Analysis of hippocampal gene expression profile of Alzheimer's disease model rats using genome chip bioinformatics.

In this study, an Alzheimer's disease model was established in rats through stereotactic injection of condensed amyloid beta 1-40 into the bilateral hippocampus, and the changes of gene expression profile in the hippocampus of rat models and sham-operated rats were compared by genome expression profiling analysis. Results showed that the expression of 50 genes was significantly up-regulated (fold change ≥ 2), while 21 genes were significantly down-regulated in the hippocampus of Alzheimer's disease model rats (fold change ≤ 0.5) compared with the sham-operation group. The differentially expressed genes are involved in many functions, such as brain nerve system development, neuronal differentiation and functional regulation, cellular growth, differentiation and apoptosis, synaptogenesis and plasticity, inflammatory and immune responses, ion channels/transporters, signal transduction, cell material/energy metabolism. Our findings indicate that several genes were abnormally expressed in the metabolic and signal transduction pathways in the hippocampus of amyloid beta 1-40-induced rat model of Alzheimer's disease, thereby affecting the hippocampal and brain functions.

INTRODUCTION

Alzheimer's disease (AD), also known as presenile dementia or senile dementia, is a type of central nervous system degenerative disease characterized by progressive cognitive impairment and memory damage. It is the most common type of dementia[12]. AD has three major pathological features: senile plaque formation as a result of amyloid beta (Aβ) accumulation around brain cells, neurofibrillary tangle formation as a result of Tau protein hyperphosphorylation within brain cells, and neuronal death. Aβ peptide accumulation is a crucial factor leading to AD, and is one of the neuropathological markers of AD[34]. However, the occurrence and development of AD is a complex pathological process involving many factors and pathways, where many genes play a synergistic (or antagonistic) role. This process includes the shearing, hydrolysis and clearing of amyloid protein precursor; imbalance of protein phosphorylation, neuronal apoptosis, DNA transcription, protein synthesis, signal transduction, and other changes of related genes[56]. It remains unknown which genes are involved in AD occurrence and development, and impedes attempts to explore the prevention and treatment of AD.

Gene chip, a recently developed technology, has been widely applied in understanding gene functions, investigating the links between genes, analyzing spatial and temporal expression of each gene[789101112], and exploring the downstream signal transduction pathways[1314]. It is a high throughput technology that is fast and requires only small amounts of samples, but yields copious information. With the application of gene chip and in-depth study of central nervous system regeneration, we have achieved great progress in the research of central nervous system diseases. Gene chip technology has been recognized as a powerful means for studying the mechanism underlying the multi-gene, multi-pathway, multi-factorial components of neurological diseases, and for elucidating the correlation of genes to neurological diseases. The present study sought to understand and diagnose central nervous system diseases, as well as to identify potential genetic therapy and drug treatment.

The hippocampus is an important brain region for neurogenesis in the adult brain, and participates in the formation of learning and memory, and the regulation of mood. A number of studies have shown that the learning and memory functions are significantly decreased in a damaged bilateral hippocampus, thus affecting learning, maintenance of defense conditioned response, and spatial acquisition capabilities[1516]. Intraventricular injection of toxic Aβ fragments can lead to a decrease in learning and memory in animals, and can partially simulate the pathological hallmarks of AD[17]. In this study, AD rat models were established through Aβ1-40 injection into the bilateral hippocampus of rats. The microarray expression profiling changes in the hippocampus of AD rats were determined with whole genome microarray analysis, and the AD-associated genes and gene expression profiles were observed in a broader attempt to elucidate the etiology of AD at the gene level, to identify the molecular genetic mechanisms underlying the Aβ1-40-induced AD, to search drug targets for the prevention and treatment of AD, and to provide laboratory evidence for screening effective drugs for the prevention and treatment of AD.

RESULTS

Quantitative analysis of experimental animals

Thirty male Sprague-Dawley rats, of specific pathogen- free grade, were maintained for 2 weeks for acclimation, and 22 of the animals that were in good condition were used in further study. A total of 22 rats were divided into a sham-operation group (n = 10) and an AD model group (n = 12) according to a random number table. Normal saline and Aβ1-40 was injected into the bilateral hippocampi, respectively. Eight rats were confirmed for AD-like behaviors by use of Morris water maze test at 4 weeks post-surgery. Finally, five rats were randomly selected from two groups for whole-genome microarray analysis of the hippocampus.

