Introduction

Chorea-acanthocytosis (ChAc) is a hereditary neurodegenerative disorder with peripheral red cell acanthocytosis and choreic involuntary movement and neuropathologically characterised by striatal degeneration. Clinically diagnosed ChAc has been considered to include disorders of various aetiologies [9]. Recently, mutations in the CHAC gene, i.e. the VPS13A gene, have been identified as a cause of ChAc [27, 37]. The VPS13A gene spans a 250-kb region on chromosome 9q21 and consists of 73 exons encoding chorein. Chorein is thought to be involved in protein trafficking at the trans-Golgi network for maintenance of the plasma membrane [27, 33]. The localisation of chorein has recently been demonstrated in wild-type mice by western blotting and immunohistochemical analyses [22]. However, the function of chorein remains unclear. Here, we report the first autopsy case of autosomal-dominant ChAc (AD-ChAc) with heterozygous VPS13A gene mutation, although most ChAc families with VPS13A gene mutations inherit this condition as an autosomal-recessive trait. In addition, we report the pathological distribution of the striatal lesions in comparison with Huntington’s disease (HD), which shows many of the same neurological manifestations and neuropathological findings as ChAc.

Case report

The patient was a 36-year-old Japanese man. Details of the clinical course of this patient and genetic analysis of his family were reported elsewhere [31, 32]. Briefly, this patient presented with generalised seizure at age 25, and developed orolingual movements with self-mutilation of the lips and tongue. He subsequently showed rapid involuntary movements of the limbs, neck and trunk, as well as motor tics at the age of 31. He was unable to sustain attention and occasionally produced snoring sounds and squeals. At the age of 33, the patient began to repetitively check his wallet, make lists and pick his nose until it bled. On admission to a hospital at age 34, he showed mild memory disturbance, attention deficit-hyperactivity disorder, obsessive-compulsive disorder, explosive speech and choreic movements of the limbs and trunk. Laboratory data revealed elevated CK level (1,179 IU/L; normal range 47–212 IU/L) and acanthocytes (10–20%) in the peripheral blood. Brain MRI demonstrated severe atrophy of the striatum bilaterally (Fig. 1). He developed progressive gait disturbance, dysphagia, dysarthria and emaciation at the age of 35. He suddenly died in our hospital at the age of 36. His father, elder sister, paternal grandfather, two paternal aunts and a cousin had been clinically diagnosed as having ChAc. His parents were not consanguineous and his mother was neurologically normal with no acanthocytes. Thus, this family had an autosomal-dominant trait of ChAc. Genetic analysis demonstrated a heterozygous mutation in exon 57 (8,295G to A) of the VPS13A gene in this patient and his elder sister [32].

Fig. 1
figure 1

Brain MR images at the age of 35. a T2-weighted image in an axial section. b T1-weighted image in a coronal section. Atrophy of the caudate nucleus and dilatation of the lateral ventricle are found. The putamen shows hyperintensity on T2-weighted images (arrows)

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Materials and methods

Neuropathological examination

An autopsy was performed 1 h after the patient’s death. The whole brain was fixed with 10% buffered formalin and the brain specimens were embedded in paraffin. Histological examinations were performed on sections, 6 μm thick using several stains: haematoxylin–eosin (HE), Klüver–Barrera (KB), Holzer, methenamine silver, Berlin blue and Gallyas–Braak. Selected sections were also immunostained using the avidin–biotin-peroxidase complex (ABC) method (Vector, Burlingame, CA, USA) with diaminobenzidine as the chromogen. The primary antibodies used were rabbit polyclonal antibodies against glial fibrillary acidic protein (GFAP; Dako, Glostrup, Denmark; 1:2,000) and ubiquitin (Dako, Glostrup, Denmark; 1:1,000), and mouse monoclonal antibodies against phosphorylation-dependent tau (AT8; Innogenetics, Ghent, Belgium; 1:200) and polyglutamine (1C2; Chemicon International, Inc., Temecula, CA, USA; 1:5000).

