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Keywords:

  • Down syndrome;
  • gene;
  • mental retardation;
  • mouse model;
  • trisomy

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. SPONTANEOUS PARTIAL TRISOMY 16 MICE AS MODELS FOR HUMAN DOWN SYNDROME
  4. DOSAGE-DEPENDENT OVEREXPRESSION OF GENES ON TRISOMIC SEGMENT OF THE DS MOUSE MODEL
  5. EXCESSIVE OXIDATIVE STRESS, IMPAIRED NEUROGENESIS AND VENTRICULAR ENLARGEMENTS IN THE BRAINS OF DS MOUSE MODELS
  6. PREFERABLE STRATEGY TO IDENTIFY THE GENES FOR DS: RESUMPTIONS OF THE COPY NUMBER OF CANDIDATE GENES IN DS MOUSE MODELS
  7. HIGHLY EFFICIENT SYSTEM FOR THE GENERATION OF DS MOUSE MODELS WITH DESIGNED PARTIAL TRISOMY SEGMENTS THAT HARBORS MULTIPLE GENE KNOCKOUTS
  8. REFERENCES

Down syndrome (DS) is the most common cause of mental retardation. Several DS mouse models with partial trisomy 16 homologous to human chromosome 21 have been developed, and our research group has been studying those mouse models. We have shown a dosage-dependent overexpression of genes in the trisomic region of the mouse. We have also described abnormalities including increased oxidative stress, increased lipid peroxidation, mitochondrial dysfunction, tau-hyperphosphorylation and overactivation of its phosphatases, impaired developmental and adult neurogenesis, histological abnormalities in brains including ventricle enlargements and minor neurodegenerations in those mice. These observations may contribute to the identification of responsible genes and understanding of molecular pathology of Down syndrome.


SPONTANEOUS PARTIAL TRISOMY 16 MICE AS MODELS FOR HUMAN DOWN SYNDROME

  1. Top of page
  2. ABSTRACT
  3. SPONTANEOUS PARTIAL TRISOMY 16 MICE AS MODELS FOR HUMAN DOWN SYNDROME
  4. DOSAGE-DEPENDENT OVEREXPRESSION OF GENES ON TRISOMIC SEGMENT OF THE DS MOUSE MODEL
  5. EXCESSIVE OXIDATIVE STRESS, IMPAIRED NEUROGENESIS AND VENTRICULAR ENLARGEMENTS IN THE BRAINS OF DS MOUSE MODELS
  6. PREFERABLE STRATEGY TO IDENTIFY THE GENES FOR DS: RESUMPTIONS OF THE COPY NUMBER OF CANDIDATE GENES IN DS MOUSE MODELS
  7. HIGHLY EFFICIENT SYSTEM FOR THE GENERATION OF DS MOUSE MODELS WITH DESIGNED PARTIAL TRISOMY SEGMENTS THAT HARBORS MULTIPLE GENE KNOCKOUTS
  8. REFERENCES

Down syndrome (DS) or trisomy 21 is the most common autosomal aneuploidy and genetic cause of mental retardation. As most of human chromosome 21, harboring ∼370 genes, is orthologous to the distal end of mouse chromosome 16, several mouse models of DS with segmental trisomy for mouse chromosome 16 have been established (Figs 1,2). Ts65Dn (Davisson et al. 1990) and Ts1Cje (Sago et al. 1998) mice carry trisomic segments of mouse chromosome 16 that contain regions orthologous to human chromosome 21 harboring ∼140 genes and ∼100 genes respectively. The Ts1Cje mouse has a smaller extra segment extending from Cu/Zn-superoxide dismutase (sod1) to znf295, but sod1 is functionally excluded because of a mutation in the gene. Ts2Cje has also been established after a fortuitous translocation of the Ts65Dn marker chromosome to chromosome 12 (Villar et al. 2005). Both Ts65Dn and Ts2Cje carry an equivalent trisomic segment extending from the gene encoding mitochondrial ribosomal protein L39 (mrpl39) to the zinc finger protein 295 (znf295) gene. Another mouse model with further narrower trisomic segment, Ts1Rhr, has also been reported (Olson et al. 2004; Belichenko et al. 2009). These trisomic segment regions have been reported to be critically involved in the DS pathology.

