Ataxin-7 aggregation and ubiquitination in infantile SCA7 with 180 CAG repeats.код для вставкиСкачать
Ataxin-7 Aggregation and Ubiquitination in Infantile SCA7 with 180 CAG Repeats Olaf Ansorge, MD,1 Paola Giunti, MD,2 Andrej Michalik, PhD,3 Christine Van Broeckhoven, DSc,3 Brian Harding, DPhil,4 Nicholas Wood, PhD,2 and Francesco Scaravilli, PhD1 Extremely long (>150) CAG repeats are often used to create models of polyglutamine diseases yet are very rare in humans where they manifest as pediatric multisystem syndromes of little specificity. Here, we describe an infant with 180 CAG repeats in the spinocerebellar ataxia type 7 gene and focus on systemic ataxin-7 aggregation. This was found in many organs, including the cardiovascular system. In the brain, the hippocampus emerged as a principal site of ataxin-7 aggregation without cell loss. We note differential ubiquitination of aggregates and discuss how this may relate to selective vulnerability. Ann Neurol 2004;56:448 – 452 Spinocerebellar ataxia type 7 (SCA7) is one of several neurodegenerative diseases that are caused by an expansion of unstable CAG repeats coding for polyglutamine (polyQ) residues (for review, see Michalik and colleagues1). The gene products of these diseases are unrelated except for the polyQ tract and are expressed throughout the body, yet each disease displays a distinct pattern of neuronal degeneration and nuclear inclusions (NIs) of aggregated mutated protein. However, the clinical phenotypes become less distinctive as the length of the polyQ tract increases. Generally, diseases manifest in adulthood at thresholds of 36 to 40 Qs and in adolescence or even infancy if the repeat number exceeds 60 to 100 Qs. Infantile SCA7 may be unique in that extreme expansions may even result in systemic disease. Particularly cardiovascular abnormali- ties have been documented clinically2– 4 but not investigated pathologically. CAG expansions of more than 150 repeats are very rare in humans but often are used to create models of polyQ diseases.5,6 Because many of the current hypotheses concerning the pathogenesis of polyQ diseases are derived from these models, it is important to document the effects of such extreme expansions in humans. Here, we present a detailed clinicopathological investigation of infantile SCA7 with 180 CAG repeats with a focus on ataxin-7 protein expression throughout the body. We observed ataxin-7 aggregates in the cardiovascular system but also in other nonneuronal tissues. In the brain, the hippocampus, generally not implicated in SCA7 pathophysiology, emerged as a principal site of ataxin-7 aggregation without neuronal loss. Finally, we note that nonneuronal inclusions, in contrast with many neuronal ones, are not detected by an ubiquitin antibody. We discuss the possibility that differential ubiquitination may contribute to selective cellular vulnerability. Case Report Patient and Family History The patient was a 29-month-old girl born into a family with known SCA7 (see pedigree, Fig 1).7 The pregnancy, birth, and first few months postpartum were uneventful. A generalized limb tremor was noted at approximately 9 months of age and developmental mile- From the 1Division of Neuropathology and 2Department of Molecular Neuroscience, Institute of Neurology, Queen Square, London, United Kingdom; 3Department of Molecular Genetics VIB8, Flanders Interuniversity Institute for Biotechnology, University of Antwerp, Belgium; and 4Department of Pathology, Great Ormond Street Hospital for Sick Children, London, United Kingdom. Received Mar 8, 2004, and in revised form May 27 and Jun 14. Accepted for publication Jun 15, 2004. Address correspondence to Dr Ansorge, Department of Neuropathology, The Radcliffe Infirmary, Oxford OX2 6HE, England. Email: firstname.lastname@example.org Published online Aug 31, 2004, in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.20230 448 Fig 1. Anonymized four-generation tree of the SCA-7 family. The 180Q-allele of the patient with infantile onset (IV:4) was paternally inherited. The patient’s father (III:3) is confirmed carrier of the SCA-7 mutation with a pathological allele of 39Q, but was clinically asymptomatic at age 54 years. (open symbols) Clinically asymptomatic; (filled symbols) symptomatic individuals. © 2004 American Neurological Association Published by Wiley-Liss, Inc., through Wiley Subscription Services stones were delayed. Soon after, marked dysphagia developed and there was a general failure to thrive. Neurological examination at 19 months showed pigmentary degeneration in both retinae. There was also downbeat nystagmus and general muscle hypotonia with head lag. Reflexes, however, were brisk and there was a positive Rossolimo sign. There was marked cerebellar ataxia. A computed tomography scan showed cerebellar and brainstem atrophy. Routine laboratory tests were