Brain, Vol. 125, No. 7, 1534-1543,
July 2002
© 2002 Guarantors of Brain
Two populations of neuronal intranuclear inclusions in SCA7 differ in size and promyelocytic leukaemia protein content
1 Laboratoire de Neuropathologie Raymond Escourolle, 2 INSERM U289, 3 U106, 4 U360, Association Claude Bernard, 5 Fédération de Neurologie and 6 Département de Génétique, Cytogénétique et Embryologie, Hôpital de la Salpêtrière, 7 CNRS UPR 9051 Centre Hayem Hôpital St Louis, Paris, 8 Institut de Génétique et de Biologie Moléculaire (IGBMC), CNRS, INSERM, Université Louis Pasteur, Illkirch, CU de Strasbourg, France, 9 Division of Neuropathology, The Jikei University School of Medicine, Tokyo, Japan and 10 Neurogenetics Unit, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden
Correspondence to: A. Brice, INSERM U 289, Hôpital de la Salpêtrière, 47, boulevard de l’Hôpital, 75651 Paris, Cedex 13, France E-mail: brice{at}ccr.jussieu.fr
Received July 19, 2001. Revised November 10, 2001. Accepted January 31, 2002.
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Spinocerebellar ataxia type 7 (SCA7) is a hereditary progressive cerebellar ataxia with retinal degeneration associated with an abnormally expanded polyglutamine stretch. Neuronal intranuclear inclusions (NIIs), as in other polyglutamine diseases, are pathological hallmarks of these disorders. NIIs in polyglutamine diseases contain not only the protein with the expanded polyglutamine stretch but also other types of proteins. Several chaperone proteins related to the ubiquitin proteasome pathway, transcription factors and nuclear matrix proteins have been detected in NIIs. The composition of NIIs might reflect the process of NII formation and part of the pathogenesis of these diseases. To investigate how these proteins relate to the pathogenesis of SCA7, we performed immunohistochemical analyses of the composition of NIIs in two cases of SCA7. We demonstrated that there are two types of NIIs in SCA7 that differ in size and immunoreactivity to promyelocytic leukaemia protein (PML), one of the essential components of nuclear bodies (NBs; also called PML oncogenic domains). Small and large NIIs contained ataxin-7, human DnaJ homologue 2 (HDJ-2) and proteasome subunit 19S. In contrast, PML was found only in small NIIs. CREB-binding protein (CBP), another component of NBs, was distributed like PML in NIIs. Our results suggest that NIIs are formed by the accumulation of ataxin-7 in NBs, which become enlarged as they recruit related proteins.
Keywords: spinocerebellar ataxia type 7; neuronal intranuclear inclusions; nuclear bodies; promyelocytic leukaemia protein; ubiquitin proteasome system
Abbreviations: CBP = CREB binding protein; DRPLA = dentatorubro-pallidoluysian atrophy; HDJ-2 = human DnaJ homologue2; NB = nuclear body; NII = neuronal intranuclear inclusion; PML = promyelocytic leukaemia protein; SCA = spinocerebellar ataxia
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Introduction |
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Spinocerebellar ataxia type 7 (SCA7) is a hereditary neurodegenerative disorder characterized by progressive cerebellar ataxia and retinal degeneration (Stevanin et al., 2000
). SCA7 is caused by an abnormally expanded polyglutamine stretch in a protein of unknown function, ataxin-7 (David et al., 1997). This type of mutation has been found in SCA1 (Orr et al., 1993), SCA2 (Imbert et al., 1996; Pulst et al., 1996; Sanpei et al., 1996), SCA3 (Kawaguchi et al., 1994), SCA6 (Zhuchenko et al., 1997), Huntington’s disease (Huntington’s Disease Collaborative Research Group, 1993), spinal and bulbar muscular atrophy (La Spada et al., 1991), dentatorubro-pallidoluysian atrophy (DRPLA) (Koide et al., 1994) and recently a CAG repeat expansion in the TATA box-binding protein gene (Koide et al., 1999; Fujigasaki et al., 2001). These disorders have several common clinical features: (i) expression of the disease phenotype when the size of the expansion reaches a given threshold, which varies according to the gene; (ii) a strong negative correlation between the age at onset and the size of the expansion; (iii) instability during transmission with a tendency to expansion, resulting in anticipation (Zoghbi and Orr, 2000). Pathologically, the presence of neuronal intranuclear inclusions (NIIs) containing the protein with expanded polyglutamine is one of the hallmarks of these disorders (Ross et al., 1998). Intensive studies have attempted to elucidate what proteins are incorporated into NIIs and how these NIIs relate to the pathogenesis of