Brain, Vol. 123, No. 12, 2519-2530,
December 2000
© 2000 Oxford University Press
Distribution of ataxin-7 in normal human brain and retina
1 INSERM U289 and 2 Laboratoire de Neuropathologie Escourolle, Hôpital de la Salpêtrière, Paris, 3 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS, INSERM, Université Louis Pasteur, Illkirch, CU de Strasbourg, France and 4 Department of Microbiology, University of Umea, Umea, Sweden
Correspondence to:
A. Brice, INSERM U289, Hôpital de la Salpêtrière, 47 boulevard de l'Hôpital, 75651 Paris, Cedex 13, France E-mail: brice{at}ccr.jussieu.fr
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Spinocerebellar ataxia 7 (SCA7) is a neurodegenerative disease caused by the expansion of a CAG repeat encoding a polyglutamine tract in the protein ataxin-7. We developed antibodies directed against two different parts of the ataxin-7 protein and studied its distribution in brain and peripheral tissue from healthy subjects. Normal ataxin-7 was widely expressed in brain, retina and peripheral tissues, including striated muscle, testis and thyroid gland. In the brain, expression of ataxin-7 was not limited to areas in which neurones degenerate, and the level of expression was not related to the severity of neuronal loss. Immunoreactivity was low in some vulnerable populations of neurones, such as Purkinje cells. In neurones, ataxin-7 was found in the cell bodies and in processes. Nuclear labelling was also observed in some neurones, but was not related to the distribution of intranuclear inclusions observed in an SCA7 patient. In this patient, the proportion of neurones with nuclear labelling was higher, on average, in regions with neuronal loss. Double immunolabelling coupled with confocal microscopy showed that ataxin-7 colocalized with BiP, a marker of the endoplasmic reticulum, but not with markers of mitochondria or the trans-Golgi network.
ataxin-7; spinocerebellar ataxia 7; polyglutamine expansion; neuronal intranuclear inclusions; immunocytochemistry
GST = glutathione S-transferase; NLS = nuclear localization signal; SCA7 = spinocerebellar ataxia 7
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Introduction |
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Spinocerebellar ataxia 7 (SCA7) is one of the autosomal dominant cerebellar ataxias (Stevanin et al., 2000b
), a group of neurodegenerative disorders characterized clinically by cerebellar ataxia associated with variable neurological signs (dysarthria, ophthalmoplegia, decreased visual acuity, extrapyramidal and pyramidal signs, deep sensory loss or dementia). Most SCA7 patients also suffer from progressive macular dystrophy and ultimately become blind. Neuropathological examination reveals moderate to severe neuronal loss and gliosis in the cerebellum and associated structures (inferior olive, cerebellar cortex, dentate nucleus, pontine nuclei) and in the motor system (globus pallidus, substantia nigra, subthalamic nucleus, red nucleus and spinal cord) (Carpenter and Schumacher, 1966; Weiner et al., 1967; Enevoldson et al., 1994; Gouw et al., 1994; Martin et al., 1994; van Schaik et al., 1997; Holmberg et al., 1998). In the retina, there is variable loss of photoreceptors, ganglion and bipolar cells (Martin et al., 1994). Neuronal loss and gliosis are also detectable in the lateral geniculate body and in the occipital cortex.
