Sca7 Research Paper

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Abstract

Autosomal dominant cerebellar ataxia with progressive macular degeneration is caused by a CAG/glutamine repeat expansion in the SCA7 gene/protein. Neuronal intranuclear inclusions were detected in the brain of an early onset SCA7 case with the 1C2 antibody directed against an expanded polyglutamine domain. Nuclear inclusions were most frequent in the inferior olivary complex, a site of severe neuronal loss in SCA7. They were also observed in other brain regions, including the cerebral cortex, not considered to be affected in the disease. Using confocal microscopy we showed that some inclusions were ubiquitinated, but to varying degrees, ranging from <1% in the cerebral cortex to 60% in the inferior olive. In addition, we also observed cytoplasmic staining using the 1C2 antibody, particularly in the supramarginal gyrus, the hippocampus, the thalamus, the lateral geniculate body and the pontine nuclei. These data confirm that the presence of intranuclear inclusions in neurons is a common characteristic of disorders caused by CAG/polyglutamine expansions, but unlike what has been reported for Huntington's disease, SCA1 and SCA3/MJD, in SCA7 the inclusions were not restricted to the sites of severe neuronal loss.

Introduction

Spinocerebellar ataxia type 7 is a neurodegenerative disorder characterized by neural loss, mainly in the cerebellum and regions of the brainstem and, particularly, the inferior olivary complex (1–3). The disease is caused by an unstable CAG repeat expansion in the 5′-translated region of a gene of unknown function and is associated with marked anticipation (4). The number of CAG/glutamine repeats in the pathological protein varies from 37 to >200 (4–7). SCA7 is the eighth disorder shown to result from a CAG/polyglutamine expansion. The group also includes the dominant ataxias SCA1, SCA2, SCA3 and SCA6, as well as spinal and bulbar muscular atrophy, Huntington's disease (HD) and dentatorubral-pallidoluysian atrophy (8–17). It has been postulated that these disorders result from a common gain-of-function of the expanded polyglutamines (18,19). In most of the other cases examined, as in SCA7 (4), the pathological genes/proteins are widely expressed in the brain and not restricted to the regions affected by disease. It was recently demonstrated, however, in patients suffering from Huntington's disease, SCA1 and SCA3 that intranuclear inclusions develop mainly in neurons of regions that are affected by the disease (20–22). The inclusions stain positively for the corresponding polyglutamine proteins as well as for ubiquitin. Furthermore, lines of HD transgenic mice that exhibit symptoms contain intranuclear inclusions in neurons of brain regions known to be affected in HD, while asymptomatic lines do not (23). The formation of nuclear inclusions precedes the neurological pheno-type in the HD animals and has been proposed to represent an important step in the neurodegenerative process (23). Why inclusions form in some brain regions but spare other areas where the mutant protein is expressed at comparable levels is not known. It has been proposed that other factors might be involved in formation of the inclusions and determine their cellular specificity. Recently it was shown that the leucine-rich acidic nuclear protein (LANP) interacts with SCA1. LANP is predominantly expressed in cerebellar Purkinje cells, a primary site of pathology in SCA1 as well as in SCA7 (24). It was also demonstrated that SCA1 and LANP are present in the same subnuclear structures when co-transfected into COS-7 cells.

In this immunohistochemical study in an early onset case of SCA7 we have used antibodies against expanded polyglutamines (1C2) (25) and against ubiquitin. We have found intranuclear inclusions in both spared and affected regions. We have also analyzed the inclusions using an antibody directed against the human homolog of LANP, pp32.

Results

Neuropathology of the SCA7 brain

Gross examination. The brain weighed 1168 g. The inferior olive was small. There was severe atrophy of the cerebellar vermis. The dentate nucleus appeared atrophic. The optic nerve was of normal size. The cerebral cortex was normal. The ventricles were not dilated.

Microscopic examination. In the cerebellum neuronal loss was severe in the Purkinje cell layer, where the Bergmann glia was prominent. Neuronal loss was also marked in the nucleus dentatus. The hilus and the mantle were pale. The superior cerebellar peduncle was atrophic, the inferior cerebellar peduncle was pale and atrophic, but the middle cerebellar peduncle was normal. In the inferior olivary complex neuronal loss was extensive and was associated with a marked astrocytic gliosis. The hilus was pale and the mantle was spared. Moderate neuronal loss and gliosis were also observed in the hypoglossal nucleus. The pyramidal tract was normal, while the median lemniscus was pale. The nucleus gracilis and nucleus cuneiformis were gliotic. The substantia nigra and the locus coeruleus were slightly pigmented; some pigment was seen in the extracellular space, associated with a mild neuronal loss. The thalamus and striatum were normal. Myelin appeared remarkably spared in the optic tract, while the lateral geniculate body was gliotic without prominent neuronal loss. A slight astrogliosis was also noted in the primary visual cortex. The rest of the cerebral cortex appeared normal. The number of neurons in the red nucleus was normal, although it appeared gliotic. Neuronal density was close to normal in the pontine nuclei, which were, however, small in size. The central region of the basis pontis was pale and vacuolar on microscopic examination. Central pontine myelinolysis was diagnosed in relation to the terminal condition of the patient.

