Human Molecular Genetics, 2001, Vol. 10, No. 21 2425-2435
© 2001 Oxford University Press
Centrosome disorganization in fibroblast cultures derived from R6/2 Huntington’s disease (HD) transgenic mice and HD patients
Division of Medical and Molecular Genetics, GKT School of Medicine, King’s College London, UK, 1Max Planck Institute for Molecular Genetics, Dahlem, Berlin, Germany and 2Imperial Cancer Research Fund, London, UK
Received June 14, 2001; Revised and Accepted July 20, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntington’s disease (HD) is a progressive neurological disorder caused by a CAG/polyglutamine repeat expansion. We have previously generated the R6/2 mouse model that expresses exon 1 of the human HD gene containing CAG repeats in excess of 150. These mice develop a progressive neurological phenotype with a rapid onset and progression. We show here that it is impossible to establish fibroblast lines from these mice at 12 weeks of age, whilst this can be achieved without difficulty at 6 and 9 weeks. Cultures derived from mice at 12 weeks contained a high frequency of dysmorphic cells, including cells with an aberrant nuclear morphology and a high frequency of micronuclei and large vacuoles. All of these features were also present in a line derived from a juvenile HD patient. Fibroblast lines derived from R6/2 mice and from HD patients were found to have a high frequency of multiple centrosomes which could account for all of the observed phenotypes including a reduced mitotic index, high frequency of aneuploidy and persistence of the midbody. We were unable to detect large insoluble polyglutamine aggregates in either the mouse or human lines. We have identified a novel progressive HD pathology that occurs in cells of non-central nervous system origin. An investigation of the pathological consequences of the HD mutation in these cells will provide insight into cellular basis of the disease.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder which develops with a variety of symptoms including psychiatric disturbances, cognitive impairment and movement disorders (1). The causative mutation is a CAG repeat expansion which results in the increased length of a stretch of polyglutamine (polyQ) residues in the huntingtin protein (2). Unaffected individuals carry repeats ranging from 6 to 35 CAGs and the pathogenic range starts at 36 (3). The neuropathology comprises a selective neuronal cell loss that is most prominent in the striatum and cerebral cortex, a widespread brain atrophy and polyQ aggregate pathology (4). These structures most likely contain a truncated version of huntingtin and can appear in the form of neuronal intranuclear inclusions, dystrophic neurites and neuropil aggregates (5–7). The aggregate load is much higher in the cortex than in the striatum, and aggregates are present in the cortex of presymptomatic individuals (5,7).
The steps in the molecular pathway that cause HD have yet to be unravelled. Huntingtin can tolerate a wide variation in the length of the polyQ tract, which, at a sharp threshold becomes pathogenic. The initiating pathogenic event must correlate with this threshold, and one molecular property fulfilling this criteria is the dramatic increase in the rate at which exon 1 huntingtin can form amyloid fibrils in vitro (8,9). Experiments conducted in a primary neuronal cell culture model of HD (10) and a mouse model of spinocerebellar ataxia type 1 (SCA1) (11) have suggested that polyQ aggregation is an epiphenomenon. However, the recent use of heat shock proteins to alleviate a detrimental phenotype in a Drosophila model of SCA3 (12), and yeast models of HD and polyQ disease (13,14) has shown that rescue correlates with an increase in aggregate solubility and loss of fibril structure. PolyQ aggregation might cause cellular dysfunction by the sequestration of cellular proteins (15,16). In support of this, transcription factors (16–19), components of the proteasome (20) and stress response (21,22), caspases (23) and the full-length huntingtin protein (24) have been found in polyQ aggregates. This could be the direct cause of neuronal dysfunction including the early downregulation of selective genes that has been observed in the brains of a mouse model of HD (25,26).
A recent publication has shown that cultured lymphocytes from HD patients are more sensitive to the induction of apoptosis by staurosporine than control cells (27) indicative of a primary deficit outside the central nervous system (CNS). To investigate this further, we elected to study fibroblast cultures established from the R6/2 HD mouse model (28) and compare these with cultures from HD patients. To our surprise, it was not possible to establish fibroblast lines from R6/2 mice at 12 weeks of age. We identified a progressive age-dependent disorganization of the centrosome and a subsequent disruption of the cell cycle leading to aneuploidy, micronuclei and dysmorphic cells. Similar, although less pronounced, deficits were observed in cell lines from HD patients.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Transgenic mouse line R6/2 expresses exon 1 of the human HD gene under the control of its own promoter sequences (28) with repeat expansions in excess of (CAG)150 (29). These mice develop a progressive neurological disease with an early onset and rapid progression. PolyQ aggregates have formed in neuronal nuclei and the neuropil prior to 4 weeks of age in some brain regions (30,31). A cognitive and motor impairment can be detected between 3 and 6 weeks on application of appropriate tests (32–34), yet neuronal cell death is not evident until 14 weeks, and then, only in selective brain regions (35). PolyQ aggregates have also been described outside the CNS in a number of post-mitotic cell types from 6 weeks of age (36).
