Effects of Honeysuckle Chlorogenic Acid on Secretory Enzymes,Lipoxygenase A4,and Biochemical Indicators inModel Mice with Aluminum Induced Alzheimer's Disease

[Objectives]To explore the effects of chlorogenic acid from honeysuckle on the secretion enzymes,lipoxygenase A4(LXA4),and blood biochemical indicators in mice with aluminum induced Alzheimer's disease(AD).[Methods]Chlorogenic acid was extracted from hon-eysuckle by ultrasound assisted alcohol extraction method.Seventy mice were randomly divided into normal group,model group,and low,me-dium and high dose groups of honeysuckle chlorogenic acid.All the mice in each group except for the normal group were given maltol aluminum by intraperitoneal injection to establish models of aluminum induced AD,continuously injected for 5 d and stopped for 2 d,totally poisoned for 8 weeks.Starting from the 5^(th) week of poisoning,the low,medium and high dose groups of honeysuckle chlorogenic acid were given honeysuck-le chlorogenc acid solution 40,80 and 160 mg/kg by gavage,respectively,while the normal group and the model group were fed with an equal volume of distilled water,all once daily,continuously gavaged until the end of the 8^(th) week.At the end of the experiment,the learning memory ability of the mice was tested by Y-type waler maze,and the number of tests required to reach the learning standard,the number of memory er-rors in 20 tests and the error rate of the mice were recorded.The brains of mice were taken to determine the contents of β-secretase,α-secre-tase,γ-secretase,LXA4 and acetylcholinesterase(AchE)in the homogenates of brain tissues by ELISA,and their blood was taken to deter-mine the biochemical indexes.[Results]Compared with the normal group,the number of learning tests,number of memory errors,error rate and the contents of β-secretase,γ-secretase and AchE in brain tissue of the mice in the model group were all significantly increased(all P<0.05),the contents of LXA4 in brain tissue were significantly decreased(all P<0.05),and the contents of α-secretase did not change significantly(all P>0.05);compared with the model group,the number of learning tests,the number of memory errors,the error rate and the content of β-secretase,γ-secretase and AchE in brain tissue were all significantly reduced(all P<0.05),the content of LXA4 in brain tissue of the high dose group of honeysuckle chlorogenic acid was significantly increased(P<0.05),and there was no significant change in the content of α-secretase in brain tissue of all groups of honeysuckle chlorogenic acid(all P>0.05).Compared with the normal group,the levels of blood glucose,TC,TG,ALT,BUN,Cr and UA in the model group and the levels of TC,TG and BUN in the low-and medium-dose groups of honeysuckle chlorogenic acid were significantly increased(all P<0.05),and the level of HDL-C in the model group and the levels of UA in the medium-and high-dose groups of honeysuckle chlorogenic acid were significantly decreased(all P<0.05);compared with the model group,the levels of blood glucose,ALT,BUN,UA in each group of honeysuckle chlorogenic acid,the levels of TC and Cr in medium and high dose groups of honeysuckle chlorogenic acid,and the level of TG in the high dose group of honeysuckle chlorogenic acid were all signifi-cantly lower(all P<0.05),while the level of HDL-C in the medium and high dose groups of honeysuckle chlorogenic acid and the level of to-tal protein in the high dose group of honeysuckle chlorogenic acid were all significantly higher(all P<0.05).[Conclusions]Chlorogenic acid from honeysuckle may improve AD induced by aluminum exposure via regulating related secretory enzymes,LXA4,and various biochemi-cal indicators.

Impaired autophagy in amyloid-beta pathology:A traditional review of recent Alzheimer's research

Alzheimer’s disease(AD) is the most prevalent neurodegenerative disorder. The major pathological changes in AD progression are the generation and accumulation of amyloid-beta(Aβ) peptides as well as the presence of abnormally hyperphosphorylated tau proteins in the brain. Autophagy is a conserved degradation pathway that eliminates abnormal protein aggregates and damaged organelles. Previous studies have suggested that autophagy plays a key role in the production and clearance of Aβ peptides to maintain a steady-state of Aβ peptides levels.However, a growing body of evidence suggests that autophagy is significantly impaired in the pathogenesis of AD, especially in Aβ metabolism. Therefore, this article reviews the latest studies concerning the mechanisms of autophagy, the metabolism of Aβ peptides, and the defective autophagy in the production and clearance of Aβpeptides. Here, we also summarize the established and new strategies for targeting autophagy in vivo and through clinical AD trials to identify gaps in our knowledge and to generate further questions.