polyglutamine disorders. It has been demonstrated that NIIs in polyglutamine diseases contain not only the protein with the expanded polyglutamine stretch but also other types of proteins. The first group of proteins is related to the ubiquitin proteasome pathway and includes heat shock proteins, ubiquitin and subunits of the proteasome, by which mutant proteins are presumed to be degraded (Cummings et al., 1998; Chai et al., 1999; Stenoien et al., 1999; Wyttenbach et al., 2000). However, there are discrepancies between the presence of NIIs and neuronal loss, as shown in several studies (Klement et al., 1998; Saudou et al., 1998; Cummings et al., 1999; Kuemmerle et al., 1999). NII formation might, therefore, be a cellular reaction to reduce polyglutamine toxicity, possibly through the ubiquitin proteasome pathway. Another group of proteins present in NIIs consists of transcription factors and their co-factors, such as TATA-binding protein, CREB-binding protein (CBP) and mSin3A (Boutell et al., 1999; McCampbell et al., 2000; Shimohata et al., 2000; Wood et al., 2000). Entrapment of these transcription regulators into NIIs might be responsible for the defective transcriptional regulation (Nucifora et al., 2001). Loss of transcription factors could increase neuronal susceptibility (Shimohata et al., 2000; Nucifora et al., 2001). The third group consists of proteins that contain a normal polyglutamine sequence (e.g. the normal counterpart of the protein with the polyglutamine expansion) (Perez et al., 1998). This group overlaps with the second group, since transcription factors such as TATA-binding protein and CBP have polyglutamine stretches.
It has been demonstrated that some of the polyglutamine proteins are associated with nuclear matrix proteins (Skinner et al., 1997; Tait et al., 1998). For example, in transfected cells, mutant ataxin-3 and mutant ataxin-7 are known to co-localize with promyelocytic leukaemia protein (PML) (Chai et al., 1999; Kaytor et al., 1999), a nuclear matrix protein and one of the essential components of nuclear bodies (NBs; also called PML oncogenic domains) (Sternsdorf et al., 1997). A number of proteins are present in NBs, many of which, such as CBP, p53 and retinoblastoma protein, regulate transcription (Zhong et al., 2000). Although the precise function of NBs remains unknown, they have been supposed to be involved in many cellular processes, such as transcriptional regulation, growth suppression and apoptotic cell death (Seeler and Dejean, 1999). More recently, it has been demonstrated that NBs are associated with the ubiquitin proteasome pathway and might be sites of protein degradation (Everett et al., 1997; Lallemand-Breitenbach et al., 2001; Mattsson et al., 2001; Reyes, 2001). Anton et al. (1999) have shown that misfolded forms of virus nucleoprotein recruit the proteasome and molecular chaperones to NBs, where misfolded protein is degraded.
These findings prompted us to hypothesize that NIIs can originate in NBs, where abnormal polyglutamine proteins are degraded. To investigate how these groups of proteins relate to the process of NII formation, we analysed the composition of NIIs in two patients with SCA7 by immunohistochemistry with antibodies against several proteins associated with NIIs. There were two types of NIIs that differed in size and PML-like immunoreactivity. We discuss the possible relationship between these two types of NIIs and the mechanism of NII formation.
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Case histories and neuropathological findings
Patient 1
The detailed clinical history and neuropathological findings in this patient have been reported previously (Holmberg et al., 1998
). Briefly, the patient was a 10-year-old boy who died 5 years after the first symptom of the disease. At the age of 5 years, learning problems and progressive visual loss were noticed. When examined at the age of 6 years, moderate cerebellar ataxia, clumsiness of the upper limbs and slight dysarthria were observed. Fundoscopy showed pigmentary degeneration. SCA7 was diagnosed by molecular analysis, which revealed the presence of an 85 CAG repeat expansion in the SCA7 gene. He died of pneumopathy after several days of coma. Neuropathological examination showed severe neuronal loss associated with gliosis in the inferior olive and cerebellar Purkinje cell layer. Neuronal loss was moderate in the dentate nucleus and mild in the external and internal pallidum and substantia nigra. The moderate neuronal loss seen in the geniculate body was considered to be a secondary alteration.
