It is well known that Down syndrome (DS) is a condition in which extra genetic material causes delays in the way a child develops, both mentally and physically. Intellectual disability is the foremost and most debilitating trait, which caused loss of cognitive abilities and the development of early onset Alzheimer’s disease (AD). Ts65Dn mice were used in this study. We isolated the hippocampus. First, we used transmission scanning electron microscopy to directly observe the hippocampus and confirm if apoptosis had occurred. Second, we customized a PCR array with 53 genes, including several important genes related to cell apoptosis. Gene expression was detected by RT-PCR. There were varying degrees of changes characteristic of apoptosis in the hippocampus of Ts65Dn mice, which mainly included the following: nuclear membrane thinning, unevenly distributed chromosomes, the production of chromatin crescents, and pyknosis of the nuclei with some nuclear fragmentation. Meanwhile, three genes (API5, AIFM1, and NF
Down syndrome (DS) is one of the most common gross chromosomal abnormalities and birth defects. It is well known that DS is a condition in which extra genetic material causes delays in child development, both mentally and physically. Intellectual disabilities are the most debilitating trait and they cause the loss of cognitive abilities. Meanwhile, it is well known that Alzheimer’s disease occurs in individuals with trisomy 21 because the gene encoding amyloid is located on this chromosome. Thus, these individuals have a higher load of amyloid which, in turn, results in a higher incidence of developing cognitive decline and Alzheimer’s dementia with aging [
Apoptosis is the mechanism by which cells die in response to a wide range of physiological and developmental stimuli. Increasing data has shown that enhanced apoptosis in Alzheimer’s disease might play a role in the mental deficiencies and neurodegeneration associated with that disease [
Down syndrome results from the presence of an extra chromosome 21, which causes changes in gene expression. It is not known whether this unbalanced gene regulation is related to apoptosis in the nervous system. However, there are so many molecules involved in the process of apoptosis that real-time quantitative PCR may not be suitable for observing it. A high-throughput platform with efficient detection capacity is needed to solve this problem. Recently, PCR arrays have been considered the most reliable tools for analyzing the expression of a focused panel of genes, especially in signal transduction pathways, biological process, and disease related gene networks [
In present study, we directly observed whether apoptosis occurred in the nervous system of Ts65Dn mice with a transmission scanning electron microscope. Meanwhile, a target PCR array related to apoptosis was constructed and applied to screen for changes in expression of genes related to apoptosis.
This study was conducted in the Changzhou Women and Children Hospital of Nanjing Medical University (Changzhou, Jiangsu, China). The animals were bred in the Animal Center of Jiangsu University (Zhenjiang, Jiangsu, China). All efforts were made to minimize the suffering and number of animals used.
Five Ts65Dn mice carrying a partial trisomy of chromosome 16 were purchased from the Jackson Laboratories (Bar Harbor, ME, USA). Five normal C57BL/6JEi mice were used as the normal control group. Both groups were matched by age and were more than 14 weeks old. The animals’ health and comfort were monitored by the veterinary service. The animals had access to water and food according to the routine methods of animal breeding in the Animal Center of Jiangsu University.
Anesthetized animals were euthanized using 10% chloral hydrate (Changzhou First People’s Hospital, Changzhou, China). We removed the brain and isolated the hippocampus.
To assess the morphological changes in the hippocampus of Ts65Dn mice, we used transmission scanning electron microscopy to directly observe whether apoptosis had occurred. The methods were performed as described by Chen’s group [
Total RNA was isolated by TRIZOL (Invitrogen) extraction. After the homogenization of tissue samples, insoluble material was removed from the homogenate by centrifugation at 12000 ×g for 10 minutes at 2 to 8°C. The homogenized samples were incubated for 5 minutes at room temperature, and 0.2 mL of chloroform per 1 mL of TRIZOL reagent was added. The tubes were shaken vigorously by hand for 15 seconds and incubated at 15 to 30°C for 2 to 3 minutes. Then, the samples were centrifuged at no more than 12000 ×g for 15 minutes at 2 to 8°C, and the colorless upper aqueous phase was transferred to a fresh tube. The RNA was precipitated from the aqueous phase by mixing with isopropyl alcohol (0.5 mL of isopropyl alcohol per 1 mL of TRIZOL reagent). Samples were incubated at 15 to 30°C for 10 minutes and centrifuged at no more than 12000 ×g for 10 minutes at 2 to 8°C. The supernatant was removed, and the RNA pellet was washed once with 75% ethanol. The samples were mixed by vortexing and centrifuged at no more than 7500 ×g for 5 minutes at 2 to 8°C. Finally, the RNA pellets were air or vacuum dried for 5–10 minutes, and RNA was dissolved in RNase-free water.
PCR arrays contained 53 genes, 6 housekeeping genes, PPC, and GDC. The 53 genes were related to cell apoptosis. The housekeeping genes were B2M, ACTB, GAPDH, RPL27, HPRT1, and OAZ1. The PPC contained synthetic DNA fragments that have no homology with the detected species and amplification primers which were used as quality control. GDC was used to detect the residual genomic DNA.
