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Neuronal atrophy and synaptic alteration in a mouse model of dentatorubral–pallidoluysian atrophy

Kenji Sakai , Mitsunori Yamada , Toshiya Sato , Masahito Yamada , Shoji Tsuji , Hitoshi Takahashi
DOI: http://dx.doi.org/10.1093/brain/awl182 2353-2362 First published online: 4 August 2006

Summary

Dentatorubral–pallidoluysian atrophy (DRPLA) is a hereditary spinocerebellar degeneration caused by expansion of a CAG repeat in the disease protein. Despite the restricted and stable brain lesions, DRPLA patients show a variety of clinical symptoms and the brain exhibits generalized atrophy. In previous studies of DRPLA, we proposed that intranuclear diffuse accumulation of the mutant protein is a significant pathological feature of neurons, and that the variable prevalence of this pathology may be relevant to the variation of symptoms observed in patients with different repeat sizes. In this study, to elucidate the pathogenesis of the brain atrophy in DRPLA, we conducted morphological and statistical analyses of neurons affected by the polyglutamine pathology in DRPLA transgenic (Tg) mice with 129 polyglutamine stretches. Golgi-impregnated pyramidal neurons in cerebral cortical layer V of 15-week-old Tg mice showed significant atrophy of the perikarya and dendrites. Dendritic spines were decreased in number and size, and showed a change in morphology resulting in dominance of stubby spines. Interestingly, dendritic arborization was preserved. Electron microscopy revealed that axons in the pyramis and corpus callosum were also atrophic. The number of axonal microtubules was preserved; however, the inter-microtubule spacing was significantly decreased. In the neuropil of cerebral cortical layers II and III, atrophy of the pre-synaptic areas and lengths of the post-synaptic density was detected, but synaptic vesicle diameter was preserved. These results suggest that neuronal atrophy is an essential feature of the cell pathology in DRPLA and that this is closely related to polyglutamine pathogenesis and development of the clinical phenotype.

  • dentatorubral–pallidoluysian atrophy
  • polyglutamine
  • pathology
  • morphometry

Introduction

Dentatorubral–pallidoluysian atrophy (DRPLA) is a hereditary spinocerebellar degeneration caused by expansion of a CAG repeat encoding a polyglutamine tract in the disease protein (Koide et al., 1994; Nagafuchi et al., 1994). For over 20 years after characterization of the disease (Naito and Oyanagi, 1982), two major clinicopathological problems have still remained unexplained. One is the discrepancy between the variety of clinical manifestations and the uniformity of the brain lesions. DRPLA patients show various symptoms, such as myoclonus, epilepsy, ataxia, choreoathetosis and dementia, and the combinations of these symptoms are determined by the age at onset (Naito, 1990). Patients with earlier onset (generally below the age of 20 years) show progressive myoclonus, epilepsy and mental retardation (juvenile type, as classified by Naito). Patients with late onset (over the age of 40 years) predominantly show cerebellar ataxia and dementia (late-adult type). Patients in whom the disease appears between the third and fifth decades belong to an intermediate type and usually show ataxia and choreoathetosis (early adult type). However, the neuropathology in these patients with different phenotypes shows a relatively uniform pattern of lesion distribution, and neuronal loss is generally restricted to the dentatorubral and pallidoluysian systems (Oyanagi, 2000). This stable pathological pattern is unlikely to account for the phenotypic variations in DRPLA, and the two-system distribution of the lesions gives no satisfactory explanation for some of the specific symptoms such as dementia or epilepsy. The other unexplained aspect of DRPLA is that the amount of CNS tissue in affected patients is quite small throughout the brain and spinal cord, despite the restricted nature of the brain lesions. Brain weights of DRPLA patients often become <1000 g (Naito and Oyanagi, 1982). Magnetic resonance imaging studies have suggested that the phenomenon is not a developmental abnormality but a progressive atrophy of the CNS (Koide et al., 1997). These clinicopathological features indicate that lesion distribution evaluated in terms of neuronal loss does not fully reflect the essential brain pathology of DRPLA. Recent studies have shown that the observed phenotypic variation depends on the expansion size of a CAG repeat in the DRPLA gene—longer expansion resulting in earlier onset and a severer phenotype (Ikeuchi et al., 1995)—suggesting that a certain neuronal pathology related to polyglutamine expansion may play a pivotal role on the development of DRPLA phenotypes.

