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Keywords:

  • Cerebellar granule neurons;
  • Apoptosis;
  • Inhibitors of apoptosis;
  • Caspases

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RNA preparation and RT-PCR
  5. N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies
  6. DNA fragmentation
  7. Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining
  8. RESULTS
  9. Expression of endogenous IAP in cerebellar granule neurons
  10. Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons
  11. Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation
  12. Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells
  13. IAPs do not protect against excitotoxic cell death
  14. DISCUSSION
  15. Acknowledgements

Abstract : The inhibitor of apoptosis (IAP) family of anti-apoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 mM potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 mM potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N-acetly-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, not at later time points suggesting that IAPS delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 μM or 1 mM glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.

Apoptosis is of fundamental importance to several biological processes including the normal development of the nervous system. Improper control of apoptosis has been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (Loo et al., 1993). Huntington's disease (Portera-Cailliau et al., 1995), amytrophic lateral sclerosis (Rabizadeh et al., 1995), and spinal muscular atrophy (SMA) (Roy et al., 1995).

Apoptosis is regulated by a number of pro- and anti-apoptotic genes. The family of caspases are key mediators in the apoptotic pathway. They do not only play an essential role in initial signaling events but are also crucial components of the apoptotic machinery (Salvesen and Dixit, 1997). In contrast to caspases, Bcl-2 is a potent suppressor of cell death. Although the Bcl-2 family of proteins has been intensively studied in mammals, relatively little is known about another anti-apoptotic protein family called inhibitor of apoptosis protein (IAP).

IAPs were originally identified in mutant baculovirus lacking p35 by their ability to rescue cells from apoptosis (Crook et al., 1993 ; Clem and Miller, 1994). Cellular homologues were subsequently identified in insects (DIAP-1 and DIAP-2/dILP) (Hay et al., 1995) and human cells [neuronal apoptosis inhibitory protein (NAIP), human IAP1 (HIAP1)/c-IAP-2/MIHC, HIAP2/c-IAP-1/MIHB, X chromosome-linked IAP (XIAP)/hILP, and survivin] (Rothe et al., 1995 ; Duckett et al., 1996 ; Liston et al., 1996 ; Uren et al., 1996 ; Ambrosini et al., 1997). Deletion mutations of NAIP were found in the hereditary neurodegenerative disorder SMA (Roy et al., 1995). The severity of SMA seems to be associated with deletions of NAIP, suggesting that the failure to inhibit apoptosis is one of the underlying mechanisms in the pathogenesis of this disorder.

All IAPs contain two to three amino acid repeats on their N-terminus, termed baculovirus IAP repeat (BIR). With the exception of NAIP and survivin, they also share a RING finger domain in their C-terminus. The preserved sequence motifs suggest a common anti-apoptotic mechanism. So far, the anti-apoptotic mechanism is not fully understood, but the available data indicate that at least one mechanism involves inhibition of caspases. XIAP, HIAP1, and HIAP2 directly inhibit caspase-3 and 7 but not caspase-8, 6, or 1 (Deveraux et al., 1997 ; Roy et al., 1997). NAIP failed to inhibit any of these caspases in vitro (Roy et al., 1997).

Previous studies demonstrate that IAPs prevent cell death induced by a variety of different stimuli such as serum withdrawal, BAX overexpression, menandione, and tumor necrosis factor (TNF) in vitro (Rothe et al., 1995 ; Liston et al., 1996 ; Uren et al., 1996), and ischemic damage in vivo (Xu et al., 1997). Apoptotic cell death triggered by staurosporine or FADD is not blocked by overexpression of IAPs (Liston et al., 1996 ; Uren et al., 1996). Whether IAPs promote survival in cultured neurons subjected to apoptotic or excitotoxic stimuli has yet to be studied. In this study, differentiated cerebellar granule neurons were used as an in vitro model of neuronal apoptosis. These cells share many biological properties with their counterparts in the brain including the development of an extensive neuronal network, the expression of excitotoxic amino acid receptors, and the production of l-glutamate (Gallo et al., 1987 ; Peng et al., 1991 ; Burgoyne et al., 1993). Cerebellar granule neurons were cultured in medium containing serum and depolarizing levels of potassium (25 mM). Removal of potassium leads to apoptotic cell death characterized by chromatin condensation, pyknosis, and nucleosomal size DNA fragmentation (D'Mello et al., 1993 ; Yan et al., 1994 ; Galli et al., 1995).