Identification of RNA in the hippocampus of Aβ1-40-induced AD model rats and sham-operated rats

Total RNA was extracted from the hippocampus of each group by using RNAEx Reagent, and quantified with a UV spectrophotometer. The ratio of absorbance at 260 nm and 280 nm was 1.8–2.0, indicating a high purity of RNA. Formaldehyde denaturing gel electrophoresis detection showed that the electrophoresis strip of RNA samples was clear and the luminance ratio of the rRNA band at 28 S to 18 S was greater than 1: 1, indicating that RNA was intact and met the requirements for expression profiling microarray and the reverse transcription-PCR test (Figure 1).

Figure 1

RNA was extracted from the rat hippocampus with 1% formaldehyde denaturing gel electrophoresis. Image was provided by the National Engineering Research Center for Beijing Biochip Technology (Beijing CapitalBio Corporation, China).

The RNA sample electrophoretic bands were clear. rRNA band intensity at 28 S to 18 S was larger than 1: 1, in accordance with the quality requirements of microarray experiments. The RNA integrity was good with fewer degraded small fragments.

1: Sham-operation group; 2: Alzheimer's disease model group.

Changes of gene expression in rat hippocampus after Aβ1-40 treatment

In this study, gene expression profile was examined with whole genome microarray analysis (Figure 2). As shown in the microarray hybridization signal intensity plot, the vast majority of hybridization signals were located around the 45° diagonal line. The ratio of Y value and X value was 0.5–2.0, indicating non-differential expression; the minor number of data points were substantially away from the 45° diagonal line. The ratio of Y value and X value was out of the range between 0.5 and 2.0, indicating differential expression (Figure 3).

Figure 2

Original image of hippocampal gene expression profile. Image was provided by the National Engineering Research Center for Beijing Biochip Technology (Beijing CapitalBio Corporation, China) who provided the original microarray data plot for the DNA microarray experiments of the samples in two groups.

(A) Sham-operation group; (B) Alzheimer's disease model group.

Figure 3

Scatter plot of gene hybridization signal intensity in two groups. Image was provided by the National Engineering Research Center for Beijing Biochip Technology (Beijing CapitalBio Corporation, China), through a paired comparison of the original microarray data in the sample analysis process.

Vertical axis represents the scaling signal (intensity) in the Alzheimer's disease model group, while the X-axis represents the scaling signal (intensity) in the sham-operation group.

Comparison of gene expression changes in the hippocampus of each group showed that the expressions of fifty genes in the hippocampus of the model group were significantly up-regulated (fold change ≥ 2), while the expressions were down-regulated in 21 genes (fold change ≤ 0.5) compared with the sham-operation group. Gene biological functions were evaluated with MAS software, and results showed that the vast majority of differentially expressed genes were associated with the immune response, and many up-regulated genes and cell cycle-related protein factors were seen. A variety of transcription-related genes were also up-regulated, due to activation of the transcription control system, and up-regulation of the genes involved in the intracellular signal transduction pathway. Furthermore, many cytokines and their receptors, intracellular enzyme- or protein-related genes were up-regulated, and the most significant difference was observed in LOC500180 (FC = 22.5), IgH-1a (FC = 12.7), and LOC4983359 (FC = 11) which all participate in the immune response (Table 1).

Table 1

Differentially expressed genes in the hippocampus of amyloid beta 1–40-injected Alzheimer's disease model rats compared with sham-operated rats (n = 5)

DISCUSSION

The recently developed gene chip technology can detect the expression of thousands of genes simultaneously, and can form a complete gene expression profile. It has become a powerful tool for the analysis of gene expression. Gene microarray can simultaneously detect the mRNA expression of different genes in varying tissues or cells at different developmental stages within the entire genome[789101112], therefore, it is an ideal approach for studying the pathological mechanisms underlying AD.

In this study, the pathological process of AD animals was simulated through injection of Aβ1-40 into the bilateral hippocampus. The hippocampal gene expression profile changes were compared between the sham-operated group and the AD model group with the whole genome microarray analysis. Results showed that the expressions were significantly up-regulated in 50 genes in the hippocampus of the model group (fold change ≥ 2), while significantly down-regulated in 21 genes (fold change ≤ 0.5) compared with the sham-operation group.

The up-regulated genes in the Aβ1-40-induced AD rats, such as nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (α-Nfkbia) (FC = 2.82), can bind with nuclear factor κB and regulate its intracellular degradation, participate in lipopolysaccharide-mediated signaling pathways and inflammatory responses, inhibit the differentiation of bone marrow stem cells and DNA binding, and reversibly control the Notch signaling pathway. The Notch signaling pathway, activated by ligand binding, can switch the conversion of the cell cycle, regulate cell apoptosis and fate, affect the nervous system formation and morphogenesis, and contribute to the differentiation and development of the nervous system[181920212223].