Recently, it was demonstrated that the neuronal intranuclear inclusions (NIIs) in DRPLA brains contain the DRPLA protein, atrophin-1, as well as expanded polyglutamine stretches (Hayashi et al., 1998; Igarashi et al., 1998; Yamada et al., 2001). Although the widespread occurrence of NIIs in the CNS suggests that neurons are affected much more widely than was recognized previously, the paucity of NIIs also makes the significance of inclusion formation in DRPLA pathogenesis unclear. Several experimental studies have suggested that NII formation may be a cellular reaction to reduce the toxic effect of the mutant protein (Everett and Wood, 2004). More recently, we have demonstrated that diffuse intranuclear accumulation of the mutant DRPLA protein affects many neurons in a wide area of the CNS including the cerebral cortex and that the prevalence of this pathology changes dynamically in relation to CAG repeat size. The results suggest that the novel lesion distribution revealed by the diffuse nuclear labelling may be responsible for a variety of clinical features, such as dementia and epilepsy in DRPLA (Yamada et al., 2001).

We have recently created transgenic (Tg) mice harbouring a single copy of full-length human mutant DRPLA gene with 129 CAG repeats (Q129), which were generated through en masse expansion of 76 CAG repeats in vivo (Sato et al., 1999a, b). The Q129 mice exhibited severe neurological phenotypes similar to juvenile-onset DRPLA patients, characterized by cerebellar ataxia, myoclonus and epilepsy, and died by 16 weeks of age. Despite the strong neurological phenotype of Q129 mice, obvious neuronal loss was not observed in any brain regions. The most striking finding was that the diffuse polyglutamine accumulation in neuronal nuclei that was detected in regions including the basal ganglia at as early as post-natal Day 4 and became more prominent and widespread with age showed tight correlation with the disease onset and progression. In contrast, the formation of NIIs that was detectable only after 9 weeks of age did not show such correlations. Interestingly, despite preservation of the neuronal population, Q129 mice showed progressive and generalized brain atrophy that commenced synergistically with the intranuclear accumulation of mutant proteins (T. Sato et al., submitted for publication). In the present study, to elucidate the pathogenesis of the brain atrophy in DRPLA, we investigated the morphological changes of cortical neurons affected by polyglutamine pathology in the Q129 DRPLA Tg mice.

Material and methods

Neuropathological examination and immunohistochemistry

For neuropathological observations, we examined the brains of Q129 Tg mice, Q76 Tg mice and non-Tg mice at 14 weeks of age. Mice were deeply anaesthetized with diethyl ether and perfused transcardially with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed from the crania and immersed in the same fixative overnight. Multiple tissue blocks were then cut from the fixed brains and embedded in paraffin. Histological examination was performed on 4-μm-thick sections stained with haematoxylin and eosin, and Klüver–Barrera. Selected sections were immunostained using a mouse monoclonal antibody against expanded polyglutamine stretches (1C2, Chemicon, Temecula, CA, USA; 1 : 16 000) as described previously (Yamada et al., 2001). Diaminobenzidine was used as the chromogen.