To investigate the possibility of using cell lines established from the R6 lines as a research tool, we set out to culture fibroblasts from R6/2 mice at 6, 9 and 12 weeks of age. Cell lines could be established from mice at 6 and 9 weeks without difficulty, but attempts to culture fibroblasts from 12-week-old R6/2 mice were continually unsuccessful. In contrast, we had no difficulty establishing cultures from wild-type mice. Migration of cells from the explant would begin as normal, but it was never possible to obtain sufficient cells to generate a confluent layer in a 25 cm3 tissue culture flask. Cells were treated with trypsin to facilitate dispersal and reseeding but would never survive beyond six to seven such treatments. This report describes the characterization of these cells using molecular and histochemical approaches. In parallel, a human fibroblast line that had been established from a juvenile HD patient (GM01917, NIGMS) with a CAG expansion in excess of 150 repeats (37) was included in the analysis. We had previously observed that this human line contained cells of unusual morphology.
The inability to establish primary fibroblast cultures from R6/2 mice at 12 weeks prompted us to conduct a histological examination of skin biopsies from R6/2 mice at the same age. Haematoxilin and eosin staining failed to uncover morphological changes and polyQ aggregates in the form of nuclear inclusions could not be detected by immunohistochemistry using either anti-huntingtin or anti-ubiquitin antibodies (data not shown).
Fibroblast lines derived from HD transgenic mice have a reduced mitotic index (MI)
The MI of fibroblast lines established from R6/2 mice at 6, 9 and 12 weeks, in addition to the human GM09197 line and controls, was determined from video footage of cells recorded over a period of 96 h by video microscopy (Table 1). Because of the length of time of each experiment, and the restricted availability of the equipment, it was not possible to monitor sufficient cell lines to conduct a detailed statistical analysis of the data. However, in all cases, irrespective of the age of the mouse from which the cultures were derived or the passage number at which the measurements were taken, fibroblasts from the non-transgenic mice had a higher MI than those derived from transgenic animals (P = 0.01). The MI of the cultures derived from the transgenic mice decreases as both the age of the mouse and the passage number of the culture increases. Therefore, the huntingtin transgene results in either a longer cell cycle or a decrease in the number of dividing cells and this effect becomes more pronounced with the age of the mouse at which the lines were established and the passage number of the cell culture. In keeping with the transgenic mouse analysis, the GM09197 fibroblast line had a lower MI than that of the human control lines.
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The reduction in MI is not associated with replicative senescence
Normal human fibroblast cultures enter a state of irreversibly arrested cell growth after a finite number of cell divisions, which correlates with a shortening of telomere repeats (38). Although mouse fibroblasts do not generally exhibit the same senescent characteristics, we set out to determine whether telomere shortening might be associated with the decrease in MI identified in both the R6/2 and GM09197 fibroblasts. Telomere repeat length was measured in DNA extracted from fibroblast cultures derived from 12-week-old transgenic (n = 5) as compared with that from non-transgenic mice (n = 2), and to DNA extracted from tail biopsy taken at 10 days of age (n = 3). Figure 1 shows a pulsed-field gel of genomic DNA extracted from the fibroblast cultures and tail biopsy that has been hybridized with an oligonucleotide, (TTAGGG)3, recognizing the telomere repeat sequence. There was no shortening of telomere length in the DNA from transgenic fibroblast cultures as compared with that from non-transgenic cells or DNA that had been extracted from tail biopsies. Similarly, there was no difference in telomere length between line GM09197 and the two human control sj and kj lines (data not shown). Human fibroblasts have been shown to express a ß-galactosidase (ß-gal) which is histochemically detectable at pH 6.0 upon senescence in culture (39). We were not able to detect any increased staining for this biomarker in either the R6/2 fibroblast derived from mice at 12 weeks of age or in the GM09197 cell line (data not shown). Therefore, the dramatic reduction in cell proliferation does not appear to be an acceleration of the senescence that naturally occurs in normal human fibroblasts after a finite number of divisions.