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Figure 1. Down syndrome model mice harboring partial trisomy 16; Ts65Dn, Ts2Cje, Ts1Cje and Ts1Rhr.

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Figure 2. Genes on the trisomic segments of Down syndrome model mice.

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DOSAGE-DEPENDENT OVEREXPRESSION OF GENES ON TRISOMIC SEGMENT OF THE DS MOUSE MODEL

  1. Top of page
  2. ABSTRACT
  3. SPONTANEOUS PARTIAL TRISOMY 16 MICE AS MODELS FOR HUMAN DOWN SYNDROME
  4. DOSAGE-DEPENDENT OVEREXPRESSION OF GENES ON TRISOMIC SEGMENT OF THE DS MOUSE MODEL
  5. EXCESSIVE OXIDATIVE STRESS, IMPAIRED NEUROGENESIS AND VENTRICULAR ENLARGEMENTS IN THE BRAINS OF DS MOUSE MODELS
  6. PREFERABLE STRATEGY TO IDENTIFY THE GENES FOR DS: RESUMPTIONS OF THE COPY NUMBER OF CANDIDATE GENES IN DS MOUSE MODELS
  7. HIGHLY EFFICIENT SYSTEM FOR THE GENERATION OF DS MOUSE MODELS WITH DESIGNED PARTIAL TRISOMY SEGMENTS THAT HARBORS MULTIPLE GENE KNOCKOUTS
  8. REFERENCES

The overexpression of genes located on the trisomic region has been assumed to be responsible for the phenotypic abnormalities of DS, but this hypothesis has not been fully confirmed and the very existence of gene dosage effects has been called into question. We have therefore investigated global gene expression profiles in Ts1Cje (Amano et al. 2004). DNA microarray analyses of six Ts1Cje and six normal littermate (2N) mouse brains at postnatal day 0 with probe sets representing approximately 11 300 genes revealed that the number of expressed genes and their identities in Ts1Cje mice were almost the same as those in 2N mice. However, the expression levels of most genes in the trisomic region were increased 1.5-fold, and the top 24 most consistently overexpressed genes in the Ts1Cje mice were all located in the trisomic region. In contrast, the expression levels of genes on other chromosomes or the euploid region of chromosome 16 were largely the same (1.0-fold) in Ts1Cje and 2N mice (Fig. 3). These results indicate that the genes in the trisomic region of Ts1Cje are overexpressed in a dosage-dependent manner and suggest that it is also the case in patients with DS.

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Figure 3. Dosage-dependent overexpression of genes in the trisomic region of Ts1Cje. The expression levels of genes in the trisomic region were increased 1.5-fold, while the expression levels of genes on other chromosomes or the euploid region of chromosome 16 were largely the same (1.0-fold) in Ts1Cje and 2N mice (Amano et al. 2004).

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EXCESSIVE OXIDATIVE STRESS, IMPAIRED NEUROGENESIS AND VENTRICULAR ENLARGEMENTS IN THE BRAINS OF DS MOUSE MODELS

  1. Top of page
  2. ABSTRACT
  3. SPONTANEOUS PARTIAL TRISOMY 16 MICE AS MODELS FOR HUMAN DOWN SYNDROME
  4. DOSAGE-DEPENDENT OVEREXPRESSION OF GENES ON TRISOMIC SEGMENT OF THE DS MOUSE MODEL
  5. EXCESSIVE OXIDATIVE STRESS, IMPAIRED NEUROGENESIS AND VENTRICULAR ENLARGEMENTS IN THE BRAINS OF DS MOUSE MODELS
  6. PREFERABLE STRATEGY TO IDENTIFY THE GENES FOR DS: RESUMPTIONS OF THE COPY NUMBER OF CANDIDATE GENES IN DS MOUSE MODELS
  7. HIGHLY EFFICIENT SYSTEM FOR THE GENERATION OF DS MOUSE MODELS WITH DESIGNED PARTIAL TRISOMY SEGMENTS THAT HARBORS MULTIPLE GENE KNOCKOUTS
  8. REFERENCES