In this study, we investigated whether virally mediated IAP expression prevents apoptotic and/or excitotoxic cell death in cerebellar granule neurons.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RNA preparation and RT-PCR
  5. N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies
  6. DNA fragmentation
  7. Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining
  8. RESULTS
  9. Expression of endogenous IAP in cerebellar granule neurons
  10. Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons
  11. Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation
  12. Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells
  13. IAPs do not protect against excitotoxic cell death
  14. DISCUSSION
  15. Acknowledgements

Cell culture

Cerebellar granule neurons were prepared from 7-day-old Sprague-Dawley rats as previously described (Weller et al., 1994b). In brief, freshly dissected cerebella were dissociated by mechanical disruption and by incubation at 37°C for 15 min in 0.3 mg/ml trypsin. Cells were plated in poly-l-lysine-precoated 35-, 60-, or 100-mm culture plates or 24- or 96-well plates and seeded at a density of 2 × 105 cells/cm2 in basal modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 20 μg/ml gentamicin, and 25 mM KCl (high K+). Cytosine arabinoside was added at a concentration of 10 μM after 24 h to prevent the growth of nonneuronal cells. Contamination with glial cells was <5% (data not shown).

Viral vectors

The preparation of recombinant adenovirus has been described recently (Miyake et al., 1996). The vectors were harvested from cell lysates and used directly or further purified on CsCl gradients (Graham and van der Eb, 1973 ; Rosenfeld et al., 1992). Determination of infectious titer was performed by plaque assay on HEK 293 cells.

RNA preparation and RT-PCR

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RNA preparation and RT-PCR
  5. N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies
  6. DNA fragmentation
  7. Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining
  8. RESULTS
  9. Expression of endogenous IAP in cerebellar granule neurons
  10. Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons
  11. Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation
  12. Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells
  13. IAPs do not protect against excitotoxic cell death
  14. DISCUSSION
  15. Acknowledgements

RT-PCR was performed according to standard protocols as described (Weller et al., 1994b). Total RNA was prepared by using the RNeasy RNA isolation kit (Qiagen, Hilden, Germany). Total RNA (1 μg) was reverse-transcribed by using Superscript II reverse transcriptase (GIBCO, Gaithersburg, MD, U.S.A.) in a 20-μl reaction volume and the volume was subsequently increased to 100 μl ; 5 μl was used as a template for PCR. PCR was performed using 0.6 U Amplitaq polymerase (Perkin-Elmer, Norwalk, CT, U.S.A.) in a 50-μl reaction volume containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.01% gelatin, 2.5 mM MgCl2, 8 mM dNTP, and 1 μM each of forward and reverse primers. Primer sequences were IAP2 up : 5′-GCTTCTGTTGTGGCCTGATG-3′ [nucleotides (nt) 1,406-1426 in human MIHB cDNA], IAP2 down : 5′-CACCT-TGGAAACCACTTGGC-3′ (nt 2,138-2,157 in human MIHB cDNA) with annealing temperature 55°C and 45 amplification cycles run, GAPDH up : 5′-ACCACAGTCCATGCCAT-CAC-3′ (nt 519-610 in rat), GAPDH down : 5′-TCCACCAC-CCTGTTGCTGTA-3′ (nt 1,042-1,023 in rat) with annealing temperature 55°C and 24 amplification cycles run. Loading dye (5 μl) was added to the reaction and 10 μl was analyzed on 1.5% agarose gels containing 0.05% ethidium bromide.

Treatment of cultures

In all experiments neurons were cultured for 7 days in 25 mMK+ and 10% fetal calf serum before use. For studies of K+ deprivation, medium was replaced by serum-free basal modified Eagle's medium containing 5 mM potassium (low K+) or 25 mM potassium (high K+) and supplemented with glutamine and gentamicin as indicated above. Dizocilpine (MK-801 ; 10 μM) and 100 mM benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) were added to either high K+ or low K+.

For studies of glutamate excitotoxicity, 100 μM or 1 mM glutamate was added to neurons cultured in high K+ and serum for 7 days, and viability was measured after 24 h.

Infection of neurons

For optimal infection of cerebellar granule neurons, 1-day-old cultures were used. The volume of conditioned medium was reduced to one-third and recombinant adenovirus was added at 50 multiplicity of infection (MOI). After allowing the virus to absorb for 1 h, the conditioned medium was added back to its original volume. Neurons were further cultured to day 7 in vitro, when experiments were performed.

Detection of β-galactosidase-positive cells

β-Galactosidase activity was measured as described previously (Price et al., 1987). Cells were fixed in 1% glutaraldehyde in phosphate-buffered saline (PBS ; pH 7.4) for 5 min at 4°C. After two washes with PBS, cells were incubated for 8 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-β-d-galactoside)].

Determination of viability

Neurons plated in 24-well plates were used for assessment of viability. Viability was measured by the capability of cells to diesterify and retain fluorescein diacetate in their cytoplasm. Medium was removed from neuronal cultures and cells were incubated at 37°C for 5 min with Locke's solution (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1 mM MgCl2, 3.6 mM NaHCO3, 5 mM HEPES, and 20 mM glucose) containing 5 μg/ml fluorescein diacetate. Cultures were washed once with Locke's solution and examined under fluorescent light microscopy. Cell numbers were determined as described previously (Schulz et al., 1996). In brief, three random fields were chosen from each well and digitized by a CCD camera connected to an image processor (MCID-IV, Imaging Research, St. Catharines, Ontario, Canada). Images were filtered and the total number of stained cells was counted automatically by MCID-IV computer software.