The SCA7 gene, localized on chromosome 3p (Benomar et al., 1995; Gouw et al., 1995; Holmberg et al., 1995), was identified by positional cloning and optimized two-dimensional RED (rapid expansion detection) (David et al., 1997; Del-Favero et al., 1998). The mutation responsible for the disease is the expansion of a trinucleotide CAG repeat in the coding region of the gene, a type of mutation previously found in four other spinocerebellar ataxias—SCA1 (Orr et al., 1993), SCA2 (Pulst et al., 1996; Imbert et al., 1996; Sanpei et al., 1996), SCA3 or MJD (Machado–Joseph disease) (Kawaguchi et al., 1994) and SCA6 (Zhuchenko et al., 1997)—and in Huntington's disease (HD) (The Huntington's Disease Collaborative Research Group, 1993), SBMA (spinal and bulbar muscular atrophy) (La Spada et al., 1991) and DRPLA (dentatorubral–pallidoluysian atrophy) (Koide et al., 1994; Nagafuchi et al., 1994). Normal alleles of the SCA7 gene contain 4–35 CAG repeats, whereas pathological alleles contain from 37 to more than 300 (Benton et al., 1998; David et al., 1998; Del-Favero et al., 1998; Gouw et al., 1998; Johansson et al., 1998; Stevanin et al., 1998). In all the SCA7 families that have been analysed, age of onset was negatively correlated with the number of CAG repeats on the pathological allele (Benton et al., 1998; David et al., 1998; Del-Favero et al., 1998; Gouw et al., 1998; Johansson et al., 1998; Stevanin et al., 2000b).
The SCA7 gene encodes an 892 amino acid protein of unknown function, ataxin-7. The transcript is widely expressed in the CNS and other tissues (David et al., 1997). Ataxin-7 contains a consensus nuclear localization signal (NLS), suggesting that the protein should normally be targeted to the nucleus. The pathological ataxin-7 protein was, indeed, detected in the nuclear fraction of lymphoblast cells from a SCA7 patient with the 1C2 antibody, which recognizes pathologically long polyglutamine tracts (Trottier et al., 1995). Intranuclear inclusions in neurones in several brain regions were labelled by this antibody as well as by a polyclonal antibody directed against ataxin-7 (Holmberg et al., 1998; Mauger et al., 1999). Expression studies in COS1 cells recently confirmed that the putative NLS directs ataxin-7 to the nucleus (Kaytor et al., 1999).
Neuronal intranuclear inclusions appear to be characteristic of polyglutamine diseases (Davies et al., 1998). They have been observed in neurones in patients with Huntington's disease (DiFiglia et al., 1997; Becher et al., 1998; Gourfinkel-An et al., 1998), SBMA (Li et al., 1998), DRPLA (Becher et al., 1998; Igarashi et al., 1998), SCA1 (Skinner et al., 1997; Duyckaerts et al., 1999), SCA2 (Koyano et al., 1999), SCA3 (Paulson et al., 1997a) and SCA6 (perinuclear inclusions) (Ishikawa et al., 1999). Intranuclear inclusions were also found in transgenic mice expressing mutated huntingtin (Davies et al., 1997), atrophin (Schilling et al., 1999) and ataxin-1 (Skinner et al., 1997), and perinuclear and nuclear aggregates were observed in cell cultures expressing the genes mutated in SCA1 (Skinner et al., 1997; Cummings et al., 1998), SCA3 (Paulson et al., 1997a), HD (Cooper et al., 1998; Hackam et al., 1998; Li and Li, 1998; Lunkes and Mandel, 1998; Martindale et al., 1998; Saudou et al., 1998; Moulder et al., 1999), DRPLA (Igarashi et al., 1998) and SBMA (Merry et al., 1998).
In order to determine the normal cellular and subcellular distribution of ataxin-7, we developed polyclonal and monoclonal antibodies directed against two different domains of the protein and analysed the distribution of ataxin-7 immunoreactivity in brain and peripheral tissues from healthy subjects.
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Material and methods |
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Antibody production
Two polyclonal antisera directed against amino acids 1–135 (SCA7-5') and 173–371 (SCA7-A) of ataxin-7 (Fig. 1
) were raised against glutathione S-transferase (GST) fusion proteins, prepared as follows. The ataxin-7-specific sequences were amplified by PCR (polymerase chain reaction) from the ataxin-7 cDNA clone A10 (David et al., 1997), containing 10 CAG repeats, using the following primers. For SCA7-A: SCA7A-5' (5'-GCG GAT CCA GCA AGC CGC CTT T-3') and SCA7A-3' (5'-GCG AAT TCT GGG TTA AGG AAT GTG T-3'). For SCA-7–5': SCA7F1 (5'-GCG AAT TCA GAT CTA TGT CGG AGC GGG CCG C-3') and SCA7R1 (5'-GCG AAT TCT TAC CCT TGA GGC CCA CAG-3'). The PCR fragments were digested with the appropriate restriction enzymes and cloned into the pGEX-2T vector (Amersham Pharmacia Biotech, Uppsala, Sweden) in-frame with the GST tag, and expressed in Escherichia coli BL21. Expression was induced with 0.125 mM isopropyl-D-thiogalactopyranoside (IPTG) for 2.5 h at 37°C. The proteins were then purified on glutathione–Sepharose 4B affinity columns according to the manufacturer's instructions (Amersham Pharmacia), and injected into rabbits to generate antisera by standard procedures.
