Tissue Transglutaminase Selectively Modifies Proteins Associated with Truncated Mutant Huntingtin in Intact Cells

Neurobiology of Disease 8, 391– 404 (2001) doi:10.1006/nbdi.2001.0390, available online at http://www.idealibrary.com on

Tissue Transglutaminase Selectively Modifies Proteins Associated with Truncated Mutant Huntingtin in Intact Cells Wanjoo Chun,* Mathieu Lesort,* Janusz Tucholski,* Peter W. Faber, † Marcy E. MacDonald, † Christopher A. Ross, ‡ and Gail V. W. Johnson* ,1 *Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0017; †Molecular Neurogenetics Unit, Massachusetts General Hospital, Charlestown, Massachusetts 02129; and ‡ Department of Psychiatry and Neuroscience, Johns Hopkins University, School of Medicine, Baltimore, Maryland, 21205-2196 Received October 11, 2000; revised January 24, 2001; accepted February 7, 2001

The cause of Huntington’s disease (HD) is a pathological expansion of the polyglutamine domain within the N-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. However, how the expanded polyglutamine repeats of mutant huntingtin cause HD is not known. Because in vitro expanded polyglutamine repeats are excellent glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. However, an association between huntingtin and tTG or modification of huntingtin by tTG has not been demonstrated in cells. To examine the interactions between tTG and huntingtin human neuroblastoma SH-SY5Y cells were stably transfected with full-length huntingtin containing 23 (FL-Q23) (wild type) or 82 (FL-Q82) (mutant) glutamine repeats or a truncated N-terminal huntingtin construct containing 23 (Q23) (wild type) or 62 (Q62) (mutant) glutamine repeats. Aggregates were rarely observed in the cells expressing full-length mutant huntingtin, and no specific colocalization of full-length huntingtin and tTG was observed. In contrast, in cells expressing truncated mutant huntingtin (Q62) there were numerous complexes of truncated mutant huntingtin and many of these complexes co-localized with tTG. However, the complexes were not insoluble structures. Further, truncated huntingtin coimmunoprecipitated with tTG, and this association increased when tTG was activated. Activation of tTG did not result in the modification of either truncated or full-length huntingtin, however proteins that were associated with truncated mutant huntingtin were selectively modified by tTG. This study is the first to demonstrate that tTG specifically interacts with a truncated form of huntingtin, and that activated tTG selectively modifies mutant huntingtin-associated proteins. These data suggest that proteolysis of full-length mutant huntingtin likely precedes its interaction with tTG and this process may facilitate the modification of huntingtin-associated proteins and thus contribute to the etiology of HD. © 2001 Academic Press Key Words: Huntington’s disease; huntingtin; tissue transglutaminase; polyamination; polyglutamine; immunocytochemistry.

INTRODUCTION

cal expansion of polyglutamine repeats in the Nterminal region of a 350-kDa protein of unknown function called huntingtin (Huntington’s Disease Collaborative Research Group, 1993). Although the pathogenic mechanism of HD has not been fully elucidated, it is likely that the abnormally expanded polyglutamine repeats in huntingtin are a primary cause of

Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disease caused by pathologi1

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392 the neurodegenerative events, as clinical manifestation of the disease occurs almost exclusively with pathological lengths of polyglutamine repeats (Vonsattel and DiFiglia, 1998). Further, expression of an amino-terminal fragment of mutant huntingtin in cultured striatal neurons (Kim et al., 1999; Saudou et al., 1998), Drosophila photoreceptor neurons (Jackson et al., 1998), or a C. elegans model (Faber et al., 1999) results in cell death. Even though mutant huntingtin is likely a fundamental cause of HD, the pattern of expression of huntingtin protein in the brain does not correlate with the extent of neuropathology (Aronin et al., 1995). The neuropathology of HD involves a characteristic pattern of neuronal death in the caudate and putamen (striatum) and at later stages of the disease in the cerebral cortex (Ferrante et al., 1985; Vonsattel et al., 1985). In striatum there is selective loss of medium spiny neurons with sparing of large and small aspiny interneurons. Therefore, it has been suggested that the expanded glutamine repeat may induce an abnormal interaction between the mutant protein and other cellular proteins which are expressed in a more context specific manner (Albin and Tagle, 1995; MacDonald and Gusella, 1996). It has been demonstrated that huntingtin interacts with more than two dozen proteins including huntingtin-associated protein 1 (HAP1) (Li et al., 1995), glyceraldehyde phosphate dehydrogenase (GAPDH) (Burke et al., 1996), a family of WW domain spliceosome proteins (Faber et al., 1998), an unidentified calmodulin-associated protein (Bao et al., 1996), a protein homologous to the yeast cytoskeleton-associated protein Sla2p (huntingtin-interacting protein 1 or HIP1) (Kalchman et al., 1997; Wanker et al., 1997), cystathione ␤-synthase (Boutell et al., 1998), transcriptional repressor proteins (Boutell et al., 1999), SH3GL3 (Sittler et al., 1998), and CREB-binding protein (Steffan et al., 2000). Although all these proteins can interact with huntingtin, they are not necessarily expressed in an appropriate context specific manner (i.e., enriched in the striatum), and in some cases the length of the polyglutamine domain does not affect their interactions with huntingtin. Cytoplasmic aggregates and neuronal intranuclear inclusions composed of the mutant huntingtin have been demonstrated in HD brain (DiFiglia et al., 1997) as well as in transgenic and transfected cell models expressing expanded polyglutamine huntingtin protein (Davies et al., 1997; Kim et al., 1999; Saudou et al., 1998; Persichetti et al., 1999), but the role of the inclusions in the pathogenesis of the disease remains inconclusive. It has been hypothesized that the expanded polyglutamine repeats may interact with each Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

