Human Molecular Genetics, 2000, Vol. 9, No. 19 2859-2867
© 2000 Oxford University Press
Intranuclear huntingtin increases the expression of caspase-1 and induces apoptosis
Department of Genetics, Emory University School of Medicine, 1462 Clifton Road NE, Atlanta, GA 30322, USA
Received 2 August 2000; Revised and Accepted 29 September 2000.
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ABSTRACT |
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Expansion of a polyglutamine repeat in huntingtin causes Huntington’s disease (HD). Although full-length huntingtin is predominantly distributed in the cytoplasm, N-terminal fragments of huntingtin with expanded polyglutamine tracts are able to accumulate in the nucleus and kill neurons through apoptotic pathways. Transgenic mice expressing N-terminal mutant huntingtin show intranuclear huntingtin accumulation and develop progressive neurological symptoms. Inhibiting caspase-1 can prolong the survival of these HD mice. How intranuclear huntingtin is associated with caspase activation and apoptosis is unclear. Here we report that intranuclear huntingtin induces the activation of caspase-3 and the release of cytochrome c from mitochondria in cultured cells. As a result, cells expressing intranuclear huntingtin undergo apoptosis. We show that intranuclear huntingtin increases the expression of caspase-1, which may in turn activate caspase-3 and trigger apoptosis. We propose that the increased level of caspase-1 induced by intranuclear huntingtin contributes to HD-associated cell death.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder resulting from expansion (>37 units) of a polyglutamine tract in huntingtin, a 350 kDa protein of unknown function (1). The N-terminal region of huntingtin contains the glutamine repeat, which is encoded by exon 1 of the HD gene. Normal huntingtin is a cytoplasmic protein and is expressed ubiquitously, but N-terminal fragments of huntingtin with expanded polyglutamine tracts are able to accumulate in the nucleus and kill cells (2–8). For example, transgenic mice (R6/2) expressing the HD exon 1 protein with >115 glutamines develop neurological symptoms and neuronal intranuclear inclusions consisting of huntingtin aggregates (2). Similar nuclear aggregates are found in patients with HD (3–5) and other polyglutamine diseases (9). However, the role of intranuclear aggregates remains elusive, though their formation correlates with disease progression in HD mice (2,10).
Despite the unclear role of huntingtin aggregates, several studies suggest that the intranuclear localization of mutant huntingtin is sufficient to cause cells to die (6–8,11). In transfected striatal neurons, cell death occurs when soluble transfected huntingtin is localized in the nucleus (6). Moreover, SCA1 transgenic mice show that the elimination of nuclear localization, but not the aggregation, of ataxin-1 with an expanded glutamine repeat can prevent neuronal degeneration (12). Our recent studies also show that intranuclear mutant huntingtin induces multiple cellular defects in PC12 cells (11).
There is growing evidence that proteins with expanded polyglutamine tracts kill cells through apoptotic pathways (6,8,13,14). Although R6/2 HD transgenic mice do not show obvious neurodegeneration, inhibition of caspase-1 can slow their disease progression (15). Thus, early apoptotic events may be involved in the neuropathology of these HD mice. However, in post-mortem HD brain tissue (16) and in HD transgenic mice that express full-length mutant huntingtin (17), apoptotic cell death has been observed. How intranuclear mutant huntingtin causes apoptotic events still remains to be investigated. Very recently, it was found that the expression of caspase-1 is increased in HD mice and that inhibition of this expression by minocycline also slows the progression of symptoms (18). Since these HD mice express the transgenic mutant huntingtin in the nucleus, an important question is whether the increased caspase activity is due to the intranuclear effect of mutant huntingtin. One possibility is that intranuclear huntingtin may affect the expression of genes important for neuronal function or cell viability (11,19–21). In the present study, we show that intranuclear huntingtin can increase the expression of caspase-1 and mediate apoptotic events.
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RESULTS |
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Apoptosis in huntingtin-transfected PC12 cells
Our previous studies established stably transfected PC12 cells expressing the HD exon 1 protein with 150 glutamines (150Q-E). These cells show altered gene expression and are susceptible to apoptotic stimuli (11). To examine whether these cells undergo apoptosis even in the absence of any exogenous insult, we let 150Q-E PC12 cells grow for 3–4 days without changing the culture medium. PC12 cells stably expressing the HD exon 1 protein with 20 glutamines (20Q-E) were included as a control. Immunostaining of huntingtin with EM48, an antibody against the N-terminal region of huntingtin, clearly shows that the majority of the 20Q-E protein is in the cytoplasm, whereas the 150Q-E protein is concentrated in the nucleus (Fig. 1a). Moreover, 150Q-E PC12 cells display obvious nuclear DNA fragmentation. To confirm this, we isolated genomic DNA from these cells and examined DNA integrity using agarose gel electrophoresis. Extensive DNA fragmentation is evident in 150Q-E PC12 cells, but not in 20Q-E PC12 cells (Fig. 1b).
