Cleavage of Atrophin-1 at Caspase Site Aspartic Acid 109 Modulates Cytotoxicity*

Lisa M. Ellerbya, Rebecca L. Andrusiaka, Cheryl L. Wellingtonbc, Abigail S. Hackambd, Stephanie S. Proppa, Jonathan D. Woode, Alan H. Sharpe, Russell L. Margolise, Christopher A. Rossef, Guy S. Salvesenag, Michael R. Haydenbh, and Dale E. Bredesenaij

From the a Programs on Aging and Apoptosis, The Burnham Institute, La Jolla, California 92037, the b Centre for Molecular Medicine and Therapeutics, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V52 4H4, Canada, the e Department of Psychiatry, Johns Hopkins University Medical School, Baltimore, Maryland 21205, and the i Department of Neuroscience, University of California, San Diego, California 92093

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Dentatorubropallidoluysian atrophy (DRPLA) is one of eight autosomal dominant neurodegenerative disorders characterized by an abnormal CAG repeat expansion which results in the expression of a protein with a polyglutamine stretch of excessive length. We have reported recently that four of the gene products (huntingtin, atrophin-1 (DRPLA), ataxin-3, and androgen receptor) associated with these open reading frame triplet repeat expansions are substrates for the cysteine protease cell death executioners, the caspases. This led us to hypothesize that caspase cleavage of these proteins may represent a common step in the pathogenesis of each of these four neurodegenerative diseases. Here we present evidence that caspase cleavage of atrophin-1 modulates cytotoxicity and aggregate formation. Cleavage of atrophin-1 at Asp109 by caspases is critical for cytotoxicity because a mutant atrophin-1 that is resistant to caspase cleavage is associated with significantly decreased toxicity. Further, the altered cellular localization within the nucleus and aggregate formation associated with the expanded form of atrophin-1 are completely suppressed by mutation of the caspase cleavage site at Asp109. These results provide support for the toxic fragment hypothesis whereby cleavage of atrophin-1 by caspases may be an important step in the pathogenesis of DRPLA. Therefore, inhibiting caspase cleavage of the polyglutamine-containing proteins may be a feasible therapeutic strategy to prevent cell death.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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To date, eight different dominantly inherited neurodegenerative diseases have been shown to be associated with polyglutamine tract expansions in their respective proteins (1-3). These include Huntington disease, spinal and bulbar muscular atrophy (Kennedy's disease), Machado-Joseph disease (SCA-3), dentatorubropallidoluysian atrophy (DRPLA),1 and spinocerebellar ataxia types 1, 2, 6, and 7 (SCA-1, SCA-2, SCA-6, SCA-7) (4-12). Expansion of the polyglutamine repeat in these disease proteins results in selective death of neurons in different regions of the brain.

Because all of these disease-associated proteins share a similar mutation, i.e. CAG expansion in the coding region causing expansion of a polyglutamine stretch, they may have a common pathological mechanism leading to neuronal cytotoxicity. Except for the polyglutamine tract, it is generally believed that the eight disease proteins are unrelated because their amino acid sequences bear no discernible sequence homology. However, recent evidence from our laboratories (13-15)2 suggest that seven of the eight identified polyglutamine repeat proteins involved in CAG expansion diseases contain caspase consensus cleavage sites (i.e. DXXD). This would imply that a second common feature of at least seven of the polyglutamine expansion disease proteins may be their involvement in the apoptotic cell death pathway as cellular substrates for the caspases. This finding has important implications because studies in vitro and in vivo indicate that the truncated forms of these proteins lead to the formation of intracellular aggregates, and thus caspase cleavage of the full-length proteins could in part explain how these cytotoxic truncated proteins are formed (16-23).

To date, we have characterized the caspase cleavage of four of the polyglutamine repeat disease proteins: huntingtin, the androgen receptor, atrophin-1 (DRPLA), and ataxin-3 (Machado-Joseph disease) (13-15). These initial studies suggested that a caspase-dependent apoptotic pathway may be a critical factor in the generation of truncated proteins in some of these polyglutamine repeat disease proteins and raised a number of questions that warrant further investigation: Is the proteolytic pathway involving caspases required to explain a common mechanism of cytotoxicity of these proteins with expanded polyglutamine stretches? How are these caspase substrates involved in the apoptotic process and neurodegeneration? Are any of the functional domains within these proteins activated or inactivated by caspase cleavage? Does the proteolytic processing by caspases form the basis for selective neuronal loss characteristic of these neurodegenerative diseases? Does the distinct cellular localization of each of these structurally and functionally unrelated proteins determine which caspase family member cleaves them? Do other proteolytic pathways contribute to the cytotoxicity of these proteins?

