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Intranuclear inclusions, neuronal loss and CAG mosaicism in two patients with Machado–Joseph disease
Journal of the Neurological Sciences 200 (2002) 19 – 25 www.elsevier.com/locate/jns
Intranuclear inclusions, neuronal loss and CAG mosaicism in two patients with Machado–Joseph disease E. Mun˜oz a, M.J. Rey b, M. Mila` c, A. Cardozo b, T. Ribalta b, E. Tolosa a,b, I. Ferrer b,* a
Neurology Service, Hospital Clinic and University of Barcelona, Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain b Neurological Tissues Bank, Hospital Clinic and University of Barcelona, Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain c Genetics Service, Hospital Clinic and University of Barcelona, Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Received 10 October 2001; received in revised form 19 April 2002; accepted 22 April 2002
Abstract The presence of neuronal intranuclear inclusions (NIIs) and neuronal mosaicism has been described in some autosomal dominant spinocerebellar ataxias (SCA), but their implication in neurodegenerative mechanisms still remains unclear. Objective: To investigate the correlation between neuronal loss and NIIs, and the size of CAG triplet expansion in selected areas of the CNS in two SCA3 patients. Material and methods: Postmortem neuropathological study was carried out, and the regional distribution of neuronal loss was compared with NIIs. CAG expansion was analysed by PCR amplification in the same regions. Results: Marked neuronal loss was seen in the anterior horn of the spinal cord, pontine nuclei and motor nuclei of the brain stem. Moderate neurone loss was found in the locus ceruleus, colliculus and substantia nigra. Loss of granule and Purkinje cells was found in the cerebellum, mainly in the vermis. NIIs were present in neurones of the involved nuclei of the anterior horn of the spinal cord, medulla oblongata and pons, but not in the locus ceruleus, substantia nigra and cerebellum. A few NIIs were found in the striatum. The number of CAG repeats was 27/70 in the first patient and 21/74 in the second patient. The variation of the expanded allele size among different cerebral areas was F 1 – 3 CAG repeats. Conclusion: The partial correlation between neuronal loss and NIIs suggests that other factors distinct from NII formation may be involved in the neuronal death. Moreover, the low degree of mosaicism between regions without neuronal loss and regions with marked neuronal loss points to the existence of selective cellular vulnerability to the genetic defect. D 2002 Elsevier Science B.V. All rights reserved. Keywords: CAG; Neuronal loss; SCA
1. Introduction Spinocerebellar ataxias (SCA) constitute a heterogeneous group of diseases clinically characterised by a progressive cerebellar syndrome, but also with symptoms and signs suggesting involvement of the brain stem, spinal cord, basal ganglia or peripheral nerves in many cases. The CAG repeat expansion in a specific locus in different chromosomes is the mutation responsible for autosomal dominant ataxias such as SCA1, SCA2, SCA3, SCA6, SCA7, dentato – rubro – pallido –luysian atrophy (DRPLA) and SCA12. The CAG expansion is translated except in SCA12 into a protein *
Corresponding author. Institut de Neuropatologia, Servei Anatomia Patolo`gica, Hospital Princeps d’Espanya, Universitat de Barcelona, carrer Feixa Llarga sn, 08907 Hospitalet de Llobregat, Spain. Tel.: +34-93-4035808; fax: +34-93-204-5065. E-mail address: [email protected] (I. Ferrer).
that is expanded in polyglutamine residues. The number of CAG repeats influences the age at onset and the disease severity in many cases. SCA3 or Machado – Joseph disease is associated with the CAG expansion on the chromosome 14q32.1 [1] that results in the abnormal expression of the polyglutamine-expanded mutant protein ataxin-3. Both the normal and mutant ataxin-3 are widely expressed in all regions of the brain [2,3]. It has been proposed that this abnormal expanded protein is the cause of the neuronal death in vitro and in vivo [4]. However, the mechanism of mutant ataxin-3-induced cell death is poorly understood. It has been suggested that the formation of neuronal intranuclear inclusions (NIIs) is a common pathogenic mechanism involved in the neurodegeneration in most of the CAG triplet-repeat disorders [5], including SCA3 [6,7]. The corresponding expanded and normal protein and ubiquitin are components of NIIs [6]. The formation of NIIs still remains unclear, but the presence of the mutated protein in
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the nucleus is required, and the interaction with the proteasome [8] and nuclear matrix [9] is probably needed. The CAG expansion size varies in different cell lines from the same individual (somatic mosaicism). The presence of neuronal mosaicism in SCA3 has been reported in some studies [2,10 –12]. However, the correlation between NII formation and the degree of mosaicism in SCA3 has not been investigated in detail. The objective of the present study was to examine whether there is any association between neuronal loss and the presence of NIIs, and the size of the CAG expansion in several CNS regions in two patients who had suffered from SCA3.