In Q129 Tg mice, polyglutamine accumulation in neuronal nuclei was observed in most of the brain regions including cerebellar Purkinje cells (Sato et al., 1999b). In Q76 mice, on the other hand, diffuse intranuclear accumulation of polyglutamine in neurons was milder compared with that of Q129 mice, and no intranuclear inclusions were formed in the brain (Sato et al., 1999a). More than 70% of neurons in the cerebral cortical layers III, IV and VI were immunolabelled for polyglutamine at 14 weeks of age; however, no labelling was observed in Purkinje cells. Therefore, to elucidate the relationship between different types of polyglutamine repeat accumulation and neuronal morphology, we selected neurons from layer VI in the primary motor cortices and Purkinje cells in the cerebellum. A total of 187 neurons from Q76 Tg mice and 190 neurons from non-Tg mice were analysed from the cerebral cortex. A total of 120 Purkinje cells from Q76 Tg mice and 118 Purkinje cells from non-Tg mice were analysed from the cerebellum. Neurons were observed using a light microscope (AX80; Olympus, Osaka, Japan) with a ×40 objective, and their digital images were captured using a digital camera (DP70; Olympus, Osaka, Japan) from sections stained with Klüver–Barrera. For each neuron, perikaryal area was evaluated using the analySIS image analysis software package, version 3.2 (Soft Imaging System, Münster, Germany).

Golgi impregnation

Our previous study of human DRPLA brains indicated the involvement of cerebral cortical neurons in the polyglutamine pathogenesis (Yamada et al., 2001) and suggests that the cortical lesions may be responsible for some symptoms such as dementia in DRPLA. Similar polyglutamine pathology was also detected in Q129 Tg brains (see the section ‘Neuropathological findings in Tg mice’ under Results). To elucidate morphological changes of soma, dendrites and axons of the same neuronal population that is affected by polyglutamine pathology in DRPLA model mouse, we selected cerebral cortical neurons for morphometric analyses by Golgi impregnation and electron microscopy.

For analysis of the perikarya, dendrites and spines of neurons, Golgi impregnation was applied to brains obtained from five Q129 Tg and five non-Tg male mice at 15 weeks of age. The mice were deeply anaesthetized with diethyl ether and perfused transcardially with PBS, followed by 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer. The brains were removed and divided into two coronal sections at the level of Ammon's horn and stored in the same fixative at 4°C. After fixation, the samples were incubated in a chromating solution of 3.5% potassium dichromate for ∼120 h. Metal impregnation was then done in 0.75% silver nitrate solution for 120 h. Finally, the brains were dehydrated in increasing concentrations of ethanol, immediately embedded in celloidin and sectioned at 50 μm on a vibratome. For the following studies, Golgi-impregnated pyramidal neurons were selected from layer V in the primary motor and primary sensory cortices.

Perikaryal area

A total of 68 neurons from Tg mice and 61 neurons from non-Tg mice were observed using a light microscope (AX80) with a ×40 objective, and their digital images were captured using a digital camera (DP70). For each neuron, perikaryal area was evaluated using the WinROOF image analysis software package, version 3.5 (Mitani, Fukui, Japan).

Dendritic diameter

A total of 60 neurons from each Tg and non-Tg mouse were observed using the same method as that for the perikaryal area. For each neuron, we evaluated the diameter of the apical dendrite at a level of 50 μm distal to its origin, using the analySIS image analysis software package, version 3.2.

Basal dendritic spine analysis

Spine density and size change as a function of distance from the neuronal soma. In Golgi-stained sections, we could not compare similar segments of apical dendrites between many neurons. Therefore, to establish spine densities in dendrites of cortical neurons, we analysed neurons whose basal dendritic trees were entirely filled with Golgi deposits as described in Benavides-Piccione et al. (2002). A total of 20 pyramidal neurons from each Tg and non-Tg mouse were observed using an AX80 light microscope with a ×40 objective, and digital images were captured using a DP70 digital camera. Two concentric circles with radii of 20 and 60 μm were drawn on the images centred on each cell body, using Sholl's concentric circle method (Sholl, 1953). For each dendritic segment between the two circles (Fig. 1A and B), the number of all visible spines and dendritic lengths were taken, and the spine density was calculated. We counted a total of 1399 and 2009 spines in Tg and non-Tg mice, respectively. Spines were classified into five types according to their shapes: thin, stubby, mushroom, wide or ramified (González-Burgos et al., 2005). For evaluation of the percentages of the various spine types, we observed a total of 10 pyramidal neurons from each Tg and non-Tg mouse using an AX80 light microscope with a ×100 objective and examined a total of 586 and 915 spines in Tg and non-Tg mice, respectively. For thin and mushroom-type spines, we further evaluated the head area and length of a total of 80 spines using the analySIS program.