Given our recent findings, we wished to address the following specific questions: Is caspase cleavage required for the cytotoxicity exhibited by these proteins? In other words, is the formation of a truncated protein containing the polyglutamine stretch via a caspase-dependent pathway required for cytotoxicity? Additionally, we wished to address whether the caspase cleavage site is required for the formation of the protein aggregates and/or altered cellular localization characteristic of these diseases. Of the four proteins we reported recently as caspase substrates, atrophin-1 is a particularly attractive candidate for our initial investigation because in all likelihood it contains only one caspase cleavage site within the entire protein. This is in contrast to ataxin-3, huntingtin, and androgen receptor, which are cleaved at multiple sites within the protein. Here we provide in vitro evidence that caspase cleavage of atrophin-1 modulates cytotoxicity, formation of protein aggregates, and its subcellular localization.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Culture and Transfection of Cells-- Cells from the human embryonic kidney cell line 293T were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum with 1% penicillin/streptomycin. Transient transfection was carried out with pcDNA3, pcDNA3-DRPLA26, pRc/CMV-LacZ, pcDNA3-DRPLA65, pcDNA3-DRPLA26D109N, and pcDNA3-DRPLA65 D109N (24). Preparation of the atrophin-1 constructs has been described previously (14, 25). Using pRc/CMV-LacZ, transfection efficiency was determined by staining for the expression of beta -galactosidase. Cell death was measured by trypan blue exclusion, acridine orange/ethidium bromide, and LacZ reporter gene cotransfection. Death was established as apoptotic based on acridine orange/ethidium bromide staining and assessment of caspase-3 activation. Cell death was measured 36-50 h after transfection. Cellular death in confluent cells was induced with tamoxifen citrate at a concentration of 35 µM 36-48 h after transfection (26). Data were collected for three to five experiments and then compared by Student's t test for statistical significance. Apoptosis was also monitored with the ApoAlert caspase assay kit according to the manufacturer's instructions with the Ac-DEVD-AFC substrate (CLONTECH).

In Vitro Translation Reactions-- Plasmids pcDNA3, pcDNA3-DRPLA26, pRc/CMV-LacZ, pcDNA3-DRPLA65, pcDNA3-DRPLA26D109N, and pcDNA3-DRPLA65 D109N were transcribed using T7 polymerase and then translated using the TNT system (Promega) in the presence of [35S]methionine. Translations (2.5 µl) were incubated with 10-50 ng caspase-3 for 2 h in the following buffer (10 µl): 20 mM PIPES, 100 mM NaCl, 1% CHAPS, 10% sucrose, 10 mM dithiothreitol, and 0.1 mM EDTA, pH 7.2 at 37 °C.

Purification of Caspase-- His-tagged caspase-3 was purified by nickel affinity chromatography as described previously (27-29).

Caspase Western Analysis-- Western blots were carried out as described previously using anti-caspase-3 mouse monoclonal antibody (Transduction Laboratories) (26).

Atrophin-1 Western Blotting-- 293T cells were transiently transfected at 40% confluence using a modified calcium phosphate protocol by mixing Qiagen-prepared DNA (Qiagen, Chatsworth, CA) with 2.5 mM CaCl2 and 2 × BBS (50 mM BES, 280 mM NaCl, 1.5 mM Na2HPO4, pH 7.0) and adding the mixture to cells immediately. After a 3-h incubation, the media were removed and replaced with fresh growth media. At 24 h post-transfection, cells were either treated with 35 µM tamoxifen (Sigma) for 4 h or left untreated. Cells were harvested by gentle scraping into the growth media and centrifugation at 4,000 × g for 4 min. Samples were washed once with PBS, centrifuged as before, and suspended in lysis buffer (20 mM HEPES, 5 mM MgCl2, 0.5 mM EDTA, 0.01% (w/v) sucrose, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, and 10 mg/ml aprotinin). Equal amounts of total cellular protein were mixed with 5 × Laemmli sample buffer, denatured at 95 °C for 5 min, and separated on 7.5% SDS-polyacrylamide gels. Protein was transferred electrophoretically to polyvinylidene difluoride membrane, immunoblotted with anti-atrophin-1 antibody, and detected using enhanced chemoluminescence (Amersham Pharmacia Biotech).

