| Alzheimer’s disease(AD)is characterized by progressive loss of memory and other cognitive functions.Typically,a decade or so passes before the illness has taken its course and patients die in a completely helpless state.Worldwide,35.6 million people have dementia and there are 7.7 million new cases every year.Alzheimer’s disease is the most common cause of dementia and may contribute to 60-70%of cases(WHO 2010 reports).AD is the leading cause of disability among older people worldwide.AD is estimated to have cost the world $604 billion in 2010 alone.It places an enormous emotional and financial burden on patients,their families,and society.AD causes a large loss of neurons,synapses,brain weight and,thus,cognitive function.Two kind of AD have been detected,early-onset familial AD and late-onset sporadic AD.The complex aetioloty and mechanisms of AD is not fully resolved.AD is probably caused by complex interactions among multiple genetic,epigenetic,and environmental factors.Mutations in three genes—amyloid precursor protein(APP),presenilin(PS)-1 and PS-2—cause early-onset(<60 years)autosomal dominant AD,which probably accounts for less than 1%of AD cases.The mutations affect APP processing,leading to altered production of different Aβ peptides and,thus,their relative ratios.Late-onset sporadic AD also has a significant genetic component,estimated at 50%-70%.Almost half that risk is conferred by the apolipoprotein E(APOE)allele.The APOE E4 allele,which is present in 10%-20%of various populations,increases the risk for AD threefold in individuals carrying one copy and 15-fold for homozygous individuals.Studies on human postmortem brain samples and peripheral leukocytes,as well as transgenic animal models,have shown that aging and AD are associated with epigenetic dysregulation at various levels,including Aβ normal DNA methylation and histone acetylation modifications.Interestingly,pharmacological inhibition of DNA methylation in the hippocampus after a learning task impaired memory consolidation in mice,and promotion of histone acetylation improved learning and memory in a mouse model of AD and increased learning-related gene expression in aged wild-type mice,suggesting epigenetic regulation of learning and memory in health and disease.Aging is the most important known nongenetic risk factor for late-onset AD.Potential environmental risk factors for late-onset AD include head injury,low educational levels,hyperlipidemia,hypertension,homocysteinemia,diabetes mellitus,and obesity.However,several of these associations remain controversial.Numerous hypotheses for Alzheimer’s disease have been introduced,among which the amyloid cascade hypothesis has dominated research in past decades.Most studies agree that the classical pathological criteria for AD,neuritic plaques and neurofibrillary tangles,can account for 40%-70%of the variance in cognition seen in elderly subjects,with additional pathologies such as cerebrovascular disease and Lewy body pathology working together with AD pathology to account for an additional 20%-30%of dementia cases.Aβ peptides,the main constituent of amyloid plaques,and various other metabolites are derived from APP by proteolytic cleavage.APP,a single-pass transmembrane protein with large extracellular domain,is produced in large quantities in neurons and is metabolized very rapidly.Multiple alternate pathways exist for APP proteolysis.APP can be proteolyzed directly by a-secretase and thenγ-secretase,a process that does not generate Aβ,or by the proteases BACE1 and thenγ-secretase,a process that results in the production of Aβ,which is then dumped into the extracellular space or degraded in lysosomes.An imbalance between production and clearance,and aggregation of peptides,causes Aβ to accumulate,and this excess may be the initiating factor in Alzheimer’s disease.Aβfibrils are acutely toxic to neurons.It induces loss of synaptic terminals,synaptic dysfunction,loss of calcium balance,mitochondria dysfunction,abnormalities on spatial memory tests,and inflammation,depletion of brain-derived neurotrophic factor(BDNF),acetylcholine release,in AD transgenic animals.Aβaccumulation can further precede and drive tau aggregation and then induce nerofibrillary tangles.Soluble oligomers and intermediate amyloids are the most neurotoxic forms of Aβ.In brain-slice preparations,dimers and trimers of Aβ are toxic to synapses.The severity of the cognitive defect in Alzheimer’s disease correlates with levels of oligomers in the brain,not the total Aβ burden.There have been only two kinds of drugs that are currently approved by the FDA for the treatment of Alzheimer’s disease.One inhibit acetylcholine esterase to increase the levels of the neurotransmitter