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Human Molecular Genetics Advance Access originally published online on August 4, 2006
Human Molecular Genetics 2006 15(18):2752-2762; doi:10.1093/hmg/ddl211
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Mitochondrial dysfunction and tau hyperphosphorylation in Ts1Cje, a mouse model for Down syndrome

Ebrahim Abdul Shukkur1, Atsushi Shimohata1, Takumi Akagi3, Wenxin Yu1, Mika Yamaguchi1, Miyuki Murayama2, Dehua Chui2, Tamaki Takeuchi1, Kenji Amano1, Karthik Harve Subramhanya1, Tsutomu Hashikawa3, Haruhiko Sago4, Charles J. Epstein5, Akihiko Takashima2 and Kazuhiro Yamakawa1,*

1 Laboratory for Neurogenetics, 2 Laboratory for Alzheimer's Disease and 3 Laboratory for Neural Architecture, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan, 4 Division of Fetal Medicine, National Center for Child Health & Development, Tokyo, Japan and 5 Department of Pediatrics, University of California, San Francisco, CA, USA

* To whom correspondence should be addressed at: Laboratory for Neurogenetics, RIKEN Brain Science Institute, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan. Tel: +81 48 467 9703; Fax: +81 48 467 7095; Email: yamakawa{at}brain.riken.jp

Received May 30, 2006; Accepted August 1, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
Trisomy 21 or Down syndrome (DS) is the most common genetic birth defect associated with mental retardation. The over-expression of genes on chromosome 21, including SOD1 (Cu/Zn superoxide dismutase) and APP (amyloid-ß precursor protein) is believed to underlie the increased oxidative stress and neurodegeneration commonly described in DS. However, a segmental trisomy 16 mouse model for DS, Ts1Cje, has a subset of triplicated human chromosome 21 gene orthologs that exclude APP and SOD1. Here, we report that Ts1Cje brain shows decreases of mitochondrial membrane potential and ATP production, increases of reactive oxygen species, hyperphosphorylation of tau without NFT formation, increase of GSK3ß and JNK/SAPK activities and unaltered AßPP metabolism. Our findings suggest that genes on the trisomic Ts1Cje segment other than APP and SOD1 can cause oxidative stress, mitochondrial dysfunction and hyperphosphorylation of tau, all of which may play critical roles in the pathogenesis of mental retardation in DS.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
Down syndrome (DS), a complex disorder affecting major body organs, results from trisomy of chromosome 21 and is the most common genetic cause of mental retardation (1). Neuropathological features of DS include decreased brain weight and neuronal number, abnormal neuronal differentiation and structural changes in synapses (2). A high degree of neurofibrillary tangle (NFT) formation, granulovacuolar degeneration and nerve cell loss have also been reported in brains of adults with DS (3). Most DS patients older than 35 years of age exhibit progressive cognitive decline, dementia and neuropathological features typically seen in much older Alzheimer's disease (AD) patients (4). NFTs represent a neurohistopathological feature shared by both DS and AD brains and are composed of phosphorylated microtubule-associated protein tau (5).

There is accumulating evidence that neuronal cell death may be induced by oxidative stress in DS (6,7). Similarly, altered free radical metabolism and impaired mitochondrial function are linked to neuronal degeneration of DS cortical neurons in culture (8,9) and may be associated with both mental retardation and AD pathology in person with DS. Neuronal death in DS has been linked to the over expression of triplicated chromosome 21 genes, including the gene encoding Cu/Zn superoxide dismutase (SOD1), which was suggested to be involved in the oxidative damage to neurons (10). Triplication of the amyloid-ß precursor protein (APP) gene, APP, could directly or indirectly cause the abnormal neuronal phenotype in DS brains, and altered APP metabolism has been proposed to be associated with mitochondrial dysfunction (9). A number of studies have described the Down syndrome critical region, which has been proposed to specifically contain genes contributing to the DS features. However, some DS patients with segmental triplications outside the DS critical region also contribute many features of DS and the role of the region still remains elusive (11).

Several mouse models have been produced to explore the etiology of DS. Ts65Dn (12) is segmentally trisomic for the segment covering only the HSA21 orthologous region of MMU16, which spans 17 Mb from App to Znf295 and contains about 136 orthologs, including Sod1, of the 364 expressed sequences of HSA21 (13). Ts65Dn displays a wide range of features in common with DS, including male sterility, severely abnormal behavior, cerebellar abnormalities, loss of basal forebrain cholinergic neurons and abnormal dendritic spines (12,14–17). They also have abnormalities of hippocampal synaptic function (18,19).

Another DS mouse model, Ts1Cje (20), carries a smaller trisomic segment than does Ts65Dn that spans from Sod1 to Znf295, which corresponds to parts of human chromosome bands 21q22.1, 21q22.2 and 21q22.3 and contains about 97 genes. Critically, the Ts1Cje trisomic region does not include App, and the third copy of Sod1 is functionally inactive (20). Ts1Cje mice are fertile, show milder behavioral abnormalities than Ts65Dn and have no obvious degeneration of basal forebrain cholinergic neurons (20). Although Ts1Cje only has a subset of the triplicated genes found in Ts65Dn, several DS phenotypic features such as craniofacial abnormalities (21) and reduced cerebellar volumes (22) are observed in both DS models. Ts1Cje mice also exhibit abnormalities in hippocampal synaptic plasticity, long-term potentiation and long-term depression, but these are less marked than in Ts65Dn (23). We recently demonstrated dosage-dependent over-expression of genes in the trisomic region of Ts1Cje (24).

