Polyglutamine diseases are inherited neurodegenerative diseases caused by the expanded polyglutamine proteins (polyQs). We have identified a novel guanosine triphosphatase (GTPase) named CRAG that contains a nuclear localization signal (NLS) sequence and forms nuclear inclusions in response to stress. After ultraviolet irradiation, CRAG interacted with and induced an enlarged ring-like structure of promyelocytic leukemia protein (PML) body in a GTPase-dependent manner. Reactive oxygen species (ROS) generated by polyQ accumulation triggered the association of CRAG with polyQ and the nuclear translocation of the CRAG–polyQ complex. Furthermore, CRAG promoted the degradation of polyQ at PML/CRAG bodies through the ubiquitin–proteasome pathway. CRAG knockdown by small interfering RNA in neuronal cells consistently blocked the nuclear translocation of polyQ and enhanced polyQ-mediated cell death. We propose that CRAG is a modulator of PML function and dynamics in ROS signaling and is protectively involved in the pathogenesis of polyglutamine diseases.

Introduction

Polyglutamine diseases are inherited neurodegenerative diseases caused by the expansion of the polyglutamine tract (Ross, 1997; Orr, 2001). Expansion of polyglutamine repeats alters the conformation or results in the misfolding of the disease-associated protein, thereby conferring a toxic gain of function that is selectively deleterious to neurons (Sato et al., 1999; Yoshizawa et al., 2000). Nuclear inclusions (NIs) with ubiquitination formed by the disease protein are a common pathological feature of polyglutamine diseases. Indeed, NIs have been observed in at least six polyglutamine diseases and many transgenic animal models and thus represent a common hallmark of polyglutamine diseases. Nuclear translocation of expanded polyglutamine protein (polyQ) was promoted efficiently in neuronal cells, suggesting that some neuronal factors containing a nuclear localization signal (NLS) may regulate the nuclear translocation of polyQ. Furthermore, polyQ nuclear aggregates have recently been demonstrated to interact with and alter the nuclear structures of the promyelocytic leukemia protein (PML), a major component of nuclear bodies (Takahashi et al., 2003). We identified a novel guanosine triphosphatase (GTPase), CRAG, which associated with PML and formed NIs in response to various stresses. We report that CRAG is involved in the mechanisms underlying nuclear translocation, ubiquitination, and inclusion body formation of polyQ.

Results And Discussion

By screening for signaling targets of repulsive axon guidance factors, semaphorins (described in Materials and methods), we identified a novel GTPase and named it CRAG, after collapsin response mediator protein (CRMP)–associated molecule (CRAM [CRMP-5])–associated GTPase. The full-length CRAG cDNA presents an open reading frame of 369 amino acid residues containing a glutamine-rich domain at the NH2 terminus, a Ras homology domain in the middle, and an NLS at the COOH terminus (Fig. 1 a). Fig. 1 b indicates a comparison of structure between CRAG and other related GTPase proteins. The amino acid sequence of CRAG shows 95% identity with centaurin-γ3 and 43% identity with the nuclear GTPase phosphatidylinositol 3-kinase enhancer, short isoform (alignment is not depicted; Ye et al., 2000). Analysis of human genomic databases suggests that CRAG may be an alternative splicing variant of centaurin-γ3. Northern blot analysis indicated that the CRAG gene was dominantly expressed in brain and slightly in heart (Fig. 1 c). A band higher than CRAG that is present in various tissues may be centaurin-γ3. Immunohistochemical analysis revealed a diffuse cytoplasmic distribution of CRAG in rat hippocampal neurons at rest (Fig. 1 d, top). Upon UV irradiation, an NI formed of CRAG was detected at 10 min (Fig. 1 d). These NIs exhibited a doughnut shape under the large-scale microscopic analysis (Fig. S1 A). This phenomenon was reproduced in UV-stimulated HeLa cells expressing HA-tagged CRAG (HA-CRAG) wild type (WT) or GTPase-deficient mutants (S114N; Fig. 1 e). In contrast, NLS-disrupted mutants of CRAG (KR342-343EE within the NLS motif) formed inclusions but were cytosolic even after UV stimulation. These results demonstrated that NLS, but not GTPase activity, was required for nuclear translocation and NI formation by CRAG.