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The specificity of each antiserum was tested by ELISA (enzyme-linked immunosorbent assay) using the fusion proteins as antigens. The antibodies were then purified with their respective antigens linked to affi-Gel 10 columns, according to the manufacturer's protocol (Bio-Rad, Hercules, Calif., USA). To eliminate possible labelling by anti-GST antibodies, part of the purified SCA7-A serum was incubated with GST-coated beads that were eliminated by centrifugation. Immunohistochemical labelling was identical to that obtained without this treatment. The polyclonal antibodies were therefore used without further purification.
The 1C1 monoclonal antibody was raised against a fusion protein consisting of residues 1–229 of ataxin-7 with 10 CAG repeats corresponding to the D1 clone (David et al., 1997) in pScreen vector (Novagen, Madison, Wis., USA). After expression in Escherichia coli BL21(DE3)LysS, the protein was purified and injected into mice, as described (Devys et al., 1993). Hybridomas were prepared and isolated by serial dilution, and the supernatants were screened first by immunofluorescence then by Western blotting on COS cells expressing ataxin-7. The 1C1 clone gave the strongest signal. The epitope recognized by the antibody was mapped between residues 66 and 119 of ataxin-7, by Western blot analysis of truncated ataxin-7 expressed by COS cells.
Verification of antibody specificity
Western blot analysis
The ability of the polyclonal antibodies to bind to their respective antigens was confirmed on Western blots of the fusion proteins, with and without the GST tags. The GST tags were cleaved from the fusion proteins (50 µg) by digestion with 1 U of thrombin protease at room temperature for 16 h. The fusion proteins (2–4 µg) and cleaved proteins (10–20 µg) were electrophoresed on a 15% sodium dodecyl sulphate (SDS)–polyacrylamide gel, then blotted onto nitrocellulose membranes (Hybond C; Amersham Pharmacia Biotech, Uppsala, Sweden). Non-specific binding sites on the membranes were blocked by incubation overnight at 4°C in 5% non-fat dry milk, 0.2% Tween in PBS (phosphate-buffered saline). The membrane was then incubated for 1 h at room temperature with the SCA7-A or SCA7-5' antibodies diluted 1 : 1000 in 5% non-fat dry milk. After three 10-min washes in PBS, the membranes were incubated for 1 h at room temperature with an anti-rabbit antiserum conjugated to horseradish peroxidase (Amersham Pharmacia Biotech) at dilution of 1 : 10 000 in 5% non-fat dry milk. After three 10-min washes in PBS, antibody binding was revealed by chemical luminescence (Super Signal Kit; Pierce Rockford, Ill., USA). The specificity of 1C1 was also tested by Western blotting using protein extracts from PC12 cells transfected with expression vectors encoding full-length normal (10 CAG repeats) and mutant (95 CAG repeats) ataxin-7.
In vitro expression and immunoprecipitation of mutant and wild-type ataxin-7
Full-length wild-type (10 CAG repeats) and mutant (95 CAG repeats) SCA7 cDNAs were tagged by PCR at the 5' end with a haemagglutinin epitope and subcloned into an appropriate vector including a T7 promoter. The corresponding 35S-labelled wild-type and mutant ataxin-7 proteins were generated using a transcription/translation kit (Promega, Madison, Wis., USA), according to the supplier's recommendations. The labelled proteins were immunoprecipitated with a monoclonal antibody against the haemagglutinin epitope (Roche, Basel, Switzerland) as described previously (Ory et al., 1994), washed in RIPA buffer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 1% sodium deoxycholate), and separated on 7.5% SDS–polyacrylamide gels.
