Protective Effect Of Idebenone On Cells And Mouse Models Of Alzheimer's Disease

Background:Alzheimer's disease(AD)is a common neurodegenerative disease,and it is clinically characterized by progressive cognitive impairment.AD is the most common type of dementia in the elderly population in our society.and The incidence tends to increase gradually,placeing a heavy burden on families and society.The etiology and pathogenesis are still unclear.In the past decades,studies have shown that multiple factors could be associated with AD.Energy deficiency and oxidative stress are considered to be the main pathophysiological changes in AD brain.According to the mitochondrial cascade hypothesis of AD,mitochondrial dysfunction are believed to be the cause of energy deficiency and increased reactive oxygen species(ROS)generation in AD,It ultimately leads to synaptic injury and neuronal death.Additionally,mitochondrial dysfunction develops with the exacerbation of amyloid beta(A?)deposition and abnormal Tau protein phosphorylation,which are typical pathological changes of AD.Previous studies show that A? cause excessive production of mitochondrial ROS.The changes of mitochondria synapses affect cognitive function.In AD,pathologic studies showed a positive correlation between hippocampal synapse loss and clinical severity.The two factors form a vicious circle,which also suggests that mitochondrial dysfunction is associated with the development of AD.There is still no effective treatment for AD.As Amyloid beta(A?)deposition increased and Tau protein abnormal phosphorylation increased,oxidative stress was further enhanced.The enhanced oxidative stress response and mitochondrial dysfunction induced by A? and other factors exist in the whole process of AD.They are important pathophysiological events.Therefore,therapeutic strategies targeting AD mitochondrial dysfunction and antioxidants,have received increasing attention,especially early intervention measures.Idebenone(IDB)is an analogue of coenzyme Q10.The difference lies in its hydroxyl decyl side chain,which makes it have a better permeation through cell membranes to target mitochondria.So IDB has a better antioxidant effect than CoQ10,even in low oxygen condition.Pharmacological studies have shown that it impact on mitochondrial complex I,complex II,complex III,acting as an electronic carrier in the brain mitochondrial respiratory chain.As an antioxidant,IDB can inhibit the brain mitochondria membrane damage caused by lipid peroxidation.IDB improves the energy metabolism during cerebral ischemia by increasing the glucose metabolism rate and increasing the production of ATP.IDB can reduce oxidative stress of mitochondria,inhibiting the activation of mitochondrial permeability transformation hole,preserving mitochondrial membrane potential and increasing ATP generation.In vitro studies,it was also reported that IDB protected mitochondrial function against Amyloid beta toxicity.In recent years,IDB has been widely used in mitochondrial diseases,such as Friedreich's ataxia(FRDA),Leber's hereditary optic neuropathy(LHON),etc.And the security is universally recognized.However,there is no consistent conclusion on the the treatment of AD.The corresponding basic studies are few.In the animal model studies,IDB are used in short term and the effect is different.The effectiveness of IDB needs to be further studied.Since typical neuropathological changes have appeared in the early stage of AD,and treatment after typical clinical symptoms occueed cannot reverse the damage.Early intervention may be effective.Therefore,in this study,we investigated whether early use of IDB could have a protective effect on AD.This study has two parts.In part one,cortical neurons cultured adding A? oligomer,as a cell model of AD,we investigated the protective effect of IDB on the mitochondria.In part two,5xFAD mice were used as the mouse model,evaluated the effects of IDB on mitochondrial function and cognitive function of 5xFAD mice in the middle and late stage of 5xFAD.Part ? Idebenone protects mitochondrial function against Amyloid beta toxicity in primary cultured cortical neuronsAims:1.To detect the effect of IDB on mitochondrial oxidative in A?-treated neurons.2.To detect the protective effect of IDB on the mitochondria bioenergetics in A?insulted environment3.To exam the influence of IDB on PKA/CREB activation in A?