Here, we describe mitochondrial dysfunction, over-generation of ROS, hyper-phosphorylation of tau and activation of tau-kinases JNK/SAPK and GSK3ß in Ts1Cje mice. The data presented here show the importance of dosage-dependent over-expression of genes in the Ts1Cje-specific trisomic segment, which exclude App and Sod1, in these abnormalities that would lead to neuronal dysfunction in Ts1Cje brain.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
Mitochondrial dysfunction in cultured Ts1Cje astrocytes
We measured the mitochondrial membrane potential of cerebral cortical astrocytes from E16 Ts1Cje and wild-type (WT) mice using JC-1 staining (Fig. 1A–F). In primary brain cell culture, neurons do not survive more than 2 weeks in DMEM media only. We observed that more than 65% cells were GFAP-positive that indicated astrocytes and remaining would be immature astroglia or other glial cells. There was no difference in morphology of cells and percentage of GFAP-positive cells between WT and Ts1Cje cultures. Primary cultures were morphologically similar after the incubation with JC-1. Under fluorescence microscopy, WT astrocytes exhibited bright red fluorescence (Fig. 1A) and a lower green signal (Fig. 1B), whereas Ts1Cje astrocytes demonstrated a marked decrease in red fluorescence intensity (Fig. 1D) and a higher intensity in green fluorescence (Fig. 1E), indicating low mitochondrial membrane potential. Moreover, whereas the merged fluorescence emissions at 590 and 530 nm of WT astrocytes exhibited bright red fluorescence (Fig. 1C), Ts1Cje astrocytes exhibited less red fluorescence (Fig. 1F). To obtain quantitative measures of changes in mitochondrial membrane potential, we analyzed the intensity ratio in JC-1-treated astrocytes using FACScan and NIH imaging software. The red/green intensity ratio of Ts1Cje astrocytes was 12% lower (P=0.030) than that of WT cells by FACScan analysis (Fig. 1G) and 8.5% lower than that of WT cells (P=0.026) by imaging analysis (Fig. 1H). Together with the fluorescence microscopy observations, these results indicate that the mitochondrial membrane potential is decreased in Ts1Cje astrocytes.


Figure 2111
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  		Figure 1. Mitochondrial impairment in fetal Ts1Cje cultured astrocytes. (A–F) Cultured astrocytes labeled with the mitochondrial membrane potential marker JC-1. WT astrocytes show bright red signals (A) and lower green signals (B) indicate normal mitochondrial potential. Ts1Cje astrocytes show lower red signals (D) and higher green signals (E) indicate compromised mitochondrial potentials. Fluorescence emissions at 590 nm (red) (A and D) and at 530 nm (green) (B and E). (C and F) Merged fluorescence emissions at 590 and 530 nm. Scale bar 50 µm. (G) Mitochondrial membrane potentials in WT and Ts1Cje astrocytes quantified by FACScan flow cytometry. Accumulative fluorescence intensity at the red/green ratio was significantly lower in Ts1Cje astrocytes relative to WT astrocytes (P=0.030). Values represent means±SE for WT, n=10, and Ts1Cje, n=9 independent experiments. (H) Quantitative analysis of mitochondrial membrane potential in astrocytes using NIH imaging software. The fluorescence intensity at the red/green ratio was significantly lower in Ts1Cje astrocytes than in WT astrocytes (P=0.026). Values represent means±SE for WT, n=14, and Ts1Cje, n=13 independent experiments. (I–L) ATP levels in cultured astrocytes and in cerebral tissues from 1-, 2- and 3-month-old mice. (I) ATP levels in cultured astrocytes from Ts1Cje were significantly lower than the levels in WT (P=0.030). Values represent means±S.E for WT, n=12, and Ts1Cje, n=14 independent experiments. (J and K) ATP levels in the cerebral cortex of 1-month (J) and 2-month-old (K) mice. The level of Ts1Cje was not altered compared with that of age-matched WT mice. Values represent means±SE for WT, n=6, and Ts1Cje, n=6 independent experiments. (L) ATP levels in the cerebral cortex of 3-month-old Ts1Cje mice were significantly lower than the levels in cerebral cortex from age-matched male WT mice (P=0.040). Values represent means±SE for WT, n=8, and Ts1Cje, n=8 independent experiments.

 
We also observed slight decreases in mitochondrial membrane potential in primary cultures of Ts1Cje hippocampal neurons; however, the difference between those of WT and Ts1Cje was not statistically significant (data not shown).

Decreased ATP levels in the Ts1Cje brain and cultured astrocytes
A decrease in mitochondrial membrane potential may be associated with defects in aerobic respiration and ATP synthesis. We checked the amount of ATP in Ts1Cje and WT mouse brains by ATP bioluminescence assay. Cultured astrocytes were prepared from E16 mice and cerebral tissues were obtained from 1-, 2- and 3-month-old mice. ATP levels were significantly decreased in Ts1Cje cultured astrocytes (20%; P=0.030) (Fig. 1I) and in the cerebral cortex of 3-month-old Ts1Cje mice (29%; P=0.040) (Fig. 1L). However, decreases in ATP levels were not statistically significant in 1- and 2-month-old Ts1Cje mice (Fig. 1J and K).

