Subcellular localization of the Huntington's disease gene product in cell lines by immunofluorescence and biochemical subcellular fractionation
Subcellular localization of the Huntington's disease gene product in cell lines by immunofluorescence and biochemical subcellular fractionationKarien E. De Rooij1, Josephine C. Dorsman2, Magda A. Smoor1, Johan T. Den Dunnen1,3 and Gert-Jan B. Van Ommen1,*
1MGC-Department of Human Genetics, 2MGC-Laboratory of Molecular Carcinogenesis, Leiden University, Leiden, The Netherlands and 3Clinical Genetic Centre Leiden, Leiden, The Netherlands
Received March 18, 1996;Revised and Accepted May 17, 1996
Huntington's disease is a progressive neurodegenerative disorder, which is caused by expansion of a polymorphic (CAG)n repeat in the coding region of the Huntington's disease gene. The function of huntingtin has not been elucidated so far. Accordingly, detailed subcellular localization studies remain useful. In an immunohistochemical study, we have reported huntingtin to be present in the cytoplasm of cells in the majority of the tissues studied. In addition, we detected a signal in the nucleus of cells in some tissues, including neuronal cells. We have further extended these studies in various mammalian cell lines, using a panel of (affinity-purified) polyclonal huntingtin antibodies in immunofluorescence, confocal laser scanning microscopy and biochemical subcellular fractionation studies. In mouse embryonic fibroblasts, human skin fibroblasts and in mouse neuroblastoma cells huntingtin was present in the cytoplasm. All five antibodies, directed against different parts of huntingtin, also showed a signal in the nucleus. This signal could be competed by the original antigen. The localization of huntingtin in both cytoplasm and nucleus, was confirmed by biochemical subcellular fractionation studies. However, in most other studies, a nuclear location for huntingtin has not been found. Our results suggest, however, that besides its function(s) in the cytoplasm, a nuclear function of huntingtin at some stages of differentiation or in some phases of the cell cycle may not be excluded.
Huntington's disease (HD) is a progressive neurodegenerative disorder with an autosomal dominant pattern of inheritance. In populations of Caucasian origin the disease affects approximately 1 in 10 000 people. The characteristic features of HD include involuntary movements, personality changes and dementia, which are caused by selective neuronal death in the caudate nucleus and putamen. The first symptoms usually occur at an age of 30-50 years and death follows in 15-20 years (1 ).
The disease is caused by the expansion of a polymorphic (CAG)n repeat to 37-120 units in the coding region of the HD gene (2 ). Further somatic expansion of the repeat is found in affected brains (3 ,4 ), but appears to be too small to cause cell death. The gene is transcribed into two transcripts of 10.5 and 13.5 kb (5 -7 ), and encodes a protein of approximately 348 kDa, designated huntingtin. The (CAG)n repeat is translated as a polyglutamine stretch, located very near the amino-terminus of the protein (8 -11 ). Both mRNA and protein are expressed in most tissues examined (5 ,7 ,8 ,10 ,12 ,13 ), although in different quantities. For example, in brain, expression is higher in neuronal than in non-neuronal cells. No differences in mRNA and protein levels have been found between healthy controls and HD patients (5 ,7 ,9 ,12 -14 ). Both the normal and the affected allele are transcribed and translated (8 ,9 ,15 -18 ).
The fact that the normal and mutant protein are expressed in a wide variety of tissues is in clear contrast with the specific pathology, which is found predominantly in the caudate nucleus and putamen in HD patients. The HD mutation is commonly thought to result in a deleterious gain of function, through interactions with components specific to affected tissue (19 ,20 ). In keeping with this model, a protein designated HAP1 has been identified, which is selectively expressed in brain, and binds to the mutant protein with increased affinity (21 ). However, the function of normal huntingtin remains elusive.
An analysis of the subcellular distribution of huntingtin in different types of cycling cells and in non-dividing cells of various origin may provide suggestions for possible roles of huntingtin. Initial immunohistochemical studies from our group (13 ), using purified polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal part of huntingtin, showed a cytoplasmic localization in all tissues tested. In addition, in several cell types including neuronal cells of freshly sectioned monkey brain and human post-mortem brain, huntingtin was also found in the nucleus (13 ). We have extended our studies in various mammalian cell lines, using a panel of (affinity-purified) polyclonal huntingtin antibodies in immunofluorescence and confocal laser scanning microscopy studies. Besides a cytoplasmic signal, we detect a characteristic nuclear signal in all cell lines tested. Biochemical subcellular fractionation studies confirm these results.
