultures were treated with 20 mm, 40 mm or 60 mm 6-OHDA for 24 h. Survival was assessed at DIV5 by



Fig. 9 Adenovirus-mediated gene transfer is not effective in cultured mesencephalic dopaminergic neurones. (a) and (b) Cultures were infected with 200 MOI of AdVLacZ or AdV-XIAP at DIV1 and processed for (a) X-gal staining or (b) immunoblotting using an antibody speci®c for XIAP at DIV4. (c±e) Cultures were infected with 200 MOI of AdV-EGFP at DIV1 and processed for immunocytochemistry at DIV4 using a TH-speci®c primary antibody and a Cy3labelled second antibody. Laser scanning images showed (c) red TH-ir (Cy3-labelled) neurones and (d) green adenovirally infected (EGFP-expressing) neurones, but (e) virtually no colocalization of these two labellings. (f) Cultures were infected with 200 MOI of AdV-LacZ or AdV-XIAP at DIV1 and treated with 40 mM of 6-OHDA for 24 h starting at DIV4. After immunocytochemical staining TH-ir cells were counted across one diameter of the coverslip, covering an area of 8 mm2. Data are mean ^ SEM, n 3.

To evaluate the functional relevance of theses ®ndings, we used a transmitter uptake assay testing primarily the functional integrity of dopaminergic nerve terminals. At DIV4 cultures were incubated with 100 mm or 200 mm of zVAD-fmk for 2 h and subsequently treated with 40 mm of 6-OHDA. Exposure to 6-OHDA for 24 h decreased the uptake of [3H]dopamine to 31% of vehicle treated controls (Fig. 8f). This reduction was not attenuated by incubation with zVAD-fmk at any concentration tested. Lack of adenovirus-mediated gene transfer in mesencephalic dopaminergic neurones causes failure of AdV-XIAP to protect against 6-OHDA toxicity As gene transfer by viral vectors might offer a clinically relevant therapeutic approach to deliver anti-apoptotic proteins to the CNS, we tested the ef®cacy of adenoviral gene transfer in this primary culture system of differentiated neurones. Cultured mesencephalic neurones were infected at DIV1 with concentrations up to 200 MOI using AdVLacZ. Cells were stained for b-galactosidase activity at DIV4, DIV5 or DIV6. In control cultures, no endogenous

b-galactosidase activity was detected (data not shown). In AdV-LacZ-infected cultures we found intense blue staining of approximately 10±20% of neurones (Fig. 9a). Transduction of mesencephalic cultures with 200 MOI of AdV-XIAP at DIV1 led to transgene expression of a protein with the molecular weight expected for XIAP as shown by western blotting (Fig. 9b). Since these labellings did not differentiate between dopaminergic and nondopaminergic neurones, we accomplished a simultaneous labelling of TH-ir neurones and cells expressing the adenovirally transmitted transgene. We combined standard immunocytochemical procedures using a Cy3-labelled second antibody with adenovirally mediated expression of EGFP. Using a laser scanning microscope, we found that there is virtually no colocalization of these two labellings (Figs 9c±e) indicating that under the conditions chosen here, dopaminergic neurones are not ef®ciently transfected or do not express the transgene in a suf®cient amount to be detected by these techniques. In accordance with these results, cell death of dopaminergic neurones induced by 40 mm of 6-OHDA was not inhibited by preceding infection with AdV-XIAP (Fig. 9f).



Discussion Today, only symptomatic but no neuroprotective therapies for the treatment of PD are available. During the course of the disease, there is a further decrease in the number of dopaminergic neurones, resulting in reduced ef®cacy of symptomatic therapy and in complications of treatment including motor ¯uctuations and dyskinesias. The characterization of the underlying cell death mechanisms will help to identify targets of treatment to achieve neuroprotection. The present ®ndings demonstrate that 6-OHDA, a toxin that has been widely used to create in vitro and in vivo models of PD, induces the morphological characteristics of apoptosis and activates caspases 3, 6 and 9 in dopaminergic cells. Although the peptide caspase inhibitor, zVAD-fmk, provides protection against 6-OHDA-induced death in SH-SY5Y cells and primary dopaminergic neurones, it does not protect against the loss of neurites and dopamine uptake sites, suggesting that primary dopaminergic neurones remain functionally impaired. The inhibition of caspases by adenovirus-mediated ectopic expression of XIAP offers an alternative to peptide inhibitors and allows targeting of transgene expression at speci®c cells with the use of cell type-speci®c promoters. However, at least in the primary cell culture system employed here, the latter opportunity appears to be limited by the reduced transfection ef®cacy of dopaminergic compared with nondopaminergic neurones. The family of caspases are key mediators in the apoptotic pathway. Caspases are a family of cysteine proteases with aspartyl protease activity (for review, see Nicholson 1999). Group I caspases comprise caspases 1, 4, 5 and 13. This pro®le is consistent with their known role in cytokine processing but excludes a major function in apoptosis. Group II caspases (2, 3 and 7) prefer a cleavage motif DExD found in many proteins cleaved during apoptosis. As a result, group II caspases are considered to be executioners of apoptosis. Group III caspases (6, 8, 9 and 10) are considered to be initiator caspases that can cleave and thereby activate group II execution caspases. To date in most studies the identi®cation of caspase activation was based on the protective effects of caspase peptide inhibitors or ¯uorogenic cleavage substrates to monitor caspase activity. Since these inhibitors and substrates only provide limited speci®city, they may not allow determination of which of the different caspases is activated. So far, only caspase 3 has been identi®ed by a speci®c antibody to be cleaved during 6-OHDA toxicity (Dodel et al. 1999). We show here for the ®rst time that in addition to the activation of caspase 3, the initiator caspases 6 and 9 are activated in response to 6-OHDA. Caspase 9 is activated in an ATP-dependent manner in a multiprotein complex composed of apoptotic protease activating factor-1 (Apaf-1), cytochrome c and procaspase 9, and de®nes the `mitochondrial pathway' of caspase activation that may be activated

