The Effect Of Gonadectomy On Brain Aging And Brain Heat Shock Proteins Expression, And Inhibitory Effect Of FLZ, A Squamosamide Derivative, On Amyloid β Production In N2a (APPswe/PS1△9) Cells And Tg Appswe/PS1 Mice

Over several decades,with the rise of average life expectancy,aging population gradually increased,which results in increased incidence of neurodegenerative disease, such as Alzheimer's disease(AD) and Parkinson's disease(PD).The public has to face the problems which aging society brings to the whole world.In order to extend the lifespan and improve the living quality of aging people,researches on aging and age related disease have raised more concerns in recent years.Brain aging and age related neurodegenerative disease is the focus of neuroscience gerontology.There are several hypotheses about mechanism of aging.Aging of reproductive system is considered to accelerate brain aging and increase the incidence of neurodegenerative diseases.Substantial evidence suggests that estrogen is a neuroprotective and neuromodulatory hormone.Besides its anti-oxidative effect, estrogen can regulate expression of a variety of genes by activation of estrogen receptors. The sharp decline of estrogen level after menopause may lead to impaired memory and cognition,and increases the incidence of AD and PD.Testosterone also has neuroprotective and neuromodulatory effect.The decline of testosterone level after middle age could lead to impaired memory and cognition and increased incidence of neurodegenerative diseases in men.During aging,brain is exposed to various stressors,which leads to accumulation of damaged proteins.The aggregation of misfolded proteins becomes a threat to cell survival,and promotes pathogenesis of neurodegenerative diseases.Heat shock proteins (HSP) serve as molecular chaperons,binding to misfolded proteins,assisting to refold misfolded proteins and eliminate irreversibly damaged proteins.Therefore,heat shock proteins are considered as endogenous cytoprotective factors,with function to protect cells against a multitude of stresses.Two of the most important heat shock proteins Hsp70 and Hsp27,can be significantly induced in many areas of brain after stresses. During aging,stress-induced expression of heat shock proteins declines in the brain, which leads to impairment of endogenous defense system.AD is a neurodegenerative disease characterized clinically by progressive impairment of memory and cognition.One of the hallmarks of AD is the presence of senile plaques in the hippocampus,which are composed primarily of aggregated extracellular deposition of Aβ_1-40 and Aβ_1-42.The progressive accumulation of Aβaggregates is widely believed to be fundamental to initiate neurodegenerative pathogenesis.Aβis generated by proteolysis of amyloid precursor protein(APP) by three specific enzymes,α-,β-,andγ-secretases.APP is enzymatically processed by two distinct and mutually exclusive pathways:in amyloidogenic pathway,cleavage of APP byβ-secretase produces Aβ.Alternatively,in nonamyloidogenic pathway,cleavage of APP byα-secretase generates sAPPαInhibition ofβ-secretase to decrease Aβproduction has been taken as target for AD drug development.In addition,substantial evidence indicates that AD transgenic mice with over-expression ofα-secretase display improved learning and memory,and less Aβdeposition in the hippocampus.Therefore,upregulation ofα-secretase mediated APP processing may be novel therapeutic applications in AD.FLZ is a synthetic cyclic analogue of natural squamosamide.Previous studies demonstrated that FLZ had significant neuroprotective effect.FLZ protected against Aβ-induced toxicity in SH-SY5Y cells.It also ameliorated the impairment of learning and memory and pathological damage to the hippocampus induced by ventricular injection of Aβin mice.Therefore,FLZ could be a promising candidate for AD treatment,and it has applied for patent.In order to elucidate the relationship between sex hormones and aging related neurodegenerative diseases,and develop novel drugs for AD treatment,three parts of study were performed and are reported in this dissertation as following:Partâ… :Establishment of gonadectomy-aecelerated brain aging model in miceIn this study,we aim to develop a gonadectomy-accelerated brain aging model. Male or female mice of 