Neuropathological examination
Tissue samples from the brain and other organs were obtained post-mortem from six control patients (Cases 1–6). Samples from an SCA1 and an SCA7 patient were also examined (Table 1). The tissue was fixed with formalin. Representative blocks were embedded in paraffin and cut into 7-µm sections. The absence of lesions was verified in each sample by neuropathological examination on contiguous slides stained with haematoxylin–eosin and Bodian silver/luxol fast blue.
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Immunohistochemistry
Primary antibodies were diluted 1 : 400 for SCA7-A, 1 : 200 for SCA7-5' (1 : 800 for detection of inclusions) and 1 : 400 for 1C1. Antibody binding was revealed with appropriate biotinylated secondary antibodies and avidin-conjugated horseradish peroxidase (Amersham Pharmacia Biotech), using diaminobenzidine as the chromogen. Sections were counterstained with Harris' haematoxylin.
The studies were performed on the following brain regions from Case 2: medulla oblongata, pons, midbrain including substantia nigra, cerebellar peduncles, cerebellum including dentate nucleus, thalamus including lateral geniculate body, subthalamic nucleus, caudate nucleus, putamen, globus pallidus, hippocampus, and frontal, insular and occipital cortices. The results were confirmed on samples from Case 6, including the medulla oblongata, cerebellum, thalamus, midbrain with substantia nigra, and several samples of the isocortex including area 4, as well as the spinal cord. Immunohistochemistry was also performed on the retina of Case 4, and on samples of heart and skeletal muscle, thyroid gland, spleen, kidney, lymph nodes, liver, intestine and testis from Cases 1, 3, 5 and 6. In addition, the three antibodies were also tested on samples of the medulla and pons from an SCA1 and an SCA7 patient. The inferior olive and the pontine nuclei have been shown to contain nuclear inclusions at high density (Holmberg et al., 1998; Duyckaerts et al., 1999).
Subcellular distribution of ataxin-7
Double immunofluorescence with antibodies against ataxin-7 and against markers of subcellular organelles was performed to determine the subcellular localization of ataxin-7. The following antibodies were used: monoclonal antibody 1C1 or polyclonal antiserum SCA7-A, as appropriate; GRP78, a monoclonal antibody (StressGen, Victoria, Canada) that labels BiP in the endoplasmic reticulum; anti-COX2 (Lombes et al., 1996), a polyclonal antibody against subunit 2 of cytochrome c oxidase to label mitochondria; a polyclonal antibody directed against the trans-Golgi network (a gift from N. Gonatas); and monoclonal antibody SMI181 against SNAP-25, a presynaptic synaptosome-associated protein of 25 kDa (Sternberger Monoclonals, Lutherville, Mass, USA).
For all double immunofluorescence labelling, the following protocol was used. Tissue sections were incubated overnight at 4°C with both antibodies. Immunolabelling with the polyclonal antibodies was revealed by incubation with a biotinylated anti-rabbit immunoglobulin antibody (diluted 1 : 400) for 2 h, followed by streptavidin-coupled CY2 (Jackson Immunoresearch, West Grove, Pa., USA), which emits green fluorescence (absorption peak 492 nm; emission peak 510 nm). The monoclonal antibody was revealed by an anti-mouse antibody coupled with CY3 (Jackson Immunoresearch), which emits red fluorescence (absorption peak 492 nm; emission peak 510 nm). The slides were examined with a Leica TCS 4D confocal microscope. Pearson correlation coefficients were calculated using Statview® software.