Site-directed Mutagenesis and Plasmid Construction-- Human DRPLA26D109N and DRPLA65D109N were created using the QuikChange site-directed mutagenesis system from Stratagene. pcDNA3-DRPLA constructs were used as templates with the following two synthetic primers according to manufacturer instructions: 5'-CCGATCTGGATAGCTTGAACGGGCGGAGCCTTAATG-3' and 5'-CATTAAGGCTCCGCCCGTTCAAGCTATCCAGATCGG -3'.

Mapping of Caspase Cleavage Sites by Radiosequencing-- Radiosequencing was performed as described previously (27, 30). Plasmid pcDNA3-DRPLA26 was transcribed and translated with T7 polymerase using the TNT system (Promega) with either [35S]methionine or [3H]leucine. The translation was treated with caspase-3, separated by SDS-polyacrylamide gel electrophoresis, and electroblotted onto a polyvinylidene difluoride membrane. After autoradiography, the position of the [35S]methionine-labeled atrophin-1 fragments was used to cut out the [3H]leucine atrophin-1 bands from the polyvinylidene difluoride membrane. The samples were subjected to automated sequencing using an Applied Biosystems 476A sequencer, and the anilinothiazolinone derivatives in each cycle were counted in a scintillation counter. Comparison of the known positions of leucines relative to the caspase cleavage site aspartate allowed identification of the atrophin-1 cleavage site.

Immunofluorescence Microscopy-- 293T cells were grown on glass coverslips and transiently transfected with the indicated DRPLA construct as described above. At 36 h post-transfection, the cells were treated with 35 µM tamoxifen for 45 min. After fixation in 4% paraformaldehyde and PBS solution for 20 min, the cells were washed and then permeabilized in 0.5% Triton X-100 PBS for 5 min. The DRPLA antibody utilized in these studies has been described by Wood et al. (31) and was raised in rabbits against synthetic peptide DRPLA425 (residues 425-439 of atrophin-1). The cells were washed twice, incubated at room temperature with anti-DRPLA antibody (1:200) for 1 h, washed three times with PBS, and then incubated in Texas red-conjugated anti-rabbit antibody (1:1,000) for 20 min. Cells were washed three times with PBS and then mounted onto slides with DAPI (4',6'-diamindino-2-phenylindole, Sigma, 0.05 µg/ml) in 90% glycerol and PBS as a nuclear counterstain. Immunofluorescence was observed using a Zeiss confocal microscope. Control experiments were performed, including incubation with secondary antibody only, and immunofluorescence of cells transfected with control plasmids.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Analysis of the Atrophin-1 Caspase Cleavage Site-- DRPLA is one of eight autosomal dominant neurodegenerative diseases with expansion of CAG trinucleotide repeats encoding polyglutamine stretches (32). This neurodegenerative disorder is characterized by progressive dementia, myoclonic epilepsy, cerebellar ataxia, and choreoathetotic movements. Like many of these disease-associated proteins, atrophin-1 is expressed ubiquitously in the central nervous system (33), and thus its expression pattern offers little clue to the relative susceptibility or resistance of certain neuronal populations of cells to undergo neurodegeneration.

We and others have demonstrated recently that atrophin-1 is cleaved by caspases (14, 34). Atrophin-1 is one of at least 40 cellular caspase substrates identified, and its function, as well as its contribution to the apoptotic process, is unknown. Atrophin-1 contains a consensus caspase-3 cleavage site (14, 35) near the NH2 terminus of the protein (106DSLD109) (Fig. 1A), and the polyglutamine tract is located in the middle of the protein (Fig. 1A). The cleavage products generated during caspase-3 cleavage of in vitro translated atrophin-1 migrated at 145 and 150 kDa for constructs with 26 and 65 glutamines, respectively (Fig. 1B, lanes 2 and 6). These COOH-terminal fragments contain the polyglutamine tract and would be expected to lack the predicted nuclear targeting sequence located at the NH2 terminus of the protein at amino acids 16-32 (see Fig. 1B). NH2-terminal sequencing of the DRPLA cleavage product confirmed that atrophin-1 was cleaved at Asp109.


