Cell Death and Differentiation (2001) 8, 377 ± 386 ã 2001 Nature Publishing Group All rights reserved 1350-9047/01 $15.00 www.nature.com/cdd Extended polyglutamine selectively interacts with caspase-8 and -10 in nuclear aggregates 1 ,1 1 1 MU, T Miyashita* , Y Ohtsuka , Y Okamura-Oho , Introduction Y Shikama1 and M Yamada1 To date, eight neurodegenerative disorders including 1 Department of Genetics, National Children's Medical Research Center, Huntington's disease (HD) and dentatorubral pallidoluysian 3-35-31, Taishido, Setagaya, Tokyo 154-8509, Japan atrophy (DRPLA), have been shown to be caused by the * Corresponding author: T Miyashita, Department of Genetics, National expansion of CAG repeats.1,2 The CAG repeats are located in Children's Medical Research Center, 3-35-31, Taishido, Setagaya, Tokyo the coding regions of the respective responsible genes and 154-8509, Japan. Tel: +81-3-3414-8121; Fax: +81-3-3414-3208; are translated into polyglutamine. These disorders, collec- E-mail: [email protected] tively called CAG-repeat disorders or polyglutamine diseases, Received 30.5.00; revised 18.10.00; accepted 21.11.00 are characterized by neuron death in the region specific to 3 Edited by DR Green each disease. The molecular mechanism underlying these disorders has yet to be clarified. However, accumulating evidence suggests that caspases, at least in part, are involved Abstract in the neurodegeneration. Caspases constitute a family of cysteine proteases that A growing number of inherited neurodegenerative disorders, play important roles in the execution of apoptosis. Each of including Huntington's disease, have been shown to be these enzymes is synthesized as a zymogen and caused by the expansion of CAG/polyglutamine repeats. The proteolytically activated by its own active form or other molecular mechanism underlying these disorders, however, members of caspases.4 Involvement of caspases in CAG- has yet to be clarified. We and others previously demonstrated repeat disorders are supported by three groups of that caspase-8 was activated by proteolysis in association observations: (i) Some of the products of the genes with the expression of extended polyglutamine. Here, we responsible for CAG-repeat disorders are substrates for further analyzed the selectivity of caspases in the process caspases, and their cleaved fragments carrying extended 5±8 mediated by extended polyglutamine. Among upstream polyglutamine tend to easily form aggregates; (ii) an caspases, caspase-10, a close homolog of caspase-8, was activated form of caspases has been detected in the also proteolytically activated, but caspase-9 was not. affected regions of the brains of patients and of model animals as well as in cultured cells of experimental Caspase-8 and -10 were recruited into nuclear aggregates of systems;9±11 and (iii) inhibition of caspases slows cell extended polyglutamine, where at least a fraction of these death or suppresses the apoptotic nuclear phenotype in caspases was converted to the activated forms. Caspase-8 model animals as well as in in vitro experimental and -10 were co-immunoprecipitated with polyglutamine only systems.10,12 when the polyglutamine was pathologically extended, We previously demonstrated that expression of extended whereas caspase-2, -3, -6, -7 and -9 were not co-immunopre- polyglutamine sequentially activated caspase-8 and then cipitated with polyglutamine regardless of its size. A caspase-3.9 SaÂnchez et al.10 also reported detection of the dominant-negative form of caspase-8 with a mutation at the proteolytically activated form of caspase-8 in the affected catalytic cysteine residue inhibited polyglutamine-mediated brain region of HD patients. Caspase-8 (MACH/FLICE/ nuclear apoptotic phenotype. These results suggest that Mch5) was originally cloned as a protein that interacts with caspase-8 and -10 are autoactivated as a result of close FADD (MORT1), a mediator of Fas (CD95/APO-1)-induced apoptosis, or as a component of death-inducing signaling proximity of the proforms of these molecules that occurs due complex (DISC).13,14 The targeted disruption of the to aggregate formation, which reveals a novel toxic gain-of- caspase-8 gene is lethal in utero and the TNF receptors, function mechanism for the pathogenesis of CAG-repeat Fas, and DR3 failed to induce cell death in embryo disorders. Cell Death and Differentiation (2001) 8, 377 ± 386. fibroblasts containing a disrupted caspase-8 gene.15 To analyze the selectivity of caspases during the process of cell death mediated by extended polyglutamine Keywords: polyglutamine; caspase; CAG repeats; Huntington's and to further investigate the mechanism of caspase disease activation, we have analyzed the interaction of polygluta- mine and various caspases. We show here that poly- Abbreviations: DISC, death-inducing signaling complex; DRPLA, glutamine preferentially interacts with and activates dentatorubral pallidoluysian atrophy; GFP, green ¯uorescent caspase-8 and its close homolog, caspase-10 (Mch4/ protein; HD, Huntington's disease; PML, promyelocytic