-stressed neurons by immunoblottingMethods:1.AD cell model and Drug treatmentC57BL/6J mice within 24 hours were used to isolate the cerebral cortex.Neurons were cultured.A? was added.The changes of mitochondrial function were detected by pretreatment with IDB.Meanwhile,the control group was set.Neurons at Div12-14 days were treated with drug.Neurons were exposed to Idebenone in different concentrations for 28 hours for cell viability assay.According to different treatment,the experimental group are as follows:vehicle,A?,sA?,IDB,IDB and A?,IDB and sA?.Neurons were pretreated with 1 ?M Idebenone for 4 hours followed by a co-incubation with 1?M A?or 1?M sA? for 24 hours for mitochondrial functional assay.2.Primary neuron culture.C57BL/6J mice born within 24 hours were used to isolate the cerebral cortex.Neurons were planted in neuromedium containing Neurobasal A,B27,L-glutamin and antibiotic,then placed in a cell incubator containing 5%CO2 at 37?.3.Oligomer A?1-42 and scramble A? preparation.A?1-42 lyophilized powder was centrifugated and then dissolved in 1,1,1,3,3,3-Hexafluoro-2-propanol(HFIP).Dissolve it in ultrasonic water bath.After centrifugation,take the supernatant and dry it overnight obtain A?1-42 film.The dried film was resuspended in DMSO,then measure the protein concentration by BCA method.4.MTT assay.Cell viability were measured by an MTT assay kit following the manufacturer's instruction.The absorbance at OD550nm was read on a microplate reade,Gene5 software was used to analyze the result.5.ATP content measurement.We used The ATP Luminescent assay kit to measure the content level of treated primary cultures following the manufacturer's instructions.Luminescent signals were read under the microplate reader and analyzed by Gene5 software.6.Mitochondrial membrane potential assay.The staining of tetramethylrhodamine was used to measure the Mitochondrial membrane potential.Neurons were incubated with 20nM TMRM.Images were collected with a Nikon inverted fluorescent microscope with on-stage incubator and were analyzed by Nikon NIS Advanced Research software.The operation was carried out according to the instructions.The staining of MitoTracker Green was carried out simultaneously with TMRM.7.Mitochondrial superoxide assay.Production of mitochondrial superoxide was detected by Mitosox Red staining following the instructions.Neurons were incubated with 2?M MitoSox Red,the images were observed under Nikon inverted fluorescence microscope and the red intensity was analyzed using nikon NIS advanced software.8.Immunoblotting analysis.Western blotting was used to detect the expression levels of PKA,CREB,pPKA and pCREB in neurons,.The membranes were imaged on the Bio-Rad Chemidoc imaging system,then was analyzed with Image Lab software(Bio-RAD).9.Statistical analysis.Statistical analysis was performed by GraphPad Prism 5 software.One-way ANOVA followed by Bonferroni post hoc analysis were used for multiple comparisons between groups.P<0.05 was considered statistically significant.Results:1.There was no significant impact on neuronal viability at concentrations up to 10?M.When the concentration up to 100?M difference was statistically significant,effecting on the activity of neurons.In reference to previous studies,neurons were treated at a concentration of 1?M in the following functional assays.2.The pretreatment of IDB alleviated A?-mediated mitochondrial oxidative stress.A? induced a substantial increase of MitoSox Red in the absence of IDB,but A?-insulted neurons pretreated by IDB exhibited a comparable level with the vehicle neurons.3.IDB protects neuronal mitochondrial bioenergetics from A? toxicity.A? induced a significant decrease in the content of ATP.The ATP levels were preserved in A?-insulted neurons with pretreatment by IDB.A? caused a substantial decrease in TMRM intensity.It was consistent with the ATP results.The TMRM staining intensity of neurons pretreated by IDB,exhibited a comparable level with the vehicle.IDB alone or scrambled A? exhibited no impact on these assays.4.The protection of mitochondrial function by IDB may be beneficial to neuronal PKA/CREB signaling.A? induced a substantial decrease in the phosphorylation levels of PKA C and a significant decrease in CREB phosphorylation at Ser133.In comparison,the pretreatment of IDB preserved PKA C alpha phosphorylation and CREB phosphorylation at Ser133 in neurons from A? insult.A? has no impact on the expression of PKA C and CREB.IDB alone or sA? has effect on PKA or CREB activation.Conclusions:IDB alleviates the production of A?