acetylcholine,which is depleted in AD brains,the other antagonize NMDA-type glutamate receptors to prevent aberrant neuronal stimulation.The impact of these drugs on disease manifestations is modest and transient,although observational studies suggest that combination treatment may increase the time before patients require nursing home care.There is no convincing evidence,though,that these agents can prevent,halt,or reverse the disease.There are also other therapeutics to decrease the production or enhance the clearance of Aβ.Drugs in the former category are designed to inhibit β-or γ-secretase,the enzymes that release Aβ from its precursor.However,it is difficult to develop drugs that penetrate the BBB and specifically inhibit the cleavage of APP without affecting the cleavage of alternative substrates such as Notch and voltage-gated sodium channel subunits.The latter efforts to lower Aβ levels through activating immunization against Aβ and then clearing amyloid plaques.Other studies light on preventing or reversing hAPP/Aβ-dependent neuronal and cognitive impairments,including reductions of tau,group IVA phospholipase A2,cyclophilin D,or Fyn;reversal of EphB2 or Nav1.1 depletions;and replacement of apoE4 with apoE3.More research is needed to further assess the therapeutic potential and safety of these approaches.Since more and more researches focus on enhancing Aβ clearance that is impaired in brains of almost sporadic AD cases,approaches targeting ApoE,the major risk factor of sporadic AD,have been detected.Efforts are made to block the interaction between apoE and Aβ and so to attenuate apoE4’s ability to promote Aβdeposition or increase apoE expression.However,these findings were obtained in mice that express murine rather than human apoE.The effect of murine apoE on Aβmetabolism clearly differs from that of human apoE in that mouse apoE promotes Aβdeposition,whereas human apoE promotes Aβ clearance.Therefore,it is conceivable that increase apoE expression or blocking the interaction between Aβ and human apoE at young ages might promote amyloid deposition.Epoxyeicosatrienoic acids(EETs)are products of the epoxygenase CYP enzymes,which catalyze the epoxidation of arachidonic acid olefin bonds and result in the production of four regioisomeric EETs:5,6-EET,8,9-EET,11,12-EET and 14,15-EET.Small amounts of 8,9-,11,12-,and 14,15-EET are present in the plasma,liver,and kidney,with 14,15-EET being the most abundant regioisomer.More than 90%of the EET contained in rat plasma is present in phospholipids,mostly in the low-density lipoproteins.EETs are fast metabolized to their corresponding DHETs by soluble epoxide hydrolase.EETs are endothelium-derived hyperpolarizing factors(EDHFs).EETs inhibit the open probability of the cardiac L-type Ca2+ channel;activate the BKCa channel and tissue plasminogen activator expression through G protein-coupled receptor;increase the intracellular adenosine 3’,5’-cyclic monophosphate content and activate PKA;activate tyrosine kinase cascade,Src kinase,MAPK,and phosphatidylinositol 3-kinase(PI-3K)/Akt pathways;inhibit cytokine-activated NF-κB-mediated transcription by inhibition of IKK phosphorylation of IκBα.Through these pathways mentioned above,EETs induce anti-inflammation,mitogenesis,angiogenesis,anti-migratory fibrinolysis,vasorelaxation,bronchodilation,and anti-platelet aggregation.However,the exact ligand of EETs is of no idea.Thus,further researches are urgently needed.The biological activity of EETs in vivo is limited since EETs are rapidly metabolized by a variety of pathways.sEH,which adds water across the epoxide to give the corresponding 1,2-dihydroxy-fatty acids,is the dominant pathway in many tissues.Thus,inhibition of this pathway will likely increase the EET concentrations in plasma and tissues significantly,whereas the other routes of metabolism ensure that,even in the absence of sEH activity,EET levels can increase only to moderate levels.Indeed,previous study has confirmed that there was no difference in brain hydrolase activity and the brain tissue EET levels between WT and sEH knockout mice.Thus,even massive doses of sEH inhibitors cannot lead to exceptionally high EET levels and limit target-related side effects.Inhibition of this enzyme is therefore a promising therapeutic strategy for many diseases.The sEH is expressed in multiple human tissues.Although it has broad distribution in the liver,its expression in other tissues such as the kidney is localized.Similarly,in the brain,sEH is highly expressed in the smooth muscles of the arterioles,as well as in the neuronal cell bodies,oligodendrocytes,and astrocytes.Both PPARa and PPARy agonists(e.g.,fibrates and glitazones)induce sEH expression.sEH is found mostly in the