2. Material and methods 2.1. Patients 2.1.1. Patient 1 This patient was a woman who consulted our service at the age of 78. The first symptoms appeared at the age of 50 years and the condition was characterised by slowly progressive gait ataxia and dysarthria. She was able to walk without help until she was 77 years old. The family history revealed the presence of a cerebellar syndrome in many family members with an autosomal dominant pattern of inheritance (Fig. 1). The clinical and neuropathological findings supporting Machado– Joseph disease in some members of the same family had been reported previously [13]. On the first neurological examination, the patient was well oriented, her speech was dysarthric and the ocular motility showed a slow velocity with paresis of the superior vertical gaze. She suffered from amyotrophy of the extremities, together with an absence of the osteotendonous reflexes in the lower extremities, and bilateral Babinski sign. In addition, she complained of reduced distal sensitivity in both legs, impaired coordination of the extremities, more severe in the legs and marked gate ataxia. A cranial magnetic resonance imaging (MRI) showed severe atrophy of the cerebellum, pons, medulla and spinal cord (Fig. 2). An electromyographic (EMG) study demonstrated severe signs
Fig. 2. Cranial T1-weighted MR sequence in the sagital plane showing atrophy of the cerebellum, pons, medulla oblongata and spinal cord.
of denervation in the extremities, and the neurographic study revealed a severe sensory-motor axonal polyneuropathy. Loss of memory appeared at the age of 82, and was followed by apraxia, aphasia and agnosia, leading to severe cognitive impairment. The patient died at the age of 86 years. 2.1.2. Patient 2 This patient was a distant relative of patient 1 (Fig. 1) who consulted at the age of 36 years because of progressive gait instability that had started when she was 32 years old. She also complained of mild dysphagia to liquids, diplopia and weakness on her legs during the year prior to admission. The neurological examination revealed slight dysarthria, nystagmus to the bilateral gaze without ophtalmoparesis and bilateral weakness of the orbicularis oculi. All tendon reflexes were brisk, but Babinski sign was absent. The ankle –shin –knee coordination was slightly impaired. Her gait was cautious, and the tandem showed bilateral deviations. She also presented slightly decreased superficial distal sensitivity in the legs. The fundoscopic examination revealed no abnormalities. A cranial tomography (CT) scan of the brain showed atrophy of the cerebellum that predominated in
Fig. 1. Pedigree of the patients’ family throughout five generations. The filled black symbols indicate individuals affected by SCA3. Patients 1 and 2 are marked by arrows. The symbols ‘‘?’’ mean that the clinical status is unknown.
E. Mun˜oz et al. / Journal of the Neurological Sciences 200 (2002) 19–25
the vermis. An EMG study revealed denervation in both legs, and the neurography disclosed moderate sensory polyneuropathy. At the age of 40, she complained of slight vertical gaze paresis, impaired coordination of the four extremities and marked axial ataxia. Babinski sign was obtained on the right extremity. Four years later, the patient was unable to walk without help. She also presented marked spasticity and severe pain in the lower extremities that required intrathecal perfusion of baclofen. At that time, a magnetic resonance imaging (MRI) showed severe atrophy of the cerebellum and pons. At the age of 48, she progressed to severe dysarthria, marked paresis of the vertical and horizontal gaze, rigidity and bradykinesia of the extremities, generalised amyotrophy, anterocollis with marked weakness for neck extension and loss of tendon reflexes in the lower extremities. A new EMG study showed denervation in the paraspinal muscles compatible with camptocormia. The patient died of aspiration bronchopneumonia at the age of 49 years. 2.2. Neuropathological study The brain and spinal cord were removed, and selected samples were obtained for morphological and biochemical studies. The postmortem delay was 13 h in the first case and 7 h in the second case. For morphological studies, the left cerebral and cerebellar hemispheres, and alternate sections of the brainstem and spinal cord, obtained every 5 mm, were fixed in 4% buffered formalin for about 3 weeks. For biochemical studies, the rest of the brain and spinal cord was frozen and stored at 80 jC until use. Representative samples of the brain and spinal cord were embedded in paraffin and sections, 8 Am thick, were obtained with a sliding microtome. Dewaxed sections were stained with haematoxylin and eosin, luxol fast blue-Klu¨ver Barrera, or processed for immunohistochemistry to glial fibrillary acidic protein (1:250; GFAP, Dako), phosphorylated neurofilament epitopes of 170 and 200 kDa (1:100 and 1:200, respectively; clones RT97 and BF10, Boehringer-Mannheim), hA4 amyloid (1:5; Boehringer-Mannheim), tau and ubiquitin (1:50 and 1:200; both from Dako) and a-synuclein (1:1000; Chemicon). In addition, formalin-fixed samples were cut with a cryostat and the sections were stained with Oil Red O and Sudan black. For comparative studies, four brains from non-neurological cases obtained at autopsy (postmortem delay between 6 and 13 h) were processed in parallel. Semiquantitative studies were carried out in diseased and control cases in the frontal cortex (area 8), head of the caudate and anterior putamen (at the level of the anterior commissure), upper cerebellar vermis, cerebellar hemisphere, superior colliculus, substantia nigra, locus ceruleus, anterior pontine area (at the level of the locus ceruleus), medulla oblongata (at midlevels of the inferior olive) and anterior horn of the cervical spinal cord. The rough numbers of neurones in every selected region in Machado – Joseph disease were compared with age-matched controls. Semiquantitative data
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of neuronal loss were represented: , absence; +, slight; ++, moderate; +++, marked. The number of NIIs was counted at a magnification of 400 in sections immunostained for ubiquitin and slightly counterstained with haematoxylin. Four different fields and three sections per region were examined in every case. The number of NIIs was expressed as a percentage of the total number of neurones. 2.3. Genetic study Frozen samples from selected brain regions (Fig. 4) were homogenised and digested with proteinase K. Samples for biochemical studies corresponded to similar regions used for morphological studies although the levels in the midbrain, pons and medulla were about 5 mm apart from the corresponding morphological sections. DNA was extracted and PCR amplification was performed using primers and conditions as described [14]. The alleles were visualised by autoradiography after electrophoresis in a 6% polyacrylamide denaturing gel.