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Fig. 1.   Panel A, primary structure of normal and mutant human atrophin-1. Caspases cleave atrophin-1 in the NH2-terminal region of the protein at Asp109. The predicted nuclear localization signal sequence is also located in the NH2-terminal region of the protein and released from the polyglutamine-containing region of the protein upon caspase cleavage. Panel B, in vitro translated DRPLA26 (lanes 1 and 2), DRPLA26D109N (lanes 3 and 4), DRPLA65 (lanes 5 and 6), and DRPLA65D109N (lanes 7 and 8) before treatment (-) or after treatment (+) with caspase-3. Panel C, Western blot of 293T cells transiently transfected with DRPLA26, DRPLA26D109N, DRPLA65, and DRPLA65D109N without (-) or with (+) a 4-h treatment with 35 µM tamoxifen. wt, wild type.

To analyze the functional significance of caspase cleavage we prepared constructs of DRPLA without a caspase cleavage site. Mutation of the caspase P1 residue in atrophin-1 from Asp109 to Asn abolished the processing of the in vitro translated atrophin-1 (Fig. 1B, lanes 4 and 8) by caspases. Because we have shown previously that atrophin-1 can be cleaved by caspase-1, caspase-7, and caspase-8 in addition to caspase-3, we evaluated whether the Asp109 mutation abolished cleavage by multiple caspases present in transfected cells by Western blotting (Fig. 1C). Caspase cleavage products were observed in 293T cells transiently transfected with DRPLA26 or DRPLA65 after tamoxifen treatment, showing that tamoxifen challenge induced caspase activation in cells transfected with these constructs. In contrast, no cleavage products were generated in cells transfected with DRPLA26D109N or DRPLA65D109N after tamoxifen treatment. These results show that we have prepared atrophin-1 proteins resistant to caspase cleavage in vitro and in transfected cells. Therefore, we utilized these constructs to test whether this site influences the cytotoxicity of atrophin-1 in cell culture.

Increased Cytotoxicity of DRPLA Mutant Protein-- We have recently developed an in vitro tissue culture model to investigate the cellular toxicity of polyglutamine repeat expansion disease proteins (15, 17). In this system, a sublethal stress is induced by tamoxifen in transiently transfected human embryonic kidney 293T cells. A sublethal stress in these studies is defined as a stress delivered by a concentration of a pro-apoptotic agent that does not result in the processing of caspase-3 in plasmid control transfected cells. As shown in Fig. 2A, treatment of 293T cells with tamoxifen at a concentration of 35 µM does not lead to processing of caspase-3 over a 24-h period of time. Higher concentrations of tamoxifen (>40 µM) result in apoptotic cell death based on acridine orange/ethidium bromide staining of the cells (data not shown) and the processing of caspase-3 (Fig. 2B).


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Fig. 2.   Effect of tamoxifen on caspase-3 processing in 293T cells analyzed by Western blot. Panel A, proteolytic profile of caspase-3 when treated with sublethal concentrations of tamoxifen (35 µM) for the indicated times. This concentration of tamoxifen is sublethal in nontransfected cells, as indicated by no capase-3 processing. Panel B, proteolytic profile of caspase-3 processing during tamoxifen-induced cell death (50 µM), forming the active 17-kDa fragment.

To investigate the cytotoxicity of atrophin-1, we transiently transfected 293T cells with expression constructs encoding the human atrophin-1 gene with a normal CAG repeat length (DRPLA26) and an expanded CAG repeat length (DRPLA65). These cells were then treated with sublethal concentrations of tamoxifen (35 µM) 36 h after transfection. Untransfected 293T cells do not express atrophin-1, and lack of expression in transfected cells was verified by immunofluorescence and Western analysis. 293T cells expressing DRPLA26 induced significantly higher proportions of apoptotic cell death than vector controls (Fig. 3A) when cells were exposed to tamoxifen. Furthermore, there was a statistically significant increase in cell death and corresponding caspase activity (Fig. 3B) when the polyglutamine repeat length was expanded (DRPLA26 versus DRPLA65), indicating a gain of function for the disease-associated form of atrophin-1 protein. Western analysis verified equal expression of each of the atrophin-1 proteins.