-mediated mitochondrial superoxide,and preserves neuronal mitochondrial bioenergetics from A? toxicity.It ameliorates inhibition of PKA/CREB signaling in cortical neurons.Part ? The protective effect of Idebenone on 5xFAD miceAims:1.To exam the effect of IDB on spatial memory in 5xFAD mice;2.To investigate the effect of preconditioning with IDB on mitochondrial productivity of 5xFAD mice;3.To detect the effect of IDB on the expression of A? in the brain of 5xFAD mice;4.To detect the effect of IDB on synaptic density of 5xFAD mice.Methods:1.Drug treatment and grouping of experiment miceThe dosage of IDB in the drug group was 100mg/kg(i.e.0.01 mL/g of IDB suspension)per day,intraperitoneal injection.The dosage was 0.01 mL/g(5%gum Arabic)in vehicle group.The mice were divided into 4 groups after genotype identification:?5xFAD-Idebenone;?5xFAD-Vehicle;?Non-transgenic mice drug group(nonTg-Idebenone);?Non-transgenic mice vehicle group(nonTg-Vehicle).Mice were treated since the age of 3 months.They were weighed weekly and the dose was adjusted accordingly.Mice at 4 months of age(the middle stage of AD)and 8 months of age(the late stage of AD)were observed.2.Maze behavior test:Using the six-arm water maze,the spatial learning and memory ability of the 5xFAD mice and Non-transgenic mice mice were examed.?3.The level f A?40 and A?42:The levels of A?40 and A?42 in brain were detected by ELLSA assay.4.Tissue section preparation and Immunofluorescence staining:The level of synaptic density in brain were detected by immunofluorescence staining in brain tissue sections.5.Statistical analysis.Statistical analysis was performed by GraphPad Prism 5 software.One-way ANOVA followed by Bonferroni post hoc analysis were used for multiple comparisons between groups.P<0.05 was considered significantly.Results:1.IDB has protective effects on spatial learning and memory function in 5xFAD mice.IDB improved spatial learning and memory in 4-month-old and 8-month-old 5xFAD mice.The spatial learning and memory ability of 5xFAD-Idbenone is better than the control group;the difference was statistically significant(p<0.05).2.IDB has a protective effect on mitochondrial productivity in 5xFAD mice.The ATP levels in the brain tissues of the mice were detected by fluorescence luminescence.ATP production in the 8-month-old 5xFAD mice was significantly reduced,but the level in 5xFAD-Idbenone group was higher than the control group.IDB had a protective effect on mitochondrial productivity and function.There was no significant difference in 4-month-old mice.3.IDB effects A? level in the brain.Early use of IDB did not effect the level of A?40 and A?42 level at age of4 months,but it did reduce the level ofA?42 in 8-month-old 5xFAD mice.These results indicate that IDB has a therapeutic effect on the level of A?42.4.IDB has a protective effect on the synaptic density in 5xFAD mice.The number of synapses was determined by vGlut1 and PSD-95 immunofluorescence staining.The numbers of synaptic density in hippocampal CA1 region,dentate gyrus and cerebral cortex were observed by confocal microscope.The results showed that 5xFAD mice of 4 months and 8 months of age all had significantly fewer synapses than non-transgenic mice.The synaptic density treated with IDB was significantly higher than the control group(c5xFAD-Idebenone group v.s.5xFAD-Vehicle group,P<0.05).Conclusions:In AD animal model,IDB can improve the spatial learning and memory ability,and has acertain protective effect on mitochondrial productivity function.IDB can also reduce the level of A? in the brain of 5xFAD mice,and has a protective effect on the synaptic density in the brain.IDB has a certain protective effect on mitochondrial function and cognitive function of AD mice.Conclusions of the full text:1.IDB alleviates the production of A?-mediated mitochondrial superoxide,andpreserves neuronal mitochondrial bioenergetics from A? toxicity.It ameliorates inhibition of PKA/CREB signaling in cortical neurons.IDB may be potential to reduce risk of AD as a preventive strategy.2.In AD animal model,IDB can improve the spatial learning and memory ability,and has acertain protective effect on mitochondrial productivity function.IDB can also reduce the level of A? in the brain of 5xFAD mice,and has a protective effect on the synaptic density in the brain.IDB has a certain protective effect on mitochondrial function and cognitive function of AD mice.Therefore,early and long-term use of IDB as a prevention strategy may have the potential of relief symptoms and slow the progression of the disease.