Increased oxidative stress in the Ts1Cje cultured hippocampal neurons and astrocytes
Hydroethidine (HEt), a freely permeable ROS-specific fluorescent dye, is oxidized to ethidium (Et) by superoxide radicals (O2–) (25). By incubating with HEt, ROS were measured in primary cultured neurons (14 div) from E16 Ts1Cje and WT mouse hippocampi (Fig. 2A and B). Under fluorescence microscopy, higher Et fluorescence intensities, were observed in primary cultures of Ts1Cje hippocampal neurons (Fig. 2B) when compared with WT neurons (Fig. 2A), indicating greater ROS generation. ROS generation was 8.5% higher in Ts1Cje neurons than that of WT neurons (P=0.030) (Fig. 2C). ROS generation was also high in primary cultures of astrocytes from E16 Ts1Cje (Fig. 2D and E). The Et intensity of Ts1Cje astrocytes was 18% higher than that of WT astrocytes (P=0.0061) (Fig. 2F). These results strongly suggest that ROS generation is significantly increased in Ts1Cje neurons and astrocytes.


Figure 2112
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  		Figure 2. Increased ROS in cultured neurons and astrocytes in the Ts1Cje brain. (A and B) Cultured hippocampal neurons (14 div) from E16 WT and Ts1Cje after incubation with HEt. (C) Fluorescence intensities of cultured neurons were quantified by NIH imaging software. WT, n=8 (80 images), and Ts1Cje, n=8 (80 images). Et intensity in Ts1Cje neurons was 8.5% higher than that in WT neurons (P=0.030). (D and E) Cultured astrocytes incubated with HEt. Ts1Cje astrocytes were intensely labeled with Et indicating elevated ROS levels. (F) Fluorescence intensities of cultured astrocytes were quantified by NIH imaging software. Et intensity in Ts1Cje astrocytes was 18% higher than that in WT astrocytes (P=0.0061). WT, n=5 (230 images), and Ts1Cje, n=6 (300 images). Scale bar: 200 µm.

 
Tau hyperphosphorylation in the Ts1Cje brain
As tau hyperphosphorylation is common to many neurodegenerative diseases and may play a critical role in the pathogenesis of AD (26), we measured tau phosphorylation levels in Ts1Cje and WT mouse brains (Fig. 3 for 3-month-old; Supplementary Material, Fig. S1 for P1, P14, 1- and 2-month-old). Using detergents of different strengths, cortical tissue was separated into soluble proteins (RAB fraction, Fig. 3A), membrane-bound proteins (RIPA fraction, Fig. 3B) and SDS-insoluble proteins recovered by formic acid treatment (FA fraction, Fig. 3C). These fractions were analyzed using western blots probed with Tau-C antibody, which recognizes both phosphorylated and unphosphorylated tau. Tau-C signals obtained in the RAB and RIPA fractions show little difference between Ts1Cje and WT mice (Fig. 3A and B; Supplementary Material, Fig. S1). Higher levels of insoluble tau (in the FA fraction) were observed in Ts1Cje samples than in WT samples, although the amounts of insoluble tau were highly variable among individual mice (Fig. 3C). The phosphorylation levels of tau in RAB fractions were investigated by probing with the three anti-phosphorylated tau antibodies, AT8, pS199 and pS400. Signals for all three probes were increased in 3-month-old Ts1Cje mice (Fig. 3D, F–H), whereas the signals obtained by Tau-1, which recognizes unphosphorylated tau, showed a decrease (Fig. 3E and I). The western blot analyses of tau phosphorylation levels in P1, P14, 1 and 2-month-old mice brain are shown in Supplementary Material, Figure S1. These results indicate that the phosphorylation of tau starts at 2–3 months stage in Ts1Cje. Tau phosphorylation was also investigated immunohistochemically in adult (3-month-old) Ts1Cje and WT cerebral cortical brain tissues using AT8, pS199 and another antibody, AT180, which recognizes tau phosphorylated at Thr-231. The signals were again higher in Ts1Cje than in WT (Fig. 3J–U) and confirmed hyperphosphorylation of tau in Ts1Cje. Signals were non-fibrillar and diffusely distributed in the neuronal cytoplasm, suggesting that the phosphorylated tau was in the stage of pre-NFTs.