Patient 2
Patient 2 developed loss of visual acuity at the age of 22 years and gait disturbance at age 23 years. At age 28 years, optic atrophy was prominent, and evoked auditory potentials indicated abnormal conduction in the pons. Brain imaging showed cerebellar and brainstem atrophy. She could walk alone until age 32 years. The symptoms progressed gradually. At age 35 years she was blind and wheelchair-bound. Neurological symptoms were cerebellar dysarthria, truncal and limb ataxia and increased reflexes in all limbs, with a bilateral extensor plantar reflex. Distal dominant atrophy with weakness of the four limbs, decreased vibration sense and deficit of superficial sensation were noted. There was dystonia in the upper extremities. Vertical and lateral gaze palsy was predominant, with abolition of voluntary saccadic eye movements. There were severe swallowing difficulties and urinary urgency. Fundoscopy revealed retinal degeneration. EMG was normal. Molecular analysis showed that the patient carried a 49 CAG repeat expansion in the SCA7 gene. She died at age 36 years of an intercurrent disease. Neuropathologically, severe neuronal loss and gliosis were seen in the dentate nucleus. Moderate neuronal loss and gliosis were also found in the inferior olive and Purkinje cell layer, whereas pathological changes in the cerebral cortex and basal ganglia were mild.
Immunohistochemistry
Sections (5 µm) of formalin-fixed, paraffin-embedded brain tissue of the two patients were cut and used for immunohistochemical staining. The following antibodies were used: anti-ataxin-7 1C1 (Yvert et al., 2000) [dilution 1 : 10 000, mouse monoclonal (G. Yvert, Strasbourg, France)], anti-ataxin-7 1261 (Yvert et al., 2000) [dilution 1 : 200, rabbit polyclonal (G. Yvert, Strasbourg, France)], anti-human DnaJ (HSP40) homologue-2 (HDJ-2) (dilution 1 : 200, mouse monoclonal; Neo Markers, Union City, Calif., USA), anti-proteasome 19S subunit (MSS-1) (dilution 1 : 200, rabbit polyclonal; Research Products, Exeter, UK), anti-PML (Daniel et al., 1993) (dilution 1 : 10, mouse monoclonal, H. de Thé, Paris, France), anti-CBP (A-22) (dilution 1 : 100, rabbit polyclonal; Santa Cruz Biotech, Santa Cruz, Calif., USA) and anti-mSin3A (AK-11) (dilution 1 : 200, rabbit polyclonal, Santa Cruz Biotech). To expose the antigenic epitopes, sections were autoclaved in 10 mM citrate buffer (pH 6.0) for 10 min. The sections were incubated with one of the primary antibodies for 48 h at 4°C, then with biotinylated secondary antibodies and horseradish peroxidase-conjugated streptavidin. 2,3-Diaminobenzidine hydrochloride was used as the chromogen (ChemoMate detection kit; Dako, Glostrup, Denmark). Several regions of the brains were examined to evaluate the incidence of NIIs and their immunoreactivity to the antibodies.
Samples from the supramarginal gyrus (Brodmann area 40) in Patient 1 and from the superior temporal gyrus (Brodmann area 22) in Patient 2 were chosen because of the high frequency of NIIs, and were used for double immunolabelling and measurement of the size of NIIs. Samples from the supramarginal gyrus (Brodmann area 40) or the superior temporal gyrus (Brodmann area 22) from five individuals (age range 8–46 years, mean age = 30.4 years) without neurological disorders served as controls. Double immunofluorescence labelling of SCA7 brain tissue was performed to examine the co-localization of ataxin-7 with the other proteins (HDJ-2, proteasome 19S subunit, PML, CBP and mSin3A). Double immunolabelling of PML and CBP was also performed. The secondary antibodies were anti-mouse immunoglobulin G or anti-rabbit immunoglobulin G antibodies coupled with FITC (fluorescein isothiocyanate; emission peak 525 nm) (Amrad Biotech, Richmond, Victoria, Australia) or with Cy3 (emission peak 568 nm) (Jackson Immunoresearch, West Grove, Pa., USA). The slides were examined with a Leica TCS 4D confocal microscope to evaluate the co-localization of the proteins in the NIIs. Ataxin-7- or PML-positive intranuclear structures in the double-immunostained sections were examined to see whether these two proteins were located in the same structures. Thirty ataxin-7-positive and twenty PML-positive intranuclear structures were examined in each case, and the structures containing each epitope were categorized according to size. The Fisher’s exact test was performed to compare the frequency of co-localization with PML in small (
2.5 µm) and large (>2.5 µm) ataxin-7-positive aggregates.