N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RNA preparation and RT-PCR
  5. N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies
  6. DNA fragmentation
  7. Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining
  8. RESULTS
  9. Expression of endogenous IAP in cerebellar granule neurons
  10. Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons
  11. Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation
  12. Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells
  13. IAPs do not protect against excitotoxic cell death
  14. DISCUSSION
  15. Acknowledgements

DEVD-amc cleavage studies were performed as described by Armstrong et al. (1997). Cells cultured in 96-well plates were lysed in 50 μl of buffer A [10 mM HEPES, pH 7.4, 42 mM KCl, 5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM 1,4-dithio-dl-threitol, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 5 μg/ml aprotinin, and 0.5% Nonidet P-40]. Subsequently, cells were incubated for 10-30 min in buffer B (25 mM HEPES, 1 mM EDTA, 0.1% Nonidet P-40, 10% sucrose, and 3 mM 1,4-dithio-dl-threitol, pH 7.5) containing 10 μM DEVD-amc. Fluorescence was measured at excitation 360 nm, emission 460 nm, using a Cytofluor fluorescent plate reader.

Immunoblot analysis

Blotting was performed essentially as described previously (Schulz et al., 1996). Cerebellar granule neurons were seeded on 60-mm dishes. Medium was removed and cultures washed once in cold PBS before the cells were lysed for 15 min on ice in lysis buffer [1% Triton X-100 and 0.1% sodium dodecyl sulfate (SDS) with 10 μg/μl leupeptin and aprotinin]. Cell debris was removed by high-speed centrifugation at 4°C. Samples containing 20 μg of protein were boiled in 1% SDS and 1% β-mercaptoethanol for 5 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% skim milk, and 2% bovine serum albumin), followed by incubation with primary antibodies [anti-NAIP, anti-RIAP1, anti-RIAP2, or anti-XIAP (Liston et al., 1996) or anti-CPP32p20Pep (Armstrong et al., 1997)] overnight at 4°C and anti-rabbit IgG horseradish peroxidase-linked antibody. Bound antibody was visualized using enhanced chemiluminescence (ECL, Amersham).

DNA fragmentation

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RNA preparation and RT-PCR
  5. N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies
  6. DNA fragmentation
  7. Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining
  8. RESULTS
  9. Expression of endogenous IAP in cerebellar granule neurons
  10. Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons
  11. Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation
  12. Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells
  13. IAPs do not protect against excitotoxic cell death
  14. DISCUSSION
  15. Acknowledgements

For quantitative DNA fluorometry (Weller et al., 1994a), neuronal cultures (30 mm dishes) were lysed in 10 mM Tris-HCl, pH 7.5, 10 mM EDTA, and 0.2% Triton X-100 for 10 min on ice. Fragmented DNA was separated from nonfragmented DNA by high-speed centrifugation for 10 min. Pellets were disrupted by 2 × 10 s of pulsed sonication. Both supernatant and pellets were incubated with RNase A, before fragmented and pelleted DNA was measured by ethidium bromide (0.5 μg/ml) fluorometry, using 530-nm excitation and 620-nm emission wavelengths (CytoFluor 2350). Percentage of fragmented DNA was calculated by dividing fragmented DNA by the total sum of fragmented and pelleted DNA.

Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RNA preparation and RT-PCR
  5. N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies
  6. DNA fragmentation
  7. Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining
  8. RESULTS
  9. Expression of endogenous IAP in cerebellar granule neurons
  10. Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons
  11. Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation
  12. Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells
  13. IAPs do not protect against excitotoxic cell death
  14. DISCUSSION
  15. Acknowledgements

TUNEL staining was performed as described previously (Schulz et al., 1997). In brief, cells were fixed for 15 min in 3% paraformaldehyde/1% glutaraldehyde at room temperature, washed with PBS, and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. After three PBS washes, cells were preincubated for 5 min with 0.1 M sodium cacodylate (TDT) buffer followed by incubation for 10 min with reaction mixture (50 U/ml terminal transferase, 10 μM dUTP-biotin, 25 mM cobalt chloride in sodium cacodylate buffer). The reaction was stopped by incubating for 10 min with 0.1 M sodium acetate buffer. After incubation for 30 min with streptavidin-alkaline phosphatase conjugate, cells were developed with 0.41 mM nitroblue tetrazolium chloride and 0.38 mM 5-bromo-4-chloro-3-indolyl phosphatase in 200 mM Tris-HCl, pH 9.5, containing 100 mM MgCl2.

For each condition three different wells were used and TUNEL-positive cells counted in three different fields. Each field contained 480 ± 46 cells.

Statistical analysis

Data are expressed as mean ± SD values. Statistical significance was assessed by one-way ANOVA followed by the Bonferroni post hoc test (comparison of more than two groups). If not otherwise stated, all experiments reported represent at least three independent replications performed in triplicate.