Figure 2113
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  		Figure 3. Tau hyperphosphorylation in the adult Ts1Cje brain western blots of RAB (A), RIPA (B) and FA (C) fractions from 3-month-old WT and Ts1Cje cortical lysates probed with Tau-C antibody. Ts1Cje FA fractions had higher levels of insoluble tau than WT samples. {alpha}-tubulin was used as internal control. (D) Blots of RAB fractions probed with antibodies, AT8 which recognizes tau phosphorylated at serine 202 and threonine 205, pS199 which recognizes tau phosphorylated at serine 199 and pS400 which recognizes tau phosphorylated at serine 400. All these antibodies showed that Ts1Cje RAB fractions have higher levels of phosphorylated tau than WT. (E) Blots of RAB fraction probed with antibody Tau1 showing decreased levels in Ts1Cje. Histograms of densitometric scanning data for the western blots are shown in (F–I). Band intensities AT8 (F), pS199 (G), pS400 (H) and Tau-1 (I) were all normalized using {alpha}-tubulin. AT8 and Ser199 levels in Ts1Cje were significantly higher (P=0.01 and P=0.0092) than that in WT, and Tau-1 levels in Ts1Cje were significantly lower (P=0.016) than that in WT. (J–U) Immunostaining of hippocampal and cortical sections from 3-month-old Ts1Cje (Images L, M, P, Q, T and U) and WT mice (Images J, K, N, O, R and S). (J–M) Anti-tau AT8 antibody-immunostained cells. (N–Q) Anti-tau AT180-immunostained cells. (R–U) Anti-tau pS199-immunostained cells. These confirm hyperphosphorylation of tau in Ts1Cje. Scale bar: 10 µm. Values represent means±S.E for WT, n=3, and Ts1Cje, n=3 independent experiments.

 
GSK3ß and JNK/SAPK activation in the Ts1Cje brain
Aberrant tau hyperphosphorylation could be due to the activation of upstream kinases such as GSK3ß (glycogen synthase kinase 3ß) (27,28) and JNK/SAPK (c-Jun N-terminal kinase) (29). The antibody for JNK/SAPK is directed against the active phosphorylated forms of JNK and recognizes all the three JNK isoforms, JNK1 JNK2 and JNK3. We checked the levels of total and activated GSK3ß and JNKs 1–3 in WT and Ts1Cje of P1 (Fig. 4A), P14 (Fig. 4B), 1 month (Fig. 4C), 2 months (Fig. 4D) and 3 months old (Fig. 4E) RAB fraction by western blot analysis. The levels of total GSK3ß and JNK/SAPK at all five stages were found to be similar in Ts1Cje and WT littermates. The levels of active GSK3ß (P-Y216) were higher in 2 and 3-month-old Ts1Cje samples than in WT samples. The level of P-JNK/SAPK was higher in 3-month-old Ts1Cje than in WT. The results of quantitative measurements by densitometry are shown in Figure 4F–I. Total GSK3ß levels were not altered in P1 to 3-month-old Ts1Cje mice (Fig. 4F). Active GSK3ß (P-GSK3ß-Y216) levels were significantly higher in 2- and 3-month old Ts1Cje compared with WT (Fig. 4G). Total JNK levels remained unaltered at P1 to 3 months in Ts1Cje mice (Fig. 4H). P-JNK/SAPK levels were higher in Ts1Cje at 3 months, but were statistically marginal (Fig. 4I). The activation of these kinases was also observed in immunohistochemical analyses of 3-month-old Ts1Cje brain tissues by using anti-PY216 GSK3ß and anti-P-JNK/SAPK antibodies (data not shown). The level of phosphorylated GSK3ß-inactive form (P-GSK3ß-S9) was not altered in Ts1Cje (Supplementary Material, Fig. S2A and D). The upregulation of GSK3ß and JNK/SAPK in Ts1Cje brains is consistent with the increased phosphorylation of tau.


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  		Figure 4. Activation of GSK3ß and JNK/SAPK in the adult Ts1Cje brain western blots of P1 (A), P14 (B), 1-month (C), 2-month (D) and 3-month-old (E) Ts1Cje and WT brain lysate RAB fractions probed with antibodies for total and activated (phosphorylated) MAP kinases, GSK3ß and JNK/SAPK. Ts1Cje and WT samples had similar levels of total GSK3ß and total JNK/SAPK at P1 to 3 months old. Levels of activated kinases, P-GSK3ß and P-JNK/SAPK, are higher in Ts1Cje than in WT at 3 months (E). (F and G) Densitometric scanning data of total GSK3ß and phosphorylated GSK3ß. (H and I) Densitometric scanning data of total JNK and phosphorylated JNK. Phosphorylated and unphosphorylated forms of GSK3ß and JNK/SAPK were all normalized using {alpha}-tubulin. Phosphorylated GSK3ß levels were significantly increased at 3 months stage. Phosphorylated JNK level was also increased at 3 months, but statistically marginal. Wt, wild-type littermate; Ts, Ts1Cje. Values represent means±SE for WT, n=5, and Ts1Cje, n=5, at P1, P14, 1 month, 2 months and 3 months. WT, n=3, and Ts1Cje, n=3 independent experiments.

 
We also performed western blot analyses using anti-ERK1/2, anti-P-ERK1/2 and anti-p25/p35 antibodies. However, we did not find any differences in the levels of total and phosphorylated ERK1/2 and p35/p25 in 3-month-old WT and Ts1Cje RAB samples (Supplementary Material, Fig. S2B, C and E–H).

Neurodegenerative changes in adult Ts1Cje brain
Light microscope (LM) and electron microscope (EM) examinations of 3-month-old Ts1Cje and WT brain tissues were performed. Figure 5A depicts the cortical areas used for sectioning. Figure 5B shows the parasagittal view of section 4. Although the number of degenerating neurons detected by LM Toluidine blue staining varied among six Ts1Cje mice analyzed, a higher frequency of darkly stained degenerating neurons was observed in Ts1Cje samples, especially in the frontal cortex in close proximity to the CA3 region of the hippocampus (Fig. 5C–F, correspond to the ‘d’-region in Fig. 5B).