We will discuss our results in the context of the results of other localization studies of huntingtin, which describe the presence of huntingtin in the cytoplasm only (9 ,10 ,18 ,22 ,23 ).
A panel of polyclonal antisera was raised against two different synthetic peptides and against several huntingtin-glutathione S-transferase fusion proteins (htt-GST). The antibodies described in this study are depicted in Figure 1 . All antibodies recognize recombinant huntingtin in immunoprecipitation and western studies (13 ; data not shown). Antibody 93 was affinity purified and detects a single protein band of approximately 350 kDa in a lymphoblastoid cell line of a healthy control, while it detects two bands in an HD patient (Fig. 2 ). These findings emphasize the specificity of this antibody preparation for huntingtin.
We have used the five antisera described in Figure 1 in indirect immunofluorescence experiments to analyze the subcellular distribution of huntingtin in various tissue culture cells of different species. The results of the analysis of mouse embryonic fibroblasts (MEFs), human skin fibroblasts and mouse neuroblastoma cells are shown in Figure 3 . Figure 3 (A-C) shows that with antibody 1356, directed against the C-terminal end of the protein, a cytoplasmic as well as a nuclear signal can be seen. The cytoplasmic signal in MEFs shows a distinctive thread-like cytoskeletal structure (Fig. 3 A), which resembles microtubules or a microtubule-associated protein. In fibroblasts and neuroblastoma cells, the cytoplasmic signal is distributed differently (Fig. 3 B,C). In non-differentiated neuroblastoma cells the cytoplasmic signal is extremely strong, masking the nucleus. The nucleus shows a distinctive pattern of an evenly distributed signal, excluding 1-8 negatively staining areas, and a number of intensely staining small foci. The spots could be most clearly observed in the MEFs (Fig. 3 A). In some instances, these foci can also be observed with serum 1359, which has been raised against another region of the protein (data not shown).
Our results show that huntingtin is localized in the cytoplasm and the nucleus, but that certain regions in the nucleus, which resemble the nucleoli, are not stained. To get more insight in the intranuclear distribution of huntingtin, we performed confocal laser scanning microscopy (CLSM) procedures. In this case, slides incubated with antisera against huntingtin were counterstained using mounting medium containing propidium iodide (PI). PI stains both RNA and DNA and with this agent the nucleoli can be visualized as heavily staining red dots. Figure 4 (A-C) shows different levels of the same cell; Figure 4 D shows the merged picture of all levels. The results show that the particulate huntingtin staining is found at all levels inside the nucleus and not specifically at the nuclear membrane and that the negatively staining areas indeed represent the nucleoli.
To determine the subcellular localization of huntingtin in an independent and complementary approach, we have performed a biochemical subcellular fractionation procedure using a protocol which separates the nucleus from the cytoplasmic remainder. The cytoplasmic and nuclear protein fractions were isolated from human skin fibroblasts, separated by SDS-PAGE and detected in western blotting procedures (Fig. 5 ). With antibody 93 against huntingtin, a band is observed in the cytoplasmic as well as the nuclear fraction (Fig. 5 ). As a control for the fractionation procedure, proteins with a well-known subcellular localization were also tested. Figure 5 also shows that the cytoplasmic proteins Raf-1, a protein kinase and [alpha]-tubulin, a component of the microtubules, are indeed present in the cytoplasmic fraction, while the nuclear proteins Creb Binding Protein (CBP) and p53 can be found exclusively in the nuclear fraction. These control results show the validity of the fractionation procedure. Specifically, they exclude the possibility that huntingtin is trapped in the nuclear fraction via tubulin, with which huntingtin might be slightly associated. In conclusion, the fractionation studies indicate that huntingtin is present both in the cytoplasm and in the nucleus. These results are in complete agreement with the findings of the immunofluorescence experiments.