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Intranuclear huntingtin activates caspases
To study the activation of caspases in huntingtin-transfected PC12 cells, we first focused on caspase-8 and caspase-3, as these caspases have been reported to be involved in huntingtin-induced apoptosis (13,14). Human cDNAs encoding caspase-8 and caspase-3 were isolated with RT–PCR based on published sequences (22,23). These caspases were synthesized in vitro and radiolabeled with [35S]methionine. The radiolabeled caspases were then incubated with extracts of 150Q-E and 20Q-E PC12 cells to examine their cleavage, a process required for caspase activation. The cleavage of caspase-3, but not capsapse-8, was caused by 150Q-E PC12 cell extracts (Fig. 2a). Activated caspase-3 is also able to cleave its substrates, such as poly(ADP-ribose)polymerase (PARP). Thus, we measured PARP cleavage products using western blots. 150Q-E PC12 cells contain more cleaved PARP (89 kDa) and less uncleaved PARP (125 kDa) than do 20Q-E PC12 cells (Fig. 2b). This finding further suggests that the activity of caspase-3 is higher in 150Q-E PC12 cells.
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Since the cleavage of caspase-8 is initiated by activating the death receptor FasR in the plasma membrane (24) and can also be activated by cytoplasmic polyglutamine aggregates (13), intranuclear huntingtin may trigger different intracellular signaling pathways that lead to the cleavage of caspase-3, but not caspase-8. A biochemical assay of caspase-3 activity showed an increased activity of the endogenous caspase-3 in 150Q-E PC12 cells, which can be inhibited by the caspase inhibitor DEVD-fmk (10 µM) (Fig. 2c). If caspase-3 is activated in 150Q-E PC12 cells, inhibition of caspases should improve cell viability. As expected, the caspase inhibitors ZVAD-fmk, Boc-D-fmk and the caspase-3-specific inhibitor DEVD-fmk all significantly improved cell viability of 150Q-E PC12 cells (Fig. 2d).
Release of cytochrome c from mitochondria is another important apoptotic event. Therefore, we examined the presence of cytochrome c in the cytosolic fraction of 20Q-E PC12 and 150Q-E PC12 cells. Cells expressing the 150Q-E protein have an increased level of cytochrome c in the cytosolic fraction (Fig. 3a). Since cytochrome c release is associated with mitochondrial oxidative stress (25), we examined the level of the mitochondrial antioxidant manganese superoxide dismutase (SOD2), which is often upregulated by mitochondrial oxidative stress (26). SOD2 was significantly higher in 150Q-E PC12 cells than in 20Q-E PC12 and wild-type PC12 cells (Fig. 3b), indicating that the 150Q-E PC12 cells were in a state of mitochondrial oxidative stress. Mitochondrial DNA rearrangement is frequently associated with oxidative stress (26). Indeed, obvious mitochondrial DNA rearrangement was observed in 150Q-E PC12 cells, but not in wild-type PC12 cells or 20Q-E PC12 cells (Fig. 3c).
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Intranuclear huntingtin induces apoptosis
To confirm that the intranuclear huntingtin-induced effects seen in 150Q-E PC12 cells are not due to the specific biological properties of the cloned cell line, we also expressed mutant huntingtin in non-neuronal 293 cells using transient transfection. Since overexpression of mutant huntingtin in 293 cells often leads to cytoplasmic and perinuclear aggregates that prevent the intranuclear accumulation of mutant huntingtin, we added nuclear localization sequences (NLSs) to the N-terminal region of huntingtin and transfected this construct into 293 cells. The NLS-tagged huntingtin proteins (NLS-150Q and NLS-20Q) were localized to the nucleus of the transfected cells, whereas the 20Q and 150Q proteins without NLSs were predominantly localized in the perinuclear or cytoplasmic region (Fig. 4a). Western blots show that both the NLS-150Q and 150Q proteins form aggregated proteins that remain in the stacking gel (Fig. 4b).