Total RNA from each sample was used for reverse transcription with an RT-PCR Kit (catalog#CTB101; CT biosciences, China) on an ABI 9700 thermocycler (ABI, Foster City, CA). For reverse transcription, 4
All data were collected and statistically analyzed using SPSS 13.0 software. The results were expressed as the means ± SD. The expression of genes in two groups was compared using
Typical synaptic structures and normal cell structures were observed in the control mice (Figure
The morphological changes in the hippocampus of Ts65Dn mice observed by TEM. (a) Normal hippocampus tissues in control mice showing typical synaptic and normal cell structure, including normal cell nuclear shapes, distributed chromatin, abundant organelles, dense mitochondria, and flat Golgi apparatus. (b) In Ts65Dn mice the nuclear membrane became thin, chromosomes appeared unevenly distributed, and chromatin crescents were produced. (c) The nuclei exhibited pyknosis and nuclear fragmentation. (d) The cell wall began to sag and cell atrophy was observed.
In present study, PCR arrays contained 53 genes related to cell apoptosis. After real-time PCR, three genes (API5, AIFM1, and NF
Differential apoptotic gene expressions in the hippocampus of Ts65Dn mice.
Gene symbol | Description of genes | Chromosome | Ts65Dn/control group | ||
---|---|---|---|---|---|
Human | Mouse | Fold changes |
|
||
API5 | Apoptosis inhibitor 5 | 11 | 2 | −11.55 | 0.00 |
AIFM1 | Apoptosis-inducing factor, mitochondrion-associated, 1 | X | X | −5.94 | 0.00 |
NF |
Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 | 4 | 3 | 3.11 | 0.02 |
Bcl-2 | B-cell CLL/lymphoma 2 | 18 | 1 | 2.75 | 0.07 |
Ntrk1 | Neurotrophic tyrosine kinase, receptor, type 1 | 1 | 3 | 2.59 | 0.09 |
Tnfrsf1a | Tumor necrosis factor receptor superfamily, member 1A | 12 | 6 | 2.52 | 0.1 |
Ripk1 | Receptor (TNFRSF)-interacting serine-threonine kinase 1 | 6 | 13 | 2.52 | 0.1 |
Rela | V-rel avian reticuloendotheliosis viral oncogene homolog A | 11 | 19 | 2.52 | 0.1 |
Map3k14 | Mitogen-activated protein kinase kinase kinase 14 | 17 | 11 | 2.52 | 0.1 |
Irak1 | Interleukin-1 receptor-associated kinase 1 | X | X | 2.52 | 0.1 |
Il1a | Interleukin-1, alpha | 2 | 2 | 2.52 | 0.1 |
Il10 | Interleukin 10 | 1 | 1 | 2.52 | 0.1 |
Ikbkg | Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma | X | X | 2.52 | 0.1 |
Ikbkb | Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta | 8 | 8 | 2.52 | 0.1 |
Igf1r | Insulin-like growth factor 1 receptor | 15 | 7 | 2.52 | 0.1 |
Fadd | Fas (TNFRSF6)-associated via death domain | 11 | 7 | 2.52 | 0.1 |
Dffa | DNA fragmentation factor, 45 kDa, alpha polypeptide | 1 | 4 | 2.52 | 0.1 |
Dapk1 | Death-associated protein kinase 1 | 9 | 13 | 2.52 | 0.1 |
Cideb | Cell death-inducing DFFA-like effector b | 14 | 14 | 2.52 | 0.1 |
Cidea | Cell death-inducing DFFA-like effector a | 18 | 18 | 2.52 | 0.1 |
Cdc2a | Cyclin-dependent kinase A-1 | 10 | 2.52 | 0.1 | |
Cd70 | CD70 molecule | 19 | 17 | 2.52 | 0.1 |
Casp-9 | Caspase-9 | 1 | 4 | 2.52 | 0.1 |
Casp-8 | Caspase-8 | 2 | 1 | 2.52 | 0.1 |
Casp-7 | Caspase-7 | 10 | 19 | 2.52 | 0.1 |
Casp-6 | Caspase-6 | 4 | 3 | 2.52 | 0.1 |
Casp-3 | Caspase-3 | 4 | 8 | 2.52 | 0.1 |
Birc2 | Baculoviral IAP repeat containing 2 | 11 | 9 | 2.52 | 0.1 |
Apaf1 | Apoptotic protease-activating factor 1 | 12 | 10 | 2.52 | 0.1 |
Akt2 | V-akt murine thymoma viral oncogene homolog 2 | 19 | 7 | 2.52 | 0.1 |
Akt3 | V-akt murine thymoma viral oncogene homolog 3 | 1 | 1 | 3.3 | 0.13 |
Il1b | Interleukin-1, beta | 2 | 2 | 6.75 | 0.14 |
Myd88 | Myeloid differentiation primary response gene 88 | 3 | 9 | 3.54 | 0.15 |
Birc3 | Baculoviral IAP repeat containing 3 | 11 | 9 | 3.63 | 0.15 |
Mapk3 | Mitogen-activated protein kinase 3 | 16 | 7 | 3.21 | 0.17 |
Mapk1 | Mitogen-activated protein kinase 1 | 22 | 16 | 6.59 | 0.29 |
Bid | BH3 interacting domain death agonist | 22 | 6 | −2.03 | 0.3 |
Fas | Fas cell surface death receptor | 10 | 19 | 3.23 | 0.43 |
Casp-4 | Caspase-4 | 11 | 9 | 1.7 | 0.48 |
Akt1 | V-akt murine thymoma viral oncogene homolog 1 | 14 | 12 | 3.44 | 0.49 |
TNF | Tumor necrosis factor | 6 | 17 | 2.01 | 0.5 |
Nf |
Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha | 14 | 12 | 2 | 0.56 |
Cd40lg | CD40 ligand | X | X | 1.64 | 0.61 |
Pik3r2 | Phosphoinositide-3-kinase, regulatory subunit 2 | 19 | 8 | −1.64 | 0.72 |
Casp-2 | Caspase-2 | 7 | 6 | −1.66 | 0.72 |
