From the
Department of Biochemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, the
Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho Showa-ku, Nagoya 466-8550, and the ¶Department of Environmental Biology, College of Bioscience and Biotechnology, Chubu University, Matsumoto-cho 1200, Kasugai 487-8501, Japan
Received for publication, March 24, 2003 , and in revised form, April 21, 2003.
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ABSTRACT |
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INTRODUCTION |
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A considerable effort has been made to find molecules that suppress polyglutamine aggregation and cell death/toxicity for therapeutic purposes (2022). In general, the misfolding and aggregation of proteins are prevented by molecular chaperones (23, 24). Some molecular chaperones such as heat shock protein (Hsp) 70 and Hsp40 have recently been identified as important regulators of polyglutamine aggregation and/or cell death in in vitro assays (25), in cultured mammalian cells (2631), in a Drosophila model (32) and in transgenic mice (33, 34). Hsp27 was also identified as a suppressor of polyglutamine-mediated cell death using a cellular model of Huntington's disease (35). However, because Hsp70/40 and Hsp27 suppressed polyglutamine-mediated death without suppressing polyglutamine aggregation in some experimental systems (35, 36), elucidation of the ways in which Hsps protect cells against polyglutamine mutations might be of relevance for other neurodegenerative conditions in which pathology is associated with protein deposition in neuronal cells.
Hsp105 is highly conserved in organisms from yeast to human (3742) and is expressed in various tissues of mammals, but especially at high levels in brain (43). Recently, Hsp105
was demonstrated to have antiapoptotic properties for neuronal survival (44). Furthermore, Hsp105
prevents the aggregation of thermal denatured protein in vitro (45). However, its role in polyglutamine-mediated cell death/toxicity has not been studied. In the present study, we examined the role of Hsp105
in the context of polyglutamine aggregation and cell death using a cellular model of SBMA and demonstrate that Hsp105
without chaperone activity protects cells against polyglutamine-mediated cell death by reducing polyglutamine-protein aggregation. These findings suggest an important role for Hsp105
in preventing neurodegenerative diseases associated with polyglutamine expansions.
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EXPERIMENTAL PROCEDURES |
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We used a construct expressing GST- and HA-tagged tAR65 (GST-tAR65-HA) in bacterial cells (47). The constructs expressing HSP105 deletion mutants in bacterial cells were generated by ligating insert DNA made by PCR using pTrcHis105-1 (45) as a template DNA and a specific set of primers (Table I) into pTrcHisA vector (Invitrogen) at the KpnI site.
Cell Culture and TransfectionAfrican green monkey kidney cells (COS-7) and human neuroblastoma cells (SK-N-SH) were supplied from Riken cell bank. COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical) supplemented with 10% fetal bovine serum. SK-N-SH cells were maintained in -minimal essential medium (Invitrogen) with 10% fetal bovine serum. For transfection of plasmid DNA, cells were grown on coverslips to 7080% confluence and washed twice with Opti-MEM (Invitrogen). Then plasmid DNA was transfected into cells with DMRIE-C reagent (Invitrogen) for 1418 h, according to the manufacturer's instructions.
Indirect ImmunofluorescenceCOS-7 cells grown on coverslips were washed with phosphate-buffered saline without Ca2+ and Mg2+ (PBS()) and fixed with 4% paraformaldehyde for 30 min at room temperature. These cells were washed with PBS() and incubated with blocking solution containing 3% bovine serum albumin (BSA) in PBS() at room temperature for 1 h. Then rabbit anti-human Hsp105 (48) or mouse anti-Hsp70 monoclonal antibody (Sigma) at a 1:300 dilution was added to the coverslips and incubated in a moist chamber at 37 °C for 1 h. After a wash with PBS(), rhodamine-conjugated goat anti-rabbit or mouse IgG antibody (Molecular Probes) at a 1:50 dilution was added to the coverslips, and they were incubated further at 37 °C for 1 h. After another wash with PBS(), cells were observed using a confocal laser scanning microscope (Zeiss).