Using five polyclonal antisera in immunofluorescence and CLSM experiments, we have shown that huntingtin is localized both in the cytoplasm and in the nucleus, in different cell lines of mammalian origin. These cells include mouse embryonic fibroblasts, human skin fibroblasts and a mouse neuroblastoma cell line. In the cytoplasm, huntingtin does not seem to colocalize exclusively with one particular structure or organelle, but instead is rather dispersed throughout the whole cytoplasm with a partial association with microtubule-like structures (see also 22 ,23 ). CLSM studies have indicated, that in the nucleus for huntingtin a characteristic signal can be observed, with an exclusion of the nucleoli.
The specificity of the observed nuclear signal in the immunofluorescence experiments was shown by preincubation/competition assays and by using an affinity-purified antibody, which recognizes normal as well as mutant huntingtin. In addition, essentially the same pattern of cellular and nuclear localization was seen using several additional polyclonal antibodies raised against various other huntingtin-GST fusion proteins (K.d.R., M.S. and J.D., unpublished results). The latter finding underscores the conclusion that the nuclear signal is not caused by spurious activities. For the human skin fibroblasts, the dual cytoplasmic and nuclear localization was confirmed using biochemical subcellular fractionation procedures.
In agreement with the analysis of tissue culture cells, previous immunohistochemical studies on tissue material also showed a cytoplasmic signal in most cell types and an additional nuclear signal in several cell types, including neurons (13 ). However, other groups have not been able to detect a nuclear localization of huntingtin in various tissues, including neuronal tissue (9 ,10 ,18 ,22 ,23 ). This contradiction may be due to differences in the antibodies used and/or differences in experimental procedures, including the preparation of brain tissue material. Different levels of metabolic and regulatory activity of cells during the preparation of brain tissue material may cause changes in the observed subcellular localization of huntingtin and influence the results.
Although for huntingtin, the attention has been focused primarily on the analysis of the expression and the subcellular localization in (parts of the) brain, in several reports the analysis of various cell lines has been described. These cell lines include the adenovirus-transformed human embryonic kidney cell line HEK-293 (24 ), SV40-transformed monkey kidney cells Cos-1 (18 ) and human lymphoblastoid cell lines (9 ). In all these cell lines huntingtin could be detected in the cytoplasm, but only in one of those cell lines, namely HEK-293, huntingtin was also found in the nucleus (24 ). In line with our experiments, the cytoplasmic signal for huntingtin does not seem to be preferentially associated with certain cytoplasmic structures or organelles, but seems present in different regions of the cytoplasm. The immunofluorescence procedures for cultured cells in our experiments were basically the same as in Bessert et al. for HEK-293 (24 ) and as in those of Trottier and coworkers for the Cos-1 cells (18 ). The conclusion derived by this last group of an exclusive cytoplasmic localization for huntingtin was based on the analysis of the subcellular distribution of overexpressed exogenous huntingtin in various transfectants. However, it is known that overexpression of a protein can significantly affect its cellular distribution, while a very strong signal of the overexpressed protein may render conclusions on the distribution of the endogenous protein difficult. Differences in the antibodies used may further contribute to the differences in the observed subcellular localization.
Our results suggest that in several mammalian tissue culture cells huntingtin is present not only in the cytoplasm but also in the nucleus. In addition, it appears that also the relative distribution of huntingtin between the cytoplasmic and nuclear compartments differs per cell type. The nuclear signal, as well as its punctate nature, was strongest in the mouse embryonic cells. Combined with the increased abundance of huntingtin in rat embryos (25 ) and the early embryonic lethality of its absence in homozygous knockout mice (26 -28 ), these results suggest that besides its cytoplasmic function(s), a nuclear function of huntingtin at some stage in differentiation or in some phases of the cell cycle may not be excluded.