Chun et al.

other through a polar zipper and thus contribute to aggregate formation (Perutz et al., 1994). Further, it has been hypothesized that tissue transglutaminase (tTG), perhaps in conjunction with the polar zipper mechanism, may be a contributing factor in the formation of these aggregates (Lorand, 1998). The transglutaminases are a family of calcium-dependent enzymes that catalyze the formation of ⑀-(␥glutamyl)lysine isopeptide bonds between substrate proteins rendering the resulting crosslinked protein complexes insoluble (Folk, 1983; Greenberg et al., 1991). Transglutaminases also catalyze the incorporation of polyamines into substrate proteins (Greenberg et al., 1991). Because the polypeptide bound glutamine is the primary determining factor for a transglutaminase-catalyzed reaction, it has been hypothesized that increasing the number of glutamines in a protein beyond a certain threshold may result in the protein becoming a transglutaminase substrate and thus be crosslinked into homo- or heteropolymers (Gentile et al., 1998; Green, 1993). Tissue TG is found within neurons (Appelt et al., 1996; Miller and Anderton, 1986) and has been implicated in a variety of processes including apoptosis (Melino et al., 1994) and axonal growth and regeneration (Eitan and Schwartz, 1993; Eitan et al., 1994; Tucholski et al., 2000). Previous it has been shown that tTG levels in SH-SY5Y cells are significantly increased by treatment with retinoic acid and further tTG can be activated by increasing intracellular calcium levels (Zhang et al., 1998a). Even though it has been demonstrated in vitro that polyglutamine repeat domains (Cooper et al., 1997; Kahlem et al., 1996) and mutant huntingtin (Kahlem et al., 1998) are substrates for tTG, it has not yet been shown that huntingtin interacts with or is modified by tTG in cells. To study the putative role of tTG in the modification of mutant huntingtin and/or huntingtin associated proteins, stable SH-SY5Y cell lines were established expressing full-length huntingtin with a physiological length of 23 glutamines (FL-Q23) or a pathological length of 82 glutamines (FL-Q82) or a truncated Nterminal huntingtin construct (residues 1–550 amino acids) with 23 glutamines (Q23) or 62 glutamines (Q62). In FL-Q23, FL-Q82, and Q23 cells huntingtin immunoreactivity was diffuse through the cytoplasm. In contrast, cytosolic, soluble complexes of huntingtin were observed in Q62 cells, and tTG was found to colocalize with huntingtin at the level of these complexes. These data suggest that tTG and truncated mutant huntingtin interact, but that tTG does not crosslink huntingtin as the complexes were SDS-solu-

tTG Modifies Truncated Mutant HTT-Associated Proteins

ble. In support of this finding, truncated huntingtin associated with tTG, and activation of tTG increased this interaction. Although tTG coimmunoprecipitated with both Q23 and Q62, activated tTG only modified proteins associated with truncated mutant huntingtin, but not huntingtin itself. These data indicate that proteolysis of full-length huntingtin may be critical for modification of mutant huntingtin-associated proteins by tTG.

MATERIALS AND METHODS Construction of expression plasmids. Expression constructs of full-length huntingtin were created in pRc/ CMV and expression constructs pRc/CMV-HDFLQ23 and pRc/CMV-HDFL-Q82 produce full-length huntingtin proteins (residue 1–3144) with 23 and 82 glutamines, respectively (Cooper et al., 1998). Expression constructs of truncated huntingtin were generated using restriction fragments and PCR products from pBS-HD-3144Q23 and pBS-HD1-3144Q62, which encode full-length huntingtin with 23 and 62 glutamines. Truncated huntingtin expression constructs were created in pcDNA3FLAG and expression constructs pcDNA3-FLAGHD1-550Q23 and pcDNA3FLAGHD-550Q62 produce 5⬘ FLAG-tagged huntingtin N-terminal fusion proteins (residues 1–550) with 23 and 62 glutamines, respectively (Faber et al., 1998). The clones were validated by DNA sequencing. Cell culture and generation of stable cell lines. Human neuroblastoma SH-SY5Y cells were transfected by electroporation (Gene Pulser II, Bio-Rad), according to the supplier’s instructions. SH-SY5Y cells stably expressing pcDNA3 vector alone, full-length huntingtin expressing 23 (FL-Q23) or 82 (FL-Q82) glutamine repeats, or truncated huntingtin expressing 23 (Q23) or 62 (Q62) glutamine repeats were selected and maintained on Corning dishes in RPMI 1640 medium supplemented with 20 mM glutamine, 10 units/ml penicillin, 100 ␮g/ml streptomycin, 5% fetal clone II serum, 10% horse serum, and 100 ␮g/ml G418 (GIBCO). To initiate differentiation, cells were grown in media containing 1% fetal clone II and 4% horse serum, containing 20 ␮M retinoic acid for 5 days. Previous studies have shown that treatment of SH-SY5Y cells with retinoic acid results in a significant increase in tTG expression (Zhang et al., 1998a). All experiments were carried out on subconfluent cultures. Immunoblotting. To evaluate the expression levels of tTG and the huntingtin proteins, extracts from cells were prepared, and quantitatively immunoblotted.