independently from cell death receptors and the recruitment of caspase-8. Since exogenous application of CD95L does not induce death of SH-SY5Y cells (S. Haid and J.B. Schulz, unpublished observation), the `mitochondrial' cytochromedependent pathway appears to be activated by 6-OHDA. This suggestion is supported by the demonstration of 6-OHDA-induced cytochrome c translocation from mitochondria to the cytosol (Dodel et al. 1999). In cytochrome c-dependent apoptotic pathways, the activation of caspase 6 is likely to occur downstream from caspase 3 (Slee et al. 1999). IAPs inhibit the group II caspases 3 and 7 (Nicholson 1999; Robertson et al. 2000) and may inhibit the group III caspase 9 (Deveraux et al. 1998, 1999), but not group I caspases (e.g. caspase 1) or other group III caspases (e.g. caspase 8). The block of caspase 6 activation by ectopic XIAP expression observed in this study may result from direct inhibition or ± more likely ± from inhibition of an upstream caspase (e.g. caspase 3 or 9). In either case, our results provide evidence that XIAP provides protection against 6-OHDA in in vitro models of PD, although it may not restore the function of dopaminergic neurones. Further indication for the importance of caspases in the death of dopaminergic neurones comes from studies showing that caspase inhibitors applied ex vivo increase survival of dopaminergic neurones grafted to hemiparkinsonian rats and thereby substantially improve functional recovery (Schierle et al. 1999). The discrepancy between the protective effects of XIAP in SH-SY5Y cells and the failure to prevent 6-OHDAinduced death of primary dopaminergic neurones is explained by the insuf®cient transgene expression in dopaminergic compared with nondopaminergic neurones. The fact that dopaminergic substantia nigra neurones can be transfected with high ef®cacy after striatal injection of the same adenoviral construct via retrograde transport argue against a general failure of adenoviral constructs to transfect postmitotic dopaminergic neurones (Eberhardt et al. 2000). Based on the data presented here, we cannot decide whether the lack of transgene expression in vitro is caused by an insuf®cient transfection or insuf®cient cellular expression. The use of different promotors and in situ-hybridization may help to answer this question in the future. Inhibition of caspases may provide promising opportunities for the treatment of acute or chronic neurodegenerative disorders (Schulz et al. 1999; Robertson et al. 2000). Although zVAD-fmk protected primary dopaminergic neurones from death, it did not prevent the degeneration of neurites and the reduction of dopamine uptake. Since 6-OHDA is selectively taken up by dopaminergic terminals, these terminals may be the primary target of 6-OHDA neurotoxicity followed by a slower and secondary death of the dopaminergic cell bodies mediated by caspase activation and apoptosis. The likely explanation for the dissociation



between the rescue of TH-ir neurones and the failure to maintain biochemical parameters of dopaminergic function is the loss of dopaminergic synaptic terminals despite treatment with zVAD-fmk treatment. Although an important role for synaptic caspase activation and apoptosis has been proposed (Mattson 2000), axonal degeneration after withdrawal of trophic support occurs without the activation of caspases in contrast to the cell death of the soma (Finn et al. 2000). Our ®ndings suggest that although dopaminergic cell somata are protected from 6-OHDA toxicity, they may be functionally impaired. Similar observations of functional impairment have been made in NGF-deprived sympathetic neurones rescued by peptide caspase inhibitors which showed smaller somata, no dendrites and maintained only basal levels of protein synthesis (Deshmukh et al. 1996). We have recently shown that gene transfer of XIAP to dopaminergic neurones protects the somata but not the neurites against MPTP toxicity in vivo. In contrast, the combination of XIAP and adenovirus-mediated GDNF expression not only rescued dopaminergic somata but also protected against the loss of nerve terminals (Eberhardt et al. 2000). Therefore, a combination of a caspase inhibitor and a neurorestorative therapy, e.g. a neurotrophic factor, may provide additive or synergistic effects. Acknowledgements This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Sch 932/2±1 to JBS). We thank Sibylle Haid for excellent technical assistance. We thank D.W. Nicholson for providing antibodies for the detection of caspases 3 and 6, Kevin Tomaselli for providing an antibody directed to caspase 9, and P. Liston for an antibody directed against XIAP.

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