| BackgroundAlzheimer’s disease(AD)is a progressive neurodegenerative disease characterized by learning and memory disorders as well as personality and behavioral changes.The incidence is high and the onset is concealed.The main clinical manifestations are learning and memory disorders,personality and behavior abnormalities,etc.It is harmful and the cause is still unknown.Therefore,it is urgent to strengthen effective prevention and treatment of disease.The main pathological feature of AD is the deposition of Aβ,highly phosphorylated Tau protein,neuronal loss and other changes.There is no effective treatment currently.Therefore,the discovery of AD effective treatment methods and its protection mechanism are hotspots and difficulties in neuroscience research today.Currently,the treatment of AD focuses on reducing the production of Aβ and Tau hyperphosphorylation,alleviating neuroinflammatory reaction and transplantation of neural stem cells in vitro.In the brain of AD patients,a large accumulation of Aβcauses neuron damage,and especially astrocytes and microglia are activated in large quantities.Activated glial cells release some neurotrophic factors and remove damaged cell debris and infectious substances to repair neurons,on the other hand release a large number of inflammatory cytokines to aggravate neuronal damage.Autophagy is involved in the development of various diseases such as tumors,cardiovascular diseases,and neurodegenerative diseases.It has been confirmed that autophagy can be disturbed during the development of inflammation,and autophagy can also negatively regulate inflammatory factors,affecting and even changing the progression and outcome of inflammation.The discovery of neural stem cells and successful culture in vitro provide new idea for functional reconstruction and nerve regeneration of neurodegenerative diseases.The complementation of exogenous neural stem cells in vitro can compensate for the functional defects of aging neurons,but its clinical application is hindered by transplant rejection and ethics.Therefore,the search for a method or a drug for activating endogenous neural stem cells to promote neurogenesis to treat neurodegenerative diseases,especially AD,has become a hotspot in recent years.γ-Aminobutyric acid(GABA)neurons in mammalian brain account for about 10-15%of the total neurons,but GABAergic neurons have important functions which regulate the activity of major neurons in the whole brain,such as participation in the formation of learning and memory.Research has shown that activation of GABAergic neurons can improve cognitive function associated with aging.Therefore,this experiment intends to use optogenetics to activate GABAergic neurons in vitro and in vivo specifically,and to observe the neuroprotective effects of AD model and its possible molecular biological mechanisms.ObjectiveWe use optogenetics to activate GABAergic neurons specifically in vitro and in vivo,and to observe the neuroprotective effects on AD model and Aβ metabolism,inflammatory response,autophagy,endogenous neural stem cell proliferation and differentiation,and other changes in brain tissue,to explore new methods of AD treatment and study its possible molecular biological mechanisms through the influence of neural network changes on brain microenvironment and cell substructure.Part Ⅰ Effect of GABAergic Neurons Activation on the Protection of AD Cell Model and Autophagy-related Protein ExpressionMethods1.The primary hippocampal neurons of newborn mice were isolated and cultured,the expression of neurons-specific marker NSE and the expression of GABAergic neurons-specific marker GAD67 were identified by immunohistochemistry.2.Lentivirus containing the GAD67 promoter and the light sensitive protein ChR2 and the green fluorescent protein EGFP were transfected into the cultured primary neurons by optogenetics.3.Aβ1-42 was used to damage the primary hippocampal neurons of mice and AD cell model was constructed.The morphological changes of cells were observed by using microscope.Effect of Aβ1-42 on cell proliferation was detected by MTT kit.4.Optogenetics was used to activate GABAergic neurons,and effect of photostimulation on the morphological changes of neurons in cell culture medium was observed by using microscope.5.Experimental groups:Normal control group,Model group(Aβ1-42 injury model),Photostimulation group(Aβ1-42 injury model+optogenetic stimulation),Inhibitor group(Aβ1-42 injury model+optogenetic stimulation+bicuculline).6.The