393 Cells were harvested in cold phosphate-buffered saline (PBS), collected by centrifugation, resuspended in a homogenizing buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and a 10 ␮g/ml concentration each of aprotinin, leupeptin, and pepstatin) and sonicated on ice. Protein concentrations of the homogenates were determined using the BCA method (Pierce) and diluted to a final concentration of 2 mg/ml with 2X reducing stop buffer (0.25 M Tris–HCl, pH 6.8, 5 mM EDTA, 5 mM EGTA, 25 mM dithiothreitol, 2% SDS, and 10% glycerol with bromophenol blue as the tracking dye). Samples (30 ␮g of protein) were resolved on 4 –7% gradient or 8% SDS–polyacrylamide gels for full-length and truncated huntingtin, respectively, and transferred to nitrocellulose. Blots were blocked in 5% nonfat dry milk in TBST (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. The blots were then incubated with the anti-tTG monoclonal antibody TG100 (1:750, Neomarkers) or with a polyclonal huntingtin antibody (raised against the N-terminal 17 amino acids of huntingtin protein, 1:20,000) (a generous gift from Dr. P. Detloff) (Lin et al., 2001) in the same buffer overnight at 4°C. The membranes were then washed three times with TBST and incubated with HRP-conjugated goat anti-mouse IgG (1:2000) for tTG or with HRP-conjugated goat anti-rabbit IgG (1:2000) for the polyclonal huntingtin antibody for 2 h at room temperature. The membranes were rinsed three times for 30 min with TBST, followed by for quick rinses with distilled water, and developed with the enhanced chemiluminescence method (ECL, Amersham). In situ tTG activity assay. Cells were labeled with 2 mM 5-(biotinamido)pentylamine (Pierce), a biotinylated polyamine, for 45 min. To increase intracellular levels of calcium and activate tTG, 2 nM maitotoxin (MTX) was added to the cells. Because tTG is a substrate of calpain (Zhang et al., 1998b), and MTX can activate calpain (Xie and Johnson, 1998), 25 ␮M NAcetyl-Leu-Leu-Methioninal (Sigma), a calpain inhibitor, was added for 15 min prior to the addition of MTX. Twenty minutes after the addition of MTX the cells were harvested in lysis buffer (10 mM Tris–HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl 2, 1 mM EDTA, 0.05% NP-40, 0.1 mM phenylmethylsulfonyl fluoride, and a 10 ␮g/ml concentration each of aprotinin, leupeptin, and pepstatin) and sonicated. A particle-free supernatant was prepared from the homogenate by centrifugation at 2000g for 5 min at 4°C and the protein concentrations were determined. Transglutaminase activity was quantified by measuring the presCopyright © 2001 by Academic Press All rights of reproduction in any form reserved.

394 ence of incorporated 5-(biotinamido)pentylamine into proteins by a microplate assay as described by Zhang et al. (1998a). To visualize the proteins into which the 5-(biotinamido)pentylamine had been incorporated, samples were electrophoresed on 8% SDS–polyacrylamide gels, transferred to nitrocellulose, and probed with HRP-conjugated neutravidin (Pierce, 1:2,000). The blots were developed as described above. Immunoprecipitation. SH-SY5Y cells stably expressing full-length huntingtin (FL-Q23 or FL-Q82) or truncated huntingtin (Q23 or Q62) were labeled with 2 mM 5-(biotinamido)pentylamine and treated with MTX as described above. The cells were subsequently harvested in immunoprecipitation buffer (0.5% NP-40, 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and a 10 ␮g/ml concentration each of aprotinin, leupeptin, and pepstatin) and sonicated. A particle-free supernatant was prepared from the homogenate by centrifugation at 2000g for 5 min at 4°C and protein concentrations were determined. Samples containing 200 ␮g of protein were immunoprecipitated overnight at 4°C with 10 ␮g of the polyclonal N-terminal huntingtin antibody. Protein A–Sepharose (Pharmacia, 40 ␮l of beads) that had been washed previously three time with the immunoprecipitation buffer and blocked with egg albumin (1 mg/ml) to decrease nonspecific binding, was added, and the incubation continued for 3 h at 4°C. After the precipitates were washed three times with IP wash buffer (1.0% NP-40, 50 mM Tris– HCl, pH 7.5, 300 mM NaCl, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and a 10 ␮g/ml concentration each of aprotinin, leupeptin, and pepstatin), 40 ␮l of 2⫻ reducing stop buffer was added to each sample and the samples were placed in a boiling water bath for 15 min before SDS–polyacrylamide gel electrophoresis and immunoblotting. Blots were probed with the monoclonal huntingtin antibody MAB2166 (Chemicon, 1:2,000), developed with ECL and then stripped by incubation in 100 mM ␤-mercaptoethanol, 2% SDS, 62.5 mM Tris–HCl, pH 7.6, at 50°C for 30 min with agitation, followed by thorough rinsing with TBST, blocking with 5% milk/TBST overnight and reprobing with HRP-conjugated neutravidin (1:2,000). Coimmunoprecipitation. Cell lysates were prepared as described above, except that the cells were not prelabeled with 5-(biotinamido)pentylamine. Samples containing 200 ␮g of protein were precleared for 1 h at 4°C with protein G–Sepharose (Pharmacia) or with protein A–Sepharose (Pharmacia) that had been washed previously three times with the immunoprecipitation buffer, for precipitation of tTG or huntingCopyright © 2001 by Academic Press All rights of reproduction in any form reserved.