Immunohistochemistry

1C2 staining in SCA7 brain neurons. With the polyglutamine-specific 1C2 antibody we stained intranuclear inclusions in neurons in several regions of the SCA7 brain (Table 1, Figs 1A–C and 2A and B), but not in an age-matched control (data not shown). The intranuclear structures were most frequent in neurons of the inferior olive, the lateral geniculate body and substantia nigra, where 17, 12 and 10% of the neurons respectively contained inclusions (Table 1). In the cerebellum, a severely affected site in SCA7, almost all Purkinje cells were lost and very few nuclear inclusions could be found in those that remained (Table 1). A substantial number of nuclear inclusions could be seen in regions of the cerebral cortex, including the supramarginal gyrus and the insula, even though no obvious neuronal loss was observed in these structures (Table 1 and Fig. 2). The nuclear inclusions were restricted to neuronal cells in all regions examined. Most frequently one inclusion was observed per nucleus, with a mean size of 3 ± 1 µm, but neurons with two nuclear inclusions could also be seen. Cytoplasmic staining was also detected in neurons in some brain regions, including the supramarginal gyrus, hippocampus, thalamus, geniculate body and pontine nuclei, but was not associated with inclusion-like structures and was not restricted to neurons containing nuclear inclusions (Fig. 3A–C). Cytoplasmic staining was not observed in the age-matched control.

Ubiquitination of SCA7 nuclear inclusions. We also found that some nuclear inclusions in the SCA7 brain stained positively for ubiquitin with an antibody against ubiquitin (Table 1). By confocal immuno-fluoroscence we confirmed the co-localization of ubiquitin and the expanded polyglutamine protein in the same nuclear inclusions (Fig. 4A–C). The degree of ubiquitination varied in the double stained sections and in six out of 15 randomly examined polygutamine-positive nuclear inclusions in the inferior olive no ubiquitin could be detected. We also calculated the number of 1C2- and ubiquitin-positive inclusions in adjacent sections of all brain regions examined. In all regions of the cerebral cortex, except the insula, very few if any of the inclusions were ubiquitinated (Table 1).

SCA7 nuclear inclusions do not contain LANP. With an antibody against human LANP, anti-pp32, we determined that LANP was not present in the nuclear inclusions of SCA7 in the inferior olive. However, in selective neurons of the brainstem in both SCA7 and control brains (data not shown) nuclear staining not associated with inclusions could be observed.

Discussion

The mechanism by which proteins with polyglutamine expansions cause neurodegenerative disease is not known. Formation of aggregates by self-association or by interaction with other factors has been proposed. Recent reports describing nuclear inclusions in brains of HD, SCA3/MJD and SCA1 patients (20–22), as well as the data presented in this report, confirm the existence of nuclear inclusions as a common feature of polyglutamine disorders. In this study we demonstrated the presence of neuronal nuclear inclusions in several brain regions of an early onset SCA7 patient, known to carry an expanded allele with 85 CAG repeats. The polyglutamine-specific antibody 1C2 has been demonstrated to specifically recognize polyglutamines of pathological size (25) and has been used to detect the expanded 130 kDa ataxin-7 protein on western blots of protein extracts from the cerebellum of the same patient (26). Whether the nuclear inclusions also contain the normal ataxin-7 protein has not yet been determined.

Figure 1

Neuronal intranuclear inclusions (see arrows) in regions of the brainstem in a juvenile case of SCA7 detected by the polyglutamine-specific mAb 1C2. (A) Inferior olivary complex. Bar 50 µm. (B) Inferior olivary complex. Bar 25 µm. (C) Pontine nuclei. Bar 25 µm.

Figure 1

Neuronal intranuclear inclusions (see arrows) in regions of the brainstem in a juvenile case of SCA7 detected by the polyglutamine-specific mAb 1C2. (A) Inferior olivary complex. Bar 50 µm. (B) Inferior olivary complex. Bar 25 µm. (C) Pontine nuclei. Bar 25 µm.

Figure 2

Neuronal intranuclear inclusions (see arrows) in regions of the cerebral cortex in a juvenile case of SCA7 detected by the polyglutamine-specific mAb 1C2. (A) Motor cortex. (B) Supramarginal gyrus. Bar 25 µm.

Figure 2

Neuronal intranuclear inclusions (see arrows) in regions of the cerebral cortex in a juvenile case of SCA7 detected by the polyglutamine-specific mAb 1C2. (A) Motor cortex. (B) Supramarginal gyrus. Bar 25 µm.

Unlike nuclear inclusions in HD, SCA1 and SCA3/MJD patients (20–22), which were mainly observed in neurons affected by disease, those in the SCA7 patient were not restricted to regions affected by disease. They were most frequent in neurons of the inferior olive, severely affected in SCA7, but could also be observed at high frequency in brain regions not known to be lesioned in SCA7, including the supramarginal gyrus and the insula. In the cerebellum, another region with extensive neuronal loss, so few Purkinje cells remained that accurate estimation of the frequency of inclusions was impossible. One might also argue that the relative absence of inclusions in highly affected brain regions was due to loss of the neurons. This would not account, however, for the inferior olive, where neuronal loss was severe and intranuclear inclusions were frequent.

Cytoplasmic staining was observed in neurons of regions, such as the supramarginal gyrus, the lateral geniculate body and the pontine nuclei, where intranuclear inclusions were frequent, although not necessarily in neurons containing inclusions. It was also observed, however, in the thalamus, where no inclusions were found and no neuronal loss or gliosis were detected. The presence of 1C2 immunoreactivity in the cytoplasm was unexpected. It has never been reported in other diseases with intranuclear inclusions caused by expanded trinucleotide repeats and our previous studies of SCA7 lymphoblastoid cells by western blot suggested that the expanded ataxin-7 protein is exclusively targeted to the nucleus (25,26). This is apparently not the case in a small subset of neurons.

Figure 3

Cytoplasmic staining of neurons in different brain regions of a juvenile case of SCA7 detected by the polyglutamine-specific mAb 1C2. (A) Hippocampus. (B) Striatum. (C) Cerebellum. Bar 25 µm.

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