| BackgroundAlzheimer’s disease (AD), an age-related neurodegenerative disorder, becomes the most common form of dementia among old people. Clinically, AD is characterized by the insidious onset and gradual progression of cognitive decline, psychiatric symptoms and loss in activity of daily living. It is a fatal cause of human death with a high incidence just inferior to the cardiovascular disease and cancer, increasingly becoming an important threat to the health of the elderly. AD is a heterogeneous disease caused by several factors, such as depositing of beta-amyloid protein (Aβ), disturbances of neuronal survival pathways, disorders of glucose metabolism, mitochondrial damage, oxidative stress and inflammation which are involved in the development of the disease. Although the pathogenesis of AD is still elusive, there is an ample consensus that the abnormal Aβ deposition in the central nerves system (CNS) plays the most crucial role, especially Aβ1-42in the form of soluble oligomers, and other pathological changes and pathogenic pathways may be secondary to the result of excessive deposition of Aβ.Brain insulin signaling pathway involves in energy metabolism and neuroendocrine. It is critical to regulate glucose and lipid metabolism, modulate synaptic genesis and remodeling, promote cell survival and growth, control inflammation, maintain mitochondrial function and improve learning and memory. The destruction of insulin signaling pathway contributes not only to diabetes and obesity but also to neurodegeneration and cognitive decline and it has been demonstrated that its injury can increase the risk of dementia. Furthermore, mammalian target of rapamycin (mTOR), one of the most important downstream targets of protein kinase B (PKB/Akt), is able to regulate the glutamate transporter expression in astrocytes. Through interactions, mTOR and Akt could also regulate synaptic activity and memory formation. Therefore, enhancing the activity of insulin signaling pathway plays an important role in neuroprotection and cognitive improvement.Astrocytes are the major glial components providing a nurturing environment for neurons in the CNS. To be specific, they contribute to synaptic functions, transfer and store information, participate in cognition, produce trophic factors, remove toxins and debris, maintain redox potential and regulate concentration of neurotransmitter and ion. Noteworthy, astrocytes express abundant insulin receptors (IRs) and might be influenced by insulin. Glutamate, the main excitatory neurotransmitter in the brain, plays a key role in learning and memory. It is released into the synaptic cleft during the neuronal action and rapidly taken up by the excitatory amino acid transporters (EAATs), among which EAAT1and EAAT2preferentially located in astrocytes are responsible for clearing the majority glutamate. Consequently, malfunctions of the EAATs may lead to aberrant glutamate accumulation and neuron injury known as excitotoxicity, which is also the reason for several neurodegenerative disorders including AD.ObjectiveThe aim of this study is as follows:first, determine the reaction of astrocytes to Aβ1-42oligomers; second, explore whether the insulin/Akt/EAAT signaling is existed in human astrocytes and whether could be influenced by Aβ1-42oligomers and insulin; third, explore the behavioral changes of the AD model rats and explore the effect of Aβ1-42oligomers to the cellular signaling proteins of insulin signaling transduction pathway in the hippocampal neurons. Overall, we aimed to further explore the pathogenesis of AD and its feasible treatment. Methods1. Preparation and determination of the Aβ1-42oligomers. We prepared the Aβ oligomers using a basic method and an improved method, respectively. But the Aβ oligomers prepared by the improved method were superior due to its relatively stability in short term. Briefly, human synthetic Aβ1-42lyophilized powder was returned to room temperature firstly and suspended in hexafluoro-isopropanol (HFIP) to make it into monomer followed by further dissolving with dimethyl sulfoxide (DMSO) to produce a homogenous suspension. Subsequently, the Ap-DMSO solution was resuspended with SDS-PBS and incubated at4℃for24h. Ultimately, the solution was diluted with PBS and continually incubated at4℃for2weeks, which was then diluted to the specific concentration with DMEM. The characterization of Aβ1-42oligomers were determined using a transmission electron microscopy (TEM).2. Groups of astrocytes and drug intervention. Astrocytes were divided into6groups which were treated with drugs after the starvation:control group and groups I to V. In brief, the control group (group C) was the negative control which was treated with DMEM without fetal bovine serum (FBS); group I was treated with100nmol/L Aβ1-42oligomers; group II was treated with1μmol/L Aβ1-42oligomers. All of the three groups were cultured for24h at the atmosphere of5%CO2and37℃. Groups