Chun et al.

tin, respectively. Precleared samples were immunoprecipitated overnight at 4°C with 1.2 ␮g of a monoclonal antibody to tTG (CUB7402, Neomarkers) or with 15 ␮g of the polyclonal N-terminal huntingtin antibody. In some experiments nonimmune rabbit IgG was included as a control. Protein G–Sepharose (50 ␮l of beads) for tTG or protein A–Sepharose (50 ␮l of beads) for huntingtin were added, and the incubation continued overnight at 4°C. After the precipitates were washed three times with IP wash buffer, 50 ␮l of 2⫻ reducing stop buffer was added to each sample and the samples were placed in a boiling water bath for 15 min before SDS–polyacrylamide gel electrophoresis and immunoblotting. Blots were probed with the polyclonal N-terminal huntingtin antibody or with the monoclonal tTG antibody TG100 and stripped as described above and reprobed with the monoclonal tTG antibody TG100 or with the monoclonal huntingtin antibody MAB2166. Cell viability assay. To determine if mutant huntingtin decreases basal cell viability, LDH release was measured in all cell lines after 24 h incubation in serum-free media (Davis et al., 1997; Decker and Lohmann-Matthes, 1988). Data were expressed as mean ⫾ SEM. Immunocytochemistry. SH-SY5Y cells stably expressing pcDNA3 vector alone, full-length huntingtin (FL-Q23 or FL-Q82), or truncated huntingtin (Q23 or Q62) were seeded on poly-d-lysine-coated cover slips in 24-well plates. Twenty-four hours later cells were fixed in 90% methanol, 50 mM EGTA, pH 6.0, for 5 min at ⫺20°C (Melan and Sluder, 1992), incubated for 10 min with 0.2% Triton X-100 in PBS, and rinsed three times with PBS, prior to incubation with 5% bovine serum albumin in PBS for 90 min to reduce the background. Cells were then incubated at room temperature for 90 min in 5% bovine serum albumin in PBS with the polyclonal N-terminal huntingtin antibody (1:20,000) or with the monoclonal FLAG antibody M2 (1:100) (UBI). Cells were then rinsed three times with PBS, incubated for 60 min at room temperature in 5% bovine serum albumin in PBS with FITC-conjugated anti-rabbit IgG (1:200) or FITC-conjugated anti-mouse IgG (1:200). For costaining studies, cells expressing full-length or truncated huntingtin were treated with 20 ␮M retinoic acid for 5 days prior to fixation and permeabilization as described above, incubated at room temperature for 90 min in 5% bovine serum albumin in PBS with the polyclonal N-terminal huntingtin antibody (1:20,000) and with the monoclonal tTG antibody CUB7402 (1:20). Cells were then rinsed three times with PBS, incubated for 60 min at room

tTG Modifies Truncated Mutant HTT-Associated Proteins

temperature in 5% bovine serum albumin in PBS with FITC-conjugated anti-rabbit IgG (1:200) and Texas red-conjugated anti-mouse IgG (1:100). Coverslips were washed extensively in PBS prior to mounting. Cells were viewed with an Olympus Fluoview confocal microscope and digitally stored images were combined and displayed with the accompanying software and Adobe Photoshop 4.0.

RESULTS Stable overexpression of huntingtin does not alter cell viability. LDH release was measured in cells expressing empty vector, full-length or truncated, wild-type or mutant huntingtin. In vector cells LDH release was 11.4 ⫾ 1.0%, for FL-Q23 it was 12.1 ⫾ 0.8, and for FL-Q82 it was 13.3 ⫾ 0.6. In Q23 cells basal LDH release was 12.0 ⫾ 0.5 and for Q62 it was 13.2 ⫾ 0.6 (n ⫽ 3 separate experiments in all cases). These data demonstrate that there are no significant differences in LDH release between groups, indicating no loss of cell viability due to the expression of the mutant huntingtin (data not shown). Further, cellular morphology and proliferation rates were not significantly different between the cell lines, and there was no evidence of increased apoptosis in the mutant huntingtin expressing cells as determined by the presence of condensed chromatin as detected by Hoescht staining (data not shown). Expression and localization of full-length and truncated huntingtin. To determine the expression and localization of full-length or truncated huntingtin, cells stably expressing full-length or truncated huntingtin (Fig. 1) were immunostained with the N-terminal huntingtin antibody. Both wild type (FL-Q23) and mutant (FLQ82) full-length huntingtin were distributed throughout the cytoplasm with some accumulation at the ends of projections (Figs. 1a and 1b); however, punctate staining was very rare in FL-Q82 cells (Fig. 1b). Immunostaining of the cells stably expressing truncated Q23 revealed a diffuse cytoplasmic staining pattern with no nuclear staining (Fig. 1c). In contrast, immunostaining of the cells stably expressing truncated Q62 revealed a punctate distribution of the protein throughout the cytoplasm as well diffuse cytoplasmic staining (Fig. 1d). As with truncated Q23, little if any huntingtin immunoreactivity was present in the nucleus of the Q62. Expression of tTG and full-length or truncated huntingtin. In SH-SY5Y cells stably transfected with fulllength, wild type (FL-Q23), or mutant (FL-Q82) huntingtin, one predominant immunoreactive band was