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Antibody characterization
On Western blots (Fig. 2A
), affinity-purified SCA7-5' and SCA7-A recognized the fusion proteins used to generate the polyclonal antibodies, as well as the corresponding SCA7-specific fragments generated by thrombin digestion of the fusion proteins (Fig. 2A). Both SCA7-5' and SCA7-A were able to immunoprecipitate in vitro synthesized wild-type (10 glutamine residues) and mutant (95 glutamine residues) ataxin-7 (Fig. 2C). In addition, the 1C1 monoclonal antibody recognized a protein with an apparent molecular weight of ~100–110 and ~140–150 kDa in extracts from PC12 cells transfected with constructs expressing the normal and expanded ataxin-7 proteins, respectively (Fig. 2B). No bands were detected in non-transfected cells. On immunohistochemical preparations, no staining was observed if the primary antibodies were omitted (not shown) or if the antibodies were preadsorbed with increasing concentrations of the GST fusion proteins (Fig. 3A and B).
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Distribution of ataxin-7
Control brains
All the brain structures studied (see Material and methods) contained neurones with different levels of ataxin-like immunoreactivity. Immunolabelling was generally punctate in appearance. Similar patterns of staining were observed with all three antibodies, but their sensitivity differed: SCA7-A was the most and 1C1 the least sensitive. When immunostaining was strong enough, the cell bodies of all neurones and some neurites were labelled, suggesting that ataxin-7 is expressed ubiquitously, although at different levels.
Neurones in the inferior olive, the subiculum and the dentate gyrus were labelled intensely, as were those of the cerebral isocortex and hippocampus, the striatum and pallidum (external and internal), the substantia nigra and the reticular formation, the nuclei pontis. Labelling was weaker in the thalamus and in the lateral geniculate body. Purkinje cells were labelled more faintly, but immunoreactivity was clearly detected both in the cell bodies and in the processes. Examples of neurones with different levels of ataxin-7-like immunoreactivity, stained with the anti-SCA7-A antibody, are shown in Fig. 3
. Figure 3C shows a low-power micrograph of the inferior olive that contains strongly labelled neurones. The neuropil was also immunolabelled. A higher magnification of the region (Fig. 3D) shows some of these neurones with labelling in the cell body and in the neuropil. Figure 3E shows a neurone with some of its dendritic processes in the nucleus gigantocellularis, which contained high levels of ataxin-7-like immunoreactivity, in contrast to the lightly stained Purkinje cell in Fig. 3F. In the granular layer of the cerebellum, the centres of the glomeruli were immunoreactive (Figs 3F and 4F). Double immunofluorescence labelling of the glomeruli in the cerebellar granule layer with SCA7-A and an antibody against the synaptic vesicle protein SNAP-25 showed that the antibodies labelled different elements within the same structure, indicating that ataxin-7 was present in the presynaptic axonal part of the glomerulus but outside the synaptic vesicles (Fig. 4F).
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In the retina, ataxin-7 immunoreactivity was detected in the optic nerve fibres, the cytoplasm and processes (inner plexiform layer) of the ganglion cells and in the cell bodies of the inner nuclear layer. Staining was also found in the inner part of the outer plexiform layer. Immunolabelling was confined to the inner segment of the rods and cones (Fig. 4A and B
). Ataxin-7 was also expressed in some non-neuronal cells. Astrocytes were stained occasionally, particularly in the transverse pontine fascicles and in the white matter of the frontal cortex (not shown), where labelling was found in the nucleus, cytoplasm and processes. This staining was not related to reactive astrogliosis, as it was not found in the inferior olive from an SCA1 patient with marked astrogliosis (data not shown). Ependymocytes were also labelled, with immunoreactivity located principally at the apex of the cell (not shown).
Peripheral tissues
Ataxin-7 was detected in almost all peripheral tissues of the controls that were studied except the liver and in the kidney, where no significant immunoreactivity could be seen. Immunostaining was particularly intense in the following structures: the Lieberkühn glands of the intestine, the follicular cells of the thyroid gland (Fig. 3O), the spermatogonia in the testis (Fig. 3N), the smooth and cardiac muscle fibres, and some lymphocytes in the lymph nodes. In the striated fibres of the skeletal muscle, staining was observed in the I band (Fig. 3M).