Thus, our results bring huntingtin back in focus as a potential regulatory factor in nuclear processes, such as transcription. A possible role in transcription is in line with some structural features of huntingtin. A number of transcriptional activators have been described which, like huntingtin, contain a polyglutamine stretch (19 ,29 ,30 ). In addition, a leucine zipper-like motif is found in huntingtin (13 ). This latter motif is also found in many DNA-binding proteins and transcriptional activators (31 ). The cytoplasmic localization, which was found in cell lines and tissues analyzed, does not disfavour an additional role for huntingtin in nuclear processes. For example, the activities of certain transcriptional activators, including the transcription factor NF-[kappa]B, are in part regulated by transport to and from the nucleus (32 ). For huntingtin a similar mechanism might be involved. Another possibility is that differential splicing of huntingtin RNA results in differences in the subcellular localization of the proteins, as has been recently described for FMR1 (33 ). The fact that huntingtin is a very large protein and thus potentially has a large interface for interactions with a variety of cellular proteins, makes it quite plausible that the protein has multiple functions in the cell.
At present we favour the model that at least one of the functions of huntingtin in cycling cells and in several differentiated cells is the interaction with certain transcriptional regulators or other important regulatory proteins (see also 13 ). Deregulation of this interaction process as a consequence of (CAG)n repeat expansion, possibly associated with aging, may contribute to the process of cell death observed in neuronal cells of affected individuals. Especially, a partially nuclear localization of a mutant huntingtin protein in cycling progenitors of neuronal cells may well cause alterations which have effects only after differentiation.
Mouse embryonic fibroblasts (MEFs) were prepared from mouse embryos obtained by hysterectomy of 13-17 days pregnant mice. Embryos were collected in phosphate buffered saline (PBS, GIBCO BRL), the head and liver were removed and the remainder of the embryos was washed several times in PBS and subsequently cut to pieces with scissors. After three rounds of 15 min of trypsinization single cells were collected and subsequently seeded at a density of 3.5 * 104/cm2. Cells were cultured on Petri dishes coated with gelatin in Dulbecco's modified Eagle's medium (DMEM) without phenol-red (GIBCO BRL) supplemented with 10% fetal calf serum (GIBCO BRL).
Human skin fibroblasts were cultured in DMEM without phenol-red (GIBCO BRL) supplemented with 10% fetal calf serum (GIBCO BRL).
Lymphoblastoid cell lines of patients and controls were maintained in RPMI-1640 (GIBCO BRL) supplemented with 15% fetal calf serum.
The mouse neuroblastoma cell line N1E-115 (34 ) was kindly provided by Dr P. van der Saag (Hubrecht laboratory, Utrecht, The Netherlands). This cell line was cultured in minimal essential medium (MEM; GIBCO BRL) supplemented with 2% fetal calf serum. Differentiation of the neuroblastoma cells was induced by adding 2% DMSO to the medium.
Polyclonal antibodies against huntingtin were raised by the injection of rabbits with synthetic peptides or a huntingtin-glutathione S-transferase (htt-GST) fusion protein. Peptide 1, corresponding to amino acid positions 3114-3141 (13 ) raised antibodies 1356 and 1358. Peptide 2 corresponds to aa 701-744 and yielded antibodies 1359 and 1495. Affinity purification of anti-peptide antibodies was performed, according to standard procedures (13 ). These four antibodies were kindly provided by Dr A.T. Hoogeveen (13 ). The huntingtin-GST fusion protein containing aa 1929-2421 resulted in antibody 93. GST antibodies were removed from serum 93 by two rounds of overnight incubation at 4oC with GST-beads. Subsequently, serum 93 was affinity purified on an immunoblot containing 50 [mu]g of the huntingtin-GST fusion protein and eluted with 0.1 M glycine pH 2.5 as described (35 ).