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Fig. 3.   Expansion of the polyglutamine repeat within atrophin-1 from 26 to 65 glutamines enhances the pro-apoptotic effect of DRPLA. Panel A, mutation of the NH2-terminal caspase cleavage site inhibits the cytotoxic effect of both wild-type and expanded atrophin-1. 293T cells were transiently transfected with plasmids pcDNA3, pcDNA3-DRPLA26, pRc/CMV-LacZ, pcDNA3-DRPLA65, pcDNA3-DRPLA26D109N, and pcDNA3-DRPLA65 D109N at 25% confluence. Death was induced 48 h after transfection with 35 µM tamoxifen, and the percent of apoptotic cells was measured with trypan blue. Panel B, caspase activity assays of 293T cells transfected with the indicated constructs and treated with 35 µM tamoxifen for 2.5 h. The difference in cell death of pcDNA3-DRPLA26- and pcDNA3-DRPLA65-transfected cells was statistically significant (p < 0.01).

Interestingly, the expression of DRPLA26 is pro-apoptotic when exposed to an apoptotic stress. Because the function of atrophin-1 protein is unknown, it is difficult to speculate how overexpression of normal atrophin-1 enhances cellular death. However, there is a growing body of literature suggesting that many of the caspase substrates can act to enhance or block apoptotic cell death upon cleavage by caspases. For example, cleavage of presenilin-2 results in the generation of an anti-apoptotic cleavage product (36). In contrast, expression of the caspase substrate mitogen-activated protein kinase kinase results in the generation of a pro-apoptotic fragment that enhances caspase activation through a positive feedback loop (27). Furthermore, we have reported recently that the truncated fragment of Huntington disease containing the normal polyglutamine repeat is pro-apoptotic (17). Our results demonstrate that DRPLA65 is more pro-apoptotic than DRPLA26, which indicates that the gain of function related to CAG length may influence downstream events in apoptosis.

The Pro-apoptotic Effect of Atrophin-1 Requires Cleavage-- Next, we assessed the effect of blocking caspase cleavage of DRPLA26 and DRPLA65 on the pro-apoptotic effects of these proteins in culture. As shown in Fig. 3, transient transfection of DRPLA26D109N and DRPLA65D109N resulted in almost complete suppression of apoptotic cell death when compared with DRPLA26 and DRPLA65. Therefore, proteins with the same CAG length which are no longer cleaved by caspases have decreased toxicity.

Increased Cytotoxicity of Atrophin-1 Correlates with Formation of Aggregates and Altered Nuclear Localization-- Because our cytotoxicity studies indicated that the caspase cleavage site is crucial to the pro-apoptotic effect of atrophin-1, we investigated whether apoptosis induction with tamoxifen modulated the formation of aggregates. Intracellular neuronal inclusions may be a common property for glutamine repeat expansion diseases (37). Aggregates have been reported recently in the brains of patients with DRPLA and are similar to those observed in Huntington disease (16, 20, 38). Immunofluorescence analysis of transiently transfected 293T cells or COS-7 cells indicated that atrophin-1 is localized at the outer border of the nucleus, consistent with its putative nuclear localization signal (Fig. 4A). This result differs from earlier work that has suggested that atrophin-1 is localized to the cytoplasm (20, 39). Some of these earlier studies used epitope-tagged proteins in which the epitope may have interfered with the nuclear targeting signal. Expression of DRPLA65, but not DRPLA26, led to formation of densely stained nuclei (granular in appearance) with altered nuclear distribution upon apoptosis induction compared with the controls (atrophin-1-transfected cells not treated with tamoxifen), as determined by confocal microscopy (Fig. 4, A and C). Normal atrophin-1 protein is localized to the outer edge of the nuclei before and after apoptotic stimulation, whereas the disease-associated form of atrophin-1 leads to dense, particulate staining throughout the nuclei during apoptotic stimulation. Further analysis (Fig. 5) of the disease-associated form of atrophin-1 demonstrates that atrophin-1 colocalizes with DAPI-stained nuclei during apoptotic stimulation with tamoxifen (Fig. 5, A-C), and confocal images under high magnification show that the aggregates are nuclear (Fig. 5, D-F). Thus, the disease-associated form of atrophin-1 shows an altered pattern of nuclear distribution compared with the normal form of atrophin-1. Modulation of aggregate formation did not occur in the absence of apoptosis induction for DRPLA65-transfected cells (data not shown) during the first 48 h after transfection. Interestingly, longer periods of time after transfection (>72 h) resulted in increased formation of cytoplasmic aggregates. These aggregates were larger in the DRPLA65-transfected cells and occurred with higher frequency (Fig. 6).