Notch signaling is not independent of the Wnt signaling pathway, as these two signaling pathways can crosstalk through Dsh. Dsh is activated by Wnt signaling and plays an antagonistic role against Notch signaling. The Wnt gene is a highly conserved family, which is involved in the body axis and germ layer formation, organogenesis and development, stem cell differentiation and cell fate in normal embryonic development. A number of studies have shown that the Wnt signaling pathway is the key pathway for the regulation of cell growth and proliferation of the vertebrate central nervous system, and especially the development of hippocampal and cortical 2/3 neurons. During embryonic nervous system development, the Wnt signaling pathway determines the spatial and temporal proliferation and differentiation of neural stem cells[24252627]. Thus, the Wnt signaling pathway has received close attention in studies regarding nervous system diseases.

The cerebral cortex, hippocampus, and entorhinal cortex, which are associated with learning, memory, and other senior functions, are the most vulnerable sites in the AD pathological process. Presenilin, Notch, and Wnt are highly expressed in these brain regions. However, Notch and Wnt signaling system disorders in neuronal plasticity may lead to neural degeneration diseases. Recent studies have shown that changes in several signaling molecules in the Wnt signaling pathway are related to the formation of Aβ and the development of AD[2829].

Lecithin cholesterol acyltransferase (FC = 3.54) is involved in lipid metabolism, and may be related to cell lipid metabolism in the nervous system. Lecithin cholesterol acyltransferase contributes to steroid and lipid metabolism, and statin signal transduction pathways, and regulates Aβ formation and brain functions[303132]. Protein kinase Cδ (FC = 2.03) is a substrate of caspase-3 and induces cellular apoptosis, and is responsible for the regulation of Wnt and G protein signal transduction pathways, while the interleukin-10/12 mediate the immune response.

The down-regulated gene in the Aβ1-40-induced AD model group, γ-aminobutyric acid (GABA) B receptor 1 subunit (FC = 0.277), can control the γ-GABA and G-protein receptor signaling pathways, and inhibit glandular nucleotide cyclase activity. GABA is an important inhibitory amino acid neurotransmitter in the mammalian central nervous system[33]. GABA modulates glutamic acid synaptic activity. Thus, abnormality of the GABAergic system can lead to a high neuronal excitability and reperfusion injury. In adult animals, GABA plays a protective role mainly through the activation of GABA receptors and increasing the flow of Cl-, resulting in the hyperpolarization of the postsynaptic membrane. Gabbr1 gene down-regulation is destined to affect nervous system signal transduction, and other normal functions

There is a negative regulator of the bone morphogenetic protein (BMP) signaling pathway, which leads to the down-regulation of the Wnt receptor signaling pathway Sostdc1 (FC = 0.31). BMP is a member of the transforming growth factor superfamily and plays an important role in skeletal development and bone morphogenetic formation. At the same time, BMP has a wide range of biological activities; it is a multifunctional protein involved in the morphogenesis of a variety of tissues and organs. Studies have shown that BMP signals are essential for neural development and its functions include the determination of the early neural ectoderm, distribution patterns and proliferation of the spinal cord, as well as embryonic and postnatal brain development[3435]. Moreover, BMP binding with the BMP receptor signaling pathways has important cellular functions in the adult central nervous system. BMP is not only involved in the nervous system development process, but also participates in neurological diseases, such as injury repairing process[363738].

Another down-regulated gene in the Aβ1-40-induced AD model group, Atxn1 (Ataxin-1), is mainly located within the nucleus and serves to influence the cellular physiological and biochemical reactions through reaction with cell components within the nucleus. This response is enhanced in parallel with increasing CAG quantity. If Ataxin-1 is expressed in the cytoplasm, it is not pathogenic. Ataxin-1 expression in the cytoplasm is another mechanism underling AD occurrence. Atxn1 (Ataxin 1 protein) contributes to brain morphogenesis, adult motor behavior, visual learning, inhibition of transcription and RNA export from the nucleus, and regulation of postsynaptic membrane potential excitability[3940].