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DNA and HD fibroblasts exhibit an abnormal cell and nuclear morphology
In order to expose the nuclear and general cell morphology, cells were immunostained with the CAG53b anti-huntingtin antibody and nuclei were counterstained with Hoechst 33258. Fibroblast cultures established from R6/2 mice at 12 weeks of age contained a high frequency of dysmorphic cells. These often appeared as large cells and frequently displayed an aberrant nuclear morphology, which ranged from membrane invagination, to the formation of highly irregular nuclear shapes, to fragmented or multiple nuclei and in rare cases the complete absence of the nucleus. The cytoplasm often contained prominent, large vacuoles and there was an increase in the number of rounded cells lacking extended processes. All of these features were also present more frequently in the human GM09197 fibroblasts than in the human control lines. Figure 2 shows examples of dysmorphic fibroblasts from R6/2 (Fig. 2B–D) and GM09197 (Fig. 2F–H) cell cultures as compared with those from their respective control lines (Fig. 2A and E). The data are quantified in Table 2.
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The centrosome becomes highly disordered in HD fibroblasts
The centrosome is the primary microtubule organizing centre (MTOC) and functions to nucleate the assembly of cytosolic microtubules. It is composed of a pair of centrioles oriented at right angles to one another, surrounded by the pericentriolar matrix, which contains
-tubulin and pericentrin (40). We first used anti-pericentrin antibodies to investigate the structure and localization of the centrosome (Fig. 3). During interphase, the centrosome appears as a single pericentrin-stained spot close to the nucleus and in the centre of the cell, whereas in mitotic cells a pair of spots can be detected, one at each pole of the spindle. The number of cells containing three or more centrosomes was significantly increased (P < 0.01) in fibroblasts derived from 12-week-old R6/2 mice [37% ± 4.0 (SEM) (n = 6 fibroblast lines, passage nos = 3–6)] as compared with age matched littermate controls [12% ± 2.5 (SEM) (n = 5 fibroblast lines, passage nos = 3–7)]. An increase in the number of centrosomes was already apparent in cell lines derived from R6/2 mice at 9 weeks of age [24% ± 1.0 (SEM) (n = 2 fibroblast lines, passage no. = 5)] as compared with those from littermate controls [9.4% ± 3.0 (SEM) (n = 2 fibroblast lines, passage no. = 5)]. Analysis of the GM09197 line identified a similar pattern with 18% of cells containing three or more centrosomes as compared with only 2% in the control lines (sj and kj). Because the increase in the number of centrosomes was such a striking phenotype, we extended the analysis to include further HD human fibroblast lines in which the number of cells containing three or more centrosomes was 7.2% (HC7), 5.5% (HD9LO) and 12.9% (HDW). These results were replicated by immunostaining with antibodies to other well characterized centrosomal proteins, cdc2 (41) in the human cell lines and -tubulin in mouse cells (data not shown), indicating that a disorganization of the centrosome in HD fibroblasts has been identified as opposed to a specific relocalization of pericentrin.
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R6/2 fibroblast exhibit a high frequency of aneuploidy
A functional consequence of centrosome disorganization might be aberrant cell division resulting in aneuploidy. To investigate this possibility, we prepared metaphase spreads from fibroblast cultures that had been derived from R6/2 and control mice at 12 weeks of age. In all cases, metaphases from the control lines (n = 2) contained the expected number of 40 chromosomes. In contrast, most metaphase spreads prepared from R6/2 fibroblast lines (n = 3) had more (98%) as opposed to less (2%) than this number (Fig. 4).
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Morphology of the cytoskeleton in R6/2 fibroblasts
The integrity of the cytoskeleton was examined by staining cells with phalloidin to detect F-actin and immunocytochemistry was performed with antibodies raised against
-tubulin and vimentin to reveal the microtubules and intermediate filaments. There was no difference in the distribution of intermediate filaments in the fibroblasts derived from the R6/2 mice and controls or between GM09197 and controls (data not shown). Figure 5 shows the distribution of F-actin and microtubules in non-transgenic and R6/2 fibroblasts. The phalloidin staining revealed the characteristic lamellipodia of a motile cell as in Figure 5A, whereas dividing cells are rounded and devoid of processes (Fig. 5D). There was a higher frequency of rounded cells in the cultures from transgenic mice at 12 weeks of age, but otherwise the overall distribution of F-actin was not dissimilar. As microtubules are nucleated by the centrosome, and we have shown above that the centrosome is disrupted in 
40% of R6/2 fibroblasts, a disorganized microtubule network might be expected. However, in the vast majority of cells the overall microtubule distribution was not abnormal. The microtubule distribution in a small number of rounded R6/2 fibroblasts had a ‘cartwheel’ appearance (Fig. 5K), a pattern that was never observed in fibroblasts from non-transgenic mice. The appearance of two daughter cells that remain connected by the midbody was a consistent observation (Fig. 5M–O). The cytoskeleton appeared normal in the GM09197 cells (data not shown).