leukemia FLICE2),16,17 and suggest a novel caspase-8- or -10- protein; TC, tetracycline; TRITC, tetramethylrhodamine isothiocya- mediated cell death pathway alternative to Fas-mediated nate; z-VAD-fmk, carbobenzoxy-Val-Ala-Asp-¯uoromethyl ketone apoptosis. Expanded polyglutamine interacts with caspase-8 and -10 MUet al 378 Results the intact form (Figure 1B). The antibody had no cross reactivity with the intact or processed forms of caspase-3, -8, Among initiator caspases, caspase-8 and -10 are -6, or -7 (Figure 1B, and data not shown). These results proteolytically activated by polyglutamine, but indicate that the caspase-9-mediated apoptotic pathway is not caspase-9 is not involved in polyglutamine-induced cell death. We and others previously demonstrated that caspase-8 was cleaved and activated in association with the expression of extended polyglutamine.9,10 In the present study, we first Caspase-8 and -10 colocalize with nuclear examined whether caspase-10, a close homolog of caspase- aggregates of extended polyglutamine 8, was also proteolytically activated during this process. To Initiator caspases such as caspase-8 and -9 were reported to address this question, we utilized the TC-controlled poly- be autoactivated by oligomerization of the molecules21,22 If glutamine expression system in IGROV cells as described procaspase-8 coaggregates with polyglutamine, caspase-8 previously.9 IGROV-EQ19 and IGROV-EQ56 cells express molecules are brought into close proximity and may cleave 19- and 56-stretches of polyglutamine fused with the one another. To test this hypothesis, we first analyzed the enhanced green fluorescent protein (EGFP) (EGFP-polyQ19 subcellular localization of caspases-8 and -10 in the presence and EGFP-polyQ56), respectively, when TC is removed from or absence of nuclear aggregates of extended polyglutamine. the culture medium. The cleaved products of caspase-8 and By immunostaining, caspase-8 and -10 were detected -10 were detected at 72 h after withdrawal of TC only in cells primarily in the cytoplasm (Figure 2B,E, thin arrows). expressing extended polyglutamine (Figure 1A, top and However, in cells with nuclear aggregates of extended middle panels). Another apical caspase that plays an polyglutamine, these caspases were primarily detected in important role in apoptosis is caspase-9 (MCH6), which is the nucleus, and co-localized with the aggregates (Figure 2B, activated by Apaf1 and cytochrome c released from E, thick arrows, and Figure 2C, F). The specificity of the mitochondria.19 In contrast to caspase-8 and -10, the amount results was demonstrated by the absence of co-localization of of the proform of caspase-9 remained constant and the caspase-3 and Bcl-2 with the aggregates (Figure 2G ± L). This cleaved form was not detected by 72 h after the TC also indicates that the nuclear signals in Figure 2B, E are not withdrawal (Figure 1A, bottom panel). The caspase-9 anti- due to the leakage of green fluorescence of GFP- body used in this experiment was confirmed to react with the polyglutamine. processed form of caspase-920 by the use of an engineered construct, caspase-9DC (amino acids 1 ± 315), in addition to Figure 1 Extended polyglutamine activated caspase-8 and -10, but not caspase-9. (A) IGROV-EQ19 and IGROV-EQ56 cells were incubated in the presence or absence of TC. Cell lysates were prepared at 72 h and Western blot analyses were performed using an anti-caspase-8 (upper panel), anti- Figure 2 Co-localization of caspase-8 and -10 with nuclear aggregates of caspase-10 (middle panel), or anti-caspase-9 antibody (lower panel). (B) extended polyglutamine. IGROV-EQ56 cells were incubated in the absence of Proforms and processed forms of caspases were translated in vitro in the TC for 3 days to induce EGFP-polyQ56 expression, and subjected to presence (upper panel) or absence (lower panel) of L-[35S]-methionine. immunostaining followed by confocal microscopy. (A,D,G,J) Green fluores- Translated products were subjected to SDS ± PAGE followed by autoradio- cence shows the presence of EGFP-polyQ56.(B,E,H,K) Red fluorescence graphy (upper panel), or Western blotting using the anti-caspase-9 antibody indicates the localization of endogenous caspase-8, caspase-10, caspase-3, (lower panel). Closed and open arrowheads indicate procaspase-9 and and Bcl-2, respectively, by immunostaining with the respective antibodies. processed caspase-9, respectively (C,F,I,L) Merged images of the left two panels. Bar: 10 mm Cell Death and Differentiation Expanded polyglutamine interacts with caspase-8 and -10 MUet al 379 We then applied the two antisera to the cells expressing Caspase-8 is activated in nuclear aggregates extended polyglutamine and forming nuclear aggregates To test whether caspases were activated in the nuclear as in the experiments shown in Figure 2. Activated aggregates, we raised antibodies that specifically react with a caspase-8 was detected in nuclear aggregates containing processed form of caspase-8 or -9. It has been demonstrated extended polyglutamine (Figure 4A ± C).