changes of LDH release in cell culture medium were detected by ELISA kit,and to observe the effect of GABAergic neurons activation on the damage of cells.7.Western-blot was used to detect the effect of GABAergic neurons activation on the expression of autophagy-related proteins Beclinl and LC3Ⅱ/LC3-Ⅰ in AD model cells.GABA-A receptor competitive inhibitor(bicuculline)was used to compare the changes of autophagy-related proteins before and after photostimulation.Results1.The isolated and cultured primary hippocampal neurons were positive for NSE expression.About 11%of NSE-positive neurons expressed both the specific markers GAD67 of GABA neurons and lentivirus.2.The effect of different concentrations of Aβ1-42 on primary hippocampal neurons viability were 97.9%,81.1%,69.5%and 54.3%,respectively.The 10μM concentrations with the stronger inhibitory and less damaging effect was applied to neurons at 12,24,36 and 48 hours,respectively.The effect of different time points on cell viability were 99.6%,91.9%,88.1%and 60.2%,respectively,in a dose-and time-dependent manner;10μM Aβ1-42 was used for 48 hours for subsequent experimental studies.3.Compared with the normal control group,the morphological damage of the neurons in the model group was observed.Compared with the model group,the photostimulation group could reduce the cell damage,and the morphological damage of the inhibitor group was not significantly improved.4.Compared with the normal control groups the LDH secretion level in the model group was significantly up-regulated(P<0.05).Compared with the model group,the LDH secretion level was down-regulated in the photostimulation group(P<0.05),and there was no change in the inhibitor group.5.Compared with the normal control group,the expression of autophagy-related proteins LC3II/LC3-I and Beclinl was decreased in the model group(P<0.01,P<0.05).Compared with the model group,the expression levels of LC3II/LC3-I and Beclinl was significantly increased in the photostimulation group(P<0.05),and there was no change in the inhibitor group.Part Ⅱ Neuroprotective Effect of GABAergic Neurons Activated in CA1 on AD Mice and its MechanismMethods1.APP/PS1(+)mice were identified by PCR amplification experiments.APP/PS1(+)male mice were paired with C57BL/6 WT female mice,and APP/PS1(+)male mice were obtained by tail clipping of offspring mice born within 7d.2.Adeno-associated virus containing GAD67 promoter and light-sensitive protein ChR2 and green fluorescent protein EGFP were transfected into GABAergic neurons of hippocampal CA1 region of APP/PS1 mice by stereotactic injection technique to prepare photo-controllable AD animal model.3.Experimental group:12 male APP/PS1(-)mice with 22-24-week-old were selected as a group,and 36 same-week-old male APP/PS1(+)mice were randomly divided into three groups:Normal control group:APP/PS1(-)mice;Model group:APP/PS 1(+)mice,Photostimulation group:APP/PS 1(+)mice+optogenetic stimulation;Inhibitor group:(APP/PS1(+)mice+optogenetic stimulation+bicuculline),4.Mouse brain frozen section and immunohistochemistry were used to detect the co-expression of GABAergic neuron-specific marker GAD67 and adeno-associated virus,and effect of activation of GABAergic neurons in hippocampal CA1 region on expression of Aβ,GFAP and IBA-1.5.Changes of Gamma and Theta oscillations in hippocampal CA1 region during activation of GABAergic neuron were detected by in vivo multi-channel electrophysiological techniques.6.Morris water maze test and Y maze test were used to evaluate the learning and memory ability of mice.7.ELISA kit was used to detect effect of activation of GABAergic neurons in hippocampal CA1 region on the formation of amyloid plaque.8.Western-blot was used to detect the effect of GABAergic neurons activation in hippocampal CA1 region on the expression of NTF,CTF,autophagy-related proteins Beclinl and LC3,neuroinflammatory factors IL-1β,IL-6 and TNF-α,and ERK1/2.9.Transmission electron microscopy was used to detect the effect of GABAergic neurons on the formation of autophagosomes in hippocampal CA1 region.Results1.Through PCR identification,mice with a target band of approximately 350 bp were APP/PS1(+)mice.2.The result of immunohistochemistry showed that some neurons in the hippocampal CA1 region of mice expressed both GAD67 and adeno-associated virus.3.Electrophysiological results showed that compared with the normal control group,Gamma(P<0.01)and Theta oscillation power(P<0.01)in the hippocampal CA1 of the