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Fig. 4.   Immunofluorescence of atrophin-1 with the expanded polyglutamine repeat shows altered nuclear distribution/and or aggregates in 293T cells during apoptotic stimulation. Panel A, localization of atrophin-1 with 26 repeats (DRPLA26) in 293T cells after tamoxifen treatment (1 h, 35 µM) shows homogeneous staining on the outer edge of the nucleus. Panel B, localization of the caspase cleavage site mutant atrophin-1 (DRPLA26 D109N) in 293T cells after tamoxifen treatment (1 h, 35 µM). Panel C, localization of atrophin-1 with 65 repeats (DRPLA65) in 293T cells after tamoxifen treatment (1 h, 35 µM) shows an altered subcellular distribution and particulate granular staining. Panel D, localization of caspase cleavage site mutant atrophin-1 (DRPLA65 D109N) in 293T cells after tamoxifen treatment (1 h, 35 µM). Images were collected with a confocal microscope.


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Fig. 5.   Subcellular localization of atrophin-1 with expanded repeats in transfected 293T cells treated with tamoxifen. Panel A, immunofluorescence of atrophin-1 with expanded repeats localizes with (panel B) DAPI-stained nuclei demonstrating a nuclear distribution. Panel C, phase-contrast microscopy of the same cells. The images in panels A, B, and C were collected at a magnification of × 400 on a Zeiss inverted microscope. Panel D, confocal image of atrophin-1 stained with atrophin-1 antibody demonstrates substantial aggregation with nuclear localization. Panel E, phase-contrast of the same cells. Panel F, overlay of panels D and E. The images in panels D, E, and F were collected using a confocal microscope at a magnification of × 1000.


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Fig. 6.   Immunofluorescence of atrophin-1 normal (DRPLA26) and disease causing (DRPLA65) 3 days after transfection in 293T cells without apoptotic stimulation. Cells expressing DRPLA65 (bottom panel) have cytoplasmic aggregates (arrows) that are found at much higher frequency and are larger than those found in cells expressing DRPLA26 (top panel). Images were collected with a confocal microscope.

Because aggregate formation was modulated by the stimulation of apoptosis, we next assessed whether mutation of the caspase cleavage site in DRPLA65 blocks formation of aggregates. Cells transfected with pcDNA3-DRPLA26 expressed atrophin-1 protein that was localized to the outer edge of the nucleus with a homogeneous pattern during apoptotic cell death with tamoxifen (Fig. 4A). Cells transfected with pcDNA3-DRPLA65 showed granular dense straining throughout the nucleus (Fig. 4C) during apoptotic cell death. In sharp contrast, cells transfected with pcDNA3-DRPLA65D109N did not show altered nuclear distribution and granular dense staining, suggesting that, at least in this system, caspase cleavage of DRPLA is required for aggregation (Fig. 4, B and D). It is of relevance to compare our results with those found for SCA1, given that it is also a nuclear protein yet does not appear to be cleaved by caspases. Interestingly, recent studies on SCA1 show that the subcellular localization of wild-type ataxin-1 differs from the mutant ataxin-1 both in vitro and in vivo. Wild-type ataxin-1 localizes to the nucleus in COS-1 cells (40), whereas mutant ataxin-1 shows a specific redistribution or disruption of the nuclear structure. In these studies there was no evidence that apoptosis modulated the formation of aggregates, which is consistent with our finding that ataxin-1 is not a caspase substrate.2 Ataxin-1 redistribution may be important for the pathogenic mechanism in this disease, and additional studies will determine whether this is similarly the case for atrophin-1.