Fig. 1

A basal dendrite of a pyramidal neuron in layer V of the primary motor cortex in DRPLA Tg (A) and non-Tg (B) mice. The number of spines is apparently reduced in A. In each panel, segments of two concentric Sholl rings (Sholl, 1953), drawn with radii of 20 and 60 μm centred on the neuronal body, are illustrated. Golgi impregnation.

Dendrite counts

We analysed neurons whose basal dendritic trees were entirely filled with Golgi deposits. A total of 11 neurons from each Tg and non-Tg mouse were observed using an AX80 inverted microscope at ×20 magnification. Fourteen to 16 serial section images of each neuron were taken using a VB-7010 CCD digital camera (Keyence, Osaka, Japan), and whole-cell images were composed using the supplied VB-7000 software (Keyence). Basal dendrites of each neuron were analysed quantitatively using Sholl's concentric circle method (Sholl, 1953) (Fig. 2). Three concentric circles centred on the cell body were drawn with radii of 20, 40 and 60 μm, and the number of basal dendrites intersecting each circle was counted (Yamada et al., 1988). The number of dendritic intersections of each concentric circle and the total number of dendritic intersections were compared between Tg and non-Tg mice.

Fig. 2

A pyramidal neuron in layer V of the primary motor cortex of a non-Tg mouse. Three concentric Sholl rings are drawn with radii of 20, 40 and 60 μm centred on the neuronal body (Sholl, 1953). Golgi impregnation; scale bar = 20 μm.

Electron microscopy

For analyses of axons and synapses, we studied five Q129 Tg and five non-Tg male mice at 14 weeks of age. The mice were deeply anaesthetized with diethyl ether and perfused transcardially with PBS, followed by 3% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer, and the brains were then removed and immersed in the same fixative overnight. Axial sections of the medulla oblongata, mid-sagittal sections of the corpus callosum and coronal sections of the cerebral cortex at the level of Ammon's horn were cut from the fixed brains. The sections were post-fixed with 1% osmium tetroxide, dehydrated with a graded ethanol series and embedded in Epon 812. From these sections, the pyramis, corpus callosum and layers II and III of the primary motor cortex were cut into ultrathin sections, stained with uranyl acetate and lead citrate and examined with a Hitachi H-7100 electron microscope. Photographs of random positions from these specimens were taken at ×10 000 magnification.

Analysis of axons

Axon diameter

We evaluated the minimum diameter of all the myelinated axons whose transverse sections were entirely observed in photographs in the pyramis and corpus callosum using the analySIS program. In Tg and non-Tg mice, 1663 and 1310 axons in the pyramis and 1907 and 1653 axons in the corpus callosum were analysed, respectively.

Distance between microtubules in axons

For examining the subcellular structural changes in axons, we evaluated the numbers and densities of microtubules and distances between the centres of nearest adjacent microtubules in axons in the pyramis. For this purpose, we selected axons in which most of the microtubules were cut perpendicularly to their axes. For each axon, we evaluated the cross-sectional area and the total number of microtubules, and then the microtubule density was calculated. The cross-sectional areas and inter-microtubule distances were measured with the analySIS program. In total, 24 and 23 myelinated axons in Tg and non-Tg mice were analysed, respectively.

Analysis of synapses

Pre-synaptic area and post-synaptic density

In layers II and III of the primary motor cortex, we evaluated pre-synaptic areas and lengths of the post-synaptic density (PSD). For this purpose, we analysed synapses that showed both pre- and post-synaptic elements in which clear PSDs were present at the synaptic junction. In the case of post-synaptic elements that contained more than one PSD, we measured their lengths separately. In Tg and non-Tg mice, 427 and 391 pre-synaptic areas and 574 and 477 PSDs were evaluated, respectively, using the analySIS program.