395 observed at approximately 350 kDa (Fig. 2). Treatment of cells with 20 ␮M retinoic acid for 5 days significantly increased tTG levels but did not alter the expression levels of the full-length huntingtin (Fig. 2). In cells stably expressing truncated wild type huntingtin, two bands at approximately 85 and 110 kDa were consistently observed in all clonal cell lines (Fig. 3). This double band is likely to be due to carboxy terminal processing of the transfected protein as an antibody to the amino terminal FLAG epitope recognizes both bands (data not shown). Q62 resulted in one predominant immunoreactive band at approximately 120 kD (Fig. 3). In situ tTG activity. To determine in situ tTG activity in both retinoic acid-treated and tTG-transfected truncated huntingtin cells, 5-(biotinamido)pentylamine was used as a probe for the tTG activity (Zhang et al., 1998a). Because tTG is a calcium-activated enzyme, intracellular levels of calcium were elevated by treating the cells with MTX, and TG activity was measured. Increasing intracellular calcium levels in cell treated with retinoic acid to increase tTG levels resulted in a significant increase in tTG activity in both full-length and truncated huntingtin cells (Fig. 4). The presence of wild-type or mutant huntingtin did not alter the basal activity of tTG or the overall activity of tTG in response to MTX treatment (Fig. 4). In addition, the profile of proteins modified by tTG in the cell did not differ between the cell lines (data not shown). Colocalization of tTG and huntingtin. To evaluate the localization of tTG and huntingtin, cells were costained with a polyclonal N-terminal huntingtin antibody and a mouse monoclonal antibody to tTG. To increase the expression of tTG, cells were treated with 20 ␮M retinoic acid for 5 days. Retinoic acid treatment did not alter huntingtin levels or distribution in cells expressing either full-length (see Fig. 5 and compare Fig. 1) or the truncated huntingtin (see Fig. 6 and compare Fig. 2). In both FL-Q23 and FL-Q82 cells huntingtin immunoreactivity was diffuse in the cytoplasm (Figs. 5a and 5c) and tTG also showed diffuse staining in the cytoplasm (Figs. 5b and 5d). In the truncated Q23 cells, both huntingtin and tTG staining were diffuse with some overlap (Figs. 6a and 6b). In contrast, distinct punctate staining was evident in the Q62 cells for both huntingtin (Fig. 6c) and tTG (Fig. 6d). In addition the majority of the punctate complexes in the Q62 cells stained for both tTG and huntingtin (Figs. 7c and 7d, arrows), clearly indicating colocalization of these two proteins. However, some of the huntingtin complexes in the Q62 cells did not Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. Immunostaining for full-length huntingtin in cells stably transfected with FL-Q23 or FL-Q82 (a, b) or truncated huntingtin in cells stably transfected with Q23 or Q62 (c, d). Huntingtin localization was detected with the polyclonal huntingtin antibody raised against the first 17 amino acids of the N-terminus of the huntingtin protein. In the FL-Q23 cells, huntingtin immunostaining was diffuse throughout the cytoplasm and showed some enrichment at the ends of projections (a). In the FL-Q82 cells, huntingtin was also diffuse in cytoplasm, and rarely showed cytoplasmic complexes (b). In the Q23 cells, huntingtin immunostaining was diffuse and entirely cytoplasmic (c). In contrast, the huntingtin staining in Q62 cells was extremely punctate demonstrating the presence of accumulated mutant huntingtin in the cytoplasm and around the nucleus (d). Original magnification for all images was 1400⫻.

show colocalization with tTG. In addition, the frequency and size of the complexes in the Q62 cells were the same in naı¨ve and retinoic acid-treated cells, demonstrating that tTG does not modulate the formation of these complexes. Tissue TG selectively coimmunoprecipitates with truncated huntingtin, but not full-length huntingtin. To determine whether tTG and huntingtin interact, and if increasing the activity of tTG alters this interaction, coimmunoprecipitation studies were carried out. Cells expressing full-length or truncated huntingtin were Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

incubated with retinoic acid for 5 days and then incubated in the absence or presence of MTX. Lysates were immunoprecipitated with a polyclonal N-terminal huntingtin antibody and probed for tTG with the monoclonal tTG antibody TG100, stripped, and reprobed with a monoclonal huntingtin antibody. In full-length huntingtin cells, tTG and full-length huntingtin interacted only very weakly and activation of tTG did not increase the association between tTG and full-length huntingtin (Fig. 7). However, in truncated huntingtin cells, tTG coprecipitates with huntingtin

tTG Modifies Truncated Mutant HTT-Associated Proteins

FIG. 2. Representative immunoblots of the expression of fulllength huntingtin (FL-Htt) (top panels) and tTG (bottom panels) in SH-SY5Y cells stably expressing Vec alone (Vec), FL-Q23, or FL-Q82. Cells were incubated in the absence (⫺) or presence (⫹) of 20 ␮M retinoic acid (RA) for 5 days prior to immunoblotting for huntingtin (Htt) or tTG. Positions at which molecular mass standards (kDa) migrate are indicated at the right.