We found that GFP fused to the NH2 terminus of CRAG WT or GTPase mutants spontaneously translocated to the nucleus and formed NI without any stimulation. The absence of nuclear localization of NLS-disrupted mutants of GFP-CRAG also supported an NLS-dependent NI formation by CRAG (Fig. 1 f). This suggested that GFP fusion at the NH2 terminus of CRAG caused a drastic conformational change in CRAG and converted it from an inactive to an active form. An in vitro GTPase assay revealed CRAG activation by UV irradiation and spontaneous activation of GFP-CRAG (unpublished data). Collectively, these data demonstrate that CRAG is a unique GTPase that forms NI under UV stress in an NLS-dependent and GTPase-independent manner, and a conformational change in CRAG may induce its intrinsic activity to form NI in an NLS-dependent manner.

Several nuclear domains have been reported, including PML body, a major component of nuclear bodies (Hodges et al., 1998; Lamond and Earnshaw, 1998). Merged images demonstrated the colocalization of endogenous CRAG and PML bodies with an enlarged ring-like structure in a UV- stimulated dorsal root ganglion (DRG) neuron (Fig. 2 a and Fig. S1 B). Similar results were obtained in UV-stimulated HeLa cells expressing HA-CRAG or unstimulated cells expressing GFP-CRAG. In contrast, HA-CRAG GTPase mutants or GFP-CRAG GTPase mutants did not colocalize with PML. Statistical analysis indicated that GTPase activity was essential for colocalization of GFP-CRAG with PML. A coimmunoprecipitation assay demonstrated that endogenous CRAG associated with PML in UV-stimulated neurons (Fig. 2 b). No association of CRAG with PML by antigen peptide against anti-CRAG antibody demonstrated the specificity of anti-CRAG antibody. Furthermore, in HeLa cell expression system, GFP-CRAG associated with HA-PML in a GTPase-dependent manner (Fig. 2 c).

We noticed that ubiquitin signals were accumulated in these GFP-CRAG–associated inclusions but not in GTPase mutants (Fig. 2 d). Therefore, we examined whether CRAG stimulated ubiquitin ligase activity in PML immunoprecipitates. An in vitro ubiquitin ligase assay revealed that the activity in PML immunoprecipitates was undetectable in the absence of GFP-CRAG, but GFP-CRAG coexpression induced ubiquitin ligase activity in immunoprecipitates of PML WT but not PML RING (really interesting new gene)–finger mutants (C51S/C54S; Fig. 2 e). For in vivo ubiquitination, ubiquitinated proteins were purified from cells coexpressing HA-PML and Flag ubiquitin with or without GFP-CRAG and analyzed by immunoblot with anti-HA and anti-Flag antibodies. As shown in Fig. 2 f, ubiquitinated proteins significantly increased by GFP-CRAG expression. Collectively, these results suggest that CRAG is an inducer for the association of unknown ubiquitin ligases with PML or a direct activator of PML-associated ubiquitin ligase. Because PML contains a RING-finger domain that confers on it an E3 ubiquitin ligase activity, it is possible that PML is an E3 ubiquitin ligase.

The fact that polyglutamine diseases are characterized by the presence of ubiquitinated, PML-associated NIs suggested the possible involvement of CRAG in polyglutamine diseases. To ascertain a possible involvement of CRAG in polyglutamine diseases, we examined subcellular localization of CRAG in the brains of Machado-Joseph disease (MJD) patients and detected specific CRAG inclusion (Fig. 3 a). Similar CRAG inclusions were observed in the brains of other MJD patients (unpublished data). To further confirm this phenomenon, we generated GFP-Q12 as control and GFP-Q69 as polyQ and examined whether CRAG could interact with polyQ. Subcellular distribution analysis revealed the diffuse cytoplasmic localization of Q12 and perinuclear aggregation of misfolded Q69 in HeLa cells (Fig. 3 b, left). Compared with the cytoplasmic distribution of both Q12 and CRAG, Q69 and CRAG translocated to the nucleus and formed NIs (Fig. 3 b, right). An immunoprecipitation assay also demonstrated that CRAG interacted with GFP-Q69 but not -Q12 (Fig. 3, c and e). Similarly, in hippocampal neurons expressing GFP-Q69 or -Q12, endogenous CRAG associated with GFP-Q69 but not -Q12 (Fig. 3 d).

