Gene Expression Profile Of Hippocampus And Cortex Of Rat In Aβ_1-40 -induced Rat Model For Alzheimer's Disease.

Alzheimer's disease (AD) is a degenerative neuropathy characterized by mental retardation. Its pathological features are extracellular senile plaque (SP) formed by the deposition of theβ-amyloid (Aβ), neurofilament tangle (NFT) formed by the aggregation of the abnormal phosphorylated Tau protein, the vascular amyloidosis, and the neuron loss in the cortex and hippocampus. All of the pathological changes are related to Aβaccording to the recent notion. With the ageing of the society, the prevalence of AD increases. But its etiology is not clear. Genetics, metabolism, and environment involve in the disease. Many studies have suggested the involvement of Aβfor neuronal cell death, the neurotoxicity of Aβis the common pathway in different causes and thus the key to the formation and development of AD. However, the molecular regulatory mechanism of the neurotoxicity is still unclear We used the animal model of AD by stereotaxic damage of CA1 area of rat hippocampus using aggressive Aβ1-40 in this experiment. The changes of their study and memory were observed, we also explore the ultramicrostructural change in different cerebral regions of AD rats from histopathological, ultramicrostructural, and molecular biological points. We also use the techniques of DNA microarray and northern blot to assay the broad spectrum of the differentially expressed genes in order to study the molecular mechanism of cerebral injury of the experimental AD rat. 1. The establishment of the rat model of hippocampal CA1 region damage by Aβand the observation of changes in behavior. The animal model of AD which mimicks the cerebral pathological changes and behavioral changes of AD patients is a very important method to study the mechanism of AD and the pharmaceutical therapy. These models are divided into three types: damage model, natural senile model, and transgenic model. Aβis the core of SP, which is the major pathological feature of AD, so the model that is directly related to Aβshows more advantages than others. The hippocampus is the most vulnerable area, and is highly related to the ability of study and memory, especially recent memory. So the rat model of hippocampal CA1 region damage by Aβis a good choice to study the pathological and behavioral changes in AD. We stereotaxically damaged hippocampal CA1 region (3.0mm behind the anterior fontanelle, 2.0mm to the right of the sagittal suture, 2.6mm under the dura mater) of AD rat using Aβ1-40 according to "The stereotaxic atlas of rat brain", and observed the changes of study and memory using water labyrinth test and jump stand test. The results showed: (1) The model had such advantages as exact localization, high success rate, and good repetition, etc. (2) water labyrinth test: AD rat showed significant decreases in the time of memory recovery (P<0.05). (3) jump stand test: AD rat showed great decreases in the ability of learning and memory, both is statistically significant(P<0.05). The results suggested: (1) Aggressive Aβ1-40 was neurotoxic, the neuron injury it caused resulted in the deterioration of study and memory, so the deposition of Aβin hippocampus is a major causes to the study and memorydysfunction of AD. Aβis highly related to the cognitive dysfunction of AD. (2) The rat model of hippocampal CA1 region damage by Aβwas a satisfactory model to explore the mechanism of AD and its pharmaceutical therapy. 2. Hippocampal CA1 region damaged by Aβ. The result showed: (1) The cortex and hippocampus of the normal group had intact structures and normal morphological cells observed by microscope with HE staining. Mild gliosis could be seen beside the needle trace in the saline group, there were not neuronal apoptosis in the cortex and hippocampus. Meanwhile, in the Aβgroup, solitary shrunken cells existed in the cortex and hippocampus, the chromatin of these round or ellipse cells aggregated to form royal blue compact plaque, the plasma also shrank and was increased eosinophilic. Karyopyknosis and karyorrhexis could be observed, the nuclear membrane disappeared, but the cellular membrane was intact. (2) Transmission electron microscopy was the best way to study the substructure of the nucleus and organelle. In the Aβgroup, pyknosis could be seen in the neuron, other apoptotic changes included unclear nuclear membrane, irregular morphologic and dissymmetric nucleus, border-aggregated chromatin, compact and densely stained nucleus and plasma matrix. Pericellular space enlarged due to the pyknosis of the neuron. Neurophil filament increased. 