3. Results 3.1. Neuropathological study 3.1.1. Patient 1 The brain weight was 1200 g. There was mild cerebral atrophy, and marked atrophy of the cerebellum, particularly the vermis, and pons. Loss of pigment was noticed in the substantia nigra and locus ceruleus. Marked neuronal loss was seen in the anterior horn of the spinal cord, motor nuclei of the medulla oblongata, facial nucleus and oculomotor nuclei. Moderate loss of neurones was observed in the periaqueductal gray matter and colliculus. Moderate neuronal loss occurred in the locus ceruleus and substantia nigra. Loss of myelin, and decreased numbers of axons, was found in the anterior roots and nerves of the spinal cord, and dorsal spinothalamic and spinocerebellar tracts. Myelin pallor was observed in the pyramidal tracts. Marked loss of granule and Purkinje cells, and axonal torpedoes, was seen in the cerebellum, mainly in the vermis. Loss of neurones was also evident in the dentate nucleus and inferior olives. Moderate to severe astrocytic gliosis occurred in parallel to neurone loss in all these regions. Ubiquitin-immunoreactive NIIs were observed in neurones of the anterior horn of the spinal cord, nuclei of the medulla oblongata and pons, and, rarely, in the caudate and putamen (Fig. 3A). No ubiquitin-immunoreactive NIIs were seen in the cerebellum, substantia nigra and locus ceruleus. In addition, typical Lewy bodies were found in the substantia nigra, locus ceruleus and nucleus of the vagus nerve. a-Synuclein-immunoreactive neurites were present in the brain stem. A few cortical-type Lewy bodies were found in the limbic system. Interestingly, Lewy bodies have been reported in other cases of Machado– Joseph
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Table1 Neuronal loss and neuronal intranuclear inclusions (NIIs) in selected regions of the CNS in SCA3 patients Patient 1 Neuronal loss Frontal cortex Caudate and putamen Cerebellar hemispheres Cerebellar vermis Midbrain (colliculus) Substantia nigra Locus ceruleus Pons Medulla oblongata Anterior horn
Patient 2 NII
Neuronal loss
+ ++ +++ ++ ++ ++ +++ +++ +++
+++ ++ +
NII +
+ ++ ++ ++ ++ +++ +++ +++
+
++ ++ ++
Neuronal loss: , absence; +, slight; ++, moderate; +++, marked. NII: , absence; +, 1% of neurones; ++, 2 – 10% of neurones; +++, more than 10% of neurones. Although NIIs correlate with neurone loss in the pons, medulla oblongata and anterior horn of the spinal cord, there is no correlation in other brain regions, including the midbrain and cerebellum, in which neurone loss is not accompanied by the presence of NIIs. In contrast, a few NIIs are found in the caudate and putamen in spite of there being no neuronal loss.
disease [15]. Yet a-synuclein pathology in the present case did not differ from that found in diffuse Lewy body disease. Finally, massive hA4 amyloid deposits in the form of diffuse and neuritic plaques occurred throughout the cerebral cortex, hippocampus, amygdala, and, to a lesser extent, the diencephalic nuclei and brain stem. Large numbers of neurofibrillary tangles were present in the entorhinal cortices, hippocampus, amygdala and isocortex. A few neurofibrillary tangles were found in the midbrain. Changes were consistent with Alzheimer’s disease stage V of Braak and Braak. 3.1.2. Patient 2 The brain weight was 1400 g. There was moderate atrophy of the cerebellum and pons. Marked loss of motor neurones was found in the spinal cord (Fig. 4A). Loss of myelin occurred in the anterior nerves of the spinal cord, and the spinothalamic, spinocerebellar and pyramidal tracts. Marked neurone loss occurred in the motor nuclei of the hypoglossus and vagus nerves, pons, facial nucleus and motor nuclei of the VI, IV and III cranial nerves (Fig. 4B). Moderate neurone loss occurred in the substantia nigra (Fig. 4C) and locus ceruleus, but a-synuclein-positive inclusions were not seen in this case. Moderate loss of granule cells and Purkinje neurones, and occasional axonal torpedoes was found in the cerebellum, particularly in the cerebellar vermis (Fig. 4D). Loss of neurones was observed in the superior colliculus. Astrocytic gliosis accompanied neurone loss in all
Fig. 5. Mosaicism of the expanded allele in several brain regions in SCA3 patients. The study was repeated three times to make sure that the results were consistent. Although in this PCR the caudate and putamen did not show amplification, they did in the other two PCR studies.