Figure 2115
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  		Figure 5. Histological abnormalities in the adult Ts1Cje brain. (A) Schematic representation of the cortical areas sampled for parasagittal sectioning, indicated by red lines. (B) Schema of antero-posterior sub-regions of cortex in a parasagittal plane. (C–F) LM images of Toluidine blue stained cortical parasagittal sections corresponding to section 4 in (A) and region d in (B). Mice: 3-month-old. (C and E) WT neurons have a well-rounded shape (arrows) and only a few intensely stained degenerating neurons were visible (arrow heads). (D and F) In Ts1Cje, a number of intensely stained neurons (arrow heads) were seen. Scale bar: 100 µm. (G–R) Representative electron micrographs of 3-month-old WT and Ts1Cje mouse brain cortices corresponding to the region d. (G) Low magnification image of WT cortex at region d. Most neurons show healthy rounded shape (arrows) and few show darkly stained degenerating features (arrowhead). (H) High magnification image of a WT neuron showing an organized nucleus (nu) and healthy smooth surface of the cytoplasm (star). (I) A WT neuron shows healthy mitochondria (m). (J) Minor WT neurons are intensely stained and show condensed cytoplasm (arrow) and nucleus (nu). (K) Low magnification image of Ts1Cje cortex at the region d. Major population of neurons show healthy rounded shape (arrow); however, significant number of darkly stained cells (arrowheads) were observed. (L) High magnification image of a healthy Ts1Cje neuron with smooth cytoplasm (star) and nucleus (nu). (M) A healthy Ts1Cje neuron with normal-shaped golgi bodies (arrowhead). (N) Mitochondria in a healthy Ts1Cje neuron. (O) A darkly stained Ts1Cje neuron with condensed cytoplasm (star) and a nucleus (nu) with shrunken membrane (arrow). (P) A Ts1Cje darkly stained neuron with condensed cytoplasm (star) and nucleus (nu). (Q) A darkly stained Ts1Cje neuron with elongated Golgi bodies (arrowhead) and nuclear membrane gap (arrow). (R) A grossly swollen mitochondria with broken cristae (stars) from a darkly stained Ts1Cje neuron. m, mitochondria; nu, nucleus. Values represent means±SE for WT, n=6, and Ts1Cje, n=6 for LM studies, and WT, n=4, and Ts1Cje, n=4 for EM studies.

 
Under electron microscopy, most neurons of WT mice showed healthy rounded shapes and organelles (Fig. 5G, arrows, H and I), although a few were darkly stained indicating degeneration (Fig. 5G, arrowhead, and J). In contrast, although most of the Ts1Cje neurons still showed healthy rounded shapes (Fig. 5L–N), an increased number of dark degenerating neurons were found (Fig. 5K, arrowheads) as had been observed in the LM study. The darkly stained cells showed abnormal nuclear shapes, chromatin margination condensation, darkened cytoplasm and condensed organelles (Fig. 5O–Q). Mitochondrial abnormalities, such as membrane blebbing, were also visible in many of these Ts1Cje dark neurons (Fig. 5R). As in the EM analyses, the number of dark neurons varied among four Ts1Cje mice analyzed.

To quantify degenerating neurons, we counted dark staining and healthy neuronal cells in Ts1Cje and WT mice brain at low magnification in the d-region mentioned in Figure 5B. The number of degenerating neurons in Ts1Cje varies among the individual mice. We found around 27.8% dark staining neurons in the Ts1Cje mice and ~72.2% healthy neurons in six independent mouse brain sections. In contrast, only a minor number of degenerating neurons (6.3%) are observed in WT mouse brains (Supplementary Material, Fig. S3A). We also quantified healthy and abnormal mitochondria from Ts1Cje and WT mice. We found increased number of abnormal mitochondria (mitochondria with broken cristae) in the Ts1Cje mice of 30.6% and healthy mitochondria of 69.4%. In contrast, a minor number of abnormal mitochondria, 7.0%, were observed in WT mice (Supplementary Material, Fig. S3B).

Absence of NFTs in the Ts1Cje brain
We used tau-antibody pS199 and Gallyas silver staining to investigate the phosphorylation status of tau and NFT formation in 3-month-old Ts1Cje mice cerebral cortices (Supplementary Material, Fig. S4). Tissues of cerebral cortex from postmortem human AD patient brain were used as a positive control. Prominent signals of pS199 for phosphorylated tau were observed in both AD cortex (Supplementary Material, Fig. S4A) and Ts1Cje cortex (Supplementary Material, Fig. S4D). In contrast, intense signals of Gallyas staining for mature NFT structures were observed only in the AD cortex (Supplementary Material, Fig. S4B), but the signals were negative in almost all areas of the Ts1Cje cortex (Supplementary Material, Fig. S4E). The pS199 and Gallyas silver staining colocalize in the AD cortex (Supplementary Material, Fig. S4C, merged images) but not in the Ts1Cje cortex (Supplementary Material, Fig. S4F, merged images). These findings again suggest that the Ts1Cje brain pathology is at the pre-tangle stage (stage 1).