and further that activation of tTG increases this association (Fig. 8a). This is particularly evident when the amount of tTG in the precipitates is compared to the amount of huntingtin in each precipitated sample. To further confirm this association between truncated huntingtin and tTG, cell lysates were immunoprecipitated with the monoclonal tTG antibody CUB7402, probed with the polyclonal N-terminal huntingtin antibody, stripped, and reprobed with the monoclonal tTG antibody TG100. The results of these experiments also demonstrate that tTG coprecipitates with both truncated Q23 and Q62 and further, calcium-stimulated activation of tTG resulted in an increased association between tTG and truncated huntingtin (Fig. 8b). Identical results were obtained with Q23 and Q62 cells transiently transfected with tTG (data not shown). Further, when cells were immunoprecipitated with non-immune IgG neither huntingtin nor tTG

FIG. 3. Representative immunoblots of the expression of truncated huntingtin (top panels) and tTG (bottom panels) in SH-SY5Y cells stably expressing Q23, or Q62. Cells were incubated in the absence (⫺) or presence (⫹) of 20 ␮M retinoic acid (RA) for 5 days prior to immunoblotting for huntingtin (Htt) or tTG. Positions at which molecular mass standards (kDa) migrate are indicated at the right.

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FIG. 4. Quantitative analysis of the tTG-catalyzed incorporation of 5-(biotinamido)pentylamine into proteins as a measure of in situ activity. SH-SY5Y cells stably expressing vector (vec), FL23, FL82, Q23 or Q62 were treated with 20 ␮M retinoic acid for 5 days, labeled with 5-(biotinamido)pentylamine, and incubated for 20 min in the absence (⫺) or presence (⫹) of 2 nM MTX. Quantitative analysis of the tTG-catalyzed incorporation of 5-(biotinamido)pentylamine into proteins using a microplate-based assay demonstrated that tTG expression induced by retinoic acid treatment resulted in significant increases in MTX-stimulated (⫹) tTG activity. Activity was normalized to the basal activity (⫺) of vec cells and presented as Mean ⫾ SEM (n ⫽ 2–3 separate experiments, each in triplicate).

were detected in the precipitates clearly demonstrating the specificity of the interaction (data not shown). Selective tTG-induced modification of certain proteins associated with truncated mutant huntingtin, but not fulllength huntingtin. The modification of huntingtin and/or huntingtin-associated proteins by tTG was examined in full-length or N-terminal truncated huntingtin cells. Cells incubated with retinoic acid were prelabeled with 5-(biotinamido)pentylamine and then incubated in the absence or presence of MTX. Cells lysates were immunoprecipitated with the polyclonal huntingtin antibody, probed with a monoclonal huntingtin antibody, stripped, and reprobed with HRPconjugated neutravidin to identify proteins modified by tTG. Treatment with MTX did not alter the levels of full-length (Fig. 9, left panel) or truncated huntingtin (Fig. 10, left panel). In full-length huntingtin expressing cells, no modification of huntingtin, either wildtype or mutant, or huntingtin-associated proteins was observed (Fig. 9, right panel). Given the fact that fulllength huntingtin did not efficiently coimmunoprecipitate with tTG (Fig. 7), this finding was not unexpected. However, certain proteins associated with the truncated mutant huntingtin, but not truncated mutant huntingtin itself, were selectively modified by activated tTG (Fig. 10, right panel). Although the amount of Q23 that was immunoprecipitated was equivalent to or slightly greater that the amount of Q62 that was precipitated (Fig. 10, left panel), tTGmodified proteins coprecipitated only with Q62 (Fig. Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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FIG. 5. Localization of full-length huntingtin and tTG. Cells stably expressing FL-Q23 or FL-Q82 were plated on coverslips and treated with 20 ␮M retinoic acid for 5 days prior to fixation and staining. All cells were double-labeled with the polyclonal huntingtin antibody and with the monoclonal tTG antibody CUB7402. In all figures, huntingtin is in green (FITC) and tTG is in red (Texas red). a and c show huntingtin staining in FL-Q23 and FL-Q82 cells, respectively. Huntingtin staining in these retinoic acid treated cells (a, c) showed the same staining pattern as the cells not treated with retinoic acid (Fig. 1). b and d show tTG staining in FL-Q23 and FL-Q82 cells, respectively. Huntingtin, either wild type or mutant, did not show any punctate staining pattern (a, c) as exhibited in truncated Q62 cells (Fig. 6c) and tTG also showed only diffuse staining (b, d).

10, right panel). This finding demonstrates that proteins interacting with truncated mutant huntingtin are selectively modified by tTG, even though tTG expression levels are equivalent in both wild type and mutant truncated huntingtin expressing cells (Fig. 4), and transglutaminase activity is equivalent in the two cell lines.