Synaptic vesicles

We evaluated the diameters of synaptic vesicles contained in the pre-synaptic elements that were analysed as described in the section ‘Pre-synaptic area and post-synaptic density’. Twelve pre-synaptic elements that contained spherical synaptic vesicles were selected randomly from each Tg and non-Tg mouse, and the minimum diameters of the vesicles were measured with the analySIS program.

Statistical analysis

Comparisons of each factor between Tg and non-Tg mice were performed using Student's t-test, Welch's t-test and Mann–Whitney U-test. The calculations were conducted using StatView version 5.0 (SAS Institute, Cary, NC, USA) on a personal computer.

Results

Neuropathological findings in Tg mice

Q129 Tg mice showed severe brain atrophy (Fig. 3A and B) including the striatum and cerebellar dentate nucleus but disclosed no apparent loss of neurons in any CNS regions. Interestingly, it was evident that neuronal densities in the grey matter including the cerebral cortex and striatum increased compared with those of non-Tg mice. Immunohistochemistry for expanded polyglutamine stretches revealed diffuse nuclear staining in many neurons in multiple brain regions (Fig. 3C). In layers II–VI of the cerebral cortex, nearly all the neurons were affected by the polyglutamine intranuclear accumulation. The brains of Tg mice showed reduction of the neuropil, and immunolabelled neurons showed deformity of the nuclei (Fig. 3D).

Fig. 3

Brain specimens of DRPLA Q129 Tg mice (A, C, D, E) and non-Tg mice (B) at 14 weeks of age. In A and B, the Tg mice show severe brain atrophy. In C, intense nuclear labelling of neurons expands throughout the cerebrum. In D, the layer V of the primary motor cortex shows diffuse labelling of all the neuronal nuclei. Nuclear deformity is seen in some neurons (arrowheads). NIIs are also detectable in many neurons (arrows). (E) Electron microscopy of neurons in the layer V of the primary motor cortex shows the presence of a filamentous intranuclear inclusion (arrow) and marked nuclear membrane indentations (arrowheads). Klüver–Barrera stainings (A and B). Immunohistochemistry for expanded polyglutamine stretches stained with monoclonal antibody 1C2 (C and D). Scale bar = 1 mm for A, B and C, 10 μm for D and 1 μm for E.

The results of morphometric analyses were summarized in Table 1. The perikaryal area of cortical neurons in Q76 Tg mice were significantly reduced to 85.7% of those in non-Tg mice, whereas there was no difference on the perikaryal area of Purkinje cells between Tg and non-Tg mice.

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Table 1

Perikaryal area

Q76 Tg (mean ± SD)Non-Tg (mean ± SD)P-value
Cortical neurons (μm2)111.2 ± 35.4 (85.7%)129.8 ± 30.1<0.0001
Purkinje cells (μm2)161.3 ± 22.3167.1 ± 27.60.0761
  • Parentheses indicate percentage compared with the value in non-Tg mice.

Perikarya, dendrites and spines of neurons

Golgi impregnation clearly revealed atrophy of pyramidal neurons in cerebral cortical layer V in Tg mice (Fig. 4A and B). The results are summarized in Table 2. The perikaryal area, dendritic diameter and spine density (Fig. 1A and B) of neurons in Tg mice were reduced to 81.3, 79.5 and 65.7% of those in non-Tg mice, respectively. Tg mice also showed a change of dendritic spine morphology, in which the percentages of thin and mushroom-type spines decreased and stubby spines became dominant. The spine head areas of thin and mushroom-type spines in Tg mice were reduced to 66.0 and 69.3% of those in non-Tg mice, respectively. The spine length of thin and mushroom-type spines in Tg mice were reduced to 69.0 and 71.3% of those in non-Tg mice, respectively. The numbers of intersections for the basal dendrites of pyramidal neurons are summarized in Table 3. Although Golgi impregnation gave an impression that basal dendrites of neurons in Tg mice were also thinner than those in non-Tg mice, the number of basal dendrites in Tg mice was preserved.

Fig. 4

Pyramidal neurons in layer V of the primary motor cortex in DRPLA Tg (A) and non-Tg (B) mice. Atrophy of the apical dendrite and a reduced number of dendritic spines are detectable in A. There is no apparent difference in dendritic arborization between A and B. Golgi impregnation; scale bars = 20 μm.