AßPP metabolism is not altered in the Ts1Cje brain
We studied AßPP metabolism in 3-month-old Ts1Cje and WT brain samples using western blots probed with the mouse monoclonal antibody 22C11 and rabbit polyclonal antibody APPC ({alpha}C-APP), which recognize the N- and C-termini of AßPP, respectively. There were no differences between Ts1Cje and WT in the levels of full-length AßPP and of the C83 (Supplementary Material, Fig. S4G–I). Similarly, there was no significant alteration in Aß40 and Aß42 levels between Ts1Cje and WT, as determined by an ELISA analysis of differentially fractionated lysates (Supplementary Material, Fig. S4J and K). These results suggest that the brain pathology in Ts1Cje arises without abnormal AßPP metabolism.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 
We have described mitochondrial dysfunction, increased oxidative stress and tau hyperphosphorylation in Ts1Cje mice. The abnormalities in brain lysates occur at about the same time, which suggests that these abnormalities are closely linked to each other. In contrast, ROS generation and mitochondrial dysfunction were seen in cultured neurons and astrocytes derived at a much earlier stage from fetal brain at E16. A possible explanation for this is that the in vitro conditions are such that they cause the abnormalities at much earlier stages than the in vivo. The present findings may contribute to a consolidated biochemical and molecular platform for the pathology underlying the cognitive anomalies in Ts1Cje mice and are explicated Subsequently.

Triplicated genes other than App and Sod1 cause oxidative stress and mitochondrial impairment, whereas AßPP metabolism remains unaltered in Ts1Cje brain
The present study has revealed that the Ts1Cje triplicated chromosomal segment, which excludes App and Sod1, can cause increased oxidative stress in Ts1Cje cell cultures. This might also be the cause in parallel to fall with persons having DS. Although abnormal AßPP metabolism has been described in DS (9), we did not find any abnormalities in the AßPP metabolism of Ts1Cje brain. This is consistent with the previous observation that Ts65Dn mice lack amyloid deposition (14), further suggesting that even the Ts65Dn trisomic segment including App and Sod1 is not enough to form Aß deposits in mice. Our results indicate that the over-expression of the genes on the triplicated Ts1Cje segment other than App and Sod1 can cause the oxidative stress and mitochondrial dysfunction without causing abnormal AßPP metabolism.

Hyperphosphorylation of tau and absence of NFTs in Ts1Cje brain
In the present study, we have described the hyperphosphorylation of tau in Ts1Cje brain, which, to our knowledge, represents the first report of tau hyperphosphorylation in segmentally trisomic DS mouse models. Phosphorylation of tau is first observed in Ts1Cje brain at 2 months of age and is accelerated at 3 months. However, frank NFTs were not present in 3-month-old Ts1Cje mice brains, which is consistent with the previous observation that no NFTs were detectable in Ts65Dn brain (14).

The pathological significance of NFTs formation in AD and other neurological diseases is still being debated. Several studies have suggested that NFTs may not be pivotal in the neurotoxic cascade, as evidenced by the observations that tau-related neurodegeneration in mice with APP over-expression occurs without frank NFT formation (30) and that, in a mouse tauopathy model, NFTs continue to accumulate after tau suppression but do not cause cognitive decline or neuronal death (31). The anomalously phosphorylated tau pretangle forms observed in Ts1Cje could be sufficiently toxic to cause moderate neurodegeneration and to initiate the impairment of cognitive function in Ts1Cje.

Activation of stress-induced MAP kinases may explain hyperphosphorylation of tau in Ts1Cje brain
Tau can be phosphorylated by multiple kinases including GSK3ß, JNK/SAPK, ERK1/2 and CDK5 (32), all of which are activated in AD brains. However, we found that GSK3ß and JNK/SAPK were activated in Ts1Cje, whereas ERK1/2 and CDK5 were not.

GSK3ß activity is induced by oxidative stress in vitro and leads to tau hyperphosphorylation (33). GSK3ß have been implicated in the pathogenic phosphorylation of tau in AD (28), and hyperphosphorylation of tau at serine 396/199 by over-activated GSK3ß has been reported to impair spatial memory in rats (34). Activated GSK3ß phosphorylates and so inhibits mitochondrial pyruvate dehydrogenase, which normally converts pyruvate to acetyl CoA (35), a key step in the synthesis of ATP in mitochondria. The upregulated GSK3ß in the Ts1Cje brain may phosphorylate tau, which may partly account for the ATP depletion observed in Ts1Cje. Furthermore, the abnormal activation of GSK3ß in Ts1Cje could play an important role in the neuronal impairments in the mice. Stress-induced kinase JNK/SAPK is also known to be activated in AD (36), the possibility of a stress-induced cascade involving GSK3ß and JNK/SAPK that may elucidate the co-activation of these factors in Ts1Cje.

ERK1/2 are mainly activated by growth factors and mitogens, but some studies show that ERK1/2 are also activated by oxidative stress (37). In AD brain, MAP kinase ERK1/2 is abnormally active and may hyperphosphorylate tau (38). Increased Cdk5 activity has also been reported in AD brain (39). However, in the present study, we did not find significant phosphorylation of ERK1/2 or any differences in p35 and p25 in Ts1Cje brain, suggesting that ERK1/2 and CDK5 activation are not responsible for the tau hyperphosphorylation in Ts1Cje.