Distributional analyses of neuronal density in the caudate nucleus

We investigated the distribution of the striatal pathology from the head to the rostral body of the caudate nucleus in this ChAc patient in comparison with three patients with HD (HD 1–3), one with polymyositis (PM1), one with mental retardation (MR1) and one with amyotrophic lateral sclerosis (ALS1) (Table 1). PM1 and MR1 showed no histological abnormalities in the central nervous system. No abnormal findings were found neuropathologically in the striatum of ALS1. A right-sided coronal section through mammillary body, amygdala and body of the caudate (CS1), and 1 and 2 cm rostral sections (CS2 and CS3) from CS1 were prepared (all sections were 6 μm thick; Fig. 2). CS2 included the caudate head, putamen, globus pallidus, anterior commissure and optic tract. CS3 contained the caudate head, putamen and accumbens nucleus. In each KB-stained coronal section, the caudate nucleus was divided into lateral and medial halves. The caudate nucleus in each section was also separated into three areas, i.e. the dorsal, middle and ventral parts. Thus, the caudate nucleus had six separated portions in each coronal section (Fig. 2). We counted the numbers of neurons in three areas of 300-μm squares chosen in a random fashion from each divided portion, and calculated cell numbers per mm2 in 18 portions of the caudate nucleus from CS1 to CS3. We identified morphologically and numbered the neurons, which contained their nucleoli in the examined sections. In this ChAc patient, a 1 cm caudal coronal section (CS0) from CS1 was also examined. CS0 contained the middle body and the tail of caudate nucleus and thalamus, lateral geniculate body and rostral substantia nigra.

Table 1 Subjects for distributional analysis of caudate nucleus and immunohistochemical studies of striatum and globus pallidus
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Fig. 2
figure 2

Scheme of divided areas to measure neuronal density in the caudate nucleus. CS1, CS2 and CS3 include the mammillary body, anterior commissure and accumbens nucleus, respectively. The interval between adjacent coronal sections is 1 cm. The caudate nucleus of each coronal section is divided into six portions. In each coronal section, two broken lines are drawn along the subependymal glial layer and the medial border of the internal capsule. The caudate nucleus is divided into the lateral and medial halves by a thick and solid line drawn along the half points between two broken lines. A thin and solid line is drawn connecting one-third points of the dorsal ends of both broken lines. Another thin and solid line is similarly drawn connecting the two-third points. These five lines divide the caudate nucleus into six areas in each coronal section: dorsolateral (dl), dorsomedial (dm), midlateral (ml), midmedial (mm), ventrolateral (vl), and ventromedial (vm) areas. The neuronal density in each area is calculated

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Immunohistochemical examination of caudate nucleus and globus pallidus

Paraffin-embedded sections 6 μm thick from CS1 of this ChAc patient, two HD patients (HD2 and HD3) and two MR patients (MR1 and MR2) were also immunostained using the avidin–biotin-peroxidase complex (ABC) method (vector) with diaminobenzidine as the chromogen. MR2 showed no histological abnormalities in the central nervous system (Table 1). The primary antibodies used were rabbit polyclonal antibodies against leucine–enkephalin ([Leu5] enkephalin, L–Enk; Cambridge Research Biochemicals, Cambridge, UK; 1:10,000), methionine–enkephalin ([Met5] enkephalin, M–Enk; Cambridge Research Biochemicals; 1:8,000) and substance P (SP; Funakoshi, Tokyo, Japan; 1:2,000). Enk and SP are neurotransmitters of the striatal neurons, which project to the globus pallidus externa and interna, respectively [6].

Results

Neuropathological findings

At autopsy, the brain weighed 1,245 g before fixation. Macroscopically, the caudate nucleus was severely atrophic and the lateral ventricles were dilated. Moderate atrophy was also observed in the putamen and the globus pallidus showed mild atrophy (Fig. 3). Pigmentation of the substantia nigra and locus coeruleus was well preserved. Microscopically, neuronal loss and astrocytic gliosis were severe in the caudate nucleus (Fig. 4a, b), and mild in the putamen and globus pallidus (Fig. 4c). In the caudate nucleus, the small and medium-sized neurons were predominantly lost rather than large-sized neurons. Not neuronal loss but atrophic neurons were diffusely observed throughout the brain except the caudate nucleus, putamen and globus pallidus (Fig. 4d). Some Betz cells showed swelling and a loss of chromatin (Fig. 4d). No senile plaque or iron deposit was found. On immunohistochemical examination, the density of GFAP-positive astrocytes was increased without significant neuronal loss throughout the cerebral cortex and white matter, thalamus, substantia nigra, cerebellum and spinal cord (Fig. 4e). Phosphorylated tau-immunopositive pretangles were found in the temporal cortex. In ubiquitin immunohistochemistry, there was no intranuclear inclusion, nuclear diffuse staining, neurite, axonal spheroid or Lewy body. There was no polyglutamine immunoreactivity throughout the brain.