Expression of endogenous IAP in cerebellar granule neurons

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RNA preparation and RT-PCR
  5. N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies
  6. DNA fragmentation
  7. Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining
  8. RESULTS
  9. Expression of endogenous IAP in cerebellar granule neurons
  10. Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons
  11. Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation
  12. Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells
  13. IAPs do not protect against excitotoxic cell death
  14. DISCUSSION
  15. Acknowledgements

To determine the endogenous messenger RNA levels of IAPs in cerebellar granule neurons in vitro, we isolated and reverse-transcribed mRNA and performed PCR analysis. We found expression of rat inhibitor of apoptosis protein 2 (RIAP2) mRNA (Fig. 1A), whereas XIAP and RIAP1 mRNA were undetectable (data not shown). NAIP mRNA was barely detectable even after 50 cycles of amplification (data not shown). IAP protein levels were examined by immunoblot analysis with rabbit polyclonal antibodies raised against full-length XIAP, RIAP1, and RIAP2, or the amino-terminal 1.0-kb fragment of human NAIP. Cultures of cerebellar granule neurons failed to display both XIAP and NAIP immunoreactivity. In contrast, RIAP2 was readily detectable by immunoblotting (Fig. 1B). Expression of RIAP1 could not be evaluated because of lack of specific antibodies.

image

Figure 1. Expression of endogenous RIAP2 mRNA and protein is not changed in cerebellar granule neurons undergoing apoptosis. Cultures were switched to low or high K+ and levels of RIAP2 and GAPDH mRNA were analyzed after 30 min and 1, 2, 4, 8, and 16 h. Total RNA (1 μg) was reverse-transcribed and the resulting cDNA was amplified for 45 and 24 cycles, respectively. PCR products were separated on agarose gels (A). SDS-polyacrylamide gel electrophoresis immunoblot comparing RIAP2 immunoreactivity of cell extracts after 0, 6, 12, and 24 h in low K+ (B). The gel shown is representative of three separate experiments.

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Next we analyzed the effect of potassium withdrawal on RIAP2 expression. At day 7, in vitro cultures were switched from medium containing 25 mM potassium and serum to either 5 mM potassium without serum (low K+) or 25 mM potassium without serum (high K+). mRNA levels of RIAP2 were analyzed by RT-PCR in the cultures at different times. Levels of RIAP2 mRNA did not change at 30 min and 1, 2, 4, 8, and 16 h after cultures were switched to low K+, demonstrating that steady-state levels of RIAP2 mRNA are not regulated by potassium withdrawal (Fig. 1A). In addition, immunoblot experiments were performed to determine whether RIAP2 is regulated posttranslationally. Immunoreactivity of RIAP2 was observed at similar intensity on immunoblots of cell lysates from neurons maintained for 0, 6, 12, and 24 h in low K+ (Fig. 1B).

Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RNA preparation and RT-PCR
  5. N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies
  6. DNA fragmentation
  7. Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining
  8. RESULTS
  9. Expression of endogenous IAP in cerebellar granule neurons
  10. Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons
  11. Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation
  12. Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells
  13. IAPs do not protect against excitotoxic cell death
  14. DISCUSSION
  15. Acknowledgements

An adenoviral vector system (AdV) was used to over-express IAPs in cerebellar granule neurons. An adenoviral construct encoding LacZ (AdV-LacZ) was used to test the efficiency of adenovirus-mediated gene transfer in our culture system. Neurons were infected at day 1 in vitro with 50 MOI AdV-LacZ and β-galactosidase activity was measured at day 7 in vitro. In control cultures, no endogenous β-galactosidase activity was detected (data not shown). In contrast, nearly all neurons (>90%) of AdV-LacZ-treated cultures were β-galactosidase positive (Fig. 2A). When cerebellar granule neurons were infected with 1 MOI only a minority of neurons became β-galactosidase positive (data not shown). We tested the virally mediated expression of NAIP, XIAP, or HIAP2 by immunoblot analysis. Cultures infected with 50 MOI of adenoviral constructs encoding NAIP, XIAP, and HIAP2 expressed a protein with the molecular weight expected for the respective IAP (Fig. 2B), demonstrating that the IAP genes had been successfully transferred into cerebellar granule neurons. Expression of HIAP1 could not be evaluated because of a lack of specific antibodies.

image

Figure 2. β-Galactosidase and IAP expression in cerebellar granule neurons after adenoviral-mediated gene transfer. At 1 day in vitro cerebellar granule neurons were infected with 50 MOI AdV-LacZ, AdV-NAIP, AdV-XIAP, AdV-HIAP1, or AdV-HIAP2 and cultured for 6 additional days. Phase-contrast images are shown of AdV-LacZ-infected (A) neurons assayed for β-galactosidase activity. Immunoblot analysis of NAIP, XIAP, and HIAP2 expression in cultures of cerebellar granule neurons (B).

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Next we analyzed whether ectopic expression of NAIP, XIAP, HIAP1, or HIAP2 reduces the apoptosis of cerebellar granule neurons. For this purpose, neurons were infected with 50 MOI recombinant adenovirus encoding NAIP, XIAP, HIAP1, or HIAP2 at day 1 in vitro and switched to high K+ or low K+ medium at day 7 in vitro. Neuronal survival was evaluated by fluorescein diacetate staining at 12, 24, and 40 h after potassium withdrawal. Control cultures were either infected with the LacZ construct or left uninfected.

Measurement of viability at 12 h in noninfected control cultures revealed that ~64% (±10%) of cerebellar granule neurons switched to low K+ were alive compared with neurons that were switched to high K+ (Fig. 3A). The viability was similar (~63 ± 13%) in cultures that were infected with AdV-LacZ (Fig. 3A). In contrast, when IAPs were transferred into cerebellar granule neurons with recombinant adenovirus, neurons were almost completely rescued. In cultures infected with AdV-NAIP, AdV-XIAP, AdV-HIAP1, and AdV-HIAP2, cell survival was ~95% (±20%), ~90% (±11%), ~77% (±11%), and ~93% (±8.5%), respectively (Fig. 3A). These results demonstrate that IAPs effectively protect against cell death in cerebellar granule neurons deprived of potassium and serum for 12 h. However, when viability was measured at later times, these effects were less dramatic. After 24 h in low K+, viability was ~54% in control cultures (AdV-LacZ), compared with 66-69% in cultures that were infected with AdV-XIAP, AdV-HIAP1, and AdV-HIAP2 (Fig. 3B). Ectopic expression of NAIP had no effect on cell death at 24 h. After 40 h in low K+, none of the IAPs afforded significant protection (Fig. 3C). Thus, adenovirus-mediated gene transfer of IAPs delayed cell death in cerebellar granule neurons but did not ultimately prevent cell death.

image

Figure 3. Virally mediated expression of NAIP, XIAP, HIAP1, or HIAP2 delays but does not block apoptosis in cerebellar granule neurons. Cerebellar granule neurons were infected with recombinant adenovirus at day 1 in vitro and switched to low K+ or high K+ at day 7 in vitro. Viability was measured after 12 h (A), 24 h (B), or 40 h (C). Data are expressed as mean ± SD values of three to six different experiments relative to high K+ controls (**p < 0.01, *p < 0.05 ; low K+ values were normalized to the respective high K+ adenovirus-treated conditions and compared with values of AdV-LacZ).

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Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RNA preparation and RT-PCR
  5. N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies
  6. DNA fragmentation
  7. Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining
  8. RESULTS
  9. Expression of endogenous IAP in cerebellar granule neurons
  10. Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons
  11. Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation
  12. Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells
  13. IAPs do not protect against excitotoxic cell death
  14. DISCUSSION
  15. Acknowledgements

Caspase-3-like caspases are activated during apoptosis in cerebellar granule cells and reach their maximal activity at 7-8 h after switching to low K+ compared with high K+ (Armstrong et al., 1997 ; Miller et al., 1997). Confirming these results, we found that 8 h after cerebellar granule neurons were switched to low K+, DEVD-amc cleavage increased fourfold compared with cultures switched to high K+ (Fig. 4). To test whether IAPs prevent the activity of DEVD-specific caspases, granule neurons were infected with recombinant adenovirus, switched to low or high K+, and DEVD-amc cleavage was measured. Ectopic expression of all four IAPs effectively blocked DEVD-specific caspases (Fig. 4A). The most pronounced result was observed in neurons infected with AdV-XIAP and AdV-HIAP2 in which DEVD-sensitive caspase activity was completely blocked (Fig. 4A). The inhibition of DEVD-specific caspases could be either due to inhibition of active caspases and/or prevention of the processing of procaspases to subunits that form active heterodimers. To test these different possibilities, we performed immunoblot experiments with a polyclonal antibody (anti-CPP32p20Pep). Cell lysates of adenovirus-infected cultures that had been deprived of potassium for 6 h were used. The p32 proenzyme form of caspase-3 is processed to a small p12 and a large p20 subunit, against which the antibody anti-CPP32p20Pep reacts (Armstrong et al., 1997). Immunoblotting of cell extracts with anti-CPP32p20Pep identified the p20 protein in low K+ but not in high K+ extracts of AdV-LacZ-infected neuronal cultures (Fig. 4B). This immunoreactive band was almost absent in extracts of cultures that were infected with AdV-XIAP, AdV-NAIP, and AdV-HIAP1 and switched to low K+ (Fig. 4B). This result demonstrates that IAPs prevent the formation of the p20 subunit in cerebellar granule neurons subjected to apoptosis. These results suggests that the effects of IAPs are upstream and/or at the level of procaspase-3 processing.

image

Figure 4. Adenoviral expression of IAPs inhibits activity of DEVD-specific caspases. Infected and uninfected cultures were switched to low K+ or high K+, and cell extracts were prepared after 8 h and monitored for DEVD-amc cleavage. Data represent mean ± SD values of triplicate measurements of two different experiments (*p < 0.001) (A). Neuronal cultures infected with AdV-XIAP, AdV-NAIP, AdV-HIAP1, or AdV-HIAP2 were switched to low K+ or high K+. Cell extracts were prepared after 6 h and subjected to immunoblot analysis with anti-CPP32p20Pep (B). The antibody only recognizes the active p20 subunit and not the procaspase.

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As caspases are involved in activating enzymes that cause DNA fragmentation, we tested whether IAPs prevent DNA fragmentation (Enari et al., 1998). Two different approaches were used to measure DNA breaks. First, the amount of fragmentation was estimated by DNA fluorometry measuring the ratio of fragmented DNA to nonfragmented DNA. When neurons were switched to low K+, ~38% (±3.6%) of the DNA was fragmented after 24 h compared with ~10% (±0.7%) in high K+ cultures (Fig. 5A). The amount of fragmented DNA was similar in low and high K+ cultures that were infected with AdV-XIAP (~12% ± 0.6%), demonstrating that XIAP expression effectively inhibited DNA fragmentation (Fig. 5A). Infection with AdV-NAIP, AdV-HIAP1, or AdV-HIAP2 leads to similar results ; however, protection from DNA fragmentation was less pronounced (Fig. 5A).

image

Figure 5. IAPs reduce DNA fragmentation in cerebellar granule neurons. DNA fragmentation was quantified by DNA fluorometry (A) or TUNEL staining (B) 24 h after switch to low or high K+. Data represent mean ± SD values of triplicates of three different experiments (*p < 0.001). TUNEL-positive cells were quantified from three different fields of triplicates (mean ± SD) (*p < 0.001) (B). Total number of cells in one field was 480 ± 46.

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Second, we also performed TUNEL staining to measure DNA strand breaks. The number of TUNEL-positive cells increased dramatically after switching neurons from high K+ to low K+ in control cultures (Fig. 5B). Infection with AdV-IAPs resulted in a marked decrease of TUNEL-positive cells, again showing that the IAP overexpression of IAPs reduces DNA damage.

Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RNA preparation and RT-PCR
  5. N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies
  6. DNA fragmentation
  7. Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining
  8. RESULTS
  9. Expression of endogenous IAP in cerebellar granule neurons
  10. Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons
  11. Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation
  12. Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells
  13. IAPs do not protect against excitotoxic cell death
  14. DISCUSSION
  15. Acknowledgements

Although adenovirus-mediated gene transfer of IAPs provides efficient protection from DEVD-specific caspase activity and reduces DNA fragmentation, cerebellar granule neurons are not ultimately protected from cell death. We therefore wondered if cerebellar granule neurons underwent secondary excitotoxic cell death. To test this, we added MK-801, an NMDA receptor antagonist, to cells that had been infected with AdV-XIAP and switched to low K+. Viability was measured 40 h after potassium deprivation, when IAPs had ceased to exert neuroprotective effect. Treatment with MK-801 did not enhance cell survival (Fig. 6), demonstrating that excitotoxic damage mediated by NMDA receptors did not contribute to the delayed cell death. We also tested whether neurons could be rescued from cell death when MK-801 and the synthetic caspase inhibitor zVAD-fmk were combined. MK-801 did not afford significant protection when combined with zVAD-fmk (Fig. 6), again showing that NMDA receptor-mediated excitotoxity was not responsible for cell death.

image

Figure 6. MK-801 does not protect from delayed cell death in XIAP-expressing cells deprived of K+. Neurons were infected with AdV-XIAP or AdV-LacZ and switched to low K+ or high K+ supplemented with 10 μM MK-801 or not. Viability was evaluated with fluorescein diacetate staining after 40 h.

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IAPs do not protect against excitotoxic cell death

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RNA preparation and RT-PCR
  5. N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies
  6. DNA fragmentation
  7. Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining
  8. RESULTS
  9. Expression of endogenous IAP in cerebellar granule neurons
  10. Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons
  11. Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation
  12. Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells
  13. IAPs do not protect against excitotoxic cell death
  14. DISCUSSION
  15. Acknowledgements

It has previously been shown that cerebellar granule neurons die by both necrotic and apoptotic mechanisms after glutamate exposure (Ankarcrona et al., 1995). We therefore analyzed whether ectopic expression of IAPs protected against excitotoxic cell death. Cerebellar granule neurons cultured for 1 day were infected with AdV-IAPs and treated with 100 μM and 1 mM glutamate for 24 h and viability was measured with fluorescein diacetate staining. All of the IAPs failed to inhibit excitotoxic cell death induced by either 100 μM or 1 mM glutamate (Fig. 7A).

image

Figure 7. Glutamate exposure does not activate DEVD-specific caspases and IAPs do not protect from glutamate-induced excitotoxic cell death. Cerebellar granule neurons were treated with 100 μM and 1 mM glutamate at day 7 in vitro. For assessment of viability cells were cultured for 7 days, treated with 100 μM or 1 mM glutamate, and stained with fluorescein diacetate after 24 h (A). Data represent mean ± SD values of two independent experiments. Cell extracts were prepared after 2, 5, 8, and 24 h and evaluated for DEVD-amc cleavage. Cell extracts from low K+ cultures were used as a positive control (B).