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The distinct subcellular localization of the NLS-150Q and 150Q proteins allowed us to examine whether intranuclear huntingtin with an expanded polyglutamine tract induces apoptotic events such as cytochrome c release. We therefore transfected NLS-150Q huntingtin into 293 cells. NLS-150Q-transfected 293 cells show the presence of cytochrome c in their cytosolic fraction (Fig. 5a). Moreover, the caspase inhibitor ZVAD-fmk, which inhibits a variety of caspases, significantly reduced the release of cytochrome c from both 293 cells expressing NLS-150Q and PC12 cells expressing 150Q-E (Fig. 5b). Since the release of cytochrome c from mitochondria can be mediated by activation of caspases (27), the inhibition of caspase inhibitors on cytochrome c release in cells expressing intranuclear mutant huntingtin suggests that mutant huntingtin in the nucleus may increase the activity of caspases to induce apoptosis.
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Intranuclear huntingtin induces the expression of caspase-1
To study how intranuclear huntingtin induces apoptosis, we wanted to examine whether intranuclear huntingtin influences the expression of genes that are involved in apoptosis. Recent studies on an HD mouse model show that caspase-1 inhibition slows disease progression (15). Our previous studies using differential display PCR and RT–PCR showed that intranuclear huntingtin affects the transcription of a number of genes (11). Further characterization of these gene products suggests that the expression of caspase-1 may be increased by intranuclear huntingtin. The level of caspase-1 transcripts is very low in cultured PC12 and 293 cells, making it difficult to detect them on northern blots. Therefore, we used RT–PCR to analyze the transcript level of caspase-1 in 150Q-E and 20Q-E PC12 cells. The result shows that caspase-1 expression is indeed higher in 150Q-E PC12 cells, whereas the level of a control mRNA (glyceraldehyde phosphate dehydrogenase, GAPDH) is almost the same in both 150Q-E and 20Q-E PC12 cells (Fig. 6a). To confirm that the protein level of caspase-1 is also higher, we performed a western blot with anti-caspase-1 antibody. As expected, the expression of caspase-1 protein is upregulated in 150Q-E PC12 cells (Fig. 6b). A small immunoreactive band (10 kDa) was also seen in 150Q-E PC12 cells, suggesting that caspase-1 is cleaved and activated. Furthermore, we examined whether transient transfection of NLS-150Q into 293 cells also increases the expression of caspase-1. RT–PCR and Southern blot analysis show that the level of caspase-1 transcripts is significantly higher in cells transfected with NLS-150Q than with NLS-20Q or the 150Q protein without an NLS (Fig. 6c). Although RT–PCR is not a very quantitative assay, the expression of GAPDH was not altered under the same PCR conditions, suggesting that the increased level of caspase-1 is specific to NLS-150Q transfection. Thus, both stable and transient transfection experiments consistently show that intranuclear huntingtin with polyglutamine expansion induces the expression of caspase-1.
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To confirm that the increased expression of caspase-1 also leads to its higher activity, we measured caspase-1 activity in huntingtin-transfected cells. Caspase-1 activity in transiently transfected 293 cells expressing intranuclear mutant huntingtin fragments (NLS-150Q) was significantly higher than that in cells expressing huntingtin with a normal glutamine repeat (NLS-20Q) or extranuclear mutant huntingtin (150Q) (Fig. 7a). To study whether the increase in caspase-1 activity occurs prior to the increase of caspase-3 activity, we measured the activities of these caspases in stably huntingtin transfected PC12 cells at 1, 2 and 3 days after culturing. Similarly, cells stably expressing intranuclear mutant huntingtin fragments (150Q-E) showed higher activities of caspases than those expressing huntingtin fragments with a normal glutamine repeat (20Q-E). More importantly, a significant increase in caspase-1 activity was found at day 2, whereas the increased activity of caspase-3 was not seen until day 3 (Fig. 7b). These results are consistent with in vitro evidence demonstrating early caspase-1 and delayed caspase-3 activation during apoptosis (28) and the in vivo findings that increased caspase-3 expression begins after caspase-1 mRNA elevation in HD animals (18).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The findings in the present study provide biochemical evidence, namely increased caspase-1 and -3 activity, cytochrome c release and higher SOD2 expression, for the involvement of intranuclear huntingtin in apoptosis. The increase in SOD2 is especially interesting, as this protein is an anti-oxidative enzyme that is upregulated when mitochondria undergo oxidative stress (26). Additional evidence supporting the presence of oxidative stress is the rearrangement of mitochondrial DNAs in 150Q-E PC12 cells. Similarly, it has been reported that HD mice expressing mutant huntingtin in the nucleus show mitochondrial dysfunction and increased oxidative stress (29,30). These findings support the hypothesis that mitochondrial oxidative stress is involved in the pathogenesis of HD (14,31).