To estimate the size of NIIs stained with different antibodies, the following procedure was used with the sections stained by 2,3-diaminobenzidine hydrochloride immunolabelling mentioned above. Microscopic fields were captured by a camera connected to a microscope, and displayed on a monitor. The long axis of NIIs were plotted manually and measured automatically with an image analyser (VisioScan Biocom, Les Ulis, France). Aggregates smaller than 0.5 µm were excluded. Fifty intranuclear aggregates stained with each antibody were examined in each section. The mean and variance of the length of the long axis of the NIIs were calculated. It was found that the range of values differed according to the antibody. The ratio of variance (F-test) was used to compare the dispersion of the size of the immunopositive nuclear structures labelled by anti-PML and other antibodies. The 50 NII that were evaluated in each case with each antibody were then categorized according to size. The number of particles in each size category from 0.5 to 5.0 µm (bin width 0.5 µm) was calculated. Subsequently, the population was divided into two categories, small (= 2.5 µm) and large (>2.5 µm). The sizes of the NIIs labelled by different antibodies in each patient were then compared using Fisher’s exact test. Since the histograms obtained for the antibodies showed different distributions, the Shapiro–Wilk test was performed to determine whether the size of distribution of the aggregates detected by each antibody deviated from normal (SAS software; SAS Institute, Cary, NC, USA).
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Distribution of the proteins in control cases
In normal neurones, ataxin-7 was found in cell bodies and in nuclei, as reported previously (Cancel et al., 2000
). HDJ-2 was present mainly in the cell bodies of neurones, accompanied by fine granular immunoreactivity in the nucleus. Proteasome subunit 19S was located preferentially in the nucleus, with a diffuse or fine granular pattern. Neurones contained several (5–15) small PML-immunoreactive dots, which correspond to NBs (Fig. 1Aa). The size of PML-immunoreactive dots was <1.0 µm. CBP had a fine granular distribution in the nucleus. Immunolabelled mSin3A was found mainly in the cytoplasm of the neurones, and diffuse or granular nuclear staining was observed in only a few neurones.
Two types of NII in SCA7
In the two patients, NIIs labelled with anti-ataxin-7 antibodies (1C1 and 1261) were observed in a subpopulation of neurones in the cerebral cortex. Generally, one large spherical NII was observed, but one or some small NIIs could also be found close to or separate from the large one. Neurones containing one or more small NIIs were also observed.
All the other antibodies examined [anti-HDJ-2, anti-proteasome 19S, anti-PML (Fig. 1Ab) and anti-CBP] labelled a variable proportion of the NIIs (Zander et al., 2001). In neurones lacking NIIs, the distribution of these proteins was similar to that observed in control cases.
Co-localization of various proteins in NIIs
The co-localization of ataxin-7 with other proteins in NIIs was demonstrated by confocal microscopy. HDJ-2 and proteasome subunit 19S were co-localized with ataxin-7 in both small and large NIIs (Fig. 1Ba–c). PML showed a characteristic co-localization with ataxin-7 in the NIIs. In large NIIs, PML-like immunoreactivity was absent or present only in small dot-like structures, whereas in small NIIs PML-like immunoreactivity was distributed throughout the whole structures (Fig. 1Bd–f), or sometimes formed a ring at the periphery. To confirm this qualitative observation, the frequency of co-localization of ataxin-7 and PML was determined as a function of the size of NIIs. No or little PML-like immunoreactivity was detected in 10 ataxin-7-positive large NIIs (>2.5 µm) randomly selected in each patient. Ninety per cent of small NIIs (0.5–2.5 µm) contained PML in both patients. The frequency of co-localization with PML was significantly lower in large ataxin-7-positive aggregates than in small ones in both patients (Fisher’s exact test, P < 0.001). The structures (>1.0 µm) that had PML-like immunoreactivity were larger than normal NBs consistently contained ataxin-7. However, four out of 10 and two out of 10 of the smaller structures with PML-like immunoreactivity (0.5–1.0 µm), which corresponded to normal NBs, were not co-localized with ataxin-7 in Patients 1 and 2, respectively (Table 1). Although CBP-like immunoreactivity was detected in both the small and large NIIs (Fig. 1Ca–c), it was sometimes weaker in the large NIIs (Fig. 1Cd–f). Smaller CBP-positive dots in the nucleus did not always co-localize with ataxin-7 (Fig. 1Cg–i). Double immunolabelling of PML and CBP demonstrated that PML was found exclusively in small NIIs (Fig. 2Aa–c). In neurones without NIIs, PML-positive dot-like staining in the nucleus was often co-localized with CBP (Fig. 2Ad–f). mSin3A-positive NIIs were rare, but the pattern of co-localization between mSin3A and ataxin-7 was characteristic. mSin3A-like immunoreactivity was contained like a seed located within or attached to the periphery of some large NIIs (Fig. 2B).