NI formation by CRAG was induced by various stress stimuli generating reactive oxygen species (ROS) such as UV. Indeed, we found that H2O2 induced CRAG nuclear translocation and that this was blocked by ROS scavenger edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one; Radicut; Fig. S2). An in vitro GTPase assay indicated that H2O2 activated CRAG GTPase (unpublished data). To understand the mechanism by which CRAG recognizes and interacts with polyQ, we focused on the role of ROS generation by polyQ in the activation of CRAG. An in vitro GTPase assay demonstrated that coexpression with Q69 but not Q12 activated CRAG GTPase, and this activation was blocked by the treatment of ROS scavenger Radicut (Fig. 3 e). Actually, Radicut inhibited Q69-mediated ROS generation (unpublished data). Furthermore, in Radicut-treated cells, CRAG failed to colocalize with Q69 and induce Q69 nuclear translocation (Fig. 3 f). A coimmunoprecipitation assay also indicated that Radicut blocked the association of CRAG with Q69 in a Radicut dose–dependent manner (Fig. 3 g). Thus, ROS may be required for CRAG activation and interaction with polyQ.

To confirm this, doxycycline (DOX)-inducible Tet-On HeLa cell lines expressing HA-CRAG WT and NLS mutants were established. Two clones each selected from WT and NLS mutants were treated with DOX, and induction of HA-CRAG expression was checked by immunoblot analysis using anti-HA antibody (Fig. 4 a). That equal amount of total protein was loaded is shown by tubulin immunoblots (Fig. 4 a, bottom). Using these cell systems, we monitored the time-dependent nuclear translocation of Q69 by CRAG. As shown in Fig. 4 b, no nuclear localization of Q69 was detected before DOX treatment, but 3 h after DOX treatment, a major part of CRAG was colocalized with Q69 aggregates at perinuclear sites and a small part of the CRAG–Q69 complex was detected in the NIs. After 6–12 h, almost all CRAG and Q69 translocated to the nucleus and formed NIs. Merged images of CRAG and Q69 (Fig. 4 b, insets, yellow) demonstrated the colocalization of CRAG and Q69 in the nucleus. As a negative control, cells treated with solvent for 12 h revealed no nuclear translocation of Q69 (unpublished data). Similar results were obtained in other Tet-On HeLa cell lines (unpublished data). On the other hand, NLS mutants of CRAG could colocalize with Q69 but not induce nuclear translocation of Q69 at all (Fig. 4 b, right). Merged images (Fig. 4 b, insets, yellow) showed the perinuclear aggregates of the CRAG–Q69 complex. Subcellular fractionation analysis also indicated CRAG-dependent nuclear translocation of Q69 and no nuclear localization of Q69 in cells coexpressing NLS mutants (Fig. 4 c). Statistical analysis also indicated that CRAG WT, but not NLS mutants, induced Q69 nuclear translocation (Fig. 4 d). These results demonstrated that CRAG promoted Q69 nuclear translocation in an NLS-dependent manner.

The disappearance of polyQ at PML body has been previously reported as a pathological finding of polyglutamine diseases (Takahashi et al., 2003). We examined CRAG-dependent degradation of Q69 using a DOX-inducible Tet-On HeLa cell line expressing HA-CRAG. Upon DOX treatment, CRAG expression was up-regulated, whereas Q69 protein level was concomitantly down-regulated. That equal amount of total protein was loaded is demonstrated by tubulin immunoblots. A distinct background band sometimes appeared as a cross-reacting protein in the anti-HA immunoblots (Fig. 4 e, left). We confirmed that DOX alone did not affect Q69 protein level in Tet-On HeLa cells without CRAG (Fig. 4 e, right). As shown in Fig. 4 f, the proteasome inhibitor MG132 blocked Q69 down-regulation by CRAG WT, suggesting that CRAG may promote Q69 degradation through the ubiquitin–proteasome pathway. Furthermore, FACS analysis of cells stained with annexin-V/propidium iodide demonstrated that CRAG WT expression suppressed Q69-induced cell death (Fig. 4 g). In contrast, DOX alone could not rescue Q69-mediated cell death in Tet-On HeLa cells without CRAG (Fig. 4 g). To confirm ubiquitination of Q69 by CRAG, an in vivo ubiquitination assay was performed. As shown in Fig. 4 h, the ubiquitinated Q69 significantly increased by CRAG expression. Degradation of Q69 was not enhanced by NLS or GTPase mutants, and DNA ladder formation assays consistently revealed that the two mutants could not rescue Q69-mediated cell death (unpublished data). Because NLS and GTPase activity in CRAG are critical for its interaction with PML, CRAG-mediated activation of PML-associated ubiquitin ligase may be responsible for this ubiquitination.