III, IV and V were treated with100nM human recombinant insulin for30min after the treatments with DMEM vehicle,100nM Aβ1-42oligomers and1μM Aβ1-42oligomers, respectively.3. Observing the reaction of astrocytes to the drugs and detecting expression and activity of the cellular signaling proteins in insulin/Akt/EAAT signaling pathway. First, we determined the mRNA of glial fibrillary acidic protein (GFAP) with real time RT-PCR, determined the GFAP protein with Western blot, and then observed the changes of cell morphology by immunofluorescence staining. Second, we detected the mRNA of IR, Akt, mTOR, EAAT1and EAAT2, detected the protein expression of IR-α, IR-β, phosphorylation of insulin receptor (p-IR, Y1361), Akt, phosphorylation of protein kinase B (p-Akt, S473), mTOR, phosphorylation of mammalian target of rapamycin (p-mTOR, S2448), EAAT1and EAAT2. Third, we further observed the expression of IR-a, IR-(3and p-IR by immunofluorescence staining.4. Detecting the cell viability of astrocytes. The cell viability of astrocytes treated with Aβ1-42oligomers and insulin was examined through conversing the methyl thiazolyl tetrazolium (MTT) to colored formazan crystals.5. Groups of animals and preparation of AD rat models.50adult male Wistar rats (3-4months old, healthy and weighing225±25g) were used.5rats were eliminated by behavioral tests preoperatively, and the remaining45rats were divided into three groups, namely the control group (group C, rats only underwent surgery, but without any injection, n=15); the normal saline group (NS group, slow and continuous infusion of normal saline into the lateral ventricle of rats, n=15) and the Aβ1-42oligomers injection group (AD group, slow and continuous injection of Aβ1-42oligomers into the lateral ventricle of rats, n=15). Briefly, the micro-osmotic pump was filled with Aβ1-42oligomer solution or normal saline firstly, and then the various parts were assembled with the micro-osmotic pump, which was implanted into the lateral ventricle with the help of a stereotaxic instrument. Adjust the flow regulator to inject Aβ1-42oligomer solution or normal saline slowly and continuously (3μL/day for30days). All operations followed the aseptic principles. Do neurological assessment until the rats were conscious and80,000units of penicillin were injected to all rats for3days routinely.6. Test of behavior. The Morris water maze (MWM) test was used to evaluate the learning and memory function of rats at the end of the drug administration. Acquisition trials were done at the31-34th days after the surgery and probe trials were done at the35th day. The platform was placed in the southern quadrant and the escape latency that the time needed to reach the platform in acquisition trials was recorded and analyzed. Then the times passing through the original position of the platform in probe trials was recorded and analyzed; the residence time and the swimming distance in the south quadrant in probe trials was recorded and analyzed; the ratio of the residence time in the south quadrant to the total time was recorded and analyzed; and also the ratio of the swimming distance in the south quadrant to the total swimming distance in probe trials was recorded and analyzed.7. Detecting the cellular signaling proteins of insulin signaling pathway in hippocampal neurons. Rats were anesthetized and the hippocampus was drawn and fixed. And then, the proteins of IR, insulin receptor substrate-1(IRS-1), Akt, B cel llymphoma/leukemia-2(Bcl-2) and cAMP response element binding protein (CREB) were detected by immunohistochemistry.8. Statistical analysis. Results are expressed as mean±SD. To determine the significance of difference among various groups, the statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s test using SPSS17.0.p<0.05was considered statistically significant in all tests.Results1. Identification of Aβ1-42oligomers. The Aβ1-42oligomers were regular and uniform spheres with a diameter of12-25nm, and no fibrillar aggregates were present in the oligomer solution.2. Reaction of astrocytes to the Aβ1-42oligomers. The mRNA level of GFAP was elevated under the treatment of Aβ1-42oligomers (p<0.05) and the protein level of GFAP was also increased (p<0.05). Moreover, both mRNA and protein levels of GFAP were changed by Aβ1-42oligomers in a dose-dependent manner. Besides, our morphological study showed that astrocytes were activated by Aβ1-42oligomers, namely enlarged cell bodies and nuclei, hypertrophic processes, and increased GFAP expression. However, there was an absence of cell proliferation.3. Changes of expression and activity of the cellular signaling proteins of insulin/Akt/EAAT signaling pathway in astrocytes.①The mRNA encoding IR was decreased by Aβ1-42oligomers in a dose-dependent manner (p<0.05), but insulin had no effect on IR mRNA (p>0.05). In addition, the Western blot result showed that lower expressions of IR-a (p<0.05), IR-β (p<0.05) and p-1R at Tyrl361(p<0.05) were potentiated with concentrations of the Aβ1-42oligomers. However, we found a significant effect on IR-a, IR-β and phosphorylation of IR after exposure to insulin compared with their respective control groups(p<0.05).