Pathologies leading to neurodegeneration and cognitive impairment in Ts1Cje
It is reasonable to assume that the dosage-dependent over-expression of genes on the Ts1Cje trisomic region (24) leads to the abnormalities that we have been discussing, which then result in cognitive impairment. However, among those abnormalities, which come(s) first and which next and which are critical and indispensable for the phenotype need to be clarified.

Some of the over-expressed genes on the Ts1Cje trisomic segment may critically affect the metabolic pathways in mitochondria, which then triggers increases in free radical generation leading to oxidative stress in Ts1Cje brain. Our results suggest that the mitochondrial dysfunction might appear first or predominantly in glial cells, which could, of course, cause and/or accelerate the dysfunction in neurons. The oxidative stress could itself aggravate mitochondrial malfunction. Thus, a vicious cycle would be set up (40), with the excessive ROS production then triggering neurodegeneration (7,8). This pro-oxidant state might also activate MAP kinases, including JNK (41), and these kinases could phosphorylate tau (32). Activation of GSK3ß might also directly cause mitochondrial dysfunction (35) as discussed above. For example, a recent study reported that tau pathology could lead to mitochondrial dysfunction and oxidative stress-induced neuronal damage, possibly distinct from that caused by ß-amyloid peptide (42). These pathways would finally terminate in neuronal damage and death, which is grossly appreciated as neurodegeneration.

The Ts1Cje trisomic segment contains about 97 genes including Dscr1, Dyrk1a and Ets2. The fly ortholog of DSCR1, nebula, has been reported to be located in the mitochondria and to play a critical role in mitochondrial function (43). DYRK1A can phosphorylate tau, but less efficiently when compared with DYRK2 (44). The transcription factor ETS-2 has been reported to promote the activation of a mitochondrial death pathway in DS neurons (45). A recent report described that increased dosage of DSCR1 and DYRK1A lead to NFAT dysregulation and DS phenotypes (46). All of these genes are interesting candidates whose aberrant expression may be responsible for the abnormalities observed in this study.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 References
 
Animals and genotyping
Ts1Cje transgenic mice were maintained by crossing carrier males with C57BL/6J females. All mice used in our experiments were of N15-N18 generations. Genotypes were determined by PCR as previously described (20). For experiments with 1-, 2- and 3-month-old mice, we exclusively used male mice.

Primary cell culture of hippocampal neurons and astrocytes
Brains were isolated from Ts1Cje and WT E16 mouse embryos and then pooled separately after genotypic confirmation. Dissected hippocampi were trypsinized and dissociated into single cell suspensions by trituration. These cell suspensions were plated in poly-L-lysine-coated plastic Petri dishes at a density of 2.5x105 cells/cm2 in neurobasal medium (with B27 antioxidant plus, 10% FBS, 0.5 mM L-glutamine and 5% penicillin/streptomycin) for the first day. On the second day, cells were transferred to serum-free medium. From the third day onwards, cultures were maintained in a serum and L-glutamine-free medium with B27 antioxidant minus for the duration of the experiment.

Astrocyte rich cultures were prepared according to standard methods (47). Briefly, cerebral cortices from E16 brain were isolated, minced and trypsinized. The triturated and dissociated cells were transferred into 75 cm2 flasks containing DMEM with 10% FBS/5% penicillin/streptomycin) and cultured for 3–4 weeks.

Assessments of mitochondrial function using JC-1 staining
Neurons and astrocytes were incubated with 0.5 µM JC-1 dye (Molecular probes, Inc.) for 30 min at 37°C and then washed with phosphate-buffered saline (PBS). Fluorescence was monitored in a multiwavelength-excitation dual wavelength-emission fluorescence Microscope (Hamamatsu Digital Camera; Meta Vue software). Levels of green and red fluorescence were coded on a scale (0–4095) representing pixel intensity and 1280x1024 pixels images were collected. 100–99 999 pixels for neurons and 200–99 999 pixels for astrocytes were used for data analysis.

Flow cytometry
WT and Ts1Cje astrocytes were trypsinized, and cells were resuspended (2.0x105 cells/cm2) in medium containing JC-1 (1 mM/ml), incubated for 30 min, pelleted, rinsed and resuspended in PBS. Analysis was done on a FACScan flow cytometer (Epics Elite ESP, Beckman Coulter, Fullerton, CA, USA) with a 488 nm argon laser. Data were analyzed in list mode with Cell Quest software and quantitatively expressed as means of red and green fluorescence intensities relative to WT (100%).

Cellular ATP determination
ATP was determined using the ATP Bioluminescence Assay kit CLS II (Boehringer Mannheim, Mannheim, Germany). Astrocyte cultures and cerebral cortices were sonicated in PBS, pelleted by centrifuging at 10 000 r.p.m. (9200g) for 2 min at 4°C, washed with PBS, resuspended in 50 µl of ice-cold ATP lysis buffer (100 mM Tris and 4 mM EDTA, pH 7.75) and incubated for 2 min at 99°C in 150 µl of boiling ATP lysis buffer. Cell lysates were centrifuged at 10 000 r.p.m. for 1 min at 4°C. ATP was measured using 20 µl of the supernatant and 80 µl of luciferase reagent. After a 20 s delay, the chemiluminescence was measured with 2 s integration time using ARVO MX/Light (1420 Multilabel Counter, Perkin Elmer).