model group were significantly decreased.Compared with the model group,the photostimulation group could be significantly improved in Gamma and Theta Oscillating power(P<0.05),and there was no change in Gamma and Theta oscillation power of the inhibitor group.4.The results of morris water maze showed that the average escape latency of the model group was prolonged on the 4th and 5th day compared with the normal control group(P<0.05);compared with the model group,the average escape latency of the mice in the photostimulation group was significantly shorter on the 4th and 5th day(P<0.05),and there was no change in the inhibitor group.In the spatial probe test on the 6th day,compared with the normal control group,the number of target crossings(P<0.01)and the percentage of time in zone in the model group(P<0.001)were reduced;compared with the model group,they increased in the photostimulation group(P<0.05),and there was no change in the inhibitor group.The results of Y maze test showed that there was no significant difference in the number of total arm entries in each group(P>0.05).Compared with the normal control group,the alternation behavior of the model group was decreased(P<0.05).Compared with the model group,the alternation behavior of the mice in the photostimulation group was increased(P<0.05),and there was no change in the inhibitor group.5.The result of immunohistochemistry showed that the expression of Aβ1-42 was significantly increased in the model group compared with the normal control group(P<0.001).Compared with the model group,the expression of Aβ1-42 was decreased in the photostimulation group(P<0.01),and there was no change in the inhibitor group.6.The results of ELISA showed that the expression of Aβ1-40 and Aβ1-42 protein in the model group was significantly increased compared with the normal control group(P<0.001);compared with the model group,the expression of Aβ1-40 and Aβ1-42 protein was significantly decreased in the photostimulation group(P<0.05,P<0.001),and there was no change in the inhibitor group.7.The results of Western-blot showed that there was no significant difference in APP content between the groups(P>0.05).Compared with the normal control group,the expression of CTF and NTF increased in the model group(P<0.05).Compared with the model group,the expression of CTF and NTF in the photostimulation group decreased(P<0.05),and there was no change in the inhibitor group.8.The results of Western-blot showed that compared with the normal control group,the expression of autophagy-related proteins Beclinl and LC3II/LC3-I in the model group was significantly decreased(P<0.01).Compared with the model group,the expression of Beclinl and LC3II/LC3-I in the photostimulation group was significantly increased(P<0.01,P<0.001),and there was no change in the inhibitor group.9.The results of transmission electron microscopy showed that the autophagosomes in the model group were significantly reduced compared with the normal control group;compared with the model group,the autophagosomes in the photostimulation group increased significantly,and there was no change in the inhibitor group.10.The results of immumohistochemistry showed that the number of GFAP+and IBA-1+ cells in the model group was significantly increased(P<0.01).Compared with the model group,the number of GFAP+and IBA-1+cells was significantly decreased in the photostimulation group(P<0.01),and there was no change in the inhibitor group.11.The results of Western-blot showed that compared with the normal control group,the expression of inflammatory factors TNF-α,IL-1β and IL-6 in the model group was significantly increased(P<0.01,P<0.05);compared with the model group,the expression of IL-1β,IL-6 and TNF-α in the photostimulation group was significantly decreased(P<0.01,P<0.05),and there was no change in the inhibitor group.12.The results of Western-blot showed that,the expression of p-ERK in the model group was significantly increased compared with the normal control group(P<0.01);compared with the model group,the expression of p-ERK in the photostimulation group was significantly decreased(P<0.05),and there was no change in the inhibitor group.Part Ⅲ Effect of GABA Neurons Activation in DG via SDF1/CXCR4 Pathway on Proliferation and Differentiation of Endogenous Neural Stem Cells in AD miceMethods1.APP/PS1(+)mice were identified by PCR amplification experiments.APP/PS1(+)male mice were paired with C57BL/6 WT female mice,and APP/PS1(+)male mice were obtained by