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Immunolabelling according to the size of NII
Since the observable size of NIIs varied depending on the antibody used, we determined the size distribution of aggregates labelled by each antibody (Fig. 3), except those labelled by anti-mSin3A antibody, which were too rare.
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Three distribution patterns were obtained: the anti-PML antibody labelled only small (
2.5 µm) intranuclear structures and anti-CBP labelled mostly small structures but also a few large (>2.5 µm) ones. All the other antibodies labelled small (2.5 µm) and large (>2.5 µm) NIIs. The dispersion of the sizes of the immunopositive structures was therefore different in these groups: the sizes were concentrated around small values when the anti-PML antibody was used. In comparison, the structures labelled by the anti-CBP and other antibodies were highly variable in size. Statistical confirmation of this observation was obtained with the F-test, which showed that the size variance of the structures with PML immunolabelling was smaller than with the other antibodies (F = 4.48–15.3; degrees of freedom = 49, 49; P < 0.001). The difference in variance was clearly due to the lack of PML-immunoreactivity in large NIIs. All the 50 PML-positive NII profiles had a size 2.5 µm. Of these 50, the number of immunoreactive structures with a size >2.5 µm was three for CBP in Patient 1 (not statistically different from PML, Fisher’s exact test, P = 0.242) and varied between 12 and 27 when the other antibodies, including CBP in Patient 2, were used (Fisher’s exact test, P 0.001). The Shapiro-Wilk test detected deviation from the normal size distribution of aggregates stained with the following antibodies: anti-ataxin-7, HDJ-2, proteasome subunit 19S and CBP in Patient 1, and anti-ataxin-7, HDJ-2 and CBP in Patient 2 (P < 0.05). Taken together, these results suggest that the anti-PML antibody labelled only small NIIs, but other antibodies labelled two populations: small and large NIIs. |
Discussion |
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The NIIs in SCA7 contained transcription factors and chaperone proteins associated with the ubiquitin proteasome pathway involved in protein degradation, as reported in other polyglutamine diseases (Shimohata et al., 2000
; Zoghbi and Orr, 2000; Nucifora et al., 2001). This suggests that these proteins are involved in a process of NII formation common to these disorders. Our results have shown that these NIIs can be divided into two types according to their size and composition. The distributions of PML and, to a lesser extent, CBP were distinct from the other proteins examined. PML was found almost constantly in the small NIIs, and large NIIs recognized by anti-PML antibody were extremely rare. The small NIIs (0.5–2.5 µm) without PML-like immunoreactivity may correspond to sections through the periphery of large NIIs.
PML is one of the main components of NBs (also called PML oncogenic domains), which are thought to be involved in many cellular functions (Seeler and Dejean, 1999). Cells typically contain 10–30 NBs per nucleus, and their diameter is between 0.2 and 1.0 µm (Koken et al., 1995). In this study, we demonstrated that PML is almost always found in small NIIs and is often more concentrated at the periphery. Ultrastructural immunohistochemistry showed that PML is also found peripherally in normal NBs (Maul et al., 2000; Lallemand-Breitenbach et al., 2001). The distribution of PML in small NIIs is, therefore, similar to that in normal NBs.