Notoginseng Saponin Rg1 Prevents Cognitive Impairment through Modulating APP Processing in Aβ1-42-injected Rats

With the intensification of the aging process of the world,Alzheimer's disease(AD),which is the main type of senile dementia,has become a primary problem in the present society.Lots of strategies have been used to prevent and treat AD in animal nlodels and clinical trials,but most of them ended in failure.Panax notoginseng saponins(PNS)contain a variety of monomer compositions which have been separated and identified.Among of the monomer compositions,notoginseng saponin Rg1(Rg1)accounts for 20%of the cultivation of panax notoginseng roots.And now PNS have been reported to be widely used to treat cardicerebrovascular diseases and have neuroprotective effects to restrain theβ-amyloid peptide(Aβ)25-35-niediated apoptosis.Moreover,it is reported that PNS could accelerate the growth of nerve cells,increase the length of axons and promote synaptic plasticity.Whether Rg1 can ameliorate the cognitive impairment and the underlying mechanism has not been elucidated.To study the preventive effect of Rg1 on cognitive impairment and the possible mechanism,we established the cognitive impairment model in rats through Aβ1-42(2.6μg/μL,5μL)injection and then treated the rats with Rg1(25,50 and 100 mg/kg)administered intragastrically for 4 weeks.We observed that Aβ1-42 could induce spatial learning and memory deficits in rats.Simultaneously,Aβ1-42 injection also resulted in the reduced neuron number in comuammonis 1(CA1)and dentate gyrus(DG)of hippocampus,as well as the increased level of hyperphosphorylatedβ-amyloid precursor protein(APP)at Thr668 site with up-regulation ofβ-APP cleaving enzyme 1(BACE1)and presenilin 1(PS1)and down-regulation of a disintegrin and metalloprotease domain-containing protein 10(ADAM 10)and insulindegrading enzyme(IDE).Administration of Rg1 effectively rescued the cognitive impairment and neuronal loss,and inhibited theβ-secretase processing of APP through reducing APP-Thr668 phosphorylation and BACE1/PS1 expression,and increasing the expression of ADAM 10 and IDE.We concluded that Rg1 might have neuroprotective effects and could promote learning and memory ability,which might be a viable candidate in AD therapy probably through reducing the generation of Aβand increasing the degradation of Aβ.

Notch pathway inhibitor DAPT enhances Atoh1 activity to generate new hair cells in situ in rat cochleae

Atoh1 overexpression in cochlear epithelium induces new hair cell formation. Use of adenovirus-mediated Atoh1 overexpression has mainly focused on the rat lesser epithelial ridge and induces ectopic hair cell regeneration. The sensory region of rat cochlea is difficult to transfect, thus new hair cells are rarely produced in situ in rat cochlear explants. After culturing rat cochleae in medium containing 10% fetal bovine serum, adenovirus successfully infected the sensory region as the width of the supporting cell area was significantly increased. Adenovirus encoding Atoh1 infected the sensory region and induced hair cell formation in situ. Combined application of the Notch inhibitor DAPT and Atoh1 increased the Atoh1 expression level and decreased hes1 and hes5 levels, further promoting hair cell generation. Our results demonstrate that DAPT enhances Atoh1 activity to promote hair cell regeneration in rat cochlear sensory epithelium in vitro.

Notch signalling pathway in development of cholangiocarcinoma

Cholangiocarcinoma(CCA)comprises of extra-hepatic cholangiocarcinoma and intrahepatic cholangiocarcinoma cancers as a result of inflammation of epithelium cell lining of the bile duct.The incidence rate is increasing dramatically worldwide with highest rates in Eastern and South Asian regions.Major risk factors involve chronic damage and inflammation of bile duct epithelium from primary sclerosing cholangitis,chronic hepatitis virus infection,gallstones and liver fluke infