Increased apoptosis of Huntington disease lymphoblasts ... - Nature

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Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization AKIRA SAWA1, GORDON W. WIEGAND2, JILLIAN COOPER3, RUSSELL L. MARGOLIS3, ALAN H. SHARP3, JOSEPH F. LAWLER JR.1, J. TIMOTHY GREENAMYRE6, SOLOMON H. SNYDER1,3,4,5 & CHRISTOPHER A. ROSS1,3,5


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Departments of Neuroscience, 2Medicine, 3Psychiatry and Behavioral Sciences and 4Pharmacology and Molecular Science, and 5Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, Maryland 21205, USA 6 Department of Neurology, Emory University, 1639 Pierce Dr. WMB 6000, Atlanta, Georgia 30322, USA G.W.B. present address: National Cancer Institute, Frederick, Maryland Correspondence should be addressed to S.H.S.

Huntington disease (HD) is a genetically dominant condition caused by expanded CAG repeats coding for glutamine in the HD gene product huntingtin1. Although HD symptoms reflect preferential neuronal death in specific brain regions, huntingtin is expressed in almost all tissues2, so abnormalities outside the brain might be expected. Although involvement of nuclei3–7 and mitochondria8–14 in HD pathophysiology has been suggested, specific intracellular defects that might elicit cell death have been unclear. Mitochondria dysfunction is reported in HD brains10–13; mitochondria are organelles that regulates apoptotic cell death15,16. We now report that lymphoblasts derived from HD patients showed increased stress-induced apoptotic cell death associated with caspase-3 activation. When subjected to stress, HD lymphoblasts also manifested a considerable increase in mitochondrial depolarization correlated with increased glutamine repeats. We evaluated mitochondrial depolarization, an initial regulatory event in cell death17 that can be quantified with the fluorescent dye JC1 (ref. 18). We assessed the effect of cyanide on mitochondrial depolarization in lymphoblasts from seven patients with juvenile onset of Huntington disease (HD) and long repeat lengths, and nine age-matched control subjects. In the absence of cyanide, control and HD mitochondrial potentials did not differ. For control cells both before and after cyanide treatment, substantially more mitochondria were polarized than depolarized. In contrast, after cyanide treatment, HD mitochondria become depolarized to a much greater extent than control samples (Fig. 1a and b). Differences were evident 0.5–6 hours after administration of 0.025–0.5 mM cyanide, with more than 1,000% greater depolarization in HD than control samples at later time points (Fig. 1b). This depolarization was evident before cell death, as shown by staining with the fluorescent dyes Hoechst 33258 and ethidium homodimer-1 (data not shown). Moreover, there was considerable correlation between CAG repeat length and mitochondrial depolarization in HD lymphoblasts (Pearson’s correlation coefficient, 0.906; P < 0.005) (Fig. 1c). We also assessed the effects of other mitochondrial toxins, including rotenone (1–10 µM), 3-NPA (0.01–0.1 mM) and antimycin A (1-5 µg/ml) (Fig. 1d). Rotenone and antimycin A, which are selective for complexes I and III respectively, caused a 1194

small amount of mitochondrial depolarization with no difference between HD and control cells, whereas azide and cyanide, which target complex IV, exerted preferential and prominent effects on HD lymphoblasts. 3-NPA, which acts on complex II, did depolarize mitochondria of HD cells preferentially, but not as much as azide and cyanide. To assess the specificity of the HD abnormality, we assessed lymphoblasts from patients with spinocerebellar ataxia type-1 (SCA-1), a condition that, like HD, involves polyglutamine repeats. Both wild-type and mutant ataxin-1, the gene product of SCA-1, are expressed in lymphoblasts19. Cyanide depolarization was the same in SCA-1 and control cells (Fig. 1b). Mitochondrial permeability transition (MPT) participates in the initiation of apoptosis15,20. MPT induces mitochondrial depolarization, and is itself augmented by depolarization. Cyclosporin A (CsA) binds to a mitochondrial form of cyclophilin21, and potently inhibits MPT. CsA reduced considerably the depolarization of HD as well as control lymphoblasts, with partial effects at 0.5 µM and maximal influences at 3 µM (Fig. 1e). We also assessed FK506, which binds to a distinct immunophilin, FK506-binding protein22, and PK11195, a drug that binds selectively and with high affinity to the mitochondrial benzodiazepine receptor of the mitochondrial outer membrane23. Both drugs failed to influence depolarization levels. The selective blockade by CsA of the repeat length-dependent mitochondrial depolarization indicates that mitochondrial abnormalities in HD cells are linked to MPT. To ascertain whether the increased susceptibility of HD cells to mitochondrial depolarization is associated with increased cell death, we used staurosporine (STS), a potent inducer of apoptosis dependent on MPT (refs. 24,25). HD lymphoblasts were more sensitive to mitochondrial depolarization elicited by STS than were control cells (Fig. 2a). STS caused cell death that seemed to be apoptotic, as demonstrated by nuclear fragmentation and DNA laddering (Fig. 2b). We evaluated the viability of cells using the dyes Hoechst 33258 and ethidium homodimer-1. In the absence of STS treatment, control and HD lymphoblasts did not differ in viability. HD lymphoblasts were significantly more sensitive to STS than were control samples, with a doubling of cell death in the HD samples (Fig. 2c). HD cells were also significantly more sensitive to cyanide than control cells, although the NATURE MEDICINE • VOLUME 5 • NUMBER 10 • OCTOBER 1999


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represent the ratios of mean levels of depolarization for HD and control groups. e, Cyclosporin A (CsA) blocks mitochondrial membrane depolarization, but FK506 and PK11195 are ineffective. NT, no treatment.

differences were less profound than those for STS (Fig. 2c). In the brain, HD is associated with intranuclear inclusions and dystrophic neurites containing aggregates of huntingtin5,6,7 (htt; GDB designation, HD). We sought to determine whether the mitochondrial abnormalities in lymphoblasts are related to nuclear aggregates in these cells. Immunofluorescent labeling of lymphoblasts showed a diffuse distribution of htt throughout the cytoplasm, with no difference between control and HD cells (Fig. 2d and e). There was no evidence for nuclear inclusions at time points equivalent to or later than the abnormal mitochondrial depolarization. In contrast, in parallel experiments, there were prominent intranuclear aggregates in HEK293 cells and N2a cells transfected with a truncated form of htt with expanded CAG repeats (Fig. 2e). These observations indicate that the HD apoptotic and mitochondrial abnormalities are not secondary to nuclear inclusions. Apoptotic cell death is often associated with activation of the cysteine protease caspase-3 (ref. 16). After STS treatment, caspase-3 activity was much greater in HD cells both 7 and 11 hours after STS treatment, with a significant increase at 11 hours compared with that of control samples (Fig. 3a). To assess specificity, we used lymphoblasts of SCA-1 patients; these did not differ from control cells (Fig. 3a). Caspase-9 is an essential part of the death signaling cascade from mitochondria to caspase-3 activation26. Caspase-9 was activated to a greater extent in HD cells than in control cells after STS stress (Fig. 3b). We attempted to monitor cytochrome c release from mitochondria to cytoplasm, but have been unable to obtain reproducible values in lymphoblast mitochondrial preparations. Involvement of capase-8 in the toxicity elicited by expanded polyglutamine proteins has been proposed27. We did not detect activation of caspase-8 after cyanide stress (Fig. 3c). Caspase-8 was activated by the addition

of Fas/Apo-1, but with no difference between HD and control cells (Fig. 3d). Caspase-3 was also activated by the addition of Fas/Apo-1; again, the extent of activation was not different between HD and control cells (Fig. 3d). These results indicate that specific subsets of caspases are activated by abnormal htt. We assessed the effects of drugs on the increased apoptotic death of HD cells. CsA (3 µM) and z-VAD-fmk (100 µM) significantly reduced STS-elicited cell death (Fig. 3e). z-VAD-fmk reportedly slows progression of HD pathology in an HD animal model28. In lymphoblasts, z-VAD-fmk blocked caspase-3 activity almost completely (data not shown). Here, we found a selective mitochondrial dysfunction in HD lymphoblasts, linked to apoptotic death. Caspases downstream of mitochondria in the death signaling cascade were selectively activated in HD cells. The HD abnormalities were not dependent on nuclear inclusions. The differences between the control and patient cells cannot be explained by adventitious factors. HD and control cells were thawed and maintained in culture in identical conditions with experiments undertaken within 2 weeks of cell thawing. It is unlikely that dietary or drug treatment differences between the groups explain the results, as during the process of cell line preparation, cells were washed and exposed to many changes in media so that drugs and other exogenous chemicals would have been washed away. Moreover, we did not find abnormalities in lymphoblasts from SCA-1 patients, whose clinical debility is similar to that of HD patients. As ataxin-1, the gene product of SCA-1, is expressed in lymphoblasts15, ataxin-1 and htt presumably have different mechanisms whereby they influence cell death. The close correlation between HD mitochondrial depolarization and CAG repeat length also supports an HD-specific basis for the abnormalities found. We are not aware of any other studies demonstrating robust

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Mitochondrial depolarization (%)

Fig. 1 Cyanide-induced mitochondrial depolarization is greater in HD than in control cells. Lymphoblasts from seven HD patients and nine control subjects were analyzed by FACS before and after cyanide stress. The extent of mitochondria depolarization after stress was compared with baseline depolarization. Data represent the means of three independent experiments. a, At 30 min after cyanide (CN) stress, mitochondria of HD cells (open bars) were more depolarized than control mitochondria (dotted bars). *, P < 0.01 at 0.5 mM and P < 0.05 at 0.2 mM. b, Only HD mitochondria (green circles) show substantial depolarization 3 or 6 h after 0.025 mM cyanide stress: *, P < 0.001 at 3 h and P < 0.0005 at 6 h. SCA-1 cells (red diamonds) are not significantly difference from control cells (diamonds). c, The extent of mitochondrial depolarization correlates with the length of CAG repeats with a threshold at 60 repeats (Pearson’s correlation coefficient, 0.906; P < 0.005). d, HD mitochondria show considerable depolarization after stress induced by complex IV inhibitors: **, P < 0.0005, cyanide; *, P < 0.0005, azide. HD mitochondria also show some susceptibility to depolarization with 3-NPA (complex II inhibitor): *, P < 0.005. There are no significant differences with rotenone (complex-I inhibitor) or antimycin A (complex-III inhibitor). Data

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Fig. 2 HD lymphoblasts have more apoptotic vulnerability than do control lymphoblasts after the addition of 20 nM staurosporine. a, HD mitochondria show greater depolarization than do control samples. *, P < 0.005. Data represent means of three independent experiments. b, Cell death induced by STS is apoptotic. Left, Ladder formation of DNA and nuclear fragmentation after STS treatment (right margin, size). Right, Nuclear morphology, evaluated with membrane-permeable, blue Hoechst 33258 dye. Data are from an experiment replicated three times. NT, no treatment; STS, staurosporin treatment. c, HD cells (open bars) are more vulnerable to STS- and cyanide (CN)-induced toxicity than are control cells (grey bars). Cell death associated with increased membrane permeability was determined by the ratio of redstained cells (ethidium homodimer-1) to blue-stained cells (Hoechst 33258). The difference between HD and control is statistically significant: *, P < 0.005

at 10 h and P < 0.001 at 20 h after STS; *, P < 0.05 at 30 h after cyanide addition. Data represent means of three independent experiments. d and e, Absence of nuclear inclusions in HD lymphoblasts before and after stress. HD cells with the longest expansion (97 repeats) and control cells (18 repeats) were assessed. Confocal microscopy was used to evaluate the image. d, Cytosolic staining of htt is detectable in HD lymphoblasts before (0 h) and after (6 h and 12 h) STS stress. The immunostaining signal by antibody against htt was visualized as green using FITC, superimposed on the phase contrast image. e, No nuclear inclusions are apparent in HD or control lymphoblasts before or after stress stained with antibody against htt. Nuclei are stained red by propidium iodide; green is the immunofluorescent signal. In contrast, N2a cells transfected with a construct containing an expanded CAG repeat show robust nuclear inclusions.

differences between HD and control peripheral cells. The limited mitochondrial complex I abnormalities reported in muscle tissue from three HD patients14 might reflect the mitochondrial aberrations described here in lymphoblasts, although our findings indicate involvement of complex IV rather than complex I. A recent study of biochemical abnormalities in HD brain concluded that complex IV inhibition may be an early event13. Lymphocytic function is presumably normal in HD patients. The overall morphology of the cultured lymphoblasts used here seemed to be the same in HD and controls, and we found no difference in growth characteristics of lymphoblasts between HD and control cells (data not shown). Accordingly, the stress-induced abnormalities we found are likely to reflect primary disturbances attributable to the CAG repeats of HD, with abnormal htt disrupting mitocondrial resistance to stress and leading to sensitivity to apoptosis. Why are lymphocytes grossly normal in HD? Unlike neurons, lymphocytes turn over fairly rapidly so that apoptotic lymphocytes are promptly replaced. Presumably, the inability to replace defective neurons explains the brain specificity of HD pathology. It has been proposed that nuclear aggregates are not essential for neuropathology in animal cells, although a nuclear localization for polyglutamine-containing proteins (htt and ataxin-1) is

important3,4. Our data in human HD patients similarly indicate that nuclear inclusions are not necessary for cell death. The relationship between the mitochondrial abnormalities we found and nuclear changes remains to be elucidated. Alhough nuclear disturbances participate in polyglutamine disease pathophysiology3,4, extranuclear factors are also involved especially in HD (refs. 8–14,29,30), consistent with our findings.

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Methods Cells. Lymphocytes were obtained from peripheral blood of seven HD patients, four SCA-1 patients and nine control subjects. HD patients were 21.7 ± 4.6 years old; control subjects were 26.4 ± 1.1 years old; and SCA-1 patients were 44.0 ± 10.2 years old. All HD cells were from female patients, and eight of nine control subjects were female. SCA-1 cells were from three male and one female patient. Lymphoblasts were immortalized by infection with Epstein-Barr virus31. The duration of freezing was the same for cells from each group. Lymphoblasts were maintained in RPMI 1640 medium (Life Technologies) plus 10% fetal bovine serum. Both control and HD cells were thawed at the same time (within 2 weeks of experiments) and maintained in exactly the same condition in culture. Chemicals. Unless otherwise noted, chemicals were obtained from Sigma. Primary antibody against the N terminus of htt (1:500 dilution) was produced in rabbits using a synthetic peptide (amino acids 1–17 of htt) as antigen, and immunopurified. Reagents not purchased from Sigma include NATURE MEDICINE • VOLUME 5 • NUMBER 10 • OCTOBER 1999


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c Activation of caspase-8 (%)

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Fig. 3 Selective activation of a set of caspases in HD lymphoblasts. Data are presented as the ratio of activity after to activity before STS treatment and represent mean values from three independent experiments (a–d). a, Caspase-3 activation is greater in HD cells (open bars) than in control cells (grey bars) after STS stimulation. SCA-1 cells (hashed bars) do not show greater activation than control cells. *, P < 0.01 at 7 h and P < 0.0001 1h at 11 h; HD compared with control. b, Caspase-9 activation is greater in HD cells (open bars) than in control cells (grey bars) after STS stimulation. *, P < 0.01 at 2 h and P < 0.01 at 4 h. Caspase-9 activation occurred before caspase-3 activation. c, There is no substantial activation of caspase-8 in HD cells (open bars) or control cells (grey bars) after 0.025 mM cyanide stress. d, Caspase-8 and caspase-3 are activated both in HD cells (open bars) and control cells (grey bars) after the addition of 100 ng/ml Fas/Apo-1, but there are no differ-

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potassium cyanide, staurosporine (Calbiochem, La Jolla, California), cyclosporin A and PK11195 antibody against Fas/Apo-1 (MBL, Nagoya, Japan). FK506 was a gift from J. Steiner (Guilford Pharmaceuticals, Baltimore, Maryland). Cell viability. Cell viability was monitored with two fluorescent dyes: a cellpermeable dye, Hoechst 33258, and a cell-impermeable dye, ethidium homodimer-1 (Molecular Probes, Eugene, Oregon). The nuclei of cells were stained blue with Hoechst 33258 (final concentration, 20 µg/ml) and red with ethidium homodimer-1 (final concentration, 10 µg/ml). The ratio of red to blue nuclei was used as an index of viability. Three independent experiments were done for all 16 cell lines. Statistical analysis compared the 21 independent values from HD cells (seven cell lines three times) as one group with 27 independent values from control (nine cell lines three times) as another group, as described32. To evaluate apoptotic cell death, Hoechst 33258 was used to visualize nuclear morphology. Visualization of DNA ladder formation. Cells were resuspended in buffer (5 mM Tris-Cl, pH 7.4, 20 mM EDTA, 0.5% Triton X-100), and left on ice for 20 min followed by centrifugation at 27,000g for 20 min. The supernatant was extracted with phenol–chloroform and nucleic acids were precipitated with ethanol. After the pellet was resuspended in 1% RNase without DNase (Boehringer) then incubated at 37 °C for 1 h, the samples were separated by 2% gel electrophoresis. DNA was visualized with ethidium bromide. Caspase assay. Caspase-3 activity was monitored by a colorimetric kit (Clontech, Palo Alto, California). The peptide substrate was conjugated with p–nitroanilide was used from the company and the peptide was used for experiment, and the extent of cleavage was monitored by the change in absorbance at 405 nm. In parallel, the protein concentration of samples was monitored with a Coomassie reagent (Pierce, Rockford, Illinois). Caspase-8 and caspase-9 activities were monitored in a similar way by a colorimetric kit (BioVision, Palo Alto, California). Statistical analysis was done as described above. NATURE MEDICINE • VOLUME 5 • NUMBER 10 • OCTOBER 1999

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ences in the extent of activation between the two cell groups. e, CsA (3 µM) and the caspase inhibitor z-VAD-fmk (100 µM) inhibit cell death induced by 20 nM STS in HD cells. NT, no treatment compared with HD with inhibitors. *, P < 0.005, CsA and P < 0.000001, z-VAD-fmk. Death was evaluated at 20 h after drug addition.

Mitochondrial membrane potential. JC1 (Molecular Probes, Eugene, Oregon) was used to indicate mitochondrial membrane potential18. JC1 produces two emissions (green and red), depending on the mitochondrial membrane potential. The green signal represents depolarized mitochondria; the red, polarized mitochondria. Cells were incubated for 30 min at 37 °C with JC1 (final concentration, 1.5 µM) before measurements by flow cytometry combined with a specific detection system (FACStar plus; Becton Dickinson, Franklin Lakes, New Jersey). Cells were adjusted to a concentration of about 1 × 106 cells/ml, and were presented to the optical path to be excited with 200 mW of monochromatic 488-nm laser light. On a cell-bycell basis, emission was divided into green and red components by placing a 560 nm short-pass dichroic filter between two detectors oriented 90° from each other. Signals from red and green light were isolated by each photomultiplier detector and processed for digitization 4,000 cells per experiment. At least three experiments were done for each sample cell line. The percentages of cells containing polarized or depolarized mitochondria were determined by histogram analysis of the ratio of the two fluorescence intensities. Immunocytochemistry. Immunocytochemistry used confocal microscopy, as described33. The nuclei of lymphoblasts were stained by propidium iodide (final concentration, 0.2 µg / ml). Acknowledgments We thank H.Y. Zoghbi for providing SCA-1 lymphoblasts, and M. McInnis for providing control lymphoblasts. We thank S. Gartner for providing her facility. We thank C. Callahan, L. Hanle, X. Luo, A. McCall, M. Delanoy for their technical assistance. We thank J. Ha, G. Thinakaran and C.D. Ferris for discussions. We thank all the members of S.H.S. and C.A.R. labs for scientific support. We also thank D. Dodson, A. Kodaira, I. Yamamoto for typing and statistical analysis. This paper was supported by USPHS grant MH-18501 and Research Scientist Award DA-00074 to S.H.S.; NS16375 from NIH and HDSA “Coalition for the Cure” to C.A.R.; and a research grant from the Brain Science Foundation (Japan) to A.S. 1197


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