Measurement of ROS production with HEt
ROS in both hippocampal neurons and astrocytes was measured by HEt staining as described previously (25). Neuron and astrocyte cultures were prepared as described in the previous section, and cultures were incubated with HEt (Molecular probes, Inc.) for 20 min at 37°C and then washed three times with PBS. All experiments were performed in a blinded manner.

Immunoblotting for tau and related-kinases
Methods for fractionation of brain lysate and immunoblotting were described previously (48). Briefly, the cortical tissues were homogenized in 4 ml/g of RAB buffer (0.1 M MES, 1 mM EGTA, 0.5 mM MgSO4, 150 mM NaCl, 0.02 M NaF, 1 mM PMSF, phosphatase and protease inhibitor, pH 7.0) and centrifuged at 40 000g for 20 min at 4°C (RAB fraction). The pellets were re-homogenized with 1 M sucrose/RAB (0.1 M MES, 1 mM EGTA, and 0.5 mM MgSO4, pH 7.0) and centrifuged at 40 000g for 20 min at 4°C. The pellets were extracted with 1 ml/g RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 0.25% sodium deoxycholate, 0.1% SDS, phosphatase and protease inhibitor, pH 8.0) and centrifuged at 40 000g for 20 min at 4°C (RIPA fraction). The RIPA insoluble pellets were extracted with 70% formic acid to recover the most insoluble cytoskeletal aggregates (FA fraction). RIPA and FA fractions were probed only with Tau-C antibody. RAB fractions were probed with the following antibodies against differentially phosphorylated tau's and tau-protein kinases: pS199 (BioSource), pS400 (BioSource), tau1 (Chemicon), mouse monoclonal antibodies against GSK3ß (BD Transduction Lab), P-JNK/SAPK (Cell Signaling), AT8 (Innogenetics) and AT180 (Innogenetics), rabbit polyclonal antibodies against PY216-GSK3ß (BioSource) and JNK (Cell Signaling).

AßPP analyses
Western blot analysis of AßPP was done according to a previously described protocol with slight modification (49). Briefly, whole brain lysates were prepared in lysis buffer (10 mM Tris–HCl, 150 mM NaCl, pH 7.4, 1 mM EDTA, 1% Triton X-100 containing protease inhibitors) and were probed with mouse monoclonal antibody 22C11 (Chemicon) and the {alpha}-C-APP antibody (recognizing both full-length and C83 fragments). For ELISA studies, TBS, 1% Triton X-100, 1% SDS and 5 M guanidine fractions were prepared. Sandwich Aß ELISAs for quantifying Aß40 and Aß42 were performed as described protocol (50).

Immunocytochemistry for phosphorylated tau
For immunohistochemistry, tissue preparation and histochemical staining were performed as described previously (51,52). Eight to 10 micrometer thick sections were processed. Deparaffinized and hydrated sections were incubated in Target Retrieval Solution (Dako, Carpinteria, CA, USA) at 70°C for 25 min and then incubated for 2 h at 4°C with 10% normal goat serum in PBS. The specimens were incubated overnight at 4°C with primary antibodies (1:100) in PBS containing 1% BSA and 0.05% Tween-20. Bound antibodies were visualized with Alexa 488-conjugated anti-mouse or Alexa 568-conjugated anti-rabbit IgG (1:1000). Gallyas silver staining was performed as previously described (51).

LM and EM analyses of cortical sections
Perfused mouse brains were immersed in 4% paraformaldehyde/2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 h at 4°C. 300 µm slices of the cerebral cortex were cut, post-fixed with 1% osmium tetroxide for 2 h at room temperature, dehydrated with ethanol and embedded in Araldite resin according to standard protocols. One micrometer semi-thin sections were cut from the 300 µm sections and stained with Toluidine blue for light microscopy. About 80–100 nm sections cut from the same resin embedded block were also stained with uranyl acetate and lead citrate for electron microscopy (LEO 912AB, LEO, Germany) according to standard protocols.

For quantitations of dark and healthy neurons in the brains of WT and Ts1Cje mice, images of six independent Toluidine blue stained brain sections of the d-region were carefully investigated at low power field. The dark neurons in brains of six mice were counted for each experiment. The total area of six mice brain sections were 5.0724 mm2 in WT and 5.0297 mm2 in Ts1Cje. For quantitative studies of mitochondria of WT and Ts1Cje mice, electron micrograph pictures were taken at magnification 26 000x. Totally, 352 images from WT (9 µm2 total area per image) and 328 images from Ts1Cje (9 µm2 total area per image) were analyzed.

Statistical analysis
All data are expressed as means±SE. Differences between study groups were compared for statistical significance by paired two-tailed Student's t test. P<0.05 were considered statistically significant.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
References
 
Supplementary Material is available at HMG online.


   ACKNOWLEDGEMENTS
 
We thank Mr K. Ohtawa for the technical assistance in flow cytometry, Ms T. Yoshida for histological studies at RRC, RIKEN Brain Science Institute, and we thank Dr N. Sahara for helpful discussions, Dr H. Omi for the technical assistance and Mr B. Singh for editing and rewriting of the manuscript and for helpful discussions. This work was supported in part by grant from RIKEN Brain Science Institute (K.Y.) and the National Institutes of Health (HD-31498 to C.J.E.).

Conflict of Interest statement. None declared.


   References
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 References
 

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