Cytochrome c released from mitochondria interacts with Apaf-1 and procaspase-9, which in turn activate caspase-3 (32). Although we did not measure the activity of caspase-9 in mutant huntingtin-transfected cells, the increased activity of caspase-3 and high level of cytochrome c in the cytosolic fraction suggest that caspase-9 is likely to be activated in these cells. The more interesting question is how cytochrome c release and caspase-3 activity are increased in cells that express mutant huntingtin in their nuclei. Caspase-8, which also increases cytochrome c release and caspase-3 activity, has been reported to be co-localized with polyglutamine aggregates in the cytoplasm of transfected cells (13). The increased activity of caspase-8 is thought to be due to recruiting this caspase by polyglutamine aggregates (13). In 150Q-E PC12 cells, mutant huntingtin predominantly accumulates in the nucleus and does not form obvious aggregates (11). In addition, the lysates of these cells do not cleave caspase-8, despite their cleavage of caspase-3. Thus, intranuclear mutant huntingtin may induce apoptosis through a mechanism different from that for cytoplasmic huntingtin. We as well as others have reported that intranuclear huntingtin affects the expression of multiple genes (11,19,20). It is very likely that intranuclear huntingtin affects the expression of the genes that are important for the function of mitochondria and caspase activities such that their altered expression contributes to mitochondrial oxidative stress and apoptosis.
Since the inhibition of caspase activity reduces the release of cytochrome c from mitochondria in cells expressing mutant huntingtin (Fig. 5b), the activation of caspases may, at least in part, contribute to cytochrome c release and mitochondrial oxidative stress seen in our huntingtin-transfected cells. Very recently, caspase-1 inhibition is found to slow disease progression in HD transgenic mice (15). Caspase-1 is also found to facilitate a transition in mitochondrial permeability that is critical for releasing cytochrome c (33,34). Thus, it is very likely that the increase in activity of caspase-1 could be an initial step toward other caspase activation and mitochondrial dysfunction. Using cells stably or transiently expressing mutant huntingtin in their nuclei, we show that the increased expression of caspase-1 and its activity are associated with intranuclear mutant huntingtin. This finding is consistent with the report that caspase-1 inhibition can slow disease progression in HD transgenic mice that express the same transgenic huntingtin as in the huntingtin-transfected cells in the present study (15). These mice show intranuclear huntingtin accumulation (3) and increased expression and activity of caspase-1. Inhibition of caspase activity or expression extends the survival of these mice and delays the onset of symptoms (15,18). Inhibition of caspase-1 also delays motor neuron degeneration in amyotrophic lateral sclerosis (ALS) (35,36) and reduces injury following cerebral ischemia (37–39). All these findings suggest that caspase-1 plays a critical role in neurodegeneration.
Caspase-1 is not activated by cytochrome c in cell-free extracts (40). Several studies suggest that caspase-1 mediates early apoptotic processes and that caspase-3 is involved in later stages of the apoptotic pathways (28,36,41,42). For example, caspase-1 activity is detected transiently in apoptotic cells, followed by a gradual increase in caspase-3 activity (28,42). In addition, the increased expression of caspase-1 mRNA precedes the elevation of caspase-3 expression in an ALS mouse model (36) and HD mouse model (18). Consistently, an increase in caspase-1 activity also occurs earlier than that for caspase-3 in huntingtin-transfected cells (Fig. 7). Furthermore, the idea that caspase-1 may act as an initiator for activating caspase-3 is supported by the finding that caspase-1 activates caspase-3 in vitro (41) and that caspase-1 expression is upregulated prior to caspase-3 in HD mice (18).
The present study suggests that intranuclear mutant huntingtin increases the expression of caspase-1 at the transcriptional level, which is also consistent with the nuclear effect of mutant huntingtin on gene expression (11,19–21). Given that intranuclear huntingtin can induce caspase-1 expression and apoptosis in cultured cells, an intriguing question is how intranuclear huntingtin contributes to the specific neuropathology of HD. Recent studies of HD-repeat knock-in mice indicate that the earliest pathologic event is the intranuclear localization of mutant huntingtin in striatal neurons that are preferentially affected in HD (43,44). Thus, cell-type specific factors in striatal neurons may promote the intranuclear accumulation of huntingtin, potentially an initial step toward a variety of neuropathological events including the increase in the expression of caspase-1 and neuronal cell death.