3. Gene expression profile alterations in hippocampus and cortex of Aβ1-40 -induced rat model for Alzheimer's disease Recently, substantial improvement in sensitivity and throughput of expression screening has been obtained by the introduction of DNA microarray technology. The study of gene expression by DNA microarray technology, which is still in development, is based on hybridization of mRNA to a high-density arrayof immobilized target sequences, each corresponding to a specific gene. Sample mRNAs are labelled by incorporation of a fluorescent nucleotide by reverse transcription. The labelled pool of sample mRNAs is subsequently hybridised to the array, where each messenger will quantitatively hybridise to its complementary target sequence. After washing, the fluorescence at each spot on the array is a quantitative measure corresponding to the expression level of the particular gene. Global changes in gene expression in a tissue may result from differences in physiology, developmental stage, pathology, or drug treatment. These changes can now be measured using DNA microarrays, which allows for a higher sensitivity, enables the parallel screening of larger numbers of genes and provides the opportunity to use smaller amounts of starting material. Many studies have suggested the involvement of amyloid β-protein (Aβ) for neuronal cell death, However, the molecular mechanism of the pathogenesis is still unclear. In order to investigate the molecular events underlying the Aβinduced neuronal impairment in rat hippocampus. In this study we used the DNA microarray to explore the gene expression patterns in cortex and hippocampus of Aβtreated rats. Of the 4,200 genes and expressed sequence tags analyzed, a group of 40 genes with altered expression were identified in Aβ1-40 treated rat hippocampus, and a group of 46 genes in Aβ1-40 treated rat cortex. Comparison of expression levels revealed that 14 genes were up-regulated (>2 fold) and 26 genes were down-regulated (<0.5 fold) by a treatment with Aβ1-40. in rat hippocampus; and 18 genes were up-regulated and 28 genes were down-regulated in rat cortex. The northern blot results correlate well with the microarray data. This differential expression profile included genes encoding proteins involved in the function of signal transduction, cell growth and/or maintenance, inflammation, regulationof transcription, apoptosis, cell motility, ion channel and neurotransmitter transporter, enzymes and others. 3.1 Cell proliferation and cell cycle related genes Recent findings from several laboratories indicate that cell cycle-related phenomena may play a key role in the formation of Alzheimer-type pathology and neuronal cell death in Alzheimer's diseases. Cell cycle re-entry and G1 phase arrest may occur in the neurofibrillary degeneration process and lead to excess apoptotic cell death. Glypican-3 (GPC3) is a novel tumor marker, which encodes cell-surface heparan-sulfate proteoglycans. GPC3 binds/modulates several growth factors such as FGF2, and BMP4, via heparan sulfate chains and acts as an inhibitor of cell proliferation and survival. Up-regulation of GPC3 by Aβtreatment plays a negative role in cell proliferation by arresting the cells at G1 phase and promoting apoptosis. POU-domain transcription factors play essential roles in cell proliferation and differentiation. Previous studies have shown that targeted deletion of POU-domain Brn3 factors in mice leads to the developmental failure and apoptosis of a unique set of sensory neurons in retina, dorsal root ganglia, trigeminal ganglia and inner ear. The POU domain transcription factor POU3F3 controls sensory neuron survival by up-regulating the expression of Trk receptors and members of the Bcl-2 family. Loss of POU3F3 leads to a dramatic increase in apoptosis and severe loss of neurons in sensory ganglia. In Alzheimer's disease, regulators spanning every phase of the cell cycle are upregulated in affected neurons, leading to successful DNA replication, but unsuccessful mitosis. The end point of this aberrant cell cycle is death. Cyclin D1is a key regulator of progression through G1 phase during the cell cycle. Increased expression of cyclin D1 in cortex suggests that the cell cycle may be disturbed during Abeta-mediated degeneration of neuron cells. 