The Role Of Astrocyte IP₃R In Synaptic Dysfunction Of Alzheimer’s Disease

Objective:Alzheimer’s disease is the most important neurodegenerative disease,and its main pathological features are age spots formed by Aβplaque aggregation,neuronal fiber tangles（NFTs）formed by excessive phosphorylation of Tau protein,and diffuse cortical brain atrophy.The calcium imbalance hypothesis is a recently proposed conjecture for the mechanism of intracellular calcium ion disorder in the pre-onset of AD,the concentration of intracellular calcium ions is much lower than that outside the cell,and calcium ions are mostly stored in the endoplasmic reticulum,mainly regulated by the inositol triphosphate receptor on the endoplasmic reticulum,and in recent studies,researchers have found that in AD model mice,many factors lead to intracellular calcium ions at high levels,such as the overopen of IP₃R in glial cells caused by long-term chronic neuroinflammation.Previous studies in this group have also shown that the administration of the IP₃R blocker Xestospongin C（Xe C）at the cellular level can effectively antagonize the neurotoxicity induced by Aβ_1-42,improve the occurrence of intracellular calcium overload and early apoptosis,and at the same time,the tripartite synaptic concept makes us pay attention to the important role of astrocytes in synaptic function,astrocytes release neuromodulators to act on synapses,regulate synaptic function,Therefore,we suspect that calcium homeostasis in astrocytes plays an important role in the pathogenesis of AD.In order to confirm this hypothesis,this experimental study selected astrocyte intracellular calcium disorder as the entry point,down-regulated the endoplasmic reticulum channel receptor IP₃R,reduced the intracellular calcium ion level of astrocytes,observed the amplitude of intracellular calcium oscillation after receiving stimulation at the cellular level,and coated with the virus,carried out intra-hippocampal injection,observed the improvement effect of AAV-IP₃R on the cognitive behavior of APP/PS1 model mice at the body level,and then detected the expression levels of synaptic-related proteins SYP and PSD-95.The morphological plasticity of synaptic morphology of hippocampal neurons was observed,and the role of ASTR IP₃ receptors in AD models was explored.Methods:1.Cell experiments:screening down-regulated sh RNA of astrocyte IP₃R and transfection to primary astrocytes cultured in advance to detect intracellular calcium levels and IP₃R expression.The astrocytes were then divided into four groups,namely:IP₃R-sh RNA+ACSF,IP₃R-sh RNA+Aβ,wildtype5’ITR+ACSF and wildtype5’ITR+Aβ,of which the 2nd and 4th groups underwent Aβincubation for24h for calcium imaging experiments to observe the degree of intracellular calcium elevation caused by acute glutamate stimulation.（1）Culture of primary astrocytes:Primary astrocytes were extracted using SD suckling mice,and astrocytes were isolated by differential adherent method and their cell purity was detected.（2）Construct sh RNA-containing plasmids and perform cell transfection:Plasmids that specifically down-regulate astrocyte endoplasmic reticulum REI IP₃R are transfected to detect their transfection rate,and their down-regulated endoplasmic reticulum IP₃R levels are detected using Western blot technology（3）Detection of calcium ions in the astrocyte endoplasmic reticulum after down-regulation of IP₃R:incubating cells with a specific endoplasmic reticulum calcium ion indicator Mag-Fluo-AM to observe the changes of endoplasmic reticulum ions after astrocyte stimulation after down-regulating IP₃R.2.vitro experiments:the screened sh RNA coated with AAV-9 virus and stereotactic injection into the mouse hippocampal CA1 area,based on which the 8-month-old APP/PS1 transgenic mice and their wild-type co-litter control mice were divided into four groups:WT+AAV-Con,WT+AAV-IP₃R,APP/PS1+AAV-Con, APP/PS1+AAV-IP₃R,12 in each group,and continued to be reared after the end of intra-hippocampal injection,and resumed observation for one month.Observe improvements in their cognitive behavior.After behavioral experiments,the levels of synaptic-associated proteins and neurotransmitters in the homogenate of hippocampal tissue were detected,and finally the brain slice observed changes in synaptic structural plasticity.（1）Hippocampal stereotactic injection:mice are fixed in stereotaxic,the scalp is cut after sterilization,the anterior fontanelle is found,the hole is drilled at 1.5 cm next to the backward 2 cm,AAV-IP₃R and its control virus are injected at 1.8 mm under the skull,and then the needle is slowly withdrawn,the wound is sutured,and reared and restored for 4 weeks.（2）Open field test:Mice are placed in the open field,the activity trajectory is recorded within 8 min,and the total distance of movement and the percentage of residence time in the central area are observed.