Catechol-O-methyltransferase is ubiquitously present in the human body, and participates in catecholamine metabolism, neurotransmitter, dopamine, S-adenosyl homocysteine acid metabolism, and synthesis of biogenic amines. Catechol-O-methyltransferase is an important biologically active substance affecting the functions of the brain. It can deactivate catechol biological activity, and its substrates are catechol hormones and neurotransmitters, such as epinephrine, norepinephrine, and dopamine. When the above-mentioned neurotransmitters are dysfunctional, they may induce nervous or mental diseases such as AD, Parkinson's disease, schizophrenia, or depression. The abnormal expression of catechol-O-methyltransferase gene is bound to affect the development and function of the nervous system, and thus, influences the occurrence and development of Parkinson's disease, AD, and other neurodegenerative diseases[41424344].

In this study, the Wnt receptor and BMP signal transduction pathway associated with nervous system development and function, and the Ras signaling pathway associated with cellular growth and differentiation, showed down-regulated expressions of several genes in the AD model group. The calcium and G protein-mediated signaling pathway, which contributes to the nervous system functions, also showed differentially expressed genes.

Based on the above findings, we conclude that the hippocampus of the AD model rats is under high stress, involving oxidative stress, inflammation via the immune response, cellular growth and differentiation, cellular cycle and apoptosis, energy metabolism, ion channel/transporter, cellular signal transduction, and other physiological and pathological processes. The effects of Aβ1-40 on hippocampal gene expression are mainly achieved through the genes involving the immune and inflammatory response and cell signaling pathway genes involved in nervous system development and function, such as Wnt, Statin, Notch, Bmp, Ras, NF-κB, G protein receptor and Ca2+. In addition, Aβ1-40 can influence energy metabolism, and regulate cellular growth and apoptosis. This study aimed to elucidate the etiology and pathogenesis of AD in a broader attempt to provide gene targets for AD gene therapy.

MATERIALS AND METHODS

Design

A randomized, controlled, animal experiment.

Time and setting

Experiments were performed from February 2008 to February 2010 in the Central Laboratory at the First Affiliated Hospital of Shenzhen University (Shenzhen Second People's Hospital) and the Animal Laboratory in the Clinical Medicine College of Jinan University (Shenzhen People's Hospital), China.

Materials

Animals

Sprague-Dawley male rats, aged 6–8 weeks, weighing 200 ± 20 g, were purchased from the Experimental Animal Center of Guangdong Province, with license No. SCXK (Yue) 2008-0002 and Guangdong Monitoring and Certificate No. 2008A020. Prior to the experiment, experimental rats were fed for 2 weeks, in a specific pathogen-free grade rearing environment, at 20–25°C, in relative humidity of 50 ± 5%, under a 12-hour day/night cycle. Experimental protocols were in strict accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China[45].

Drugs

Aβ1-40 (Sigma, St. Louis, MO, USA; 2 mg) dissolved in 1 mL normal saline after autoclaving was used to prepare a 2 μg/μL solution and incubated at 37°C constant temperature incubator for 7 days for the aging process, then stored at 4°C for further use.

Methods

Establishment of AD model animals with Aβ1-40

In the AD model group, rats were weighed before surgery after 12-hour fasting and anesthetized via intraperitoneal injection of pentobarbital sodium (45 mg/kg) for 10 minutes. Rats were fixed in a stereotaxic instrument (RWD Life Science Co., Ltd., Shenzhen, China) and the head was surgically cut to expose the skull. According to the positions (anterioposterior -3.0 mm, mediolateral 2.0 mm) in the Rat Brain Stereotaxic Atlas[46], the rat skull was drilled at a diameter of 1.0–2.0 mm, and needles were slowly inserted (1 mm/min) into the bilateral hippocampi (dorsoventral 2.9 mm), while maintaining the integrity of the dura mater. AD animals were established through the injection of fore-processed condensed Aβ1-40 (1 μL/min; supplementary Figure 1 online). Each rat was injected with Aβ1-40 50 ng/g; total volume in each injection was no more than 10 μL. After injection, the needle was retained for 5–10 minutes and slowly withdrawn (1 mm/min), followed by the suture of the scalp. Rats were postoperatively injected with penicillin 480 000 U/kg per day for 3 days and housed in separated cages. They were fasted for 12 hours post-surgery and received a regular diet on the next day (supplementary Figure 2 online). Sham-operated rats were injected with 10 μL sterile saline.

Behavioral test was performed using the Morris water maze (Panlab Co., Barcelona, Spain) at 4 weeks after the Aβ1-40 injection, to observe and compare the decline of learning and memory abilities in the animals, and to screen out the successful AD model rats. The test included space navigation and space probe sections. Escape latency, swimming time and distance, and search time were recorded. The time and route to each quadrant were automatically analyzed with water maze software, then the percentage of the time and route of rats swimming in the original platform quadrant to the total duration and searching distance was calculated[47]. The average escape latency of each rat was calculated to screen out eight qualified rats, according to the determination criteria that takes +2 times of the standard deviation of the mean escape latency of normal controlled rats as the lower limit, while +1 time of the standard deviation of their mean escape latency was the maximum. The hippocampal tissues were randomly harvested from five of the eight rats.