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Integrity of the secretory and endocytic pathways in R6/2 fibroblasts
It has been reported previously that the endosome/lysosome pathway is disrupted in primary neurons that have been transfected with huntingtin constructs (42) and that huntingtin accumulates in lysosome-like organelles in the HD brain (43). Therefore, we used immunocytochemistry to examine the integrity of the endosome/lysosomal pathway in the R6/2 fibroblast cell lines and in line GM09197. Cells were immunolabelled with antibodies raised against the Golgi network (GalT, mostly trans), endoplasmic reticulum [ER (calnexin)], early (Rab5) and late (Rab7) endosomes, lysosomes (LAMP1 and cathepsin D), and clathrin-coated pits and vesicles (clathrin and
-adaptin). Representative examples of the staining pattern detected with a subset of these markers are shown in Figure 6. Even in cells that are highly dysmorphic, the subcellular localization of the structures detected by these antibodies was not dramatically changed with respect to wild-type cells. In cells containing multiple nuclei, the Golgi was positioned between and shared by the nuclei. Occasionally, the Golgi in R6/2 fibroblasts was widely dispersed throughout the cell (Fig. 6F) but this could just reflect the stage of the cell cycle and in most cases, the placement of the Golgi appeared normal. The ‘cartwheel’ distribution of microtubules (Fig. 5K) is also seen for the ER and endosomes in a small number of cells (Fig. 6L). Cathepsin D staining indicated that a small number of cells (<1%) had an increased number of lysosomes that were also larger in size (Fig. 6S). The absence of staining with monodansylcadaverine (44) would suggest that these vacuoles are not autophagic (data not shown). The overall integrity of the secretory and endocytic pathway supports the conclusion that the cytoskeleton is largely intact and functional.
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Huntingtin distribution in the fibroblast cultures
It was not possible to identify the presence of large insoluble polyQ aggregates in fibroblast cells derived from 12-week-old R6/2 mice or in the GM09197 cell line by western blotting or the filter retardation assay (Fig. 7 and 8). To investigate huntingtin distribution in the R6/2 and HD fibroblasts, immunocytochemistry was performed with huntingtin antibodies S830, CAG53b, EM48, AG51 and 4C8. This panel of antibodies failed to detect polyQ aggregates in the form of nuclear or cytoplasmic inclusions in any of the fibroblast lines (Figs 2 and 7). Prior to the formation of large aggregations, a change in huntingtin distribution in the form of a diffuse staining occurring in the nucleus or in the processes, detected with antibody EM48 (7,31) or a perinuclear redistribution (43) has been noted. We were unable to detect either a consistent redistribution of huntingtin in the R6/2 fibroblasts or line GM09197 (Fig. 7) or an association of huntingtin with the centrosome (data not shown).
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To investigate the possibility that the centrosome abnormalities arise as a consequence of a disruption of huntingtin function, immunocytochemistry was performed with two antibodies to the huntingtin interacting protein HAP1 (huntingtin associated protein) (45,46). HAP1 in turn interacts with the p150glued component of the dynactin complex which is involved in the retrograde motility of vesicles (47,48). The dynein–dynactin interaction is important for the assembly of pericentrin and
-tubulin into centrosomes (49) and the organization of microtubules (50,51). Therefore, antibodies to dynactin (C-20) and the dynein heavy chain (R-325) and the intermediate chain (MAB1618) were used. The distribution of these proteins was not altered in either the fibroblast lines from R6/2 mice at 12 weeks of age or line GM09197 as compared with controls (data not shown). Similarly a redistribution of the huntingtin interacting protein (HIP1) (52,53) could not be detected. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have discovered that it is not possible to derive fibroblast cell lines from R6/2 mice at 12 weeks of age, whereas they can be established without difficulty from R6/2 mice aged 6 or 9 weeks. We have uncovered a severe progressive phenotype of a proliferating non-CNS tissue by growing cells in culture. Fibroblast cells derived from mice at 12 weeks showed pronounced morphological abnormalities including large cells, cells with large nuclei, multinucleate cells, rounded cells lacking processes and the persistence of the midbody between daughter cells. An extensive immunocytochemical analysis of cytoskeletal proteins and those comprising the secretory and endocytic pathways revealed that the most prominent and consistent abnormality was that many cells contained more than two centrosomes. This ranged from 35% cells (after three trypsin treatments) to 45% cells (after six trypsin treatments) in cells from 12-week-old mice and 24% of cells (passage no. 5) in cells from mice at 9 weeks of age. The number of cells containing supernumerary centrosomes was also found to be increased in fibroblast lines derived from HD patients.