2-month old were orchiectomized(ORX) or ovariectomized (OVX) bilaterally,and were kept for 10 months.All the mice were divided into 4 groups: female-sham(12-month old),female-OVX(12-month old),male-sham(12-month old) and male-ORX(12-month old).2-month old female(female-young) and male(male-young) mice were obtained before determination of brain aging-related parameters.The results of water maze show that spatial learning and memory was impaired in gonadectomized mice as compared with young mice.The results of western blotting and immunostanning show increased expression of Aβ,BACE1 and phospho-Tau,decreased expression of NGF,BDNF,NT3 and TrkA as well as increased expression of p75NTR in gonadectomized mice,as compared with young mice.The result of Nissl staining indicates that the amount of Nissl bodies decreased in the brain of gonadectomized mice. In addition,as compared with young mice of the same gender,the changes of brain aging related parameters were more obvious in ovariectomized mice than that in orchiectomized ones.There are no significant changes of these parameters in the brain of sham-operated mice as compared to young mice.As a result,gonadectomy can accelerate brain aging which is displayed as impaired spatial learning and memory,increased Aβexpression as well as decreased neurotrophins level in the brain.Partâ…¡:The role of estrogen in modulating expression of brain heat shock proteins in ovariectomized miceHeat shock proteins serve as endogenous cytoprotective factors.The impaired thermotolerance in old people is probably due to diminished expression of heat shock proteins,and heat shock proteins also play a role in the pathogenesis of AD and PD.In order to elucidate the effect of ovariectomy and the modulatory effect of estrogen on expression of brain heat shock proteins,in this study,we investigated the regulatory effect of estrogen on heat stress-induced brain heat shock protein expression in ovariectomized mice and its underlying mechanism.Female mice of 2 months old were ovariectomized bilaterally or sham operated,and were kept for 10 months.Some ovariectomized mice were supplemented with 17β-estradiol(0.5 mg/kg/day every other day for 14 days,S.C.).Mice were divided into four groups:young(2-month old),old-OVX (12-month old),old-sham(12-month old) and OVX+17β-estradiol(E2)(12-month old),and were subjected to heat stress at 44℃in an oven for 1 hour followed by 3 hours at room temperature.The results of western blotting and RT-PCR show that in ovariectomized mice,heat stress-induced brain HSP70 expression and mRNA transcription are significantly lower than that of young mice.And the result of gel mobility shift assay shows that the binding activity of heat shock factor with heat shock element was also significantly impaired in the brain of ovariectomized mice.In the brain of mice from OVX+E2 group,supplement of estrogen restored heat stress-induced brain HSP70 expression,and attenuated heat stress-induced brain DNA fragmentation, caspase-3 activation and mitochondrial cytochrorne C and AIF leakage.The results suggest that in ovariectomized mice,heat stress-induced brain Hsp70 expression decreased and the mechanism was related to impaired binding of heat shock factor with heat shock element.Estrogen supplement restored heat stress-induced heat shock protein expression and protected against heat stress-induced brain injury in ovariectomized mice. Partâ…¢:A squamosamide derivative FLZ reduces amyloidβproduction by increasingα-secretase mediated non-amyloidogenic APP processingAs mentioned above,upregulation ofα-secretase mediated APP processing leads to decreased Aβproduction,which may be novel therapeutic applications in AD.The neuroprotective effect of FLZ has been proved in several models.In this study,we investigate the effect of FLZ onα-secretase mediated APP processing.We found that Aβproduction was reduced by FLZ in Aβ-expressing N2a(APPswe/PS1Δ9) neuroblastoma cells,which was paralleled with an increase in sAPPαin the medium.Moreover,the result of western blotting show that the active form of ADAM10 and APP expression were elevated at the cell surface of FLZ-treated cells,consistent with immunostaining result showing an enhanced co-localization of ADAM10 and APP at the membrane of FLZ treated cells.Pretreatment with protein