3.2 Inflammation related genes Inflammation is one of the neuropathological features that accompany progression of the Alzheimer's disease. Prostaglandin D2 synthase (PTGDS) is a major protein constituent of cerebrospinal fluid (CSF). PTGDS level increased response to administration of a bacterial endotoxin lipopolysaccharide (LPS) or proinflammatory cytokine IL-1 and participates in neuronal apoptosis. The molecular mechanism of L-PGDS-induced apoptosis appears to be at least in part via the inhibition of the PI3-K pathway and the subsequent inhibition of the phosphorylation of pro-apoptotic gene Bad. Cathepsins B is widely expressed cysteine proteases implicated in both intracellular proteolysis and extracellular matrix remodeling. It has been implicated in a variety of diseases involving tissueremodeling states, such as inflammation, parasite infection, and tumor metastasis. Release of cathepsin B in the conditioned medium from microglial cells activated by LPS induces neuronal apoptosis and activation of caspase 3. Metallothioneins1 (MT1) are low molecular weight, metal-binding proteins with established antioxidant capabilities. MT-I overexpression stimulated astroglia and increased immunostaining of antiinflammatory IL-10, growth factors, and neurotrophins (basic fibroblastic growth factor, transforming growth factor-beta, nerve growth factor, brain-derived neurotrophic factor, glial-derived neurotrophic factor) in hippocampus. Accordingly, MT-I has function that likely contribute to the neuroprotection during Aβ1-40-induced excitotoxicity.ITGAM is a major surface antigen family on human leukocytes which promote adhesion of granulocytes to each other and to endothelial cell monolayers. ITGAM is widely expressed in many tissues including vessels,hippocampus and neurofibers. Injecton of Aβ1-40 in rat hippocampus resulted in significantly increased production of ITGAM and contribute to the inflammatory response and subsequent neuron damage observed in AD brain. 3.3 Signal transduction related genes G protein-coupled receptors represent one of the largest families of cell surface receptors in nature. These receptors play fundamental roles in diverse physiological processes such as cells communication, cell migration, cell differentiation and growth as well as cytoskeletal rearrangement. It is interesting to note that two of the known genes were G protein-related molecules. They include neuronal chimaerin (n-chimaerin) and endothelial differentiation sphingolipid G-protein-coupled receptor 1(EDG1). Although none of them have been reported in relation to the AD pathology, the expressional alterations of these G proprotein-related genes in vivo would suggest a certain abnormality in the intracellular signal transduction which might lead to neuronal cell death. For example, n-chimaerin is a Rho GTPase-activating protein regulatingcdc42/Rac 1. Although there is no report of a direct relationship between n-chimaerin and neuronal cell death, Rac 1 is a MAP kinase superfamily recently discussed in connection with the regulation pathways of apoptosis. Moreover, the decrease in expression of n-chimaerin would imply the disturbance of the regulation in RhoGTPase family resulting in the acceleration of the apoptosis-inducing pathway. EDG-1 is a heterotrimeric guanine nucleotide binding protein-coupledreceptor (GPCR) for sphingosine-1-phosphate(S1P). The S1P regulates cell proliferation, apoptosis, motility, and neurite retraction. Its actions may be both intracellular as a second messenger and extracellular as a receptor ligand. S1P induced neurite retraction in N1E-115 cells via EDG1 receptor-dependent pathway that involves contraction of the actin cytoskeleton. In neuronal cells, neurite retraction is one of the first steps in the apoptotic process. 3.4 Growth factors and neurotrophins The abilities of neurotrophins to control the fate of neurons in the developing nervous system, stimulate neurite outgrowth, protect neurons from apoptosis and increase neurotransmitter release have been discussed previeously. Brain-derived neurotrophic factor (BDNF) is a prosurvival factor induced by cortical neurons that is necessary for survival of striatal neurons in the brain. In cultured neurons Aβinterferes with the BDNF-induced activation of the Ras-mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. The functional consequences of Aβinterference with signaling are robust, causing increased vulnerability of neurons, abrogating BDNF protection against DNA damage-and trophic deprivation-induced apoptosis. Insulin-like growth factor-I (IGF-1) is implicated in the development, survival and maintenance of function of sympathetic and sensory neurons. In our present study we observed a significant decrease of IGF-1 receptor (IGF-1R) mRNA in the cortex and hippocampus of Aβ1-40-treated rats. Accumulated evidence indicates that IGF-1, apart from regulating growth and development, protects neurons against cell death induced by amyloidogenic derivatives via theactivation of intracellular pathways implicating phosphatidylinositide PI3/Akt kinase and CREB phosphorylation or modulate the production of free radicals. Therefore, the down-regulation of IGF-1R mRNA in our study may explain the sensitivity and cell death in this rat model for AD. Insulin-like growth factor binding protein 2 (IGFBP-2) play an important part in regulating the bioavailability