All of the abnormal phenotypes occurring in the R6/2 fibroblasts could be a consequence of multiple centrosomes. The centrosome is the primary MTOC and is the site for the nucleation and organization of microtubules that form the interphase cytoskeleton and the mitotic spindle (54). In dividing cells the centrosome duplicates once to ensure the formation of a bipolar spindle and the inheritance of one centrosome to each of the daughter cells. Three types of microtubules emanate from the centrosome during mitosis. The kinetochore microtubules attach to and position the chromosomes on the metaphase plate, the aster microtubules radiate to the cell cortex where they help to position the mitotic apparatus and to determine the cleavage plane during cytokinesis and the polar microtubules interdigit with the polar microtubules from the opposite pole. The high frequency of tetraploid cells in the R6/2 fibroblast cultures indicates that chromosome replication has occurred and yet cell cleavage has not taken place, possibly because the cleavage plane cannot be positioned in cells containing multiple centrosomes. This is supported by: (i) an increase in the number of rounded cells containing two nuclei; (ii) cells containing micronuclei in which the nuclear envelop has reformed without cell cleavage; and (iii) cells containing large nuclei in which the reformation of the nuclear envelope may have failed. The consequential reduction in the number of cells successfully completing the cell cycle would result in a reduction in the MI as observed here.
Prior to the abnormalities arising as a result of multiple centrosomes, the ability of the centrosome to nucleate microtubules does not appear to be impaired as the cytoskeleton did not appear to be disorganized. The centrosome is the major organizing structure in the cell and helps determine the organization of microtubule-associated structures and organelles e.g. the Golgi complex and endoplasmic reticulum. In cultured fibroblasts the MTOC is strikingly at the centre of the cell and the Golgi complex is concentrated near the MTOC. During mitosis the Golgi complex breaks into small vesicles that are dispersed throughout the cytosol (55). When the cytosolic microtubules reform during interphase, the Golgi vesicles move along these microtubule tracks towards the MTOC where they reaggregate to form large membrane complexes. Therefore, the appearance of a dispersed Golgi in some cells probably reflected the cell cycle disturbances discussed above. In addition, the distribution of the constituents of the secretory and endocytic pathways is not abnormal in most cells, even those with a highly abnormal appearance which is also indicative of an intact and functional cytoskeleton.
The R6/2 fibroblasts express a exon 1 huntingtin protein with a long polyQ tract (the N-terminal 3% of human huntingtin) and two normal copies of mouse huntingtin, whereas the GM09197 cell line expresses human huntingtin with a normal and an expanded polyQ tract. It is possible that the abnormal phenotypes described in this paper occur as a result of a progressive disruption of normal huntingtin function. However, for this to occur in the R6/2 fibroblasts, exon 1 huntingtin would have to be capable of targeting to the correct subcellular compartments. Alternatively, it might disrupt huntingtin function from an ectopic location, e.g. by sequestration of normal huntingtin into polyglutamine aggregates. However, we did not find any evidence of aggregated huntingtin in these cultured cells. Data arising largely from protein interaction studies highlight a role for huntingtin in receptor-mediated endocytosis and vesicle trafficking. A function associated with receptor mediated endocytosis is indicated by localizations to or interactions with clathrin coated vesicles (56), -adaptin, (57), SH3GL3 (58), HIP1 (52,53,59) and Rab8 through interaction with FIP2 (60). Whilst dysmorphologies of the endosomal/lysosomal pathway have previously been observed in primary neuronal cultures transfected with huntingtin constructs (42), such abnormalities were not replicated in this study. Huntingtin is also involved in vesicle trafficking as revealed by its interaction with HAP1 (45) which in turn interacts with the p150glued component of the dynactin complex which is involved in the retrograde motility of vesicles (47,48). The dynein–dynactin interaction is important for the assembly of pericentrin and -tubulin into centrosomes (49) and the organization of microtubules (50,51). We were unable to find evidence of an abnormal distribution of HAP1 or components of the dynein–dynactin complex and so currently have no support for a centrosome disruption being mediated through these interactions.