cytosol but also in the peroxisomes of some organs.The N-and C-terminal regions of sEH are separated by a proline-rich linker.The C-terminal contains the EH activity,and the smaller N-terminal is a phosphatase that apparently acts on lysophosphatidic acids.Several single-nucleotide polymorphisms of sEH have been identified,two of which have been associated with various cardiovascular diseases:K55R(17%of the population)and R287N(8-14%of the population).These.observations point to an essential role of sEH in human health.There are lots of drugs that have been found to inhibit sEH.Current sEHIs inhibit the epoxide hydrolase activity of the C-terminal domain without affecting the phosphatase activity of the N-terminal domain.The first-generation sEHIs were potent competitive inhibitors and included chalcone oxides and glycidols.Unfortunately,these alternative substrates are rapidly inactivated by glutathione and glutathione transferases,making them difficult to use in tissue samples and in vivo.The first report to show in vivo biological effects of a sEHI was achieved with CDU,which had antihypertensive actions when injected intraperitoneally for 4 days.AUDA,which has been widely used in cultured cells and animals,can be orally administered.But it requires DMSO for in vitro experiments,and a considerable amount of 2-hydroxylpropyl β-cyclodextrin for it to be administered in drinking water for in vivo studies.Efforts were made to increase the water solubility of the inhibitor without reducing potency.The application of this concept was used to produce other drug-like sEHI molecules,including t-AUCB,TPAU,AEPU,APAU,and others that have both excellent potency and efficacy in many species.Although APAU was taken through Phase IIA trials,the more metabolically stable and potent modern compounds TPPU and t-TUCB may represent a better clinical candidate.TPPU and t-TUCB show low-nanomolar potency,exhibit good oral bioavailability and pharmacokinetics(in rodents,canines,felines,and primates),and are easier to formulate.The compounds tested show little off-target activity on targets such as cytochrome P450s and hERG Interestingly,the registered Raf kinase inhibitor sorafenib is a low-nanomolar sEHI that is similar to t-CUPM.Although most sEHIs are not kinase inhibitors,researchers have prepared a variety of compounds that are good sEHIs with interesting selectivity profiles on kinases.Studies showed much biological effect of sEH inhibitors on disease,include anti-inflammation in various inflammatory models;analgesic both to inflammatory pain and neuropathic pain;anti-hypertension;anti-ischemia;reduction of cardiac hypertrophy and arrhythmia;anti-atherosclerosis;protection against renal failure;reduction of platelet aggregation;treating pulmonary hypertension;and normalizing dysregulated glycemic states.The deletion of the sEH gene also increases the tissue levels of EETs and so has the similar effect as sEHIs mentioned above.However,there is still no feasible therapy for AD that can both ameliorate the pathology and recollect the memory with acceptable side effect.Think of the beneficial effect of sEHI,we suppose sEH inhibition would be an appropriate approach for AD therapy.In current research,western blotting was used to detect the expression of sEH in the brains of 5×FAD transgenic mice(Tg6799).Then we apply a modern sEHI,TPPU,to 5×FAD transgenic mice and compare the amyloid burden to the solvent vehicle mice.Then we cross 5×FAD transgenic mice with sEH knockout mice and compare the amyloid deposition and behavior phenotype between APP/PS1/sEH+/+ mice and APP/PS1/sEH-/-mice.It shows that sEH gene deletion can release the amyloid burden of 5×FAD transgenic mice.However,it failed to recollect the impaired memory of 10 months old mice.Different months-aged mice were sacrificed for Aβ immunohistochemistry of the amyloid deposition.We can see that there is not any Aβ plaque in the brain of 1 month old mice nor WT mice,the amyloid occurs at 2 months and grows to a large mount at 6 months and most serious at 9 months.Except for the pathologic changes,we test the memory using Radial Arm Water Maze model,a space and working memory model especially worked in AD mice model.From the results we can see that Tg6799 mice(n=10)made much more errors than WT mice(n=10)when escaping to the platform.Difference of mean was evaluated using repeated measures ANOVA.Mauchly’s Test of Sphericity:W=0.0367,p=0.3188,no adjust was needed;Tests of Between-Subjects Effects:F=4.5132,p=0.0477.To identify the sEH null mice,PCR-based genotyping and Western blotting-based protein expression was applied.PCR genotyping results showed 295bp DNAs as knockout homozygous and 338bp DNAs as WT.Both two straps are yielded