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Table 2

Measurements of the perikarya, dendrites and spines of cerebral cortical neurons

Q129 Tg (mean ± SD)Non-Tg (mean ± SD)P-value
Perikaryal area (μm2)176 ± 54.3 (81.3%)216 ± 50.7<0.0001
Dendritic diameter (μm)2.22 ± 0.437 (79.5%)2.80 ± 0.528<0.0001
Spine density (per μm)0.467 ± 0.151 (65.7%)0.711 ± 0.131<0.0001
Spine shape percentage (%)
    Thin23.4 ± 8.1232.5 ± 2.48<0.05
    Stubby50.2 ± 4.0428.7 ± 3.15<0.001
    Mushroom22.9 ± 6.2532.7 ± 4.65<0.01
    Wide3.23 ± 2.495.41 ± 1.850.0815
    Ramified0.381 ± 0.8140.733 ± 0.6970.2006
Spine head area (μm2)
    Thin0.202 ± 0.0861 (66.0%)0.306 ± 0.107<0.0001
    Mushroom0.239 ± 0.0751 (69.3%)0.345 ± 0.121<0.0001
Spine length (μm)
    Thin0.855 ± 0.256 (69.0%)1.24 ± 0.380<0.0001
    Mushroom0.756 ± 0.185 (71.3%)1.06 ± 0.355<0.0001
  • Parentheses indicate percentages compared with the values in non-Tg mice.

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Table 3

Number of intersections for basal dendrites of pyramidal neurons in the primary motor or primary sensory cortices

Distance from theperikaryon (μm)Q129 Tg (mean ± SD)Non-Tg (mean ± SD)P-value
2012.5 ± 2.313.0 ± 2.90.7129
4018.1 ± 3.415.8 ± 3.00.1345
6018.8 ± 5.116.4 ± 4.30.1382
Total49.5 ± 9.545.3 ± 9.30.3732

Axons and microtubules

Electron microscope revealed atrophy of myelinated axons in both the pyramis and corpus callosum in Tg mice (Fig. 5A and B), although the thickness of myelin sheaths or the sizes of mitochondria showed no apparent differences between Tg and non-Tg mice. The results of axon diameter measurements are summarized in Table 4. Axon diameters in the pyramis and corpus callosum in Tg mice were reduced to 92.1 and 90.9% of those in non-Tg mice, respectively. To elucidate the reason for the axonal atrophy, we further conducted a morphological study on microtubules. There was no apparent difference in microtubule diameters between Tg and non-Tg mice. Because axonal areas were reduced and the number of microtubules was preserved in Tg mice, the density of microtubules in this mouse model became high and reached 160% of that in non-Tg mice. It was also demonstrated that distances between adjacent microtubules in Tg mice were reduced to 82.9% of those in non-Tg mice (Table 5).

Fig. 5

Electron micrographs of axons in the pyramis (A and B) and layer II of the primary motor cortex (C and D) obtained from DRPLA Tg (A and C) and non-Tg (B and D) mice. Axons (arrow) and neurites (arrowheads) are atrophic in the Tg mouse. Asterisks indicate axon terminals at synapses. Scale bar = 1 μm.

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Table 4

Axon diameter

Q129 Tg (mean ± SD)Non-Tg (mean ± SD)P-value
Pyramis (nm)690 ± 268 (92.1%)749 ± 292<0.0001
Corpus callosum (nm)528 ± 131 (90.9%)580 ± 177<0.0001
  • Parentheses indicate percentages compared with the values in non-Tg mice.

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Table 5

Measurements of axons and microtubules

Q129 Tg (mean ± SD)Non-Tg (mean ± SD)P-value
Axonal area (μm2)0.597 ± 0.3530.848 ± 0.367<0.05
Number of microtubules25.7 ± 10.123.2 ± 5.220.4687
Density of microtubules (per μm2)49.9 ± 15.3 (160%)31.2 ± 12.0<0.0001
Distances between microtubules (nm)71.2 ± 22.0 (82.9%)85.9 ± 41.3<0.0001
  • Parentheses indicate percentages compared with the values in non-Tg mice.