Analysis of Aggregation in VivoCOS-7 cells transfected with expression plasmid for tAR24, tAR65, or tAR97 were washed with PBS() and fixed with 4% paraformaldehyde for 30 min at room temperature. Cells on coverslips were washed with PBS() and stained with 10 µM Hoechst 33342 for 15 min at room temperature. The cells were washed with PBS() and then examined using a confocal laser scanning microscope. The number of transfected cells with visible aggregates and the number of transfected cells without aggregates were counted independently in randomly chosen microscopic fields in different areas of a coverslip. Approximately 300600 transfected cells were analyzed for data in each experiment.
Detection of the Apoptotic CellsThe apoptotic cells were identified by their nuclear morphology and the terminal nucleotidyl transferase-mediated UTP nick end labeling (TUNEL) method (29). Nuclear morphology was examined by staining with Hoechst 33342. The TUNEL method was performed using a DeadEndTM apoptosis detection kit (Promega) according to the manufacturer's instructions. Briefly, cells were fixed with 4% paraformaldehyde at 72 h after transfection. Fixed cells were incubated with biotinylated deoxynucleotides, then stained with streptavidin-rhodamine conjugate (Molecular Probes) and Hoechst 33342. Cells were then observed by confocal laser scan microscopy.
Western Blotting AnalysisCells were lysed with a solution containing 0.1% SDS at 72 h after transfection. The cellular proteins (15 µg) were separated by 7.5% SDS-PAGE and blotted onto a nitrocellulose membrane. The membrane was incubated with rabbit anti-human Hsp105 (37) or mouse anti-Hsp70 (Sigma) antibody, then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz) for Hsp105 or anti-mouse IgG (Santa Cruz) for Hsp70 at a 1:2,000 dilution. These proteins were detected using the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences).
Protein PurificationGST-tAR65-HA was expressed in BL21 bacterial cells on addition of 1 mM isopropyl--D-thiogalactopyranoside. Cells were collected and resuspended in ice-cold TEGM buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10% glycerol, and 10 mM sodium molybdate). Cells were then sonicated for 1 min and centrifuged for 30 min at 10,000 x g. To purify GST-tagged proteins, the supernatants were mixed with glutathione-Sepharose 4B (Amersham Biosciences) and incubated at 4 °C for 1 h. The Sepharose beads were then washed with PBS() and eluted with 10 mM reduced glutathione. Wild-type Hsp105
and its mutants were purified as His-tagged proteins by successive Ni2+-agarose (Invitrogen) and Mono Q anion exchange column (Amersham Biosciences) chromatographies, as described previously (45).
Detection of Aggregates of Truncated AR in VitroGST-tAR65-HA (1 µM) was incubated with Hsp105, its mutants, or BSA in 20 µl of buffer A (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) at 30 °C for 12 h in the presence of 2 mM ADP. The reaction was stopped by the addition of 20 µl of a solution containing 2% SDS and 100 mM dithiothreitol, and the mixtures were heated at 98 °C for 5 min. After the addition of 200 µl of a 1% SDS solution, the mixture was filtered through a 0.2-µm cellulose acetate membrane (Advantec). Aggregates on the membrane were incubated with anti-HA tag antibody (1:500, Santa Cruz) then with peroxidase-conjugated anti-mouse IgG antibody (1:2000) and detected with an ECL detection system (Santa Cruz).
ImmunohistochemistryWe perfused 20 ml of a 4% paraformaldehyde fixative in 0.1 M phosphate buffer, pH 7.4, through the left cardiac ventricle of SBMA transgenic mice (49) deeply anesthetized with ketamine-xylazine, postfixed tissues overnight in 10% phosphate-buffered formalin, and processed tissues for paraffin embedding. Then, 4-µm thick tissue sections were deparaffinized, dehydrated with alcohol, and treated in formic acid for 5 min at room temperature and with trypsin (Dako) for 20 min at 37 °C. The tissue sections were blocked with normal goat serum (1:20) and incubated with rabbit anti-mouse Hsp105 antibody (1:100). The sections were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories), and immune complexes were visualized using streptavidin-horseradish peroxidase (Dako) and 3,3'-diaminobenzidine (Dojindo) substrate and counterstained with methyl green. For the immunohistochemistry of tissues of SBMA patients, paraffin-embedded sections of the spinal cord and scrotal skin from nine patients with clinicopathologically and genetically confirmed SBMA (age 5184 years, mean 64.3 years) were examined using rabbit anti-human Hsp105 antibody (1:100).