these regions. Ubiquitin-immunoreactive NIIs were found in the anterior horn of the spinal cord, medulla oblongata and pons (Fig. 3B), but not in the cerebellum, substantia nigra and locus ceruleus. No other abnormalities were noticed in the cerebral cortex and diencephalic nuclei excepting a few NIIs in the caudate and putamen, and superior colliculus. No Alzheimer’s disease changes were present. A comparison of neuronal loss and NIIs in selected regions of the CNS in these cases is summarised in Table 1. 3.2. Genetic study The mean number of CAG repeats was 27/70 in the first patient and 21/74 in the second patient. The variation of the expanded allele size among different CNS regions was F 1– 3 CAG repeats in both patients (Fig. 5). The size of the
Fig. 3. Neuronal intranuclear inclusions, as revealed with ubiquitin immunohistochemistry, in patient 1 (A) and 2 (B). Paraffin section, slight haematoxylin counterstaining 1000. Fig. 4. Neuropathological findings in patient 2. Neuronal loss in the anterior horn of the spinal cord (A), motor nuclei of the vagus nerve (B) and substantia nigra (C). An axonal torpedo (asterisk) is observed in the vermis (D). Haematoxylin and eosin staining 200.
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normal allele did not show variation in the different CNS regions studied.
4. Discussion The main clinical symptoms and neuropathological findings were similar in both cases and consistent with previous observations in Machado –Joseph disease in the same family [13]. One of the patients was very old and had additional lesions of Alzheimer’s disease and diffuse Lewy body disease. The major severity of lesions in the cerebellum in this case is probably related to the more prolonged duration of the disease. We have only found a partial correlation between the neuronal loss and the presence of ubiquitin-immunoreactive NIIs in different CNS areas in both patients with Machado– Joseph disease. Lack of complete correlation was not due to concomitant lesions in the first case. Although it can be hypothesised that in certain areas NIIs might not be ubiquitinated, the absence of NIIs in the surviving neurones of the cerebellum, one of the more severely damaged structures in SCA3, does not support a major role of ubiquitinated NIIs in neurodegeneration. Moderate neuronal loss was also observed in the substantia nigra and locus ceruleus in the absence of ubiquitinated NIIs. A poor correlation between the localisation of NIIs and the distribution and severity of neurodegenerative changes has been previously reported in SCA2 and SCA7 [16,17]. Furthermore, although high level expression of expanded full-length ataxin-3 in vitro causes cell death and formation of NIIs in neurones, there is no close correlation between cell death and NIIs [18]. Finally, recent studies in SCA1 transgenic mice suggest that the presence of ataxin-1 in the nucleus, but not NII formation, is essential to induce the neuronal death [19]. Complementary data have been obtained from Huntington disease, another inherited disease with specific CAG repeat expansion producing abnormal huntingtin expression. The capacities to form NIIs and to induce neurodegeneration are suppressed in transgenic cultured neurones with the expansion for Huntington’s disease after blocking the nuclear localisation of mutant huntingtin [20]. Interestingly, conditions that only suppress NIIs formation without preventing the mutant protein transportation into the nucleus result in a higher neuronal loss [20], thus suggesting that NII formation may reflect a neuroprotective strategy against the mutant protein. The present study has also shown a low degree of mosaicism between different regions of the CNS, and a lack of correlation between the presence of mosaicism and neurone loss and NIIs in specific CNS regions. These findings agree with and complement previous SCA studies showing no correlation between the variation in the CAG repeat size and severity of neurodegeneration, although the expanded CAG repeats were shorter in the cerebellum than in other regions [11,12,21]. These findings suggest that
mosaicism reflects the different cell composition of different CNS regions, but that it is not the cause of the selective neuronal death observed in SCA [10]. In the same line, it is worth stressing that mutant ataxin-3 is widely expressed throughout the brain but its expression is not restricted to regions that show NIIs and neurodegeneration SCA [22]. Taken together, these findings suggest that other mechanisms may be implicated in neuronal vulnerability. It has been shown that the mutant protein has the capacity to interact with the proteasome [8], other nuclear proteins such as the leucine-rich acidic nuclear protein [23], chaperones [24] and proteins involved in DNA repair [25]. Recent experimental studies have shown that cAMP response element binding protein (CREB-binding protein), a histone acetyltransferase, is sequestered by mutant polyglutamines leading to altered protein acetylation in neurones [26,27]. Interestingly, it has been found that the cell death induced by nuclear accumulation of polyglutamines can be mitigated by overexpression of full-length CREB-binding protein [27] or by the administration of other inhibitors of histone deacetylase [26]. It is clear that further studies are necessary to elucidate the mechanisms that modulate ataxins in neurodegeneration.