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Table 6

Measurements of synapses and synaptic vesicles

Q129 Tg (mean ± SD)Non-Tg (mean ± SD)P-value
Pre-synaptic area (μm2)0.366 ± 0.204 (84.5%)0.433 ± 0.272<0.0001
PSD length (nm)258 ± 105 (88.2%)293 ± 139<0.0001
Vesicle diameter (nm)51.1 ± 9.451.5 ± 7.80.7216
  • PSD = post-synaptic density. Parentheses indicate percentages compared with the values in non-Tg mice.

Synapses

Electron microscopy showed that in cerebral cortical layers II and III of Tg mice, the pre-synaptic terminals and diameters of neurites, including both axons and dendrites, were smaller than those in non-Tg mice (Fig. 5C and D). The results of morphometric analysis are summarized in Table 6. Pre-synaptic areas and PSD lengths in Tg mice were reduced to 84.5 and 88.2% of those in non-Tg mice, respectively. However, there was no significant difference in the size of synaptic vesicles between Tg and non-Tg mice.

Discussion

The results of the present morphological studies on the DRPLA Tg mouse indicate that neuronal atrophy is an essential aspect of the cell pathology that is induced during the course of polyglutamine accumulation. This atrophic change involves the neuronal somata, dendrites and axons. Although, in the present study, the change was evaluated in pyramidal neurons of layer V of the primary motor and primary sensory cortices, it is likely that neuronal cell atrophy occurs rather ubiquitously in the CNS, because Tg mouse brains showed extensive and proportional atrophy without neuronal loss in any of the brain regions, and polyglutamine accumulation affected multiple brain regions. This speculation may be partly supported by the extensive atrophy of neurites (dendrites and axons) and pre-synaptic terminals in layer II of the cerebral cortex (Fig. 5C), because neuroanatomically most of these structures are derived from outside the pyramidal neurons in layer V. Interestingly, in contrast to the generalized atrophy of the pyramidal neurons, there was no apparent difference in dendritic arborization between Tg and non-Tg mice. Immunohistochemistry revealed that polyglutamine accumulation in neuronal nuclei first appeared at P4 in several brain regions including cerebral cortical layer VI, and then gradually extended throughout the brain (T. Sato et al., submitted for publication). This post-natal progression of polyglutamine pathogenesis might allow the neuronal dendrites to develop to some extent. It will be necessary to elucidate how polyglutamine accumulation influences the development or maintenance of dendritic arborization temporally and quantitatively.

As regards the neuronal cytoskeletal changes in the DRPLA mouse brain, morphometric analysis revealed that axonal microtubules lost their normal inter-microtubule spacing and were relatively compacted in axons, suggesting the presence of certain alterations of microtubule-based function. Because microtubules are the essential cytoskeletal elements that play a pivotal role in protein transport within dendrites and axons, DRPLA mice may have problems associated with intra-axonal transport. Recently, it has been proposed that protein transport problems may lead to the disease pathology observed in several neurodegenerative disorders, including polyglutamine diseases (Gunawardena et al., 2003; Szebenyi et al., 2003). In the present study, in addition to the axonal changes, atrophy of dendrites, pre-synaptic terminals and PSD was also evident. Thus, it will be important to study whether in DRPLA the transport pathways are related to the process of disease progression. The distances between microtubules in dendrites and axons are mainly regulated by microtubule-associated protein (MAP) 2 and tau, respectively (Chen et al., 1992). In addition, MAP1A and MAP1B are necessary for bridging between the microtubules in axons (Hirokawa et al., 1985). Future studies of MAPs may help in clarifying the molecular mechanism of cytoskeletal abnormality leading to generalized neuronal cell atrophy in DRPLA.