Subcellular localization of ataxin-7 in neurones
Cytoplasmic labelling
Double immunofluorescence labelling in control Case 6, examined with confocal microscopy, showed that ataxin-7, labelled by SCA7-A or 1C1, colocalized with BiP (Fig. 4C), a protein in the lumen of the endoplasmic reticulum, but not with markers of the mitochondria (Fig. 4D) or trans-Golgi network (Fig. 4E). The localization of the protein in the endoplasmic reticulum is consistent with the punctate appearance of immunoreactivity.
Nuclear labelling
Normal ataxin-7 in control brain.
Neurones were labelled consistently in the cytoplasm, but nuclear labelling was also observed in some cells. Both the frequency and the intensity of nuclear labelling varied among brain regions (Table 2). The frequency and the intensity were not correlated. The frequency of ataxin-7-positive neurones with stained nuclei reached 95% in the red nucleus but only 5% in the inferior olive and pontine nuclei (Table 2). Nuclear labelling was faint in the substantia nigra, hippocampus, caudate nucleus and superior colliculus but strong in the red nucleus and internal pallidum. Examples of nuclear staining are shown in Fig. 3G–I. Figure 3G shows neurones in the external pallidum with a very high level of nuclear immunoreactivity that appears to be uniformly distributed. In Fig. 3H, a neurone in the red nucleus is shown in which nuclear labelling was less intense, revealing the inhomogeneous distribution of ataxin-7; in addition to the nuclear envelope, small spots were labelled that were particularly dense around the nucleolus, which remained unstained. This can also be seen in Fig. 3I, where the nucleus of one pontine neurone is moderately labelled. Labelling was absent or very light in the neighbouring cells. This emphasizes the variability of the nuclear labelling even in the same structure.
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Ataxin-7 in an SCA7 patient.
The frequency of nuclear labelling in several brain regions differed from that in controls (Table 2
). The nuclei of Purkinje cells that were not labelled in the control brain were labelled in 32% of the cells in the SCA7 patient. In contrast, the proportion of labelled nuclei in the red nucleus decreased from 95% in the control case (Case 2) to 2% in the SCA7 patient (Case 7). The mean proportion of immunolabelled nuclei was significantly higher in regions with neuronal loss (21%) than in regions that were spared (4%) (t = 2.14; P < 0.05).
Ataxin-7 in intranuclear inclusions.
In the samples from the patient with SCA7, where numerous intranuclear inclusions had previously been detected with an antibody against the expanded polyglutamine tract (Table 2), the inclusions were labelled only by the SCA7-5' (Fig. 3K) and 1C1 (Fig. 3L) antibodies directed against the extreme N-terminal of ataxin-7, but not by the SCA7-A antibody (Fig. 3J) directed against an epitope further downstream. The immunoreactivity in nuclear inclusions in the SCA7 patient was specific to the ataxin-7 protein, since the anti-ataxin-7 antibodies did not label nuclear inclusions in a patient with SCA1 (not shown). The distribution of nuclear inclusions was not correlated with the diffuse nuclear labelling observed both in the control (r = –0.10; P = 0.69) and in the SCA7 case (r = 0.35; P = 0.15) (Table 2). There was no correlation between the proportion of cells with labelled nuclei in the SCA7 case and in the control (r = 0.30; P = 0.24). There were no correlations between the extent of neuronal loss in the SCA7 case and the proportion of neurones with inclusions.
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Discussion |
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Spinocerebellar ataxia 7 is one of the nine known neurodegenerative disorders caused by a polyglutamine expansion in the disease-causing protein (Stevanin et al., 2000a
). As in other such disorders (Onodera et al., 1995; Servadio et al., 1995; Trottier et al., 1995; Paulson et al., 1997b), ataxin-7 expression was ubiquitous in neurones of the nervous system, although only specific subsets of neurones are known to be lost in patients. Ataxin-7 protein was widely expressed in normal brain and retina, and also in a number of peripheral tissues, such as striated muscle, heart, testis, Lieberkühn glands, lymphocytes and thyroid gland, although, to the best of our knowledge, except for congestive heart failure in a patient with very early onset and an extremely large expansion (Benton et al., 1989), extraneurological deficits have not been reported in SCA7 patients. Ataxin-7 immunoreactivity was found mostly in neurones, but was also present in some astrocytes. Labelling varied in intensity from region to region, but also from cell to cell. In all neurones, labelling was found in the cytoplasm, particularly in the endoplasmic reticulum, including the nuclear envelope, according to double-labelling experiments with subcellular markers, but not in mitochondria or the trans-Golgi network. In some neurones, diffuse intranuclear labelling was also observed, which differed from the labelling of intranuclear inclusions in brain samples from patients.