Acknowledgements This work was supported in part by a grant FIS 99-1118. We wish to thank T. Yohannan for editorial assistance.
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Intranuclear inclusions, neuronal loss and CAG mosaicism in two patients with Machado–Joseph disease E. Mun˜oz a, M.J. Rey b, M. Mila` c, A. Cardozo b, T. Ribalta b, E. Tolosa a,b, I. Ferrer b,* a
Neurology Service, Hospital Clinic and University of Barcelona, Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain b Neurological Tissues Bank, Hospital Clinic and University of Barcelona, Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain c Genetics Service, Hospital Clinic and University of Barcelona, Institut d’Investigacions Biome`diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Received 10 October 2001; received in revised form 19 April 2002; accepted 22 April 2002
Abstract The presence of neuronal intranuclear inclusions (NIIs) and neuronal mosaicism has been described in some autosomal dominant spinocerebellar ataxias (SCA), but their implication in neurodegenerative mechanisms still remains unclear. Objective: To investigate the correlation between neuronal loss and NIIs, and the size of CAG triplet expansion in selected areas of the CNS in two SCA3 patients. Material and methods: Postmortem neuropathological study was carried out, and the regional distribution of neuronal loss was compared with NIIs. CAG expansion was analysed by PCR amplification in the same regions. Results: Marked neuronal loss was seen in the anterior horn of the spinal cord, pontine nuclei and motor nuclei of the brain stem. Moderate neurone loss was found in the locus ceruleus, colliculus and substantia nigra. Loss of granule and Purkinje cells was found in the cerebellum, mainly in the vermis. NIIs were present in neurones of the involved nuclei of the anterior horn of the spinal cord, medulla oblongata and pons, but not in the locus ceruleus, substantia nigra and cerebellum. A few NIIs were found in the striatum. The number of CAG repeats was 27/70 in the first patient and 21/74 in the second patient. The variation of the expanded allele size among different cerebral areas was F 1 – 3 CAG repeats. Conclusion: The partial correlation between neuronal loss and NIIs suggests that other factors distinct from NII formation may be involved in the neuronal death. Moreover, the low degree of mosaicism between regions without neuronal loss and regions with marked neuronal loss points to the existence of selective cellular vulnerability to the genetic defect. D 2002 Elsevier Science B.V. All rights reserved. Keywords: CAG; Neuronal loss; SCA
1. Introduction Spinocerebellar ataxias (SCA) constitute a heterogeneous group of diseases clinically characterised by a progressive cerebellar syndrome, but also with symptoms and signs suggesting involvement of the brain stem, spinal cord, basal ganglia or peripheral nerves in many cases. The CAG repeat expansion in a specific locus in different chromosomes is the mutation responsible for autosomal dominant ataxias such as SCA1, SCA2, SCA3, SCA6, SCA7, dentato – rubro – pallido –luysian atrophy (DRPLA) and SCA12. The CAG expansion is translated except in SCA12 into a protein *
Corresponding author. Institut de Neuropatologia, Servei Anatomia Patolo`gica, Hospital Princeps d’Espanya, Universitat de Barcelona, carrer Feixa Llarga sn, 08907 Hospitalet de Llobregat, Spain. Tel.: +34-93-4035808; fax: +34-93-204-5065. E-mail address: [email protected] (I. Ferrer).
that is expanded in polyglutamine residues. The number of CAG repeats influences the age at onset and the disease severity in many cases. SCA3 or Machado – Joseph disease is associated with the CAG expansion on the chromosome 14q32.1 [1] that results in the abnormal expression of the polyglutamine-expanded mutant protein ataxin-3. Both the normal and mutant ataxin-3 are widely expressed in all regions of the brain [2,3]. It has been proposed that this abnormal expanded protein is the cause of the neuronal death in vitro and in vivo [4]. However, the mechanism of mutant ataxin-3-induced cell death is poorly understood. It has been suggested that the formation of neuronal intranuclear inclusions (NIIs) is a common pathogenic mechanism involved in the neurodegeneration in most of the CAG triplet-repeat disorders [5], including SCA3 [6,7]. The corresponding expanded and normal protein and ubiquitin are components of NIIs [6]. The formation of NIIs still remains unclear, but the presence of the mutated protein in
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the nucleus is required, and the interaction with the proteasome [8] and nuclear matrix [9] is probably needed. The CAG expansion size varies in different cell lines from the same individual (somatic mosaicism). The presence of neuronal mosaicism in SCA3 has been reported in some studies [2,10 –12]. However, the correlation between NII formation and the degree of mosaicism in SCA3 has not been investigated in detail. The objective of the present study was to examine whether there is any association between neuronal loss and the presence of NIIs, and the size of the CAG expansion in several CNS regions in two patients who had suffered from SCA3.