DISCUSSION The hypothesis that tTG contributes to the etiology of HD was first proposed several years ago by Green Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

(1993). With the discovery of neuronal intranuclear inclusions and cytoplasmic inclusions of mutant huntingtin in HD brain (DiFiglia et al., 1997), it was further hypothesized that tTG may facilitate the formation of these aggregates by selectively cross-linking mutant huntingtin (for a review see (Cooper et al., 1999)). In vitro constructs with polyglutamine expansions are excellent substrates of tTG (Cooper et al., 1997; Kahlem et al., 1998, 1996). Additionally, tTG levels and activity are significantly increased in HD brain (Karpuj et al., 1999; Lesort et al., 1999) in a grade and region dependent manner and there was a significant increase in

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FIG. 6. Colocalization of truncated huntingtin and tTG. Cells stably expressing Q23 or Q62 were plated on coverslips and treated with 20 ␮M retinoic acid for 5 days prior to fixation and staining. All cells were double-labeled with the polyclonal huntingtin antibody and with the monoclonal tTG antibody CUB7402. In all figures, huntingtin is in green (FITC) and tTG is in red (Texas red). a and c show huntingtin staining in Q23 and Q62 cells, respectively. Huntingtin staining in these retinoic acid treated cells (a, c) showed the same staining pattern as the cells not treated with retinoic acid (Fig. 2). b and d show tTG staining in Q23 and Q62 cells, respectively. The huntingtin complexes in Q62 cells showed colocalization with tTG (arrows in c, d), but some huntingtin accumulations did not show colocalization with tTG. Q23 cells, which showed only diffuse staining of huntingtin (a) and tTG (b) did not show any punctate costaining pattern as exhibited in Q62 cells.

the tTG immunoreactivity in specific neuronal populations in HD brain (Lesort et al., 1999). Although these findings are intriguing, there has been no demonstration of an interaction between huntingtin and tTG in intact cells, nor has it been demonstrated that mutant or normal huntingtin is modified by tTG in intact cells. Therefore, the goals of this study were to demonstrate an association between huntingtin and tTG, and to determine whether huntingtin was modified by tTG in cells. In this study we demonstrate that tTG selectively associates with truncated, but not full-length, huntingtin. Further, we show that in intact cells neither wild type nor mutant huntingtin is a tTG substrate. How-

ever, proteins that are associated with mutant truncated huntingtin are modified by tTG. In the FL-Q23 and FL-Q82 cells, huntingtin immunoreactivity was diffuse throughout the cytoplasm and excluded from the nucleus. Further, there were no complexes or aggregates in the FL-Q82 cells. These findings are in agreement with previous studies (Hackam et al., 1998; Kim et al., 1999; Persichetti et al., 1999). Because full-length mutant huntingtin rarely forms complexes and does not accumulate in the nucleus, it has been hypothesized that proteolytic cleavage of mutant huntingtin is an important step in the pathogenesis of the disease facilitating nuclear and Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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FIG. 7. No significant in situ association between full-length huntingtin and tTG in response to MTX treatment. Cells stably expressing FL-Q23 or FL-Q82 were treated with 20 ␮M retinoic acid for 5 days and incubated for 20 min in the absence (⫺) or presence (⫹) of 5 nM MTX. Huntingtin in lysates was immunoprecipitated with the polyclonal huntingtin antibody and the precipitates were immunoblotted with the monoclonal tTG antibody TG100 (top panel). The immunoblots were stripped and reprobed with the monoclonal huntingtin antibody MAB2166 (bottom panel). Positions at which molecular mass standards (kD) migrate are indicated at the right.

cytoplasmic aggregate formation and compromising cell viability (Martindale et al., 1998). Huntingtin immunoreactivity was also diffuse throughout the cytoplasm in the cells expressing Q23; however, cells expressing the mutant truncated huntingtin exhibited distinct punctate huntingtin staining in the cytoplasm, although no nuclear staining in was observed. Q62 is 550 amino acids and therefore is approximately the size of a fragment of huntingtin that is generated by caspase-3 in vitro (cleavage sites have been identified at D513 and D552) (Wellington et al., 1998). Transient transfection of a similar sized mutant huntingtin fragment (548 aa) into HEK 293T cells has been shown to form perinuclear aggregates (but at a very low frequency) and sensitize cells to tamoxifen-induced cell

Chun et al.

death (Hackam et al., 1998). In the Q62 cells no loss of cell viability was observed under basal conditions; however, preliminary studies indicate that the expression of mutant truncated huntingtin may sensitize cells to specific apoptotic stresses (data not shown). It is also important to note that the accumulations observed in the Q62 cells were not insoluble aggregates, i.e., no SDS-resistant huntingtin immunoreactivity was observed at the top of the gels. It also should be noted that in previous studies the aggregates formed from the longer forms (⬎500 aa) of truncated mutant huntingtin were also likely to be soluble, as no SDSresistant material was observed (Hackam et al., 1998). This indicates that further processing is required to form SDS-resistant mutant huntingtin aggregates and to efficiently localize to the nucleus (Cooper et al., 1998). It has been well documented that in vitro polyglutamine constructs and mutant huntingtin are substrates for tTG (Cooper et al., 1997; Kahlem et al., 1998, 1996). However, the ability of tTG to modify mutant huntingtin in intact cells has not been demonstrated, and the putative role of tTG in aggregate formation is still controversial. It was reported that TG inhibitors suppressed aggregate formation and apoptosis in cells expressing truncated DRPLA protein with an expanded polyglutamine domain (Igarashi et al., 1998). However, both TG inhibitors used in this study can also inhibit other enzymes, and in some cases the TG inhibitors reduced apoptotic cell death, but not aggregate formation (Lorand, 1998). In another study, tTG overexpression was reported to increase the aggregate formation of synthetic fusion proteins containing 36 or 46 glutamines. However, the percentage increase in

FIG. 8. In situ association between truncated huntingtin and tTG in response to MTX treatment. Cells stably expressing truncated Q23 or Q62 were treated with 20 ␮M retinoic acid for 5 days and incubated for 20 min in the absence (⫺) or presence (⫹) of 5 nM MTX. (a) Huntingtin in lysates was immunoprecipitated with the polyclonal huntingtin antibody and the precipitates were immunoblotted with the monoclonal tTG antibody TG100 (top panel). The immunoblots were stripped and reprobed with the monoclonal huntingtin antibody MAB2166 (bottom panel). (b) tTG in lysates was immunoprecipitated with the monoclonal tTG antibody CUB7402 and the precipitates were immunoblotted with the polyclonal huntingtin antibody (top panel). The immunoblots were stripped and reprobed with the monoclonal tTG antibody TG100 (bottom panel). Positions at which molecular mass standards (kDa) migrate are indicated at the right.