In this study, we show that cells transfected with expression constructs encoding atrophin-1 undergo enhanced apoptotic cell death that is mediated by a pro-apoptotic caspase cleavage product. In vitro mutagenesis of caspase cleavage site Asp109 blocks production of this pro-apoptotic fragment and reduces cellular toxicity dramatically. This is consistent with our recent findings that expression of truncated huntingtin fragments resulted in significantly more cell death than the full-length huntingtin (17, 22). Our results suggest that caspase cleavage is required for modulation of aggregate formation but does not determine whether aggregation is required for cellular toxicity. Recent work on ataxin-1 indicates that nuclear localization is critical for pathogenesis but not aggregation (41).

The results described here, along with our recent work (13-15, 17), suggest that one common feature shared among at least seven of the polyglutamine repeat disease proteins is that they are cleaved by caspases to produce pro-apoptotic fragments. In this model, initial cleavage by caspases or other proteases would produce a toxic fragment with a gain of toxic function, e.g. aggregation or altered protein-protein interactions. This generation of a toxic fragment would lead to increased activation of caspases through a feedback loop. In other words, the toxic fragments may function as caspase amplifiers. This amplification loop would be highly dependent upon the cellular context such as caspase/inhibitor distribution within the cell as well as protein-protein and/or protein-ligand interaction with each type of polyglutamine repeat protein (14). Further, amplification would also be dependent upon the ability of a particular cell type to evoke proteolytic pathways that remove this toxic caspase-amplifying fragment. These experiments have not addressed what is the initial trigger for caspase activation in the disease process but suggest that proteolysis is important for cytotoxicity. However, physiological stresses that are otherwise sublethal may in the presence of a caspase amplification mechanism lead to cell death. In addition, the results in this study do not exclude additional mechanisms for proteolytic cleavage of atrophin-1 or the other CAG-containing gene products generating a smaller toxic fragment.

Furthermore, the results do not offer an explanation for the specific pattern of neuronal loss in CAG repeat diseases. It is possible that alterations in caspase expression, caspase inhibitors, partner proteins, or downstream targets may determine the selective vulnerability for each of the CAG repeat diseases. Further studies directed at identifying the specific caspase family members that process the CAG repeat disease proteins, as well as a study of the regional specificity of the caspases in the brain, should shed light on this question. Finally, because blocking the cleavage of atrophin-1 inhibits its pro-apoptotic effect, such a strategy may prove useful for the treatment of neurodegenerative diseases associated with polyglutamine repeat expansions. Extension of these results to animal models of polyglutamine expansion diseases should prove useful.

    FOOTNOTES

* This work was supported in part by Grants AG12282 and CA69381 from the National Institutes of Health (to D. E. B) and by the Glendorn Foundation (Catherine Dorn, trustee).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

c  Hereditary Disease Foundation postdoctoral fellow.

d  Medical Research Council of Canada postdoctoral fellow.

f  Supported by Grants NS34172 and NS16375 from the NINDS, National Institutes of Health.

g  Supported by Grant NS37878 from the National Institutes of Health.

h  Supported by operating grants from the Canadian Networks of Centers of Excellence (NCE-Genetics) and Medical Research Council of Canada. Established investigator of the British Columbia Children's Hospital.

j  To whom correspondence should be addressed: Program on Aging, The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-455-6480; Fax: 619-646-3192; E-mail: dbredesen{at}ljcrf.edu.

2 L. M. Ellerby, R. L. Andrusiak, C. L. Wellington, A. S. Hackam, S. S. Propp, J. D. Wood, A. H. Sharp, R. L. Margolis, C. A. Ross, G. S. Salvesen, M. R. Hayden, and D. E. Bredesen, unpublished data.

    ABBREVIATIONS

The abbreviations used are: DRPLA, dentatorubropallidoluysian atrophy; SCA, spinocerebellar ataxia; CMV, cytomegalovirus; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; PBS, phosphate-buffered saline; DAPI, 4',6'-diamindino-2-phenylindole.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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