of IGF-1 by competitively binding IGFs and preventing their binding to IGF-1R, thereby inhibiting the neuroprotection and antiapoptotic action of IGFs. 3.5 Ca2+ homeostasis related genes Previous study suggests that Aβaccumulation may have a key role in the pathogenesis of AD as a result of deregulation of ER Ca2+ homeostasis. The inositol 1,4,5-trisphosphate receptor (IP3R) is one of the important channels responsible for Ca2+ release from intracellular Ca2+ stores. One of the members of the IP3R family, IP3R1, plays crucial roles in several neuronal functions, including synaptic plasticity, neurite extension, and nerve growth cone guidance. Thus, the precise regulation of Ca2+ release through IP3R1 is one of the important factors in these physiological functions in the brain. Our findings, thus, suggest that the IP3R1 expression level is an important determinant of the sensitivity of Ca2+ stores to IP3 in hippocampal neurons. It was shown that an endoplasmic reticulum (ER)-specific apoptotic pathway mediated by caspase-3 and mitochondrial cytochrome c release, which is activated upon the perturbation of ER Ca2+ homeostasis, may contribute to Aβtoxicity. 3.6 Neurotransmitter transporter related genes Our observation of the increase of GABA-A receptor α1 gene expression in Aβ-treated rat cortex can not be fully understood. In a recent study it was foundthat GABA-A receptor α1 subunit may act as a dynamic element during channel-gating transitions and play an active role in the formation of GABAergic synapses.. 　-Amino butyric acid (GABA) is not only the primary inhibitory neurotransmitter in the central nervous system but also acts as a neurotrophin which antagonized the neurotoxic effect of glutamite. It is found in almost every region of brain, and is formed through the activity of the enzyme glutamic acid decarboxylase (GAD). Aβ-induced GAD reductions likely reflect decreases in both metabolic and pre-synaptic GABA levels suggesting a plastic down-regulation of normal adult inhibitory GABA neurotransmission and a enhancement of excitotoxic damage of glutamate. Glutamate transporters are involved in the maintenance of synaptic glutamate concentrations. Because of its potential neurotoxicity, clearance of glutamate from the synaptic cleft may be critical for neuronal survival. Inhibition of glutamate uptake from the synapse has been implicated in AD. Evidence for the involvement of glutamate transporters in AD is its inactivation and inhibition through oxidative damage from reactive oxygen species and lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE). Here we have shown for the first time that Aβtreatment in rats induces a significant increase in glutamate transporter mRNA expression in the cortex. as a result of persistence to protect the neurons from cell death. 3.7 Other genes Other genes were less obvious; however, they might imply novel pathways worthy of investigation. Dystrophia myotonica kinase is a dystrophin-associated protein found in dystrophin-associated protein complexes in the brain. Given thatone-third of Duschenne muscular dystrophy patients, a disease caused by mutations in the dystrophin gene, have a mild dementia, it is possible that altered expression of dystrophia myotonica kinase could be related to dementia of the Alzheimer type. CD81 is a member of the tetraspanin family of proteins. This group of small membrane proteins is associated with the regulation of cell migration and mitotic activity. CD81 is upregulated by astrocytes and microglia after traumatic injury to the rat central nervous system (CNS). KCNH2 overexpression has been indicated in Alzheimer's disease. It was shown that the Aβneurotoxicity in fibroblast was prevented using potassium channel openers (KCOs) in neuronal cell culture. Therefore, up-regulation of the channel may be an attempt to protect the neurons from cell death. Thymosin beta(10) is a monomeric actin sequestering protein that regulates actin dynamics. Recent studies show that thymosin beta(10) is not only a cytoskeletal regulator, but that it also acts as a potent inhibitor of angiogenesis and tumor growth by its interaction with Ras. We hypothesize that overexpressed thymosin beta(10) significantly inhibited vascular endothelial growth factor-induced endothelial cell proliferation, migration, invasion, and tube formation in vitro. ENO2 and PGAM2 encoding enzymes involved in glycolysis and gluconeogenesis, down-regulation of ENO2 and PGAM2 expression suggests Aβinterferes with the energy motablism in neuron cell. The current study demonstrated for the first time a concerted alteration of gene expression in the cortex, and hippocampus of Aβ-rat model for AD. The broad spectrum for the distribution of the differentially expressed genes indicated...