Elevated oxidative stress has been suggested to be associated with the features of Down syndrome (DS). We reported that Ts1Cje brain shows decreases of mitochondrial membrane potential and ATP production, increases of reactive oxygen species (ROS), hyperphosphorylation of tau without neurofibrillary tangle (NFT) formation, increase of tau kinases (GSK3β and JNK/SAPK) activities, unaltered AβPP metabolism, and moderate neurodegenerations (Shukkur et al. 2006). 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. We also examined lipid peroxidation (LPO), a typical marker of oxidative stress, in the brains of Ts1Cje and Ts2Cje (Ishihara et al. 2009). Accumulations of proteins modified with the LPO-derived products, 13-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPODE) and 4-hydroxy-2-nonenal (4-HNE) were markedly increased in Ts1Cje and Ts2Cje brains. Analysis with oxidation-sensitive fluorescent probe also showed that ROS themselves were increased in Ts1Cje brain. However, electron spin resonance (ESR) analysis of microdialysate from the hippocampus of Ts1Cje showed that antioxidant activity remained unaffected, suggesting that ROS production was accelerated in Ts1Cje. Proteomics approaches with mass spectrometry identified the proteins modified with 13-HPODE and/or 4-HNE to be involved in either ATP generation, the neuronal cytoskeleton or antioxidant activity. These results are consistent with our previous observations (Shukkur et al. 2006), and that Ts1Cje segment, which is common to Ts2Cje, harbor the responsible gene(s) for the increase of oxidative stress. Structural or functional impairments of these proteins by such modifications may contribute to the DS features such as cognitive impairment that are present in the Ts1Cje mouse.

We also described ventricular enlargements in Ts1Cje and Ts2Cje adult brains at a similar degree (Ishihara et al. 2010). Both mice also showed decreases of the number of doublecortin-positive neuroblasts and thymidine-analog BrdU-labeled proliferating cells in the subventricular zone of the lateral ventricles and in the hippocampal dentate gyrus at a similar degree, suggesting impaired adult neurogenesis. Additionally, at embryonic day 14.5, both strains of mice, when compared to diploid littermates, had smaller brains and decreased cortical neurogenesis that could possibly contribute to the ventricular enlargements observed in adulthood. Our findings suggest that the trisomic segment of the Ts1Cje mouse, which is shared with Ts2Cje, contains the gene(s) that are responsible for these abnormal phenotypes and would be relevant to the mental retardation associated with DS. All of these abnormalities could possibly be relevant to the impairments of learning and memory in the DS mouse models and also the mental retardation phenotype observed in patients with Down syndrome as well.

PREFERABLE STRATEGY TO IDENTIFY THE GENES FOR DS: RESUMPTIONS OF THE COPY NUMBER OF CANDIDATE GENES IN DS MOUSE MODELS

  1. Top of page
  2. ABSTRACT
  3. SPONTANEOUS PARTIAL TRISOMY 16 MICE AS MODELS FOR HUMAN DOWN SYNDROME
  4. DOSAGE-DEPENDENT OVEREXPRESSION OF GENES ON TRISOMIC SEGMENT OF THE DS MOUSE MODEL
  5. EXCESSIVE OXIDATIVE STRESS, IMPAIRED NEUROGENESIS AND VENTRICULAR ENLARGEMENTS IN THE BRAINS OF DS MOUSE MODELS
  6. PREFERABLE STRATEGY TO IDENTIFY THE GENES FOR DS: RESUMPTIONS OF THE COPY NUMBER OF CANDIDATE GENES IN DS MOUSE MODELS
  7. HIGHLY EFFICIENT SYSTEM FOR THE GENERATION OF DS MOUSE MODELS WITH DESIGNED PARTIAL TRISOMY SEGMENTS THAT HARBORS MULTIPLE GENE KNOCKOUTS
  8. REFERENCES

To evaluate each ch.21 gene, several DS mouse models have been generated by several groups using multiple distinct systems including introducing BAC, PAC, or YAC human or mouse genomic DNA, cDNA clones with artificial promoters. Even though some of those mice showed DS-like phenotypes such as memory dysfunction, human ch. 21 genes or mouse ch. 16 genes may be highly overexpressed, ectopically expressed, or expressed in the wrong developmental timings in those mice, and therefore it is very difficult to compare these results impartially. Instead, other groups including ours are analyzing DS mouse models with partial trisomy mated with mice with gene knockouts in those the copy number of the corresponding genes were resumed to two copies while the other genes on the trisomic segment are remained to be three copies (Fig. 4). In this system, physiological significance of the candidate genes can be impartially compared in a common platform.