The centrosome has an additional significance for diseases of protein misfolding or aggregation (61). Mammalian cells have a microtubule-based apparatus for the sequestration of protein aggregates within the cytoplasm. Aggregated proteins are specifically delivered to inclusion bodies by dynein-dependent reterograde transport on microtubules to a microtubule dependent inclusion body called an aggresome (62). For example, heterologous expression of CFTR leads to the formation of SDS-insoluble aggregates that accumulate in a single aggresome that co-localizes with centrosomal markers (62,63). The formation of such structures by aggregated forms of presenilin 1 (62), the peripheral myelin protein PMP22 (64) and mutant forms of the cytosolic protein super oxide dismutase (65) have also been described. In these aggresome containing cells, vimentin is redistributed to form a cage-like structure wrapped around the exterior of the inclusion (62). The formation of aggregates is a concentration dependent process that is probably influenced by expression levels, compartmentalization and dilution due to cell division. In keeping with this, the non-neuronal cells in the R6/2 mouse in which aggregates have been detected have been post-mitotic (36). However, the aggregation of polyQ containing proteins can occur in fibroblasts as nuclear aggregates have been identified in the cell nuclei of skin biopsies from patients with spinal and bulbar muscular atrophy (SBMA) (66). Therefore, it is tempting to speculate that the centrosome disruption seen in the R6/2 fibroblasts occurs through the directed accumulation of misfolded or aggregated forms of N-terminal huntingtin at the centrosome. However, we could find no evidence for the redistribution of huntingtin or vimentin or of aggregated forms of the N-terminal protein in these fibroblasts. Therefore, the mechanism underlying the centrosome disruption currently remains obscure.
We identified a progressive pathology in the skin fibroblasts from R6/2 transgenic mice that is exposed by growing the cells in culture. Expression of the HD mutation in this mouse model leads to the formation of multiple centrosomes with consequential abnormalities in cell division. These abnormalities are progressive and their detection depends on the stage in the disease at which the cultures are established. We show that the same phenotype is present in a fibroblast cell line that had been established from a juvenile HD patient with an extremely long CAG repeat expansion. A limited analysis indicated that multiple centrosomes are also more frequent in other fibroblast lines that had been established from HD individuals than in controls. Therefore, we have most likely identified an HD pathology occurring in a non-CNS tissue. A well-controlled study in fibroblast lines derived from HD patients at different stages of disease progression will be required to determine the extent of the HD fibroblast pathology. The identification of a non-neuronal system that exhibits pathological consequences of the HD mutation will be invaluable in unravelling the cellular basis of HD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Transgenic mice, cell lines and cell culture
Transgenic mouse line R6/2 (28) can be obtained from the Induced Mutant Resource, The Jackson Laboratory, Bar Harbor [Code: B6CBA-TgN(Hdexon1)62]. The colony was maintained by backcrossing to (C57BL/6 x CBA)F1 mice. Genotyping and CAG repeat sizing was as described previously (29). Fibroblast cell lines were established from the skin of R6/2 mouse ears and from age and sex matched littermate control mice at 6, 9 and 12 weeks of age. The HD fibroblast line (GM09197) was obtained from the NIGMS Human Genetic Mutant Cell Repository Camden, NJ and lines HC7, HDW and HD9LO were from Joy Delahanty (University College London, UK). The CAG repeat sizes were: GM09197, 151/21; HC7, 65/16; HDW, 44/20; HD9L0, 43/16. Normal fibroblasts control lines were provided by Tina Slade (King’s College, London, UK) and had CAG repeats: control-sj, 22/18; control-kj, 28 /13. Cells were grown in DMEM (Gibco BRL) supplemented with 1% L-glutamine, 1% penicillin-streptomycin and 20% fetal bovine serum (Gibco BRL) at 37°C with 5% CO2.
Genotyping, Southern blotting and hybridization
The isolation of genomic DNA and determination of CAG repeat size were as previously described (29,67). DNA was digested to completion with Sau3A (New England Biolab), fractionated on a 1.0% agarose gel by pulsed-field gel electrophoresis (PGFE) using a CHEF DR II (Biorad) at 5 V/cm with switch times increasing from 0.5 s to 10 s over 20 h. DNA was transferred to nylon membrane (Hybond-N+, Amersham) by Southern blotting and hybridization was performed with telomere repeat oligomer (TTAGGG)3 in SSSark, composed of 4 x SSC (1x SSC = 150 mM NaCl, 15 mM sodium citrate pH 7.0) and 7% N-lauroyl sarkosyl at 30°C for 16 h. Filters were rinsed in SSSark at room temperature for 10 min and then washed twice in SSSark at 65°C for 15 min.