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RESULTS |
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Under these conditions, apoptotic cells with condensed chromatin were observed in 12 and 18% of all COS-7 cells expressing GFP at 48 and 72 h after transfection of the expression plasmid for tAR97, respectively (Fig. 2A). In contrast, cells expressing tAR24 showed little apoptotic morphology after the transfection (Fig. 2A). Most apoptotic cells had aggregates of tAR97 in the nucleus, whereas cells containing cytoplasmic aggregates exhibited few apoptotic features (Fig. 2B). Furthermore, when the presence of fragmented DNA was assessed by the TUNEL method, the cells with intranuclear aggregates, but not cells with cytoplasmic aggregates, were found to be positive (Fig. 2C). These findings suggested that the intranuclear aggregates induced apoptosis in COS-7 cells. Similar results were obtained with SK-N-SH cells (data not shown). Thus, although both non-neuronal and neuronal cells could be utilized as a cell model of SBMA, we used the COS-7 model for further study because the cells expressed the transfected plasmids markedly well.
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Colocalization of Hsp105 and Hsp70 with Aggregates of Truncated ARWe next examined the cellular distribution of Hsp105
and Hsp70 in COS-7 cells by indirect immunofluorescence using anti-human Hsp105 and anti-Hsp70 antibodies. Under nonstressed conditions, endogenous Hsp105
and Hsc70/Hsp70 were localized mainly in the cytoplasm of cells (Fig. 3A). When tAR24 was transiently expressed in COS-7 cells, both endogenous Hsp105
and Hsp70 were also detected in the cytoplasm of the cells (Fig. 3, B and C). In contrast, when tAR97 was transiently expressed in COS-7 cells, endogenous Hsp70 was colocalized to the aggregates of tAR97, whereas endogenous Hsp105
was not. However, when overexpressed with tAR97 in cells, Hsp105
was colocalized to the aggregates of tAR97 (Fig. 3D), whereas Hsp105
was localized to the cytoplasm of cells in which tAR97 was not expressed. Thus, the increased amounts of Hsp105
in cells seemed necessary for the interaction with and binding to the tAR containing an expanded polyglutamine tract.
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Overexpression of Hsp105 Reduced Aggregation of tAR97 Hsp105
prevents the aggregation of denatured protein in vitro (45) and suppresses apoptotic cell death induced by various forms of stress in neuronal PC12 cells (44). Because the overexpressed Hsp105
colocalized to intracellular aggregates of tAR97 (Fig. 3), we next examined the effects of Hsp105
on the aggregation of tAR containing an expanded polyglutamine tract. When expression plasmids for Hsp105
and tAR97 were cotransfected into COS-7 cells, the proportion of cells with tAR97 aggregates was reduced to
50% of that transfected without Hsp105
(Fig. 4, A and B). Overexpression of Hsp70 or Hsp40 also suppressed the formation of aggregates similarly to Hsp105
, and Hsp70 and Hsp40 in combination suppressed the formation strongly. However, the suppression of aggregation by Hsp70 and Hsp40 was not enhanced by the coexpression of Hsp105
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When cellular toxicity was analyzed by examining the nuclear morphology of cells stained with Hoechst 33342, numbers of apoptotic cells with condensed chromatin were found to be markedly reduced by coexpression of Hsp105 with tAR97 (Fig. 4C). Overexpression of Hsp70 and/or Hsp40 also suppressed apoptotic cell death caused by expression of tAR97. Furthermore, when various amounts of Hsp105
were coexpressed with tAR97, the aggregation of tAR97 and apoptosis were both suppressed depending on cellular levels of Hsp105
(Fig. 5). Under these conditions, cellular levels of endogenous Hsp70 were not changed by overexpression of Hsp105
(Fig. 5, lower panel). These findings strongly suggested that when overexpressed, Hsp105
suppressed effectively not only the formation of aggregates but also the expanded polyglutamine-mediated cellular toxicity.