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We also tested to see whether DEVD-specific caspases were activated during 100 μM or 1 mM glutamate exposure. We did not observe any increase in DEVD-amc cleaving activity at 2, 5, 8, or 24 h after glutamate treatment (Fig. 7B).

DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RNA preparation and RT-PCR
  5. N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies
  6. DNA fragmentation
  7. Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining
  8. RESULTS
  9. Expression of endogenous IAP in cerebellar granule neurons
  10. Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons
  11. Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation
  12. Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells
  13. IAPs do not protect against excitotoxic cell death
  14. DISCUSSION
  15. Acknowledgements

In this study, we investigated the anti-apoptotic activity of the novel family of human IAPs in an in vitro model of neuronal apoptosis. Cultured cerebellar granule neurons are a useful model of neuronal apoptosis. These cells die synchronously when potassium is withdrawn. We took advantage of an adenovirus-based vector to target XIAP, NAIP, HIAP1, and HIAP2 into primary cultures of cerebellar granule neurons.

This vector is ideally suited for this purpose, because it offers long-term expression of foreign proteins without disturbing survival, electrophysiological function, or cytoarchitecture of neuronal cells (Slack et al., 1996). Recombinant adenovirus expression systems have previously been used successfully to target foreign genes into cerebellar granule neurons (Fritz et al., 1997). The human glutamic acid decarboxylase gene, for example, was transferred into nearly 100% of the neurons in rat cerebellar granule neurons (Fritz et al., 1997). In line with these results, we showed that recombinant adenovirus can infect >90% of the cells and detectable levels of protein are expressed for at least 7 days after infection.

Here, we asked whether virally mediated gene transfer of IAPs promotes survival of potassium-deprived cerebellar granule neurons. We showed that overexpression of IAPs protects from cell death up to 24 h after potassium and serum withdrawal but does not offer any protection at later times, indicating that IAPs delay rather than block cell death in cerebellar granule neurons. In addition, we investigated the mode of action of these proteins in the apoptotic pathway of neurons. It has previously been shown that caspase-3-like caspases are activated during apoptosis of cerebellar granule neurons (Armstrong et al., 1997 ; Eldadah et al., 1997). We demonstrate that virally mediated expression of IAPs leads to efficient inhibition of DEVD-specific caspase activity and prevents the formation of the active subunit of caspase-3. In addition, we show that IAPs reduce DNA fragmentation in cerebellar granule neurons. Our results suggest that IAPs function upstream or at the level of DEVD-specific caspases in potassium-deprived cerebellar granule neurons. These findings are in agreement with existing data on the anti-apoptotic mechanisms of IAPs in peripheral cells or in vitro assays (Duckett et al., 1996 ; Deveraux et al., 1997 ; Roy et al., 1997). The first members of the IAP family, baculovirus IAPs, were initially identified by their ability to functionally complement the activity of p35-deficient virus (Crook et al., 1993 ; Clem and Miller, 1994). p35 is known to exert its function by inhibiting members of the caspase family, suggesting that IAPs may function in a similar manner. Indeed, it has been demonstrated that XIAP, HIAP1, and HIAP2 but not NAIP act by inhibition of caspase-3 and 7 in cell-free assays (Deveraux et al., 1997 ; Roy et al., 1997). In addition, HIAP2 acts on a second level of the anti-apoptotic cascade by its ability to bind TRAF and thereby activate NF-κB, which in turn regulates several anti-apoptotic genes (Chu et al., 1997). The anti-apoptotic mechanism of NAIP has so far not been clarified. In contrast to results obtained in a cell-free assay, which showed that a glutathione S-transferase-NAIP fusion protein did not block activity of purified caspase-3 and 7, we demonstrate that virally mediated expression of NAIP inhibits DEVD-specific caspases. One possible explanation for this discrepancy is that NAIP does not directly inhibit caspase-3 and 7, but instead acts on an “upstream” level of the apoptotic pathway, which eventually leads to inhibition of DEVD-sensitive caspases.