Video microscopy
Video microscopy was performed using a CCD camera and time-lapse controller (Iris Ely electronics) fitted to an Olympus microscope. 2 x 104 fibroblast cells were placed in 35 mm culture dishes (Corning) and growth was monitored from 24 to 85 h after seeding under normal culture conditions. Microscope images were collected at a rate of 10 frames every 3 min on a Sony betacam video recorder. Individual frames were transferred from videotape to a Macintosh computer with a frame grabber, processed using the Radius Edit software package and analysed to determine the growth rate of the fibroblast cultures. The number of cell divisions in a given time span and thus the MI was calculated (cell division/h) (Table 1). Statistical analysis ANOVA (general linear model).
Antibodies
The source and dilution of antibodies that were used for immunocytochemistry was as follows. Clathrin (1:100), mAb (monoclonal antibody) (E.Wanker, Berlin); CAG53b (1:200), pAb (polyclonal antibody) (8); AG53 (1:150), pAB (9); 4C8 (1:100), mAb (68); GalT (Golgi network—mostly trans) (1:100), mAb, (DTS); -tubulin (1:200), mAb (Sigma); -tubulin (1:500), mAb (Sigma); pericentrin (1:150), pAB (BAbCO); Rab5B (1:100), pAB (Santa Cruz Biotechnology); Rab7 (1:40), pAB (Santa Cruz Biotechnology); LAMP1 (1:20), pAB (Santa Cruz Biotechnology); calnexin (1:300), pAB (StressGen Biotechnologies); Cathepsin D (1:300), pAB (Dako); phalloidin (1:40) (Texas red-conjugated) (Molecular Probes); HAP1 (1:150), pAB (EEW); HAP1 antisera (1:16), pAB, (46); -HIP1 (1:150), pAB, (53); dynactin (1:50), goat pAB (Santa Cruz Biotechnology); dynein heavy chain (1:75), pAB, (Santa Cruz Biotechnology); dynein intermediate chain, (1:50), mAB (Chemicon).
Immunocytochemistry
Fibroblast cells were plated onto coverslips and allowed to grow for 24 h. Cells were rinsed briefly with *PBS+, fixed for 15 min in ice-cold methanol at –20°C (for anti-pericentrin and calnexin antibodies) or fixed for 20 min in 4% paraformaldehyde in PBS+ at room temperature for other antibodies except phalloidin. For phalloidin, cells were rinsed in cytoskeleton buffer (pH 6.1) (10 mM MES, 150 mM NaCl, 5 mM EGTA, 5 mM MgCl2, 5 mM glucose and 100 mg/l streptomycin and with NaOH) briefly and fixed in 1.0% glutaraldehyde (Agar Scientific) for 1 min followed by 0.1% glutaraldehyde for 10 min (69). After fixation, cells were washed three times with ^PBS– and permeabilized with ice-cold 0.1% Triton X-100 in PBS– for 5 min. Cells were washed with PBS– and blocked with 0.2% gelatin in PBS– (fish skin gelatin, Sigma) by three rinses of 2 min prior to incubation of primary antibodies. All single and double primary antibodies were diluted in blocking solution, incubated for 1 h at room temperature and followed by three short rinses in 0.2% gelatin in PBS–. Secondary antibodies were used at 1:200 dilution for Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes Inc.) and 1:100 dilution for Texas red-conjugated goat anti-rabbit IgG (Molecular Probes). Either single or pooled antibodies were incubated for 30–45 min and followed by three rinses as described previously. Controls in which the primary antibody had been omitted were included. The nuclei of the cells were counterstained with Hoechst 33258 (Sigma) (1 µg/ml) in PBS– for 5 min and washed three times in PBS–. Coverslips were mounted with mowiol (Calbiochem). Cells were examined using conventional fluorescence microscopy (Zeiss AxioPlan II). Image processing was performed with Smart Capture VP and IP lab software packages (Zeiss).
*PBS+, contains 1 mM Ca2+ and 0.5 mM Mg2+. ^PBS–, free of Ca2+ and Mg2+.