tail clipping of offspring mice born within 7d.2.Adeno-associated virus containing GAD67 promoter and light-sensitive protein ChR2 and green fluorescent protein EGFP were transfected into GABAergic neurons of hippocampal DG region of APP/PS1 mice by stereotactic injection technique to prepare photo-controllable AD animal model.3.Experimental group:12 male APP/PS1(-)mice with 22-24-week-old were selected as a group,and 36 same-week-old male APP/PS1(+)mice were randomly divided into three groups:Normal control group:APP/PS1(-)mice;Model group:APP/PS1(+)mice,Photostimulation group:APP/PS1(+)mice+optogenetic stimulation;Inhibitor group:(APP/PS1(+)mice+optogenetic stimulation+bicuculline).4.Mouse brain frozen section and Immunohistochemistry assay were used to detect the expression of GABAergic neuron-specific marker GAD67 and adeno-associated virus,and effect of activation of GABAergic neurons in hippocampal DG region on Nestin and NSE expression.5.Changes of Gamma and Theta oscillations in hippocampal DG region during the activation of GABAergic neuron were detected by in vivo multi-channel electrophysiological techniques.6.Morris water maze test and Y maze test were used to evaluate the learning and memory ability of mice.7.Western-blot was used to detect the effect of GABAergic neurons activation in hippocampal DG region on SDF1/CXCR4 signal transduction pathway.Results1.Through PCR identification,mice with a target band of approximately 350 bp were APP/PS1(+)mice.2.The results of immunohistochemistry showed that some neurons in the hippocampal DG region of mice expressed both GAD67 and adeno-associated virus.3.Electrophysiological results showed that compared with the normal control group,Gamma and Theta oscillation power(P<0.01)in the hippocampal DG of the model group were significantly decreased.Compared with the model group,Gamma and Theta Oscillation power could be improved in the photostimulation group(P<0.05),and there was no change in Gamma and Theta oscillation power of the inhibitor group.4.The results of morris water maze showed that the average escape latency of the model group was prolonged on the 4th and 5th day compared with the normal control group(P<0.05,P<0.01);compared with the model group,the average escape latency of the mice in the photostimulation group was significantly shorter on the 4th and 5th day(P<0.05),and there was no change in the inhibitor group.In the spatial probe test on the 6th day,compared with the normal control group,the number of target crossings(P<0.01)and the percentage of time in zone in the model group(P<0.05)were reduced;compared with the model group,they increased in the photostimulation group(P<0.05),and there was no change in the inhibitor group.The results of Y maze test showed that there was no significant difference in the number of total arm entries in each group(P>0.05).Compared with the normal control group,the alternation behavior of the model group was decreased(P<0.01).Compared with the model group,the alternation behavior of the mice in the photostimulation group was increased(P<0.05),and there was no change in the inhibitor group.5.The results of immunohistochemistry showed that the number of Nestin+and NSE+cells in the model group was significantly lower than that in the normal control group(P<0.01).Compared with the model group,the number of Nestin+and NSE+cells in the photostimulation group significantly increased(P<0.05,P<0.001),and there was no change in the inhibitor group.6.The results of Western-blot showed that the expression of SDF1 and CXCR4 in the model group was significantly lower than that in the normal control group(P<0.05).Compared with the model group,the expression of SDF1 and CXCR4 in the photostimulation group was significantly increased(P<0.05,P<0.01),and there was no change in the inhibitor group.Conclusion1.The activation of GABAergic neurons by optogenetics can protect hippocampal neurons,which may be related to the induction of autophagy.2.Activation of GABAergic neurons in hippocampal CA1 region can improve learning and memory function,induce autophagy,and thereby promote Aβdegradation and alleviate inflammation,which may be related to the ERK1/2 signaling pathway.3.Activation of GABAergic neurons in hippocampal DG region can promote the proliferation and differentiation of NSC and improve the learning and memory function of AD mice,which may be related to the SDF1/CXCR4 signaling pathway. |
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