We next examined the effect of CRAG knockdown by RNA interference method on nuclear translocation of polyQ in neurons. Several small interfering RNA (siRNA) oligonucleotides specifically targeted to the CRAG sequence were generated, and their inhibitory effects were estimated using the COS-7 cell expression system. All three RNA interference oligonucleotides, but not scramble oligonucleotides, suppressed the expression of HA-CRAG (Fig. 5 a, left). Among them, one siRNA (No. 1) showing the strongest inhibitory effect on CRAG expression was used for the following experiments. This siRNA or scramble with Q69 plus pEGFP vector as a marker were introduced into cultured rat hippocampal neurons, and the inhibitory effect on endogenous CRAG expression was evaluated by CRAG immunoblotting and immunostaining. As shown in Fig. 5 (a [right] and b) this siRNA, but not scramble, was found to suppress endogenous CRAG expression, indicating that this siRNA against the CRAG gene is useful to assess CRAG function.

To determine whether endogenous CRAG is involved in nuclear translocation, inclusion body formation, and decreased cell toxicity of polyQ in neuronal cells, the effects of CRAG knockdown on subcellular distribution and cell toxicity of Q69 were examined. In scramble siRNA–cotransfected DRG neurons, endogenous CRAG and Q69 translocated to the nucleus and formed NIs (Fig. 5 c, top). In contrast, perinuclear aggregations of Q69 were dominantly detected in siRNA-mediated CRAG-deficient cells (Fig. 5 c, middle). Moreover, almost all CRAG-deficient cells revealed a typical apoptotic phenotype including chromatin condensation as shown in Fig. 5 c (bottom). Statistical analysis indicated that siRNA-mediated CRAG depletion blocked the nuclear translocation of Q69 in DRG neurons (Fig. 5 d, top). In addition, analysis of a cell death assay, judging from chromatin condensation, showed that >80% of CRAG-deficient cells underwent cell death, whereas only 30% of scramble siRNA–transfected cells died by Q69 expression 48 h after transfection (Fig. 5 d, bottom). CRAG knockdown by siRNA did not cause death in cells that were not expressing Q69 (unpublished data). These results demonstrate that endogenous CRAG mediates nuclear translocation and NI formation by polyQ and confers resistance to cell death under the conditions of polyQ accumulation. A schematic model of CRAG action on polyQ is shown in Fig. 5 e.

Patients with polyglutamine diseases and transgenic mouse model carrying polyQ showed the late-onset and gradually progressive neurological pathology (Turmaine et al., 2000; Katsuno et al., 2003). Indeed, CRAG expression is very high in the developing brain and decreased thereafter in the adult brain (Fig. S3). This developmentally regulated expression of CRAG may be closely related to the appearance of polyglutamine disease; a decreased level of CRAG expression fails to scavenge unfolded proteins at PML bodies and permits an accumulation of polyQ aggregates in the nucleus, thereby conferring a toxic gain of function that is selectively deleterious to neurons. If so, CRAG is a rate-limiting factor in the degradation of pathological forms of polyQs and targeted expression of CRAG is a potential gene therapy for polyglutamine disease.

Materials And Methods

Materials

Anti-Flag M2 monoclonal and anti–α-tubulin antibodies were obtained from Sigma-Aldrich. Anti-HA high affinity, anti-HA affinity matrix, and anti–c-myc mouse mAbs were obtained from Roche. Anti-GFP rabbit polyclonal antibody, annexin V, propidium iodide (PI), and secondary antibodies conjugated with Alexa Fluor 488, 594, and 647 were obtained from Invitrogen. Anti-6×His monoclonal, anti-GFP mouse monoclonal, and Hoechst 33258 were obtained from Nacalai Tesque. DOX and mouse MTN blot were obtained from CLONTECH Laboratories, Inc. HA-probe was purchased from Santa Cruz Biotechnology, Inc. Nuclear mitotic apparatus protein antibody 1 was purchased from Biocarta. Anti-ubiquitin antibody was obtained from Santa Cruz Biotechnology, Inc. Anti-HA mouse mAb was purchased from Covance. Anti-PML mouse mAb was purchased from Santa Cruz Biotechnology, Inc.