The specificity of the immunohistochemical labelling of ataxin-7 was confirmed by the observations that (i) the same pattern of expression was obtained with three different antibodies, in terms of both the brain structures labelled and the relative intensities of labelling in the different structures, and (ii) preabsorption of the antibodies with their corresponding antigen abolished the staining.
Ubiquitous expression in the brain and in peripheral tissues has also been observed for ataxin-1 (Servadio et al., 1995), ataxin-2 (Huynh et al., 1999) and ataxin-3 (Paulson et al., 1997b; Trottier et al., 1998). Like ataxin-7, these three proteins were expressed in all the brain areas studied, although the cellular localization and the levels of expression of each protein differed. Ataxins 2, 3 and 7 were found predominantly in the cytoplasm of neurones, whereas ataxin-1 was labelled only in the nuclei of neurones, except for Purkinje cells, where both the nucleus and the cytoplasm were immunostained. Within the same structure, the intensity of the labelling also varied according to the type of ataxin. For example, strong staining was observed in Purkinje cells with antibodies against ataxin-1 and -2, whereas labelling with anti-ataxin-7 antibodies was faint. Although the intensity of staining may depend on the antibody used, the relative intensities in different brain structures suggest that, despite the wide distribution of the ataxin proteins in the brain, each appears to have a specific pattern of expression.
The relationship between the expression of ataxins, normal or mutant, and neuronal loss is still unclear. In the present study, as in studies on other spinocerebellar ataxias, there were no relationships between either the number of stained neurones or the intensity of cytoplasmic staining in a given structure in normal brain and neuronal loss in patients (Carpenter and Schumacher, 1966; Weiner et al., 1967; Enevoldson et al., 1994; Gouw et al., 1994; Martin et al., 1994; van Schaik et al., 1997; Holmberg et al., 1998). Cytoplasmic labelling was strong in the external pallidum but faint in the Purkinje cells, two areas that are usually severely affected in patients. Intense labelling was also observed in numerous neurones in the putamen, which is usually spared in the disease (Enevoldson et al., 1994; Holmberg et al., 1998). In the retina, however, strong staining of ataxin-7 in bipolar and ganglion cells as well as in the mitochondrion-rich inner segment of photoreceptors correlates with the severe retinal lesions seen in the patients (Weiner et al., 1967).
One explanation for the lack of correlation between protein expression and neuronal death in patients may be that the immunoreactivity seen post-mortem is the net result of synthesis and catabolism, and only partially reflects the function of the protein in the cell, where rapid turnover could prevent its accumulation. The distribution of ataxin-7 in the nuclei differed between the control case and the patient, suggesting that the polyglutamine stretch not only induces the formation of inclusions but also alters the translocation of the protein to the nucleus.
There was also no relationship between the distribution of neurones with diffuse nuclear ataxin-7 immunoreactivity in the normal brain, and neuronal loss or even with the presence of intranuclear inclusions in patients. Nuclear labelling ranged from <1% in neurones in the frontal cortex and in the Purkinje and granular cells in the cerebellum to over 95% in the red nucleus and pallidum. No nuclear labelling was detected in the Purkinje and granular cells in the control subject, although these structures were known to be affected in the SCA7 patient. Furthermore, in the red nucleus, which was severely affected, and in the pallidum, which was only slightly affected, almost all neuronal nuclei were positive for ataxin-7 in the control. However, the proportion of nuclear labelling in a given structure was different in the control and in the patient, possibly because of a change in the nuclear targeting in ataxin-7 in SCA7. In the SCA7 case, the proportion of neurones with nuclear labelling was significantly higher in regions with neuronal loss. This suggests a link between nuclear labelling and neuronal loss. However, neuronal loss also occurred without significant nuclear labelling, as seen in the lateral geniculate body and in the granule cells of the cerebellum. In both instances of these cases, cell loss might be explained by another mechanism, such as trans-synaptic degeneration.