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Identification the Domain of Hsp105 Required for Suppression of tAR97 AggregationHsp105
is composed of N-terminal ATP binding, central
-sheet, loop and C-terminal
-helix domains, similar to the Hsp70 family proteins. To determine the domain of Hsp105
essential to suppress the aggregation caused by expansion of the polyglutamine tract, we constructed expression plasmids for various deletion mutants of Hsp105
, as shown in Fig. 6A. When coexpressed with tAR97 in COS-7 cells, the Hsp105
mutant C3 or
L significantly suppressed the aggregation of tAR97 as did wild-type Hsp105
(Fig. 6B). However, other deletion mutants failed to suppress the aggregation of tAR97. Because the C3 and
L mutants contain both
-sheet and
-helix domains, these domains seemed to be essential to suppress the aggregation caused by expansion of the polyglutamine tract.
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Hsp105 Inhibits Aggregation of GST-tAR65 in VitroTo examine further whether Hsp105
can directly suppress aggregation of the expanded polyglutamine tract, we analyzed the effects of Hsp105
on the aggregation of tAR65 in vitro (Fig. 7). GST-tAR65-HA was incubated with or without Hsp105
or its mutant, and insoluble aggregates that formed during the incubation were collected on cellulose acetate membranes. Hsp105
suppressed the aggregation of tAR65 in a dose-dependent manner (Fig. 7A). Furthermore, the aggregation was suppressed by wild-type Hsp105
and the mutants C3 and
L but not by other deletion mutants (Fig. 7B). Thus, it was suggested that Hsp105
itself suppressed the aggregation of truncated AR containing an expanded polyglutamine without other cellular components and that both the
-sheet and
-helix domains of Hsp105
seemed necessary for the suppression in vitro as well as in vivo.
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Immunohistochemistry of Hsp105 in Nuclear Inclusions in the Tissues of SBMA Patients and Transgenic MiceNuclear inclusions containing mutant and truncated AR with an expanded polyglutamine have been shown to occur in residual motor neurons in the brain stem and spinal cord (4) and also in the skin, testis, and some other visceral organs of SBMA patients (5). We next examined whether Hsp105
localizes in the nuclear inclusions in these tissues of SBMA patients. As shown in Fig. 8, A and B, Hsp105
staining was observed in nuclear inclusions in neurons of the spinal anterior horn and scrotal skin epidermal cells. Furthermore, when male transgenic mice carrying a full-length AR with an expanded polyglutamine (97 repeats) tract and showing neuropathologic changes equivalent to human SBMA (49) were examined immunohistochemically, Hsp105
was also detected in nuclear inclusions in neurons of the spinal anterior horn and muscle cells (Fig. 8, C and D). However, although Hsp105
was commonly observed in nuclear inclusions in scrotal skin epidermal cells of SBMA patients and in muscle cells of the transgenic mice, only a few Hsp105-immunoreactive nuclear inclusions were observed in neurons of the spinal anterior horn of either patients or mice.
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DISCUSSION |
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Hsp70 and Hsp40 have recently been identified as important regulators of polyglutamine aggregation and/or cell death in cellular models of polyglutamine disease (29). Hsp70 promotes protein folding by an ATP-dependent process involving polypeptide segments enriched in hydrophobic residues (50, 51) and cooperates in this function with members of the Hsp40 family (52). The binding of Hsp70 to substrate proteins may prevent protein aggregation directly by shielding the interactive surfaces of nonnative polypeptides. Suppression of polyglutamine-induced neurotoxicity by expression of Hsp40 alone is most likely caused by the ability to activate the endogenous Hsp70 for suppression of the neurotoxicity. Here, we showed that overexpression of Hsp105 alone suppressed the aggregation of truncated AR containing an expanded polyglutamine tract similarly to Hsp70 or Hsp40, whereas Hsp70 and Hsp40 in combination suppressed the aggregation much more markedly. However, because the suppression of aggregation by Hsp70 and Hsp40 was not enhanced by the coexpression of Hsp105
, Hsp105
and Hsp70/Hsp40 seem to suppress the polyglutamine-induced neurotoxicity by similar mechanisms.