unremarkable. Analysis of blood DNA revealed an expansion of 180 CAG repeats in the SCA7 gene. The pathological allele was inherited from the father who had 39 repeats but was clinically asymptomatic when last examined at age 54 years (III:3, see pedigree). The patient died 20 months after clinical onset. With consent of next of kin, a postmortem examination was conducted. Genetic Analysis DNA was extracted from peripheral blood lymphocytes by standard methods. Analysis of the SCA7 (CAG)n expansion was done by polymerase chain reaction.8 Allele repeat size was determined by polyacrylamide gel electrophoresis using an ABI 377 automatic sequencer and Genescan software (PE Applied Biosystems, Foster City, CA). Immunohistochemistry Formalin-fixed, paraffin-embedded tissue samples were cut into 5m sections and stained with hematoxylin and eosin or Luxol fast blue. Tissue from a 27-monthold female patient who died of encephalitis was used as a control. Microwave antigen retrieval (8 minutes) was used for polyclonal ataxin-7 antibody CM1899 (1:2,000) and polyclonal ubiquitin antibody (1:400; Dako, Glostrup, Denmark). Primary antibodies were incubated for 1 hour at room temperature. Appropriate biotinylated secondary antibodies were applied for 30 minutes, followed by avidin-biotin complex and 3⬘,3diaminobenzidine as chromogen. Sections were photographed with a digital camera mounted on an Olympus microscope. Results General Pathological Findings The body was small for age (height, 82.5cm; weight, 7.4kg). The head circumference for age was below the tenth percentile, and individual organ weights were low for age: brain, 850gm (normal mean, 1,064gm); kidneys, 23 and 26gm (normal, 50gm); liver, 306gm (normal, ⬎400gm);unfortunately, the weight of the heart was not recorded. Peripheral organs were macroscopically normal; there was no evidence of a cardiac malformation or patent ductus arteriosus. The brain showed macroscopically severe olivopontocerebellar at- rophy and thinning of the spinal cord. In contrast, the neocortex, hippocampi, and central gray nuclei appeared relatively preserved. Distribution of Inclusions in Neural Tissue and Relationship to Neuronal Loss Ataxin-7 NIs were seen throughout the central, peripheral, and autonomous nervous system (for summary and examples, see Table 1 and Fig 2). Very large nuclear inclusions could be detected on routine hematoxylin and eosin preparations (see Fig 2D). Inclusions were clearly not limited to areas of severe cell loss such as the retina, olive, or cerebellum. In fact, NIs tended to be most frequent in areas not affected by neurodegeneration. This was particularly remarkable in the hippocampus which showed neither obvious neuronal loss nor gliosis despite the presence of ataxin-7–positive NIs in 93% of pyramidal cells (see Fig 2A, B). Ubiquitinated NIs were detected in only 52% of hippocampal pyramidal neurons compared with 93% of surviving olivary neurons. Table 1. Summary of Nervous System Distribution of Ataxin-7 Protein Neuronal Nuclear Inclusions in Relation to Neuronal Loss Nervous System Region Cortex Frontal neocortex Hippocampus Anterior cingulate Deep gray nuclei Caudate nucleus Globus pallidus Thalamus Lateral geniculate Brainstem Substantia nigra Pontine nuclei Inferior olive Oculomotor nucleus Cerebellum Purkinje cells Golgi cells Granule cells Dentate nucleus Spinal cord Anterior horn cells Sensory ganglia Sympathetic ganglia Retina Neurons with NIs Neuronal Loss ⫹⫹ ⫹⫹⫹a ⫹ ⫹ ⫺ ⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹b ⫹⫹ ⫹ ⫹⫹ ⫹⫹⫹ ⫹ ⫺c ⫹⫹⫹ ⫹ ⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹ ⫹ ⫹⫹⫹ a 100 pyramidal neurons were assessed for NIs: 93 showed ataxin-7 NIs, 52 contained ubiquitinated NIs. Of 60 surviving olivary neurons 58 (97%) contained ataxin-7 NIs and 56 (93%) ubiquitinated NIs. c Hardly any Purkinje cell was left for assessment. b Semiquantitative rating: ⫺ ⫽ absent; ⫹ present at low frequency/ degree; ⫹⫹ present at moderate frequency/degree; ⫹⫹⫹ present at high frequency/degree. Ansorge et al: Ataxin-7 in 180Q SCA7 449 Fig 2. Ataxin-7 inclusions in neuronal and nonneuronal cells in infantile (Q180) SCA7. There is no loss of hippocampal neurons (A, Luxol fast blue cresyl violet) despite ataxin-7 nuclear inclusions (NIs) in virtually all pyramidal cells (B); however, an ubiquitin antibody labels fewer NIs (C). Some NIs are so large that they can easily be detected as paranucleolar eosinophilic spheroids on routine stains: (D) olivary neuron (hematoxylin and eosin); compare with E, anterior horn cell (ataxin-7). (F) Oligodendroglial ataxin-7 NIs in the brainstem. Nonneuronal ataxin-7 NIs are present in endothelial cells (G), cardiac (H) and skeletal (I) muscle, exocrine pancreas (J), and epithelial cells of Brunner’s glands of the duodenum (L) (K, hematoxylin and eosin for comparison). Original magnifications ⫻40 (A), ⫻400 (B, C, K, L), ⫻600 (D–J). Distribution of Inclusions in Nonneural Tissues We found widespread yet regionally selective nuclear aggregation of ataxin-7 protein in nonneuronal cell 450 Annals of Neurology Vol 56 No 3 September 2004 types (summarized in Table 2, examples in Fig 2G–L). The presence of nuclear ataxin-7 inclusions was the only distinguishing feature in peripheral organs be- Table 2. Summary of Peripheral Organ Distribution of Ataxin-7 Protein NIs Tissue/Organ Endocrine/exocrine Anterior pituitary Pancreas Adrenal gland Thyroid gland GI system Liver Stomach Intestine Muscle Skeletal muscle Cardiac muscle Smooth muscle (gut) Other Vascular system Kidney Lung Spleen Nuclear Inclusions Cell Types ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫺ Endocrine epithelium Exocrine epithelium, islet cells Cortex ⬎ medulla n/a ⫺ ⫹ ⫹⫹⫹ n/a Chief and neuroendocrine cells Brunner’s gland epithelium, Auerbach and Meissner plexus ⫹⫹ ⫹ ⫺ Myocytes Myocytes n/a ⫹⫹ ⫹⫹ ⫺ ⫺ Endothelial cells Tubular epithelium, glomeruli n/a n/a Semiquantitative rating as in Table 1. n/a ⫽ non applicable. tween patient and control material. However, skeletal muscle showed occasional atrophic fibers, some of which were angulated. In contrast with neuronal inclusions, we could not detect ubiquitin epitopes in any of the nonneuronal inclusions. Discussion The CAG repeat associated with SCA7 is extremely unstable in the male germline which may result in massive intergenerational expansions not commonly seen in other CAG/polyQ diseases.7,8 Therefore, a pediatric neurologist may be confronted with an infantile-onset, rapidly progressive, complex neurological syndrome in a child of an apparently healthy father, as illustrated in this study, where we observed an intergenerational expansion of 141 CAG repeats (see Fig 1). Although the salient diagnostic features of SCA7 (retinal degeneration, cerebellar ataxia) are usually present in the infantile form, nonspecific neurological as well as systemic symptoms have been reported.2– 4 Our proband with 180 CAG repeats showed a failure to thrive, muscle weakness, and internal organs small for age. When we probed the peripheral tissues with antibody CM189, which preferentially detects aggregated (N-terminal) ataxin-7,9 we found widespread NIs. Particularly intriguing was the finding of very frequent NIs in endothelial cells (see Fig 2G), as capillary leakage syndrome and multiple hemangiomas have been reported in SCA7 infants with even higher repeat numbers (⬎325 CAGs).4 Ataxin-7 aggregates were also present in cardiac and skeletal muscle, tissues with high transcript levels,8 that are also implicated clinically in infantile SCA7 (atrial septum defect, patent ductus ar- teriosus and congestive heart failure were noted in children with ⬎230 repeats).2– 4 These tissues showed no evidence of NI formation in a patient with 60 CAG repeats.10 Skeletal muscle weakness in SCA7 therefore may reflect loss of anterior horn cells compounded by a direct myopathic effect of mutated ataxin-7 in cases with extremely large repeat expansions.11 The most interesting finding in the brain of our patient was the presence of ataxin-7 NIs in almost all pyramidal neurons of the hippocampus in the absence of obvious cell loss. This has not been reported before in human SCA7 to our knowledge but was a feature in a recently created 266Q knock in model of the disease,6 where it was associated with mild impairment of short-term synaptic plasticity. Hippocampal NI formation is a feature that only emerges with extreme CAG expansions, because even with a relatively high number of 85 CAGs only very rare NIs (⬍1%) were seen,12 and even fewer repeats were associated only with diffuse nuclear staining.10 The role of the large visible NIs in the pathogenesis of the polyQ disorders is still debated.13 The dynamics14 and toxicity15 of NI formation may vary considerably not only between different cell types but also in homogeneous populations. Postmitotic cells (neurons, myocytes) appear to be more vulnerable than proliferating cells15; however, NIs may induce cell cycle arrest in the latter.16 An attractive hypothesis postulates that NIs may engage the ubiquitin-proteasome system in a futile attempt of refolding and degradation, leading to demise of the cell.16 In this context, it is, however, noteworthy that not all NIs (as defined by staining for the disease protein) are ubiquitinated, and that ubiq- Ansorge et al: Ataxin-7 in 180Q SCA7 451 uitination of neuronal NIs is a late process6,17 that may vary between brain regions.18 An intriguing observation in our study as well as previous12 studies is that ubiquitination of neuronal NIs appears to be more frequent in areas with severe cell loss (olive) than in those without (hippocampus). 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