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MATERIALS AND METHODS |
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Transfected cells
PC12 cells stably transfected with huntingtin constructs were obtained in our previous studies (11). Transient transfection of 293 cells was performed with lipofectamine. Cells were transfected for 24–48 h and collected for analysis. For immunofluorescent staining, transfected cells grown in chamber slides were fixed in 4% paraformaldehyde and treated with 0.2% Triton X-100. Hoechst dye (1 µg/ml) was used to label the nuclei. A Zeiss fluorescent microscope (Axioskop 2) and video system (Dage-MTI, Michigan City, IN) were used to capture images. The captured images were stored and processed using Adobe Photoshop software.
DNA fragmentation
Cultured cells were collected and lyzed in 1 ml of lysis buffer (20 mM Tris–HCl, 10 mM EDTA and 0.5% Triton X-100 pH 8.0) for 10 min at room temperature. Genomic DNA was extracted with phenol–chloroform and precipitated with 0.3 M sodium acetate and 70% alcohol. The pellet was resuspended in 10 mM Tris–HCl pH 8.0 and 1 mM EDTA. After digestion with RNase (0.1 mg/ml) at 37°C for 1 h, samples were electrophoresed through a 1.2% agarose gel. Mitochondrial DNA rearrangement was examined using a method described previously (26). Briefly, total genomic DNA was purified from cultured cells and amplified by PCR with two primers specific for mitochondrial DNA (rMito DNA 15134, 5'-AATCGGAGGCCAACCAGTAGAACACC-3'; and rMito DNA 14527, 5'-GTAGCTCCTCAGAATGATATTTGTCCTC-3'). The PCR reaction contained genomic DNA (200 ng), 200 nM of each primer and 4 U of Taq polymerase (Boehringer, Mannheim, Germany) in 50 µl total volume. Annealing was performed at 68°C for 10 min and the reaction was run for 35 cycles. Intact mitochondrial DNA appears as a 16 kb band, whereas rearranged DNA with truncations or deletions gives rise to smaller bands on a 1% agarose gel.
Subcellular fractionation and western blot analysis
Subcellular fractionation and mitochondrial isolation from cultured cells were performed as described previously (26). Briefly, cells were suspended in H-buffer (210 mM mannitol, 70 mM sucrose, 1 mM EGTA, 5 mM HEPES, 0.5% bovine serum albumin, pH 7.2). The cells were then homogenized in a Douncer with 10 strokes. The homogenate was transferred into a cold 1.5 ml Eppendorf tube and centrifuged at 1300 g at 4°C for 5 min to remove nuclei, unbroken cells and large membrane fragments. The supernatant was then transferred to a new, cold 1.5 ml Eppendorf tube and was centrifuged at 14 000 g at 4°C for 15 min. The mitochondrial pellet was washed in cold H-buffer, recentrifuged at 14 000 g and then resuspended in 100 µl of H-buffer. The high-speed (14 000 g) supernatant was used to measure cytochrome c released from mitochondria (26). Protein samples (50 µg) were then resolved by 10 or 15% SDS–PAGE. GST fusion protein antibody EM48, which is specific to the N-terminal region (amino acids 1–256) of human huntingtin, was obtained in our previous studies (5). Antibodies against caspase-1 (M-20; Santa Cruz Biotechnology, Santa Cruz, CA), tubulin (Sigma, St Louis, MO), cytochrome c (Transduction Laboratories, Lexington, CA), PARP (Enzyme Systems, Dublin, CA), SOD2 and the adenine nucleotide translocator (ANT-2) (26; provided by Dr B.A. Cottrell, Emory University) were also used for western blots.