Total RNA extraction from rat hippocampus

Five rats in each group were randomly selected. The brains were conventionally harvested under sterile condition (supplementary Figure 3 online). The surface fascia tissue was removed after washing with PBS and bilateral hippocampal tissue was carefully isolated (supplementary Figure 4 online). The specimens were immediately transferred to cryopreservation tubes or wrapped in aluminum foil in gauze bags, and stored in liquid nitrogen for further use.

Before the extraction of RNA, the tissues were ground for 8–10 minutes in a mortar that had been pre-cooled with liquid nitrogen, and liquid nitrogen was repeatedly added to avoid evaporation to dryness. The amount of the tissue for grinding was no more than 300 mg. The ground tissue powder was mixed with 2.0–3.0 mL Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and Trizol reagent was then transferred to 1.5-mL centrifugation tubes. Total RNA was extracted according to the standard use of Trizol reagent[48]. Tissues were centrifuged at 12 000 r/min for 5 minutes, and the precipitates were discarded. Specimens were mixed with 200 μL chloroform and placed on ice for 15 minutes, and centrifuged at 4°C, at 12 000 r/min for 15 minutes. The upper aqueous phase was collected and transferred to another centrifuge tube, mixed with equal volume of pre-cooled isopropanol and stored at −20°C for more than 2 hours or overnight. Specimens were centrifuged at 4°C, 12 000 r/min for 10 minutes and the supernatant was discarded. Specimens were mixed with 1 mL 70–80% ethanol and the centrifuge tube was moderately oscillated until the precipitates were suspended. Specimens were again centrifuged at 4°C, 8 000 r/min for 5 minutes, and the supernatant was discarded. RNA precipitating at the bottom of tube was dried at room temperature or under vacuum for 5–10 minutes. The harvested RNA was dissolved in 20–30 μL DEPC-treated deionized water.

The concentration and purity of 2–5 μL RNA was detected with a UV-visible spectrophotometer (U-3310, Hitachi, Tokyo, Japan), and its integrity was assayed with formaldehyde denaturing gel electrophoresis[49]. Total RNA (0.3 μg) was mixed with 1/5 volume of 5 × loading buffer, and was heated to 65°C for 5 minutes, and then quenched on ice to remove the RNA secondary structures. RNA samples before the sampling were treated with 0.5–1.0 μL of ethidium bromide (1.0 mg/mL). Formaldehyde denaturing gel (1.2%) was placed in 1 × formaldehyde denaturing gel electrophoresis buffer for 15 minutes. RNA samples were subjected to electrophoresis for 30 minutes at 5–10 V/cm voltage.

Microarray analysis

Gene microarray analysis was performed in the CapitalBio Corporation (the National Engineering Research Center for Beijing Biochip Technology, China). The target samples were prepared with a single-cycle cRNA amplification and biotin labeling method for microarray hybridization. Samples were eluted and stained with Fluidics Station 450 (Affymetrix, Santa Clara, CA, USA), and the chip was scanned with Affymetrix GeneChip Scanner 3000 (Affymetrix), and images were analyzed with Affymetrix GeneChip Operating Software Version 1.4 software. The image signal was transformed into digital signals, then the images were corrected and normalized with dChip software (Cheng Li Lab, Harvard University, Cambridge, MA, USA). The fluorescence signal intensity and ratio were analyzed with SAM 2.10 (Significance analysis of microarrays 2.10, written by Balasubramanian Narasimhan and Robert Tibshirani, Stanford University, Stanford, USA). Finally, the differentially expressed genes were screened out according to the criteria of two-fold differences (q value (%) < 1 %, FC ≥ 2 or ≤ 0.5)[50]. The cluster analysis of the signal value of each sample was performed with cluster3.0 (written by Michael Eisen, Stanford University, Palo Alto, CA, USA; updated in 2002 by Michiel de Hoon, University of Tokyo, Tokyo, Japan), and calculated with a hierarchical, average linkage algorithm. The function and signal transduction pathway of differentially expressed genes were determined with MAS (CapitalBio Molecule Annotation System V 4.0, CapitalBio Corporation/the National Engineering Research Center for Beijing Biochip Technology, China).