Finally, as we showed in a previous study in which the inclusions were detected with antibodies directed against ubiquitin and the 1C2 antibody, which recognizes long stretches of glutamine (Holmberg et al., 1998), the presence of nuclear inclusions did not coincide with the pathology of the disease (Table 2). Numerous nuclear inclusions in the patient were always found in areas where cytoplasmic ataxin-7 immunoreactivity was intense. In the control, however, strong cytoplasmic ataxin-7 immunoreactivity was not always correlated with a high density of inclusions in the same region in the patient; ataxin-7 immunoreactivity, for example, was strong in the putamen, where inclusions were rare. Furthermore, the frequency of ataxin-7-positive nuclei in normal brain tissue was not correlated with the presence of nuclear inclusions in the patient. In the pallidum, for example, where 96% of the nuclei were labelled by the SCA7-A antibody in the control subject, few inclusions were found in the patient (Table 2).
Intranuclear inclusions, such as those seen in SCA7, are characteristic of diseases caused by polyglutamine expansions, but the mechanisms underlying their formation and their role in the pathogenic process are not understood (Kim and Tanzi, 1998). The first questions that must be answered are (i) how, and under what conditions, normal ataxin-7 is addressed to the nucleus, since this occurs in only a subset of neurones, and (ii) what determines its distribution, which is not homogeneous. The observation in the present study that nuclear inclusions in brain tissue from a patient with SCA-7 were labelled only with the SCA7-5' and 1C1 antibodies raised against the N-terminal domain, including the pathological polyglutamine tract, but not SCA7-A antibody, which was raised against a domain that is downstream of the polyglutamine repeats, suggests that the ataxin-7 that is present in the inclusions has been processed and no longer contains the epitope recognized by the SCA7-A antibody, or that a change in conformation masks the epitope. The latter is more probable, as the putative nuclear localization signal is located even further downstream than the peptide sequence used to generate SCA7-A. However, we cannot exclude the possibility that the mutant ataxin-7 enters the nucleus by a mechanism different from that used by the normal protein.
In the future, it will be of major importance to elucidate the mechanism underlying the formation of nuclear inclusions and to determine their role in the pathogenesis of SCA7. This may also shed light on the pathogenesis of other polyglutamine diseases. In addition, since the normal cellular and intracellular distribution and the level of expression of ataxin-7 are not sufficient to explain the pattern of neurodegeneration observed in SCA7 patients, it is possible that the normal and mutated proteins have different topographies, both regional and cellular. The antibodies that we have generated will now permit us to determine the regional and subcellular distributions of ataxin-7 in brain samples from additional patients. This should clarify the relationship between the expression of pathological ataxin-7 and neuronal loss. Finally, susceptibility to the mutated protein may be high in some neurones, such as Purkinje cells, and low in others, e.g. those of the putamen. Differential susceptibility might be conferred by interactions with other proteins that are specific to the vulnerable neuronal populations. Studies in this direction are under way.
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Acknowledgments |
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We wish to thank O. Roussaouen and C. Weber for technical assistance, the IGBMC monoclonal facility for their help in generating 1C1 antibody, Y. Trottier and D. Devys for advice and for technical assistance, Dr S. El Mestikawi for helpful discussions, Dr G. Stevanin for critical reading of the manuscript and Professor N. Gonatas (Philadelphia) for the gift of the anti-trans-Golgi antibody. This study was supported by the Association Franciaise contre les Myopathies (AFM) and the Verum Foundation for Behaviour and Environment, Munich. G.C. and C.Z. were supported by fellowships from the Verum Foundation and AFAF (Association Franciaise contre l'Ataxie de Friedreich), respectively.
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Received February 10, 2000. Revised June 16, 2000. Accepted August 15, 2000.
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