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Figure 4. Down syndrome mouse models with partial trisomy mated with mice with gene knockouts. In these mice, the copy number of the corresponding genes is resumed to two copies while the other genes on the trisomic segment are remained to be three copies. Abnormal parameters for Down syndromes are evaluated in these mice.

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HIGHLY EFFICIENT SYSTEM FOR THE GENERATION OF DS MOUSE MODELS WITH DESIGNED PARTIAL TRISOMY SEGMENTS THAT HARBORS MULTIPLE GENE KNOCKOUTS

  1. Top of page
  2. ABSTRACT
  3. SPONTANEOUS PARTIAL TRISOMY 16 MICE AS MODELS FOR HUMAN DOWN SYNDROME
  4. DOSAGE-DEPENDENT OVEREXPRESSION OF GENES ON TRISOMIC SEGMENT OF THE DS MOUSE MODEL
  5. EXCESSIVE OXIDATIVE STRESS, IMPAIRED NEUROGENESIS AND VENTRICULAR ENLARGEMENTS IN THE BRAINS OF DS MOUSE MODELS
  6. PREFERABLE STRATEGY TO IDENTIFY THE GENES FOR DS: RESUMPTIONS OF THE COPY NUMBER OF CANDIDATE GENES IN DS MOUSE MODELS
  7. HIGHLY EFFICIENT SYSTEM FOR THE GENERATION OF DS MOUSE MODELS WITH DESIGNED PARTIAL TRISOMY SEGMENTS THAT HARBORS MULTIPLE GENE KNOCKOUTS
  8. REFERENCES

Although the above-mentioned strategy is promising, the generations of a number of knockout mice is still huge task. To further narrow down the responsible chromosomal regions, we are also trying to develop a highly efficient system for the generation of mice with freely designed partial trisomic segments by using cells that give highly effective gene conversion (Fig. 5) (paper in preparation). This system is not only highly efficient but also makes it possible to introduce multiple gene inactivation's into the mice, which is very difficult or even not possible in the standard partial trisomy versus knockout mice mating protocol. Because the cumulative effects of multiple ch.21 genes (e.g. the collaborative effect of DSCR1 and DYRK1 on NFAT deregulation [Arron et al. 2006]) have been expected in the DS pathology, the new system should become a strong weapon against DS pathology.

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Figure 5. Highly efficient system for the generation of mice with freely designed partial trisomic segments and genes knockouts. In X cells, highly effective gene conversion occur.

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DS molecular and cellular pathology remains unveiled. Our observations on the DS mouse models together with our new system mentioned above should contribute to the identification of responsible gene(s) on trisomic segments of the mice and on human chromosome 21 and to the understanding of DS molecular pathomechanism.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. SPONTANEOUS PARTIAL TRISOMY 16 MICE AS MODELS FOR HUMAN DOWN SYNDROME
  4. DOSAGE-DEPENDENT OVEREXPRESSION OF GENES ON TRISOMIC SEGMENT OF THE DS MOUSE MODEL
  5. EXCESSIVE OXIDATIVE STRESS, IMPAIRED NEUROGENESIS AND VENTRICULAR ENLARGEMENTS IN THE BRAINS OF DS MOUSE MODELS
  6. PREFERABLE STRATEGY TO IDENTIFY THE GENES FOR DS: RESUMPTIONS OF THE COPY NUMBER OF CANDIDATE GENES IN DS MOUSE MODELS
  7. HIGHLY EFFICIENT SYSTEM FOR THE GENERATION OF DS MOUSE MODELS WITH DESIGNED PARTIAL TRISOMY SEGMENTS THAT HARBORS MULTIPLE GENE KNOCKOUTS
  8. REFERENCES