trafficking inhibitor brefeldin blocked FLZ-induced translocation of ADAM10 and APP to the cell surface;it also reversed FLZ-induced increase in.sAPPαrelease and decrease in Aβproduction.Furthermore,oral administration of FLZ to APPswe/PS1 transgenic mice significantly reduced Aβlevel and increased ADAM10 level in hippocampus.These findings suggest that decreasing effects of FLZ on Aβproduction may be mediated by its promotion ofα-secretase mediated APP non-amyloidogenic processing,which provides evidence for therapeutic applications of FLZ in AD.Establishing good animal models of aging is very important for the research on aging and neurodegenerative disease.The ultimate goal of research on aging and neurodegenerative disease is to find novel therapeutic applications.To summarize the study:(1) As compared with young mice,gonadectomized mice had impaired spatial learning and memory,increased expression of Aβ,BACE1 and phospho-Tau,decreased Nissl bodies and decreased expression of NGF,BDNF,NT3 and TrkA as well as increased expression of p75NTR.All of these results indicate that long-term sex hormone deficiency by gonadectomy can accelerate brain aging.(2) Heat stress-induced brain Hsp70 expression decreased in ovariectomized mice and the mechanism is related to impaired binding of heat shock factor with heat shock element.It suggests that deficiency of estrogen during aging can exacerbate impairment of endogenous defense system in brain.Estrogen supplement restored heat stress-induced heat shock protein expression and protected against heat stress-induced brain injury in ovariectomized mice.Therefore, the modulatory effect of estrogen on heat shock protein expression provides a new evidence for its neuroprotective effect.(3) FLZ decreased Aβproduction by promotingα- secretase mediated APP non-amyloidogenic processing.Our study provides new evidence for therapeutic potential of FLZ for AD.In addition,in order to develop novel drugs for the treatment of acute liver failure,we investigated the protective effect and mechanism of bicyclol derivative WLP-S-10 against acetaminophen-induced acute liver failure in mice.The results are reported as follow:Acute liver failure is a severe emergency with rapid deterioration of liver function. Overdose of acetaminophen is the most common cause of drug-induced liver failure. Bicyclol is a novel anti-hepatitis drug and has been widely used to treat chronic viral hepatitis and drug-induced liver injury in China.Pharmacological investigation demonstrated that bicyclol has protective action against liver injury in several models, however,bicyclol is not water soluble;it is difficult to make injectable preparation for treatment of acute liver failure patients at present.In this study,a number of water soluble bicyclol derivatives were synthesized and screened in mice using CCl₄, acetaminophen or D-galactosamine plus LPS induced liver injury models.Among ten compounds,a methionine derivative of bicyclol(WLP-S-10) was shown to be the most effective one in lowering serum ALT level and reducing the mortality of mice.WLP-S-10 was,therefore,further studied in acetaminophen induced acute liver failure in mice.A single dose of WLP-S-10 200mg/kg was intraperitoneally injected 1 hour before administration of acetaminophen 450mg/kg.Pretreatment of mice with WLP-S-10 200mg/kg significantly reduced mortality of mice due to acute liver failure caused by lethal dosage of acetaminophen.The liver necrosis,serum ALT and AST elevation,and GSH depletion induced by injection of acetaminophen were all reduced.WLP-S-10 200mg/kg inhibited mitochondrial swelling,breakdown of membrane potential,depletion of mitochondrial ATP,and release of cytochrome C and AIF from mitochondria.In addition,the result of HPLC shows that WLP-S-10 200mg/kg inhibited metabolism of acetaminophen in liver microsomes.WLP-S-10 is converted into bicyclol and methionine. All the results indicate that WLP-S-10 is a novel potential compound against acetaminophen induced acute liver failure in mice,and its active mechanism is related to a summation of inhibiting bioactivation of acetaminophen and increasing liver GSH level, and subsequently attenuating mitochondrial dysfunction and hepatocyte death.The paper has been published in Liver International,2008 Nov;28(9):1226-35.