2. Material and methods 2.1. Patients 2.1.1. Patient 1 This patient was a woman who consulted our service at the age of 78. The first symptoms appeared at the age of 50 years and the condition was characterised by slowly progressive gait ataxia and dysarthria. She was able to walk without help until she was 77 years old. The family history revealed the presence of a cerebellar syndrome in many family members with an autosomal dominant pattern of inheritance (Fig. 1). The clinical and neuropathological findings supporting Machado– Joseph disease in some members of the same family had been reported previously [13]. On the first neurological examination, the patient was well oriented, her speech was dysarthric and the ocular motility showed a slow velocity with paresis of the superior vertical gaze. She suffered from amyotrophy of the extremities, together with an absence of the osteotendonous reflexes in the lower extremities, and bilateral Babinski sign. In addition, she complained of reduced distal sensitivity in both legs, impaired coordination of the extremities, more severe in the legs and marked gate ataxia. A cranial magnetic resonance imaging (MRI) showed severe atrophy of the cerebellum, pons, medulla and spinal cord (Fig. 2). An electromyographic (EMG) study demonstrated severe signs
Fig. 2. Cranial T1-weighted MR sequence in the sagital plane showing atrophy of the cerebellum, pons, medulla oblongata and spinal cord.
of denervation in the extremities, and the neurographic study revealed a severe sensory-motor axonal polyneuropathy. Loss of memory appeared at the age of 82, and was followed by apraxia, aphasia and agnosia, leading to severe cognitive impairment. The patient died at the age of 86 years. 2.1.2. Patient 2 This patient was a distant relative of patient 1 (Fig. 1) who consulted at the age of 36 years because of progressive gait instability that had started when she was 32 years old. She also complained of mild dysphagia to liquids, diplopia and weakness on her legs during the year prior to admission. The neurological examination revealed slight dysarthria, nystagmus to the bilateral gaze without ophtalmoparesis and bilateral weakness of the orbicularis oculi. All tendon reflexes were brisk, but Babinski sign was absent. The ankle –shin –knee coordination was slightly impaired. Her gait was cautious, and the tandem showed bilateral deviations. She also presented slightly decreased superficial distal sensitivity in the legs. The fundoscopic examination revealed no abnormalities. A cranial tomography (CT) scan of the brain showed atrophy of the cerebellum that predominated in
Fig. 1. Pedigree of the patients’ family throughout five generations. The filled black symbols indicate individuals affected by SCA3. Patients 1 and 2 are marked by arrows. The symbols ‘‘?’’ mean that the clinical status is unknown.
E. Mun˜oz et al. / Journal of the Neurological Sciences 200 (2002) 19–25
the vermis. An EMG study revealed denervation in both legs, and the neurography disclosed moderate sensory polyneuropathy. At the age of 40, she complained of slight vertical gaze paresis, impaired coordination of the four extremities and marked axial ataxia. Babinski sign was obtained on the right extremity. Four years later, the patient was unable to walk without help. She also presented marked spasticity and severe pain in the lower extremities that required intrathecal perfusion of baclofen. At that time, a magnetic resonance imaging (MRI) showed severe atrophy of the cerebellum and pons. At the age of 48, she progressed to severe dysarthria, marked paresis of the vertical and horizontal gaze, rigidity and bradykinesia of the extremities, generalised amyotrophy, anterocollis with marked weakness for neck extension and loss of tendon reflexes in the lower extremities. A new EMG study showed denervation in the paraspinal muscles compatible with camptocormia. The patient died of aspiration bronchopneumonia at the age of 49 years. 2.2. Neuropathological study The brain and spinal cord were removed, and selected samples were obtained for morphological and biochemical studies. The postmortem delay was 13 h in the first case and 7 h in the second case. For morphological studies, the left cerebral and cerebellar hemispheres, and alternate sections of the brainstem and spinal cord, obtained every 5 mm, were fixed in 4% buffered formalin for about 3 weeks. For biochemical studies, the rest of the brain and spinal cord was frozen and stored at 80 jC until use. Representative samples of the brain and spinal cord were embedded in paraffin and sections, 8 Am thick, were obtained with a sliding microtome. Dewaxed sections were stained with haematoxylin and eosin, luxol fast blue-Klu¨ver Barrera, or processed for immunohistochemistry to glial fibrillary acidic protein (1:250; GFAP, Dako), phosphorylated neurofilament epitopes of 170 and 200 kDa (1:100 and 1:200, respectively; clones RT97 and BF10, Boehringer-Mannheim), hA4 amyloid (1:5; Boehringer-Mannheim), tau and ubiquitin (1:50 and 1:200; both from Dako) and a-synuclein (1:1000; Chemicon). In addition, formalin-fixed samples were cut with a cryostat and the sections were stained with Oil Red O and Sudan black. For comparative studies, four brains from non-neurological cases obtained at autopsy (postmortem delay between 6 and 13 h) were processed in parallel. Semiquantitative studies were carried out in diseased and control cases in the frontal cortex (area 8), head of the caudate and anterior putamen (at the level of the anterior commissure), upper cerebellar vermis, cerebellar hemisphere, superior colliculus, substantia nigra, locus ceruleus, anterior pontine area (at the level of the locus ceruleus), medulla oblongata (at midlevels of the inferior olive) and anterior horn of the cervical spinal cord. The rough numbers of neurones in every selected region in Machado – Joseph disease were compared with age-matched controls. Semiquantitative data
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of neuronal loss were represented: , absence; +, slight; ++, moderate; +++, marked. The number of NIIs was counted at a magnification of 400 in sections immunostained for ubiquitin and slightly counterstained with haematoxylin. Four different fields and three sections per region were examined in every case. The number of NIIs was expressed as a percentage of the total number of neurones. 2.3. Genetic study Frozen samples from selected brain regions (Fig. 4) were homogenised and digested with proteinase K. Samples for biochemical studies corresponded to similar regions used for morphological studies although the levels in the midbrain, pons and medulla were about 5 mm apart from the corresponding morphological sections. DNA was extracted and PCR amplification was performed using primers and conditions as described [14]. The alleles were visualised by autoradiography after electrophoresis in a 6% polyacrylamide denaturing gel.