②Both mRNA and protein levels of total Akt were not altered by Aβ1-42oligomers and insulin (p>0.05). However, Akt phosphorylation at Ser473was significantly decreased by the soluble Aβ1-42oligomers (p<0.05). Moreover, we also observed that insulin enhanced the phosphorylation of Akt compared with the control groups (p<0.05).③Aβ1-42oligomers and insulin had no effect on mRNA and protein levels of mTOR (p>0.05). However, the expression of p-mTOR was decreased by the Aβ1-42oligomers (p<0.05) and insulin could reverse the reduction in p-mTOR (p<0.05).④Aβ1-42oligomers and insulin had no effect on the mRNA levels of EAAT1and EAAT2(p>0.05). However, the protein expressions of EAAT1and EAAT2were both lowered by the Aβ1-42oligomers (p<0.05), whereas they were increased by insulin in group III, group IV and group V compared with the control groups (p<0.05).4. Cell viability of the astrocytes. Incubation with Aβ1-42oligomers at100nmol/L and lμmol/L for24h caused a significant decrease of MTT reduction (p<0.05), whereas insulin exposure induced an increase of MTT reduction compared with the control groups (p<0.05).5. Assessment of animal behavior. The escape latency in the acquisition trials could react the learning ability of rats and the times of finding the location of the platform, the ratio of time in the south quadrant to the total time and the ratio of distance in the south quadrant to the total distance to find the platform in the probe trials could react the memory capacity of rats.①The escape latency of AD rats was87.40±6.70s, which was significant longer than the NS group (15.23±4.65s, p<0.05) and the control group (14.00±6.01s, p<0.05); however, there was no significant difference between the NS group and the control group (p>0.05).②The frequency of passing through the original position on the platform in AD group were significantly less than the NS group and the control group (p<0.05,p<0.05).③The ratio of time (AD group vs. NS group:0.24±0.09s vs.0.34±0.07s,p<0.05; AD group vs. group C:0.24±0.09s vs.0.35±0.01s,p<0.05) and the ratio of distance (AD group vs. NS group:0.27±0.05vs.0.35±0.03, p<0.05; AD group vs. group C:0.27±0.05vs.0.35±0.06, p<0.05) to find the original position on the platform in the AD group were also significantly less than the NS group and the control group, while no significant difference was detected between the NS group and the control group (p>0.05).6. Changes of the cellular signaling proteins of insulin signaling pathway in hippocampal neurons:①The expression of IR was reduced by Aβ1-42oligomers (AD group vs. NS group:0.25±0.02vs.0.38±0.03, p<0.05; AD group vs. group C:0.25±0.02vs.0.40±0.02, p<0.05).②The level of IRS-1protein in AD group was significantly lower than the NS group and group C (AD group vs. NS group:0.22±0.02vs.0.35±0.03, p<0.05; AD group vs. group C:0.22±0.02vs.0.29±0.06, p<0.05), while no significant difference was found between the NS group and the group C (p>0.05).③The amount of Akt protein in AD group was significantly lower than the NS group (0.20±0.03vs.0.37±0.03, p<0.05) and the group C (0.20±0.03vs.0.38±0.03,p<0.05), while there is no significant difference between the NS group and the group C (p>0.05).④The protein expression of Bcl-2was decreased by Aβ1-42oligomers (AD group vs. NS group:0.20±0.02vs.0.4±0.04,p<0.05; AD group vs. group C:0.20±0.02vs.0.40±0.06,p<0.05).⑤The level of CREB in AD group was similar with the NS group (AD group vs. NS group:0.43±0.03vs.0.40±0.03) and the group C (AD group vs. group C:0.43±0.03vs.0.41±0.03).Conclusions1. The modified method for preparing oligomeric Aβ1-42is stable for storage and viable in the preparation of AD models.2. Aβ1-42oligomers are toxic to the astrocytes and hippocampal neurons. The continuous injection of Aβ1-42oligomers to the lateral ventricle could decrease the ability of learning and memory in rats. 3. Insulin/Akt/EAAT signaling pathway exists in the astrocytes and Aβ1-42oligomers at100nmol/L can induce abnormal expressions of the cellular signaling proteins in the signaling pathway. The insulin signaling transduction pathway of hippocampal neurons in AD rat can be disturbed by the Aβ1-42oligomers. Overall, disorders of the insulin signaling transduction pathway in astrocytes and neurons may be one of the important pathogenesis of AD.4. Although chronic and persistent high level of insulin in plasma is harmful to brain tissue, insulin at100nmol/L could protect insulin/Akt/EAAT signaling pathway in astrocytes after30min. In addition, it is also very important for the activity of the signaling pathway. This indicates that a correct way to give appropriate concentration of insulin might give a therapeutic effect on AD. |
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