from heterozygous mice.Western blots demonstrates that sEH protein is abundantly expressed in the brain tissue of WT(+/+),but is not expressed in homozygous sEH null(-/-)mice.To detect the effect of sEH deletion on the memory of mice,the sEH null mice and their littermate WT mice were subjected to Fear conditioning test.The results showed that sEH null mice(n=9)showed more freezing time than WT mice(n=9).Difference of mean was evaluated using Independent samples T-test,t=-3.065,p=0.007.This indicates that sEH null mice have got much stronger fear memory than WT mice.To know the relation of Aβ and sEH,we detected the expression level of sEH in Tg6799 mice.It was significantly higher in 6 months old Tg6799 mice(n=3)than their littermate WT mice(n=3).Difference of mean was evaluated using Independent samples T-test,t=-7.026,p=0.002.Whileas the level of sEH expression did not change in 2 months old mice(n=3 for each group),t=1.099,p=0.333.TPPU,a sEHI,was orally administrated through drinking water(1mg/kg weight per day)for executive 7 days.Then the mice were sacrificed and the brains were removed and sectioned for following thioflavin S staining.Thioflavin S stained amyloid plaques were counted and the number was analyzed using Independent T-test.We find that Thioflavin S stained amyloid plaques in both hippocampus and cortex were largely decreased after TPPU treatment(n=10 for each group),t=2.761,p=0.03 for hippocampus;and t=2.70,p=0.032 for cortex.In our project we crossed the Tg6799 mice with sEH null mice and APP/PS1/sEH+/+,APP/PS1/sEH-/-mice were tested.10 months old mice were used for RAWM test and sacrificed for amyloid plaque analysis later.Unfortunately,we did not get positive result in this test.There is no difference in error times between APP/PS1/sEH-/-(n=10)and APP/PS1/sEH+/+(n=8)mice.Repeated measures ANOVA was used for evaluating the difference of mean(F=2.790,p=0.055).3,6,and 10 months old mice were sacrificed for Thioflavin S staining,Aβ IHC,and Aβ ELISA assay.Thioflavin S stained amyloid plaques in the brain were counted and the number was compared between APP/PS1/sEH+/+mice and APP/PS1/sEH-/-mice.The result showed that the number of amyloid plaques is significantly decreased in both hippocampus and cortex of 6 months old APP/PS1/sEH-/-mice(n=4)compared to APP/PS1/sEH+/+mice(n=4).Difference of mean was evaluated using Independent samples T-test.t=4.3,p=0.005 for hippocampus and t=6.22,p=0.001 for cortex.But there is no difference between the two group neither in hippocampus nor in cortex of 3 months old mice(n=4 for each group),though it tends to be lowered t=1.331,p=0.232 for hippocampus and t=1.4,p=0.2 for cortex.And the plaque number did not change in the brain of 10 months old mice(n=6 for each group).t=0.583,p=0.573 for hippocampus and t=0.16,p=0.88 for cortex.Aβimmunohistochemistry assay was used to measure the Aβ burden in hippocampus and cortex.Aβ area fraction was calculated using Image J software by comparing Aβ area to the hippocampus or cortex area.The results showed that the area fraction of Aβ is significantly decreased in both hippocampus and cortex of 3 months old APP/PS1/sEH-/-mice(n=4)compared to APP/PS1/sEH+/+ mice(n=4),t=2.852,p=0.029 for hippocampus and t=2.949,p=0.049 for cortex.Difference in area fraction of Ap between APP/PS1/sEH-/-(n=4)and APP/PS1/sEH+/+(n=4)mice of 6 months old is also significant,t=3.148,p=0.02 for hippocampus and t=7.905,p=0.0002 for cortex.However,there is no difference in Aβ area fraction in the brain of 10 months old mice(n=6 for each),t=-0.077,p=0.94 for hippocampus and t=-0.36,p=0.727 for cortex.ELISA assay was also used to measure the level of Aβ in brain tissue of 6 months old mice.The result showed that the RIPA solved Aβ1-40 and Aβ1-42 in the brains of APP/PS1/sEH-/-mice(n=4)is significantly lower than APP/PS1/sEH+/+mice(n=4),t=2,891,p=0.028 for Aβ1-40 and t=2.567,p=0.042 for Aβ1-42,whileas there is no difference in SDS solvent Aβ1-40 and Aβ1-42 between the two genotype of mice,t=0.891,p=0.407 for hippocampus and t=-0.706,p=0.507 for cortex.We detected whether APP expression and processing was changed.The results showed there is no difference in APP expression(p=0.238)and formation of carboxy(C)-terminal fragments(CTFs)(CTFs:t=-0.24282,p=0.818,P-CTF:t=0.365,p=0.73,α-CTF:t=-0.996,p=0.365)between APP/PS1/sEH+/+(n=4)and APP/PS1/sEH-/-mice(n=3).These results showed that sEH expression level was increased in AD mice.sEH inhibition and deletion can attenuate Aβ deposition and amyloid plaque formation.The reduction of Aβ deposition is probably not due to changes of APP expression and processing.sEH inhibition may be a novel therapeutic target for AD. |
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