All incubations were performed at room temperature. Cells were grown on coverslips and were fixed in 2% paraformaldehyde in PBS (PBS: 150 mM NaCl, 9.9 mM Na2HPO4.2H2O, 1.6 mM KH2PO4) for 10 min and permeabilized in methanol during 20 min or in PBS/0.1% Triton X-100 twice 10 min. After blocking in PBS containing 0.5% bovine serum albumin (BSA) and 20 mM glycine (PBS/BSA/Gly) for 5 min, cells were incubated with polyclonal huntingtin antibodies in the appropriate dilution in PBS/BSA/Gly for 1 h (1356 and 93 1:100; 1358, 1359 and 1495 1:50; 93 affinity-purified 1:2.5). Cells were washed quickly with PBS/BSA/Gly and subsequently two times 5 min with the same buffer. The cells were incubated with swine anti-rabbit IgG-FITC (Dakopatts) in a 1:80 dilution in PBS/BSA/Gly for 45 min and washed 3 times with PBS/BSA/Gly for 5 min and once with PBS. After rinsing, the coverslips were mounted on glass slides in Vectashield (Vector) or mounting medium containing DABCO [1,4-di-azabicyclo-(2,2,2)-octane] and when appropriate propidium iodide. Immunofluorescence staining was observed using a Leitz Aristoplan microscope equipped with a *63/1.32 objective. Standard control experiments were performed (see also 35 ), including incubation with only the secondary antibody, with non-immune rabbit serum followed by incubation with the secondary antibody and with peptide competition experiments. For the latter experiment, antibody 1356 was titrated. Subsequently, antibody 1356 was diluted 1:1600 and incubated with 1 mg/ml specific or control peptides for 1 h prior to application to the cells. The cells were processed as described above.
Slides were prepared as described above and examined using a Bio-Rad MRC 600 confocal system mounted on a Nikon microscope with a *60/1.4 Planapo objective. Images were processed using the Comos software package (Bio-Rad).
Human skin fibroblasts were harvested in PBS, collected by centrifugation at 6000 r.p.m. for 2 min, and allowed to swell in hypotonic buffer (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA) containing freshly added protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride/PMSF, 0.5 mM trypsin inhibitor and 0.05% leupeptin) for 15 min on ice. Lysis is achieved by the addition of 0.63% Nonidet-P 40 and vortexing for 10 s. Nuclei are collected by centrifugation at 14 000 r.p.m. for 15 s and subsequently incubated in lysis buffer (20 mM Hepes, 0.4 M NaCl, 1 mM EDTA, 10% glycerol) supplemented with protease inhibitors for 15 min on ice. The supernatant was cleared by centrifugation at 13 000 r.p.m. for 15 min at 4oC and the resulting pellet was suspended in Laemmli sample buffer (35 ). Protein concentrations of the cytoplasmic and nuclear fractions were determined by the Bradford assay (BioRad).
For immunoblot analysis, 70 [mu]g of each subcellular fraction was loaded on 8% SDS-polyacrylamide gels and electrophoresed and transferred to nitrocellulose (Schleicher & Schuell) or Immobilon-P (Millipore) using the wet electrophoretic transfer procedure, as described (35 ). Prestained low and high molecular weight markers (BioRad) were run in lanes adjacent to the fractions. For the detection of the mutated protein in HD patients, cell extracts were electrophoresed on a 7.5% SDS-polyacrylamide gel containing a low concentration of bisacrylamide (ratio 30:0.135) until the 200 kDa marker reached the bottom of the gel of the BioRad minigel system (16 ). This gel was blotted as described above.
The membranes were blocked in TBST (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 2% Tween-20) containing 5% non-fat dry milk for 3 h at room temperature and incubated with the primary antibodies diluted in TBST for 1-3 h at room temperature or overnight at 4oC (affinity-purified polyclonal antibody 93 against huntingtin, 1:250; monoclonal antibody against [alpha]-tubulin (Sanbio, kindly provided by Dr B. van de Water), 1:400; polyclonal antibody A-22 against Creb Binding Protein/CBP (Santa Cruz), 1:1000; monoclonal antibody #122 against p53, 1:3; polyclonal antibody C12 against Raf-1 (Santa Cruz), 1:500). Subsequently, the membranes were washed four times with TBST for 5 min and incubated with goat-anti-rabbit or goat-anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz) at 1:2000 in 5% non-fat dry milk in TBST for 30-60 min at room temperature. After washing the membranes in TBST for four times 15 min, the antibodies were detected in an enhanced chemiluminescence procedure, essentially as described by the manufacturer (Amersham).
We thank Dr R. Willemsen and Mrs N. Meyer for technical assistance, Dr A.T. Hoogeveen and Dr B. van de Water for the gift of antibodies, Dr P. van der Saag for the neuroblastoma cell line N1E-115, and Dr R. Dirks, Dr E. de Heer and Dr W. de Priester for helpful discussions.
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*To whom correspondence should be addressed
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