（3）New object recognition experiment:mice explore two identical objects,and after 8 h, replace one of the objects with different colors and shapes,so that they can explore the same time and record the new object recognition index（NOI）.（4）Y-maze experiment:Record the number of arm advances and spontaneous alternating accuracy rates of each group of mice within 8 min,and each third arm entry is different from the previous two times as a correct arm entry.（5）Morris water maze experiment:the localization navigation experiment recorded the daily evasion incubation period of each group of mice,and the space exploration experiment recorded the percentage of time and the number of times each group of mice in the target quadrant.（6）Western blot experiment:after the end of the behavioral experiment,anesthetize the mice,perfuse the hippocampal tissue homogenate,and detect the synapse-related protein content.（7）Transmission electron microscopy experiment:the number and morphology of synapses in each group of tissues were observed under 6000x microscopy.（8）Gorky staining experiment:Brain slices observe the number of neuronal dendrites and dendritic spine density in the hippocampus region.（9）ELISA:To detect the expression content of Glu and GAD in the homogenate of hippocampal tissue.Results:（1）In cell experiments,IP₃R-sh RNA can significantly reduce the intracellular basal calcium ion concentration,after administration of glutamate stimulation,the intracellular calcium ion concentration increase decreased,and the increase rate decreased,and the expression level of GRP78 in the IP₃R-sh RNA group decreased, indicating that down-regulating IP₃R can slow down the level of endoplasmic reticulum stress caused by Aβ.（2）In the open field experiment,the down-regulation of IP₃R did not change the motor ability of mice,and the intrahicampal injection virus experiment had no significant effect on the motor function of mice（P>0.05）.（3）In the new object experiment,there was no significant difference in the exploration time between the two objects in the four groups of mice in the familiarization stage （n=12,P>0.05）,and the NOI index of APP/PS1 mice in the testing stage was significantly lower than that of WT mice（P<0.01）,and the NOI index increased after the downgrading of IP₃R（P<0.01）.（4）In the Y-maze experiment,mice in the APP/PS1 group had a significant improvement in the intersection of spontaneous alternating accuracy after down-adjusting IP₃R in the AAV-Con group（P<0.05）.（5）In the water maze experiment,the WT+AAV-Con group had a shorter evasion incubation period（P<0.05）compared with the APP/PS1+AAV-Con group,and the long-term learning function of APP/PS1 mice was improved after IP₃R downregulation.Mice in the APP/PS1+AAV-Con group had significantly lower dwell times and times of pitting in the target quadrant than in the WT+AAV-Con group（P<0.01）,indicating that the APP/PS1 transgenic mice had less swimming time in the target quadrant,while the target quadrant dwell time and number of pit-throughs in the APP/PS1+AAV-IP₃R group increased（P<0.05）.（6）The expression of synapse-related proteins in the APP/PS1 group decreased,and the expression of PSD-95 and SYP in the hippocampal tissue homogenization of bi-rotating mice showed an upward trend after down-regulation of IP₃R.（7）Transmission electron microscopy results showed that the number of synapses per unit area in the APP/PS1+AAV-Con group was significantly reduced compared with that in the WT+AAV-Con group（P<0.01）,and the synaptic gap was wide and short, and the synaptic morphology tended to normalize after the down-regulation of IP₃R, and the number of synaptic density increased.（8）Gorky staining results showed that the number of neuronal dendrites in APP/PS1 bi-rotating mice was small and the dendritic bifurcation was small,and the dendritic bifurcation within 50μm was improved after the IP₃R was down-regulated,and the average dendritic spine density increased（P<0.01）.（9）ELISA experimental results show that the GAD concentration is higher and the Glu expression content is lower in the APP/PS1 model mice,and the expression of GAD decreases after down-regulating IP₃R,and the Glu content increases,and it is speculated that IP₃R may be stable by down-regulating the expression of GAD.Conclusion:In this study,the cognitive behavior of APP/PS1 bitransmitter mice was improved by injecting AAV virus down-regulating IP3R in the brain,the endoplasmic reticulum stress response in AD mice was alleviated,the expression of synaptic-related proteins was improved and synaptic morphology was improved,and the effect of specific down-regulated astrocyte IP3R on the learning and memory function of APP/PS1 mice on the learning and memory function and synaptic structure function in the brain was confirmed,thus confirming that the calcium homeostasis of astrocytes in the triple recombinant synaptic structure in the brain was the target.Will likely be an effective strategy for early prevention and treatment of AD.