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tTG Modifies Truncated Mutant HTT-Associated Proteins

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FIG. 9. No selective modification of full-length huntingtin or associated proteins by tTG. Cells stably expressing FL-Q23 or FL-Q82 were treated with 20 ␮M retinoic acid for 5 days, labeled with 5-(biotinamido)pentylamine, and incubated for 20 min in the absence (⫺) or presence (⫹) of 5 nM MTX. Huntingtin in lysates was immunoprecipitated with the polyclonal huntingtin antibody and the precipitates were immunoblotted with the monoclonal huntingtin antibody MAB2166 (left panel). The immunoblots were stripped and reprobed with neutravidin-HRP, which recognizes proteins that are modified by tTG as measured by the incubation of 5-(biotinamido)pentylamine (right panel). Positions at which molecular mass standards (kDa) migrate are indicated at the right.

aggregate formation induced by tTG overexpression was only ⬃10 –15% (de Cristofaro et al., 1999). In another study, a TG inhibitor had no effect in mutant huntingtin aggregate formation, and when the inhibitor was used at higher concentrations, it was cytotoxic

(Kim et al., 1999). These findings indicate the need for further investigations on the role of tTG in the etiology of HD. In the present study, soluble, cytoplasmic complexes were abundant in undifferentiated SH-SY5Y

FIG. 10. Selective tTG-catalyzed modification of proteins associated with mutant truncated huntingtin, but not huntingtin itself. Cells stably expressing Q23 or Q62 were treated with 20 ␮M retinoic acid for 5 days, labeled with 5-(biotinamido)pentylamine, and incubated for 20 min in the absence (⫺) or presence (⫹) of 5 nM MTX. Huntingtin in lysates was immunoprecipitated with the polyclonal huntingtin antibody and the precipitates were immunoblotted with the monoclonal huntingtin antibody MAB2166. The immunoblots were stripped and reprobed with neutravidin-HRP, which recognizes proteins that are modified by tTG as measured by the incubation of 5-(biotinamido)pentylamine (bottom right blots). Positions at which molecular mass standards (kDa) migrate are indicated at the right.

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402 cells stably expressing Q62. Increasing the levels of tTG significantly by treatment with retinoic acid, did not alter the abundance or size of the aggregates, or result in the formation of SDS-insoluble structures. Further, huntingtin was not modified by tTG in situ. These findings would seem to indicate that it is unlikely that tTG plays a role in aggregate formation in HD brain. Interestingly, tTG and huntingtin showed a striking colocalization in Q62 cells, in that almost all the cytoplasmic complexes were immunopositive for both proteins. Further, tTG co-precipitated with truncated huntingtin, again demonstrating an association between the two proteins. Nonetheless, no modification of huntingtin by tTG was observed. Instead, several proteins that coprecipitated with truncated mutant, but not physiological, huntingtin were modified by tTG. This finding leads to the intriguing possibility that tTG may contribute to the etiology of HD by modifying proteins that preferentially associate with truncated mutant huntingtin. More than 2 dozen proteins that associate with mutant huntingtin have been identified (Bao et al., 1996; Boutell et al., 1999, 1998; Burke et al., 1996; Faber et al., 1998; Kalchman et al., 1997; Li et al., 1995; Sittler et al., 1998; Steffan et al., 2000; Wanker et al., 1997). Although HAP 1 and GAPDH associate with mutant huntingtin (Burke et al., 1996; Li et al., 1995), and GAPDH can be crosslinked into a GST-polyglutamine fusion protein in vitro (Cooper et al., 1997), neither of these proteins were modified by tTG in situ (data not shown). It also needs to be considered the crosslinking and polyamination reactions catalyzed by tTG are competing reactions (Greenberg et al., 1991). The levels of polyamines in the brain are in the millimolar range (Morrison et al., 1995), and in vitro 1 mM putrescine effectively inhibits tTG-catalyzed crosslinking of proteins (Miller and Johnson, 1995). These data would suggest that in vivo tTG is more likely to polyaminate proteins, rather than crosslink them. Numerous proteins are polyaminated in intact cells, and polyamination can modulate the function and metabolism of proteins (for a review see (Lesort et al., 2000)). Therefore, it is tempting to speculate that tTG-catalyzed polyamination of certain proteins that associate with truncated mutant huntingtin may contribute to the etiology of HD. Current studies are under way to identify these proteins, which associate with mutant huntingtin and are modified by tTG. Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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ACKNOWLEDGMENTS This work was supported by NIH Grant AG12396 (G.V.W.J.) and a fellowship from the Hereditary Disease Foundation (M.L.). We thank Dr. Peter Detloff for the generous gift of the polyclonal N-terminal huntingtin antibody.

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