In polyglutamine diseases such as spinocerebellar ataxia type 3/Machado-Joseph disease, Hsp70 colocalizes in intracellular aggregates, whereas Hsp105 is not found in the aggregates (27). In the present study, Hsp105 was detected in nuclear inclusions in neurons of the spinal anterior horn and scrotal skin epidermal cells of SBMA patients and also in neurons of the spinal anterior horn and muscle cells of SBMA transgenic mice, although only a few Hsp105-immunoreactive nuclear inclusions were observed in neurons of the spinal cord of the patients and mice. On the other hand, in a cellular model of SBMA, endogenous Hsp70 but not endogenous Hsp105
localized to aggregates of tAR97, although the overexpressed Hsp105
localized to the aggregates of tAR97. Because Hsp70 exists in much larger amounts than Hsp105
in cells, Hsp70 may interact preferentially with truncated AR containing an expanded polyglutamine tract. However, when Hsp105
was overexpressed in the cells, reaching high levels, it seemed to bind and localize to truncated AR containing an expanded polyglutamine tract like Hsp70. Thus, the existence of molecular chaperones at high concentrations in cells may be essential to prevent the aggregation of truncated AR containing an expanded polyglutamine tract.
The intranuclear aggregation of truncated AR containing an expanded polyglutamine tract and apoptotic cell death coincided in the cellular model of SBMA, and both processes were suppressed by overexpression of Hsp105. As to the mechanism by which Hsp105
suppresses the apoptosis caused by expansion of the polyglutamine tract, one possibility is that suppression of aggregation by Hsp105
mediates suppression of apoptosis. Key components of the transcription apparatus, such as cAMP response element binding protein-binding protein, p53, and TAFII130, are sequestered in polyglutamine-containing inclusions, then the expanded polyglutamine tract causes altered gene transcription (30, 5357). By preventing the formation of intranuclear aggregates, Hsp105
may suppress the alteration of gene transcription caused by an expanded polyglutamine tract and eventually apoptotic cell death.
Another possibility is that the abilities of Hsp105 to suppress aggregate formation and cellular toxicity caused by expansion of the polyglutamine tract are independent. Recently, molecular chaperones, such as Hsp70, Hsp40, and Hsp27, were shown to suppress an expanded polyglutamine-mediated cellular toxicity independently of suppression of aggregation (35, 36). Although the relationship between the aggregation and the induction of apoptosis remains unknown, the suppression of cellular toxicity by molecular chaperones may be caused by the ability to inhibit apoptosis. Hsp105
suppresses heat shock-induced apoptosis in neuronal cells by preventing the activation of c-Jun N-terminal kinase (44). Because c-Jun N-terminal kinase is activated by the expanded polyglutamine tract (58), Hsp105
may also suppress the cellular toxicity by its ability to inhibit apoptosis.
In conclusion, we identified Hsp105 as a novel molecule that reduces aggregation and cellular toxicity caused by an expansion of the polyglutamine tract. Molecular chaperones, such as Hsp105
, Hsp70, Hsp40, and Hsp27, seem to suppress cell toxicity caused by an expansion of the polyglutamine tract. These findings suggest that increasing the expression levels or enhancing the function of chaperones in neurons may open up a promising approach to the treatment of polyglutamine diseases, although more studies are required to determine the precise mechanism of neurodegeneration of CAG repeat diseases.
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FOOTNOTES |
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|| To whom correspondence should be addressed. Fax: 81-75-595-4758; E-mail: hatayama{at}mb.kyoto-phu.ac.jp.
1 The abbreviations used are: SBMA, spinal and bulbar muscular atrophy; AR, androgen receptor; BSA, bovine serum albumin; GFP, green fluorescent protein; HA, hemagglutinin; PBS, phosphate-buffered saline; tAR, truncated androgen receptor; TUNEL, terminal nucleotidyl transferase-mediated UTP nick end labeling.
2 N. Yamagishi, K. Ishihara, and T. Hatayama, unpublished data.
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REFERENCES |
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