3. Results 3.1. Neuropathological study 3.1.1. Patient 1 The brain weight was 1200 g. There was mild cerebral atrophy, and marked atrophy of the cerebellum, particularly the vermis, and pons. Loss of pigment was noticed in the substantia nigra and locus ceruleus. Marked neuronal loss was seen in the anterior horn of the spinal cord, motor nuclei of the medulla oblongata, facial nucleus and oculomotor nuclei. Moderate loss of neurones was observed in the periaqueductal gray matter and colliculus. Moderate neuronal loss occurred in the locus ceruleus and substantia nigra. Loss of myelin, and decreased numbers of axons, was found in the anterior roots and nerves of the spinal cord, and dorsal spinothalamic and spinocerebellar tracts. Myelin pallor was observed in the pyramidal tracts. Marked loss of granule and Purkinje cells, and axonal torpedoes, was seen in the cerebellum, mainly in the vermis. Loss of neurones was also evident in the dentate nucleus and inferior olives. Moderate to severe astrocytic gliosis occurred in parallel to neurone loss in all these regions. Ubiquitin-immunoreactive NIIs were observed in neurones of the anterior horn of the spinal cord, nuclei of the medulla oblongata and pons, and, rarely, in the caudate and putamen (Fig. 3A). No ubiquitin-immunoreactive NIIs were seen in the cerebellum, substantia nigra and locus ceruleus. In addition, typical Lewy bodies were found in the substantia nigra, locus ceruleus and nucleus of the vagus nerve. a-Synuclein-immunoreactive neurites were present in the brain stem. A few cortical-type Lewy bodies were found in the limbic system. Interestingly, Lewy bodies have been reported in other cases of Machado– Joseph
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Table1 Neuronal loss and neuronal intranuclear inclusions (NIIs) in selected regions of the CNS in SCA3 patients Patient 1 Neuronal loss Frontal cortex Caudate and putamen Cerebellar hemispheres Cerebellar vermis Midbrain (colliculus) Substantia nigra Locus ceruleus Pons Medulla oblongata Anterior horn
Patient 2 NII
Neuronal loss
+ ++ +++ ++ ++ ++ +++ +++ +++
+++ ++ +
NII +
+ ++ ++ ++ ++ +++ +++ +++
+
++ ++ ++
Neuronal loss: , absence; +, slight; ++, moderate; +++, marked. NII: , absence; +, 1% of neurones; ++, 2 – 10% of neurones; +++, more than 10% of neurones. Although NIIs correlate with neurone loss in the pons, medulla oblongata and anterior horn of the spinal cord, there is no correlation in other brain regions, including the midbrain and cerebellum, in which neurone loss is not accompanied by the presence of NIIs. In contrast, a few NIIs are found in the caudate and putamen in spite of there being no neuronal loss.
disease [15]. Yet a-synuclein pathology in the present case did not differ from that found in diffuse Lewy body disease. Finally, massive hA4 amyloid deposits in the form of diffuse and neuritic plaques occurred throughout the cerebral cortex, hippocampus, amygdala, and, to a lesser extent, the diencephalic nuclei and brain stem. Large numbers of neurofibrillary tangles were present in the entorhinal cortices, hippocampus, amygdala and isocortex. A few neurofibrillary tangles were found in the midbrain. Changes were consistent with Alzheimer’s disease stage V of Braak and Braak. 3.1.2. Patient 2 The brain weight was 1400 g. There was moderate atrophy of the cerebellum and pons. Marked loss of motor neurones was found in the spinal cord (Fig. 4A). Loss of myelin occurred in the anterior nerves of the spinal cord, and the spinothalamic, spinocerebellar and pyramidal tracts. Marked neurone loss occurred in the motor nuclei of the hypoglossus and vagus nerves, pons, facial nucleus and motor nuclei of the VI, IV and III cranial nerves (Fig. 4B). Moderate neurone loss occurred in the substantia nigra (Fig. 4C) and locus ceruleus, but a-synuclein-positive inclusions were not seen in this case. Moderate loss of granule cells and Purkinje neurones, and occasional axonal torpedoes was found in the cerebellum, particularly in the cerebellar vermis (Fig. 4D). Loss of neurones was observed in the superior colliculus. Astrocytic gliosis accompanied neurone loss in all
Fig. 5. Mosaicism of the expanded allele in several brain regions in SCA3 patients. The study was repeated three times to make sure that the results were consistent. Although in this PCR the caudate and putamen did not show amplification, they did in the other two PCR studies.