Fig. 3
figure 3

Gross observation. a convex view of the fixed brain. The frontal lobe is mildly atrophic, b coronal section through the anterior commissure. The caudate nucleus and putamen are severely atrophic, c coronal section through the mammillary body. Atrophy is severe in the caudate head and moderate in the putamen and globus pallidus. The lateral ventricles are dilated. Cerebral cortices show no abnormalities (a–c bars 1 cm)

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Fig. 4
figure 4

Histological and GFAP immunohistochemical observations. a severe neuronal loss and proliferation of astrocytes are found in the caudate nucleus. Small- and medium-sized neurons are affected to a greater extent than large-sized neurons. Remaining neurons are atrophic, b there is remarkable proliferation of GFAP-immunoreactive astrocytes in the caudate nucleus, c the globus pallidus interna shows neuronal loss of mainly small- and medium-sized neurons, d a chromatolytic and swelling neuron (arrow head) and an atrophic neuron (arrow) are found in the 5th layer of the precentral gyrus, e radialised astrocytes are observed in the precentral gyrus. a, c Haematoxylin–eosin; b, e GFAP; d Klüver–Barrera (bars a, b, d 50 μm; c, e 100 μm)

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Distribution of neurons in the caudate nucleus

Neuronal densities in the caudate nucleus from CS1 to CS3 were 52.3 mm−2 in this ChAc patient, 69.8, 123.1, 163.9, 309.6, 286.4, 266.1 mm−2 in HD1, HD2, HD3, PM1, MR1 and in ALS1, respectively. The neuronal density in the caudate nucleus in this ChAc patient was reduced in comparison with all of the HD and the disease control cases. Comparing the divided areas from CS1 to CS3 of this ChAc patient, the neuronal densities in all the dorsal areas were reduced more severely and were lower than those in the ventral areas (Fig. 5a). On the ventral side, the neuronal density in CS2 was relatively preserved in comparison with CS1 and CS3 (Fig. 5a). Neurons were scarce in the middle body and the tail of the caudate nucleus in CS0 of this ChAc patient, showing a neuronal density of 7 mm−2. PM1, MR1 and ALS1 revealed small variations in the neuronal densities in the divided areas and in CS1 to CS3 of the caudate nucleus; these neuronal densities ranged from 200 to 360 mm−2 except in a few portions (Fig. 5e–g). Therefore, the neuronal densities in this ChAc patient were less than 10% in the dorsal caudate nucleus and about 0–80% in the ventral caudate nucleus in comparison with the disease controls.

Fig. 5
figure 5

Neuronal densities at 18 portions of the caudate head. a this patient with chorea-acanthocytosis (ChAc), b Huntington’s disease patient 1 (HD1), c Huntington’s disease patient 2 (HD2), d Huntington’s disease patient 3 (HD3), e polymyositis patient 1 (PM1), f mental retardation patient 1 (MR1), g amyotrophic lateral sclerosis patient 1 (ALS1). CS1 Coronal section through mammillary body, CS2 the next coronal section 1 cm rostral from CS1, CS3 the next coronal section 1 cm rostral from CS2

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In three HD patients, neuronal densities of the caudate nucleus were lower than those in the disease control patients, and the degree of the reduction tended to be more marked in the dorsal and medial sides as compared with the ventral and lateral sides (Fig. 5b–d). In the caudate nucleus, especially in the middle and ventral areas of the HD patients, the neuronal densities of the more caudal sides were lower than those of the rostral sides as follows: CS1 < CS2 < CS3 (Fig. 5b–d). Thus, the distribution of preserved neurons in the caudate nucleus of HD patients tended to be different from that of this ChAc patient (Fig. 5a).