Caspase cleavage, activity and cell viability assays
Human caspase-8 and caspase-3 cDNAs were isolated by RT–PCR using primers based on published sequences (22,23). Human caspase-8 cDNA was amplified from nucleotides 257–1730 using primers 5'-ATGGATCCATGGACTTCAGCAGAAATCTTTA-3' (BamHI) and 5'-CATCTAGATCAATCAGAAGGGAAGAC-3' (XbaI). Human caspase-3 cDNA was amplified from nucleotides 224–1065 using primers 5'-ATGGATCCATGGAGAACACTGAAAACTCAG-3' (BamHI) and 5'-CTTCTAGAAACCACCAACCAACC-3' (XbaI). The PCR products were subcloned into the PRK vector and sequenced at the Emory DNA core facility. In vitro translation of these cDNAs was performed with TNT in vitro translation kit (Promega, Madison, WI) and SP6 RNA polymerase in the presence of [35S]methionine. The in vitro synthesized caspases were incubated with lysates of huntingtin stably transfected cells at 30°C for 30 min using the method described previously (45). Cleaved caspase products were resolved on SDS–PAGE and subjected to autoradiography.
Caspase activity was measured using colormetric and fluorometric assays (46). Briefly, cultured cells were placed in 96-well plates. To each well 200 µl of assay buffer (20 mM HEPES pH 7.5, 10% glycerol, 2 mM dithiothreitol) was added. Caspase-1 activity was measured on a fluorescence plate reader (Fluostar Galaxy; BMG Labtechnologies, Durham, NC) set at 390 nm excitation and 460 nm emission and caspase-3 activity was assayed using a colormatric kit (ApoAlert Caspase-3; Clontech, Palo Alto, CA) with a microplate reader (SPECTRAmax Plus; Molecular Devices, Sunnyvale, CA). Peptide substrates for caspase-3 (Ac-DEVD-AMC) or caspase-1 (Ac-YVAD-AMC) (Alexis Biochemicals, San Diego, CA) were added to each well to a final concentration of 25 ng/µl. When the caspase-3 inhibitor (DEVD-fmk) was used, it was added to cell lysates at a concentration of 10 µM for 30 min before the addition of the caspase-3 substrate. Cell lysates (20 µg of protein) were added to start the reaction. Background was measured in wells containing assay buffer, substrate and lysis buffer without the cell lysate. Assay plates were incubated at 37°C for 1 h for measurement of caspase-3 and 3 h for measurement of caspase-1, based on preliminary measurements of the time course (0.5–6 h) of caspase activities. Caspase activity was calculated as [(mean value from triplicate wells) – (background value)]/µg of protein. Statistical significance was assessed with Student’s t-test. P < 0.05 was considered significant.
Cell viability was determined by a modified 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTS) assay (Cell Titer 96; Promega), which is based on the conversion of tetrazolium salt 3-(4,5-dimethyl thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) 2-H-tetrazolium by mitochondrial dehydrogenase to a formazan product, as measured at an absorbance of 490 nm. A microplate reader (SPECTRAmax Plus) was used to assess cell viability, as described previously (11). For caspase inhibitor treatment, cells were incubated with 50 µM ZVAD-fmk, 10 µM Boc-D-fmk, and 10 µM DEVD-fmk (Enzyme Systems) for 48 h before the MTS assay was performed.
Gene expression studies
RT–PCR and Southern blot analysis were performed as described previously (11). RT–PCR was performed for human GAPDH (11), human caspase-1 (GenBank accession no. NM001223) from nucleotides 452–1127 (5'-TCGGCAGAGATTTATCCAATAATG-3' and 5'-ATCTGGCTGCTCAAATGAAAATCG-3') and rat caspase-1 from nucleotides 90–1086 (5'-AGAGAAGAGAGTCCTGAACCAG-3' and 5'-TCTGAAAATGTCCTCCAAGTCA-3') (22). First strand cDNA was generated from RNA of cultured PC12 cells. PCR conditions were 95°C for 45 s, 60°C for 45 s and 72°C for 1 min with 35 cycles. PCR products were electrophoresed on a 1.2% agarose gel and visualized with ethidium bromide. Caspase-1 PCR products were subcloned and sequenced. Some PCR products were also analyzed with Southern blotting. The blots were hybridized with [32P]human caspase-1 cDNA probe in 50% formamide and 5x SSPE hybridization buffer at 42°C and washed with 0.2x SSC/0.5% SDS at 55°C before autoradiography.
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ACKNOWLEDGEMENTS |
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We thank Drs L.A. Esposito, B.A. Cottrell and D.C. Wallace at the Center for Molecular Medicine at Emory University for advice and providing antibodies to SOD2 and ANT-2. This work was supported by National Institutes of Health grant NS36232 and the Hereditary Disease Foundation Cure HD Initiative.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 404 727 3290; Fax: +1 404 727 3949; Email: xiaoli@genetics.emory.edu
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REFERENCES |
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