Identification of CRAG in CRAM immunoprecipitates from developing rat brain

CRAM (CRMP-5) has been implicated in semaphorin signaling (Inatome et al., 2000). We have searched for CRAM-interacting proteins from developing rat brain. The purification procedure was performed by using anti-CRAM antibody affinity column chromatography and specific elution with antigen peptides for anti-CRAM antibody. Among purified proteins, we focused on a 42-kD protein, and partial amino acid sequence analysis revealed several peptide sequences, including KSALVHRYLTGTYVQEESPEGGRF. Based on this information, we cloned a novel gene encoding a GTPase from the mouse brain cDNA library (available from GenBank/EMBL/DDBJ under accession no. AB078345).

Expression constructs

CRAG WT, GTPase mutants, NLS mutants, and murine PML1 isoform (available from GenBank/EMBL/DDBJ under accession no. BC020990) cDNA tagged with the HA epitope at the NH2 terminus were subcloned into pCMV5 expression vectors. Murine PML1 shows 67% homology to human PML1 at the amino acid level. CRAG GTPase mutation was generated by missense mutation S114N. CRAG NLS mutation was generated by missense mutation KR342-343EE. The plasmid encoding NH2-terminal–truncated ataxin-3 with Q69 together with the COOH-terminal myc epitope and the NH2-terminal HA epitope was described previously (Yoshizawa et al., 2000). In this plasmid, 286 amino acid residues of ataxin-3 were deleted from the NH2-terminal side. For our experiment, the HA epitope was removed.

The Q12 gene was created by PCR using the primers 5′-CCCAAGCTTGGGATGGCCTACTTTGAAAAACAG-3′ and 5′-GGGGTACCCCAGGGAATGAAGAATAATG-3′ and subcloned into pcDNA3.1- myc His. A RING-finger domain mutation of PML was generated by the missense mutation C51S/C54S using the PCR primers 5′-CCTGCACACGCTGAGCTCCGGAAGCCTGGAGGCGC-3′ and 5′- GCGCCTCCAGGCTTCCGGAGCTCAGCGTGTGCAGG-3′.

Immunoprecipitation, immunoblotting, and immunofluorescence microscope

These procedures were performed as described previously (Mitsui et al., 2002; Hotta et al., 2005). Fluorescence images were analyzed on a confocal microscope (LSM 510 META; Carl Zeiss MicroImaging, Inc.) equipped with three lasers (UV.Ar. 364, Ar. 488, and HeNe 543) using Plan-Apochromat 63× oil-immersion (NA 1.40) objective. LSM 510 META 3.0 software (Carl Zeiss MicroImaging, Inc.) was used for image acquisition from confocal microscopy. Photoshop 6.0 software (Adobe) was used for minor adjustments to contrast and overlaying.

Immunohistochemistry with pathological specimens

Postmortem brain tissues were obtained from two MJD patients and analyzed as described previously (Yoshizawa et al., 1990).

GTPase assay for CRAG

The GTPase assay was performed as described previously (Der et al., 1986).

In vitro ubiquitin ligase assay

Immunoprecipitates were washed three times with lysis buffer and once with ubiquitination buffer (50 mM Tris-HCL, pH 7.4, 10 mM MgCl2, 5 mM ATP, and 2 mM dithiothreitol) and incubated in 50 μl of the same buffer supplemented with 100 ng E1 (Honda et al., 1997), 500 ng E2ubcH5(a,b,c) (Kobirumaki et al., 2005), and 2.5 μg His-tagged ubiquitin (Calbiochem) for 30 min at 25°C. Samples were analyzed with anti-His antibody.

Online supplemental material

Fig. S1 a shows the hollow, doughnut-like nuclear bodies of CRAG in UV-irradiated hippocampal neurons, and Fig. S1 b shows colocalization of CRAG doughnut-like bodies with PML1 in a UV-irradiated DRG neuron. Fig. S2 shows NIs of CRAG in response to H2O2 stimulation in HeLa cells. Fig. S3 shows a comparison of CRAG expression in adult and developing mouse brains.

Acknowledgments

We thank Dr. S. Jahangeer for critical reading of this manuscript.

This study was supported in part by Grants-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology and the Japan Society for the Promotion of Science.

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Q. Qin and R. Inatome contributed equally to this paper.

Abbreviations used in this paper: CRAM, CRMP-associated molecule; CRMP, collapsin response mediator protein; DOX, doxycycline; DRG, dorsal root ganglion; GTPase, guanosine triphosphatase; MJD, Machado-Joseph disease; NI, nuclear inclusion; NLS, nuclear localization signal; PML, promyelocytic leukemia protein; polyQ, polyglutamine protein; RING, really interesting new gene; ROS, reactive oxygen species; siRNA, small interfering RNA; WT, wild type.

Supplementary data