Immunohistochemical findings of caudate nucleus and globus pallidus

On immunohistochemical analysis for M–Enk and L–Enk, the caudate nucleus of our ChAc patient showed marked decreases in granular immunoreactivity and immunopositive neurons (Fig. 6a). The caudate nucleus of two HD patients (Fig. 6b) showed a similar pattern but a smaller number of immunoreactive neurons in comparison with the MR patients, which showed diffuse extracellular granular positive patterns and cytoplasmic immunoreactivity in some of the medium-sized neurons (Fig. 6c). The external globus pallidus showed M–Enk and L–Enk immunoreactivities in a linear pattern (Fig. 6d–f). The linear Enk immunoreactivity in the external globus pallidus was markedly decreased in this ChAc patient (Fig. 6d) and moderately decreased in the HD patients (Fig. 6e) in comparison with the MR patients (Fig. 6f).

Fig. 6
figure 6

Immunohistochemical studies for methionine–enkephalin (M–Enk) and substance P (SP) in the caudate nucleus and globus pallidus. a The caudate nucleus of this patient with chorea-acanthocytosis (ChAc) reveals poor M–Enk immunoreactivity. b The caudate nucleus of Huntington’s disease patient 3 (HD3) shows granular M–Enk immunoreactivity and M–Enk-positive an atrophic neuron (arrow). c The caudate nucleus of mental retardation patient 1 (MR1) shows granular M–Enk immunoreactivity and M–Enk positive medium-sized neurons (arrows). d The globus pallidus externa of this ChAc patient shows linear M–Enk-immunoreactivity. e The globus pallidus externa of HD3 shows granular and linear immunoreactivity of M–Enk. f The globus pallidus externa of MR1 shows linear immunoreactivity of M–Enk, which is stronger than in this ChAc patient and HD3. g The globus pallidus interna of this ChAc patient shows poor immunoreactivity for SP. h The globus pallidus interna of HD3 shows sparse SP immunostaining in granular and linear patterns. i The globus pallidus interna of MR1 shows strong positive immunoreactivity in a linear pattern for SP. a–f M–Enk; g–i SP (bars a–i 100 μm)

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On SP immunohistochemistry, the internal globus pallidus showed a linear immunopositive pattern (Fig. 6g–i). SP immunoreactivity was scarce in our ChAc patient (Fig. 6 g), and moderately decreased in the HD patients (Fig. 6 h) in comparison with the MR patients (Fig. 6i).

Discussion

This is the first report of an autopsy case of AD-ChAc in which a heterozygous VPS13A mutation was confirmed. Besides AR- and AD-ChAc, HD [24, 42], dentatorubro-pallidoluysian atrophy [42], HD like-2 [17, 25, 41], neuroferritinopathy [7, 8], McLeod syndrome [4, 40] and pantothenate kinase-associated neurodegeneration [18] show chorea as a major clinical sign and remarkable neuropathological changes in the striatum. Molecular genetics and neuropathological features of these disorders are shown in Table 2. The neuropathological features of clinically diagnosed ChAc reported previously include marked atrophy, neuronal loss and gliosis of the caudate nucleus and putamen [2, 3, 5, 19, 21, 29, 30, 38]. However, ChAc is a syndrome that includes some aetiologically and genetically different disorders [9]. VPS13A gene mutations are the major causes of ChAc and have been found in several ChAc families [26, 27, 37]. Although most ChAc families with VPS13A gene mutations are homozygous for the mutated alleles and show autosomal recessive transmission, the present family showed an autosomal-dominant trait [31, 32]. This AD-ChAc patient showed marked atrophy, neuronal loss and gliosis in the striatum and mild changes in the internal and external globus pallidus. Our findings were similar to those of earlier reports of autosomal recessive ChAc (AR-ChAc) cases with VPS13A mutations, although the sites of the mutations in VPS13A have not been described in these reports (Table 3) [2, 5, 9, 19, 29, 30, 38]. Furthermore, this patient showed a broad distribution of atrophic neurons and astrocytic gliosis throughout the whole brain along with ballooning and a loss of chromatin in Betz cells. In addition, GFAP-positive astrocytes showed diffuse proliferation throughout the whole brain and spinal cord. Such extensive neuropathological involvement as observed in this patient has not been described in previous reports of ChAc with VPS13A mutation and may be specific to this patient. Differences in neuropathological findings between AD-ChAc and AR-ChAc may reflect the different pathomechanisms of neurodegeneration.