Our study also revealed that in the brain of the DRPLA Tg mouse dendritic spines were decreased in both number and size and showed morphological changes that resulted in dominance of stubby-type spines. Dendritic spine loss is observed in many neurological conditions, such as neurodegenerative diseases, mental retardation and brain trauma (Fiala et al., 2002; Spires et al., 2004). Among the many causes of spine loss, it is well known that epilepsy leads to a decrease of spine density (Isokawa, 2000; Swann et al., 2000). In a slice culture model of epilepsy, the number of spines on CA3 pyramidal cell dendrites decreased by 40%, but there were no significant differences in dendritic length or in the branching index between control and epilepsy cultures (Drakew et al., 1996). Because Q129 Tg mice showed epilepsy that commenced from 11 weeks of age and continued until death (Sato et al., 1999b), these myoclonic seizures could be a major cause of the spine loss. Alternatively, the loss of spine density might be a pathological change related to polyglutamine pathogenesis, as suggested by a study of brains from humans with Huntington disease and Huntington disease model mice (Spires et al., 2004). In the latter, spine loss was associated with reduction in the length of thin and mushroom-type spines; however, spine morphology was preserved. Thus, the increased percentage of stubby spines we observed in the present study might be unique to the DRPLA Tg mice. It has been reported that an absence of normal levels of pre-synaptic activity results in loss of spine volume (Fiala et al., 2002), as well as an increase of stubby spine density (Petrak et al., 2005). It is possible that atrophy of pre-synaptic terminals may result in decreased afferent inputs, leading to the characteristic spine alterations observed in DRPLA mice. Because spine morphology and density are closely related to memory and learning functions (Kasai et al., 2003), it is important to study synaptic functions in this mouse model to elucidate the pathomechanism of mental retardation or dementia in DRPLA patients. A recent study has disclosed age-dependent and region-specific electrophysiological abnormalities including synaptic dysfunctions in the Q129 mice (T. Sato et al., submitted for publication).

Recently, it has been reported that polyglutamine induces transcriptional abnormality without aggregate formation (Takahashi et al., 2005). As regards the toxic effects of expanded polyglutamine proteins, several hypotheses have been suggested, such as impaired gene expression, dysfunction of cytoskeletal, vesicle and axonal transport, and alteration of metabolic pathways (Ross, 2002; Gunawardena et al., 2003; Szebenyi et al., 2003; Everett and Wood, 2004). Although the molecular mechanisms involved in polyglutamine pathogenesis seem to be complex, transcriptional dysregulation has been proposed as one of the major causes of neuronal degeneration (Shimohata et al., 2000; Freiman and Tjian, 2002; Luthi-Carter et al., 2002; Zhang et al., 2002). Much attention has been given to the ‘protein–protein interaction’ between expanded polyglutamine proteins and transcription factors that possess non-pathogenic polyglutamine repeats. These factors include the TATA box binding protein (TBP) (Schaffar et al., 2004), cyclic AMP response element (CRE) binding protein (CREB) binding protein (CBP) (Nucifora et al., 2001) and TBP-associated factors (TAFII130), a co-activator for CREB-regulated transcriptional activator (Shimohata et al., 2000). Mutant polyglutamine proteins bind directly to CBP, TBP and TAFII130, preventing activation of the transcription machinery. Interestingly, MAP1B also has a CRE and two TATA boxes in its promotor region (Liu and Fischer, 1996). As the MAP1B-deficient mouse shows a reduction in body weight (Edelmann et al., 1996; Meixner et al., 2000), the impairment of inter-microtubule spacing observed in the Q129 DRPLA mouse may depend on transcriptional abnormality involving MAP1B.

Acknowledgments

We thank S. Egawa, T. Ichikawa, Y. Ohta, C. Tanada, J. Takasaki, N. Kaneko and K. Kobayashi for their technical assistance, and M. Machida and A. Kobayashi for their secretarial assistance. This research was supported by a grant from the Research Committee for Ataxic Diseases, the Ministry of Health, Labor and Welfare, Japan, a Grant-in-Aid for Scientific Research (16390104, 17300109) and a Grant-in-Aid for Scientific Research on Priority Areas—Advanced Brain Project (15016044) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References

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