Although it not completely clear whether all IAPs work by similar mechanisms, they all possess anti-death activity in a variety of peripheral cell lines using different death signals. The delay rather than the block of apoptosis in cerebellar granule neurons might reflect multiple, independent parallel processes that occur in these cells. Another possibility may be secondary excitotoxic damage after inhibition of caspase activity. However, block of NMDA receptors with MK-801 did not afford protection in cells that were switched to low K+ and infected with AdV-XIAP or treated with zVAD-fmk. Recently, Miller et al. (1997) provided evidence for an alternate death pathway in these cells that proceeds in the presence of caspase inhibitors and requires BAX. Although synthetic caspase inhibitors only delayed cell death in cerebellar granule neurons, cultures from BAX -/- rats were resistant to potassium withdrawal-induced apoptosis. In a similar manner, Xiang and colleagues (1996) reported that overexpression of BAX leads to apoptosis in Jurkat cells, which could not be blocked by a panel of protease inhibitor including caspase, serine, cysteine, granzyme B, and proteasome inhibitors. However, there are also numerous reports that show that caspase inhibitors promote long-term survival. For example, the viral caspase inhibitor CrmA or p35 blocks apoptosis in chicken dorsal root ganglion and Rat1 fibroblasts (Gagliardini et al., 1994 ; Wang et al., 1994). Treatment with synthetic or viral caspase inhibitors prevents the death after serum withdrawal in lumbar spinal motorneurons (Martinou et al., 1995) and blocks Fas- and TNF-induced killing in rat fibroblasts (Enari et al., 1995 ; Los et al., 1995). In some instances, the death signal may directly activate or recruit caspases, in which case the inhibition of caspases might be sufficient to prevent all manifestations of apoptosis. For example, the initiation of death via Fas and TNF receptor 1 leads to direct activation of caspase-8 on the cytosolic side of the TNF receptor (Boldin et al., 1996 ; Muzio et al., 1996). At present, however, these pathways have not been described during neuronal apoptosis.

Although overexpression of IAPs protects a variety of cells from apoptosis, it is not yet clear which role endogenous IAP plays in controlling the death process. We determined the levels of endogenous IAP mRNA and protein in cerebellar granule neurons and found only detectable expression of RIAP2, but not XIAP, NAIP, and RIAP1. We also analyzed the effects of potassium withdrawal on RIAP2 expression. Expression of RIAP2 did not change during apoptosis, suggesting that RIAP2 is not involved in controlling potassium-induced apoptosis in cerebellar granule neurons. A recent study demonstrates that transient forebrain ischemia selectively elevates levels of NAIP in rat neurons that are resistant to ischemic damage. These results suggest that endogenous NAIP has a functional role as an anti-apoptotic protein in ischemia-induced neuronal injuries (Xu et al., 1997).

It is still undetermined whether glutamate-induced excitotoxicity induces apoptosis. Ankarcrona et al. (1995) have suggested a delayed apoptotic cell death in cultures of cerebellar granule neurons, beginning at 8-12 h after glutamate exposure, and Du et al. (1997) observed caspase activation and DNA fragmentation after low-dose glutamate exposure. In contrast, treatment of cerebellar granule neurons with 300 μM or 3 mM glutamate did not induce caspase-3 activity and could not be blocked by treatment with synthetic caspase inhibitors (Armstrong et al., 1997). We did not observe any caspase-3 activity up to 24 h after exposure to either 100 μM or 1 mM glutamate. These results show that caspase activation is not necessarily an essential part of excitotoxic cell death, but may only occur under certain conditions. We also investigated the protective effects of virally mediated expression of IAPs after 100 μM and 1 mM glutamate exposure. Exposure to 100 μM and 1 mM glutamate for 24 h resulted in ~70 and ~80% cell death in cerebellar granule cultures, respectively. When IAPs were transferred into the neurons, no reduction in cell death was observed, demonstrating that IAPs do not protect against excitotoxicity at the dosages used in this study. The results suggest that IAP requires caspase activation to exert neuroprotective effects.

In summary, we show that adenovirus-mediated gene transfer of IAPs delays apoptotic neuronal cell death by inhibition of caspases. Further studies are required to characterize the caspase-independent pathway leading to cell death after potassium withdrawal in cerebellar granule neurons.

Acknowledgements

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RNA preparation and RT-PCR
  5. N-Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin (DEVD-amc) cleavage studies
  6. DNA fragmentation
  7. Terminal transferase-mediated dUTP-biotin DNA nick end-labeling (TUNEL) staining
  8. RESULTS
  9. Expression of endogenous IAP in cerebellar granule neurons
  10. Virally mediated overexpression of NAIP, XIAP, HIAP1, and HIAP2 delays but does not block cell death in cerebellar granule neurons
  11. Virally mediated expression of NAIP, XIAP, HIAP1, and HIAP2 blocks caspase activity and reduces DNA fragmentation
  12. Inhibition of NMDA receptors does not protect from delayed cell death in AdV-XIAP-infected cells
  13. IAPs do not protect against excitotoxic cell death
  14. DISCUSSION
  15. Acknowledgements

This study was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 430, B8, and Schu 932/2-1 to J.B.S.). We thank K. J. Tomaselli for providing the CPP32-p20Pep antibody.

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