Table 2 Molecular genetics and neurpathological features of diseases presenting with chorea
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Table 3 Neuropathological findings of chorea-acanthocytosis with a mutation or mutations of the VPS13A gene
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Recently, we reported the results of an immunohistochemical study with anti-chorein antibodies in skeletal muscles from three ChAc patients with heterozygous VPS13A mutations including the present AD-ChAc patient [33]. In this previous study, the skeletal muscles from AD-ChAc patients showed uneven and discontinuous chorein-immunoreactivity along the sarcolemma, while chorein-immunoreactivity was found in a linear distribution along the sarcolemma and appeared as speckles in the sarcoplasm in HD, McLeod syndrome and in a normal control subject [33]. We suggested that abnormalities in the function or distribution of chorein may be responsible for neurodegeneration in AD-ChAc. Immunohistochemical analyses of paraffin-embedded brain sections for chorein are currently underway in our laboratory.

In the striatum, small and medium-sized neurons were predominantly lost in this patient, as previously reported in AR-ChAc and HD patients [21, 24]. The neuronal density of the caudate nucleus in this patient was higher on the ventral than on the dorsal side. These observations suggest that degeneration of the caudate nucleus in AD-ChAc extends from the dorsal to the ventral side, which is similar to observations in HD [24]. Interestingly, the caudate nucleus of CS2 through the anterior commissure showed the highest neuronal density in this ChAc patient, while that of CS3 through the accumbens nucleus showed the highest neuronal density in HD patients, especially at the areas of more ventral sides. These findings suggest that the caudate nucleus in this AD-ChAc patient would be more vulnerable on both caudal and rostral sides, while the caudal caudate nucleus would be more degenerated in HD. These differences in vulnerability patterns might reflect differences in the degenerative mechanisms between AD-ChAc and HD. In earlier pathological studies of AR-ChAc, neuronal distributions in the striatum have not been investigated or described [2, 5, 19, 29, 30, 38]. Further studies on the other AD-ChAc patients are needed to confirm whether AD-ChAc show unique patterns of neurodegeneration.

Previous studies have confirmed that the medium-sized GABA/Enk-containing spiny striatal neurons, which project to the globus pallidus externa, are most vulnerable in HD [1, 11, 14, 16, 26, 39], while there is selective sparing of interneurons containing somatostatin, neuropeptide Y, and NADPH-diaphorase [10, 12, 13, 20]. Although it was considered that cholinergic interneurons were spared in HD, several studies have suggested dysfunction of cholinergic system in HD without apparent loss of cholinergic neurons in the striatum [15, 35, 36]. The medium-sized GABA/Enk-containing spiny striatal neurons are located in the dorsomedial striatum and the preferential loss of these neurons is thought to be correlated with the appearance of choreic movement [1, 28] and neuropsychiatric problems, including obsessional behaviour [34]. However, the correlation between huntingtin and the vulnerability of these medium-sized GABA/Enk-containing neurons remains unclear. As ChAc shares most of the clinical and pathological findings with HD, both ChAc and HD may share some neurodegenerative process. Our AD-ChAc patient showed more extensive involvement of SP as well as Enk-containing neurons as compared with the two HD patients, suggesting that there might be differences in degeneration of the striatal neurons.

Moreover, experiments using a gene-targeted mouse model of AR-ChAc revealed an increased level of gene expression of gephyrin, which is known to be a GABAA receptor-anchoring protein, in comparison with wild-type controls [23]. To determine the pathomechanisms underlying the neuropathology of AD-ChAc as well as AR-ChAc, further studies of ChAc brains are necessary, including analyses of the expression of chorein and related molecules.