Tnfrsf10b | Tumor necrosis factor receptor superfamily, member 10b | 8 | 14 | 1.49 | 0.75 |
Ppp3cc | Protein phosphatase 3, catalytic subunit, gamma isozyme | 8 | 14 | 1.58 | 0.8 |
Xiap | X-linked inhibitor of apoptosis | X | X | −1.05 | 0.85 |
Ppp3r1 | Protein phosphatase 3, regulatory subunit B, alpha | 2 | 11 | 1.22 | 0.89 |
Bad | Bcl-2-associated agonist of cell death | 11 | 19 | 1.15 | 0.9 |
Pik3ca | Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha | 3 | 3 | 1.21 | 0.93 |
Endog | Endonuclease G | 9 | 2 | 1.05 | 0.96 |
Pik3cg | Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit gamma | 7 | 12 | −1.01 | 0.99 |
Note:
Fold change in gene expression between the Ts65Dn mice and the control group. Compared with the control group, the expression of API5 and AIFM1 showed decreased levels, and NF
Comparison of the expressions of API5, AIFM1, and NF
In present study, we examined the genes of the caspase family (including casp-2, casp-3, casp-4, casp-6, casp-7, casp-8, and casp-9). However, they showed no significant differences in expression levels. Meanwhile, several well-known genes related to cell apoptosis, such as Bcl-2, Bad, Bid, Fas, and TNF, also did not differ in expression in the hippocampus of Ts65Dn mice compared to the normal control group (Table
In this study, we showed cell apoptosis in the nervous system of Ts65Dn mice by changes in cell morphology. Then, using a target PCR array, we found that three genes related to cell apoptosis showed different levels of expression in the hippocampus of Ts65Dn mice.
Apoptosis, or programmed cell death, is a regulated physiological process that leads to cell death and is characterized by cell shrinkage, membrane blebbing, and DNA fragmentation. The changes associated with apoptosis often occur in multiple stages. First, there is a decrease in cell volume, and then the cytoplasmic density appears to increase, followed by a decrease in the mitochondrial membrane, an increase in permeability, chromatin condensation, nuclear nucleolar fragmentation, DNA degradation, and finally the formation of apoptotic bodies. In the present study, we also found the typical characteristics of cell apoptosis as stated above. For example, the nuclear membrane thinned, chromosomes appeared unevenly distributed, chromatin crescents were produced, the nuclei exhibited pyknosis, and nuclear fragmentation was observed. It is still uncertain whether cell apoptosis occurs in the brains of DS patients. Through the observation of transmission electron microscopy, we could preliminarily confirm that cell apoptosis occurs in the nervous system of Down syndrome model mice.
Currently, the mechanism of cell apoptosis is not fully understood. However, it is well known that the process is controlled by multiple genes, including the Bcl-2 family, caspase family, TNFR family, C-myc, and P53. In the present study, we included 53 genes in one customized PCR array, including several important genes related to cell apoptosis. For example, we included seven members (casp-2, casp-3, casp-4, casp-6, casp-7, casp-8, and casp-9) of the caspase family in the PCR array. We hoped to discover which gene plays an important role in cell apoptosis of Down syndrome. In contrast to our expectations, all members of the caspase family, including Bcl-2, Bad, Bid, Fas, and TNF, showed normal expression in the hippocampus of Ts65Dn mice compared with the normal control group. Only three genes, API5, AIFM1, and NF
We found that API5, AIFM1, and NF
In conclusion, we found that there were varying degrees of changes characteristic of apoptosis in the hippocampus of Ts65Dn mice. Meanwhile, by PCR array, including several important genes related to cell apoptosis, three genes (API5, AIFM1, and NF
The study design and protocol were reviewed and approved by the Ethics Committee of Changzhou Woman and Children Health Hospital Affiliated to Nanjing Medical University.
The authors declare that they have no competing interests.
The authors thank all of the project participants for their contributions. This study was supported by grants from the Changzhou Research Program of Applied Basic (CJ20140055), the major projects of Jiangsu Maternal and Child Health (F201217), the project of the health department of Jiangsu Province (H201352), and the youth project of Health Bureau of Changzhou City (QN201405).