Karyotyping
The fibroblast cells were plated in 25 cm2 culture flasks and allowed to grow until 70–80% confluence. Colcemid (Gibco BRL) was added to the medium to a final concentration of 75 ng/ml and incubated for 2 h in the same culture conditions. Cells were washed twice with PBS and trypsinized. Three millilitres of hypotonic solution [10% donor calf serum (Gibco BRL) in distilled deionized water] was added to the cells, which were transferred to a 15 ml tube and incubated at 37°C for 20 min. Ten drops of fresh fixative (3:1, methanol:glacial acetic acid) was added and cell were pelleted 1K (MSC bench top centrifuge) for 5 min. The supernatant was removed, and the cells fixed twice by resuspension in 3 ml fixative followed by centrifugation at 1K for 5 min. The cell pellet was resuspended in 0.5 ml fixative and incubated at –20°C for a minimum of 1 h. The cells were dropped on a methanol washed slide at 50°C and allowed to dry. The slides were baked at 50°C overnight, stained with Hoechst 33258 (Sigma) (1 µg/ml) in PBS– for 5 min. and washed in PBS– for 10 min. The slides were allowed to dry and mounted with mowiol.
ß-Galactosidase staining
Fibroblast cells were plated onto coverslips and allowed to grow for 24 h. ß-Gal staining was performed using a Senscence ß-gal staining kit (Cell Signaling Technology) and the procedure was performed according to the manufacturer’s recommendations.
Western blotting and filter retardation assay
Total protein was extracted from PBS-washed fibroblast cells in four different lysate buffers: RIPA buffer [150 mM NaCl, 1% IGEPAL (Sigma), 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris–HCL pH 8.0, 1 mM ß-mercaptoethanol, 1 mM PMSF, complete protease inhibitors (Boeringer Mannheim)]; SDS buffer 8 [50 mM Tris–HCl pH 8.0, 10% glycerol, 5 mM EDTA, 150 mM KCl, 1 mM PMSF, complete protease inhibitors (Boeringer Mannheim)]; SDS buffer 6.8 [50 mM Tris–HCl pH 6.8, 10% glycerol, 5 mM EDTA, 150 mM KCl, 1 mM PMSF, complete protease inhibitors (Boeringer Mannheim)]; Trottier’s buffer [100 mM Tris–HCl pH 9.0, 2% SDS, 5% ß-mercaptoethanol, 15% glycerol, 1 mM PMSF, complete protease inhibitors]. The cells were scraped into the buffer, incubated on ice for 30 min followed by sonication for 1 min. Lysates (30 µg unless otherwise stated) were boiled for 5 min in Laemmli loading buffer (50 mM Tris–HCl pH 6.8; 100 mM DTT; 2% SDS; 0.1% bromophenol blue; 10% glycerol), separated on a 6% SDS–polyacrylamide gel and transferred onto immobilon membranes (Amersham). Proteins were detected by chemiluminescence (ECL kit, Amersham) following incubation with primary antibodies and horseradish peroxidase conjugated secondary antibodies. Primary antibodies were S830 (1:1000), affinity-purified sheep pAB raised against exon1 huntingtin with 53Q, CAG53b (1: 3000) (8), AG53 (1: 1000) (9), EM48 (1: 1000) (7,70) and 1C2 (1: 5000) (68). The filter retardation assay was performed with the above protein lysates as described (71).
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ACKNOWLEDGEMENTS |
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We wish to thank Marian DiFiglia, Carl Hobbs and Roman Gonitel for helpful discussions, Emma Hockly for guidance with statistics, Ghazala Mirza, Kalyani Pal and Ioannis Ragoussis for help with karyotyping and Tina Slade, Tony Fensom and Joy Delahanty for fibroblast lines. The EM48 antibody was provided by Xiao-Jiang Li, antibodies to HAP1 were from Marian DiFiglia and Alan Sharp and dynein and dynactin antibodies were from Lizzy Fisher. This work was supported by grants from the Wellcome Trust (051897), Human Frontiers Science Programme (RG0132), Medical Research Council (G9800001), Hereditary Disease Foundation (in the form of an award from Harry Lieberman) and the Huntington’s Disease Society of America.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Division of Medical and Molecular Genetics, GKT School of Medicine, 8th Floor Guy’s Tower, Guy’s Hospital, London SE1 9RT, UK. Tel: +44 020 7955 4485; Fax: +44 020 7955 4444; Email: gillian.bates@kcl.ac.ukPresent address: Fabien Bertaux, Genoway, Lyon, France
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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