Double immunostaining with PML and CBP, one of the components of NBs (LaMorte et al., 1998), confirmed that they are co-localized in discrete nuclear structures compatible with normal NBs, and also in ataxin-7-positive small NIIs. These findings suggest that, in the SCA7 brain, mutant ataxin-7 initially accumulates in NBs, which are thought to be sites of protein degradation. A previous report demonstrating that mutant ataxin-7 co-localizes with PML in the same nuclear structures in transfected cells also supports this hypothesis (Kaytor et al., 1999). The small NIIs contain heat shock protein HDJ-2 and proteasome subunit 19S, suggesting that these molecules are recruited into NBs to assist the refolding or degradation of mutant ataxin-7 through the ubiquitin proteasome pathway.
PML-positive NIIs were larger than the normal size of NBs. This could result from accumulation of ataxin-7 and other proteins in the NBs. Although the precise function of PML still remains unknown, PML has a RING finger domain, which is generally associated with an E3 ubiquitin ligase function in ubiquitin-mediated proteolysis (Freemont, 2000; Reyes, 2001). PML could therefore be associated with the degradation of mutant ataxin-7. Reduction in activity of the ubiquitin-proteasome system is thought to accelerate the pathology of polyglutamine diseases. Loss of Purkinje cells in SCA1 transgenic mice, for example, was exaggerated by inactivation of the ubiquitin-proteasome pathway, even though the prevalence of intranuclear aggregates decreased (Cummings et al., 1999). There is, thus, a possibility that NIIs are formed to sequester harmful mutant proteins and that NBs serve to facilitate the process of detoxification of mutant proteins by accumulating the proteins of the ubiquitin–proteasome system.
The link between small and large NIIs remains to be elucidated. Since both types of NII contain the same ubiquitin proteasome pathway components, both may be formed during the degradation process of mutant ataxin-7, and the difference in size may only reflect the time course of NII formation. Ultrastructurally, large NIIs in SCA7 consisted of granular and filamentous material (data not shown), as in other polyglutamine disorders (DiFiglia et al., 1997; Hayashi et al., 1998). Small NIIs probably grow larger with time by accumulating mutant ataxin-7 and other related proteins. If ataxin-7 initially accumulates in NBs, their destruction, caused by further accumulation of ataxin-7, could lead to the formation of large NIIs with little PML-like immunoreactivity. If PML is involved in the degradation of ataxin-7, this function is probably abolished when NIIs become larger.
Unlike PML, CBP was also detected in some of the large NIIs, indicating that the incorporation of CBP can continue, even when NIIs have grown larger. It has been supposed that polyglutamine proteins are incorporated into NIIs through polyglutamine–polyglutamine interaction (Perez et al., 1998; Nucifora et al., 2001). CBP, which contains a polyglutamine stretch, is probably recruited into large NIIs through a similar mechanism in SCA7 brains.
We have shown that ataxin-7 co-localizes with PML and CBP in what appear to be NBs. We have also observed that a few ataxin-7-positive NIIs contained mSin3A. The relationship between the PML-positive and the mSin3A-positive structures remains to be determined. In DRPLA brains, it has also been shown that a certain population of NIIs also contain PML (Yamada et al., 2001). However, when atrophin-1, the polyglutamine-containing protein responsible for DRPLA, is expressed in transfected cells, it co-localizes instead with mSin3A in sites that are not NBs (Wood et al., 2000). These observations in SCA7 and DRPLA suggest that aggregation of proteins with mutant polyglutamine might be initiated in different ways, one of which will predominate according to the nature or the function of the protein involved.
In conclusion, this study suggests that PML-containing NBs accumulate ataxin-7 and other proteins, such as chaperones and proteasome subunits, forming small NIIs that gradually lose their characteristic features as the mutant polyglutamine-containing protein accumulates and the NIIs increase in size. Further studies are needed to determine whether the sequential mechanism of NII formation proposed here is applicable to all polyglutamine diseases.
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Acknowledgements |
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We thank Dr Merle Ruberg for critical reading of the manuscript and Dr Patrice Verpillat for statistical analyses. This work was supported by the VERUM Foundation and l’Association Française contre les Myopathies (A.B.). J.T. was supported by a fellowship from the Association of Claude Bernard and the Japan Society for the Promotion of Science (JSPS), H.F. by a fellowship from EGIDE and C.D. by JSPS.
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