these regions. Ubiquitin-immunoreactive NIIs were found in the anterior horn of the spinal cord, medulla oblongata and pons (Fig. 3B), but not in the cerebellum, substantia nigra and locus ceruleus. No other abnormalities were noticed in the cerebral cortex and diencephalic nuclei excepting a few NIIs in the caudate and putamen, and superior colliculus. No Alzheimer’s disease changes were present. A comparison of neuronal loss and NIIs in selected regions of the CNS in these cases is summarised in Table 1. 3.2. Genetic study The mean number of CAG repeats was 27/70 in the first patient and 21/74 in the second patient. The variation of the expanded allele size among different CNS regions was F 1– 3 CAG repeats in both patients (Fig. 5). The size of the
Fig. 3. Neuronal intranuclear inclusions, as revealed with ubiquitin immunohistochemistry, in patient 1 (A) and 2 (B). Paraffin section, slight haematoxylin counterstaining 1000. Fig. 4. Neuropathological findings in patient 2. Neuronal loss in the anterior horn of the spinal cord (A), motor nuclei of the vagus nerve (B) and substantia nigra (C). An axonal torpedo (asterisk) is observed in the vermis (D). Haematoxylin and eosin staining 200.
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normal allele did not show variation in the different CNS regions studied.
4. Discussion The main clinical symptoms and neuropathological findings were similar in both cases and consistent with previous observations in Machado –Joseph disease in the same family [13]. One of the patients was very old and had additional lesions of Alzheimer’s disease and diffuse Lewy body disease. The major severity of lesions in the cerebellum in this case is probably related to the more prolonged duration of the disease. We have only found a partial correlation between the neuronal loss and the presence of ubiquitin-immunoreactive NIIs in different CNS areas in both patients with Machado– Joseph disease. Lack of complete correlation was not due to concomitant lesions in the first case. Although it can be hypothesised that in certain areas NIIs might not be ubiquitinated, the absence of NIIs in the surviving neurones of the cerebellum, one of the more severely damaged structures in SCA3, does not support a major role of ubiquitinated NIIs in neurodegeneration. Moderate neuronal loss was also observed in the substantia nigra and locus ceruleus in the absence of ubiquitinated NIIs. A poor correlation between the localisation of NIIs and the distribution and severity of neurodegenerative changes has been previously reported in SCA2 and SCA7 [16,17]. Furthermore, although high level expression of expanded full-length ataxin-3 in vitro causes cell death and formation of NIIs in neurones, there is no close correlation between cell death and NIIs [18]. Finally, recent studies in SCA1 transgenic mice suggest that the presence of ataxin-1 in the nucleus, but not NII formation, is essential to induce the neuronal death [19]. Complementary data have been obtained from Huntington disease, another inherited disease with specific CAG repeat expansion producing abnormal huntingtin expression. The capacities to form NIIs and to induce neurodegeneration are suppressed in transgenic cultured neurones with the expansion for Huntington’s disease after blocking the nuclear localisation of mutant huntingtin [20]. Interestingly, conditions that only suppress NIIs formation without preventing the mutant protein transportation into the nucleus result in a higher neuronal loss [20], thus suggesting that NII formation may reflect a neuroprotective strategy against the mutant protein. The present study has also shown a low degree of mosaicism between different regions of the CNS, and a lack of correlation between the presence of mosaicism and neurone loss and NIIs in specific CNS regions. These findings agree with and complement previous SCA studies showing no correlation between the variation in the CAG repeat size and severity of neurodegeneration, although the expanded CAG repeats were shorter in the cerebellum than in other regions [11,12,21]. These findings suggest that
mosaicism reflects the different cell composition of different CNS regions, but that it is not the cause of the selective neuronal death observed in SCA [10]. In the same line, it is worth stressing that mutant ataxin-3 is widely expressed throughout the brain but its expression is not restricted to regions that show NIIs and neurodegeneration SCA [22]. Taken together, these findings suggest that other mechanisms may be implicated in neuronal vulnerability. It has been shown that the mutant protein has the capacity to interact with the proteasome [8], other nuclear proteins such as the leucine-rich acidic nuclear protein [23], chaperones [24] and proteins involved in DNA repair [25]. Recent experimental studies have shown that cAMP response element binding protein (CREB-binding protein), a histone acetyltransferase, is sequestered by mutant polyglutamines leading to altered protein acetylation in neurones [26,27]. Interestingly, it has been found that the cell death induced by nuclear accumulation of polyglutamines can be mitigated by overexpression of full-length CREB-binding protein [27] or by the administration of other inhibitors of histone deacetylase [26]. It is clear that further studies are necessary to elucidate the mechanisms that modulate ataxins in neurodegeneration.
Acknowledgements This work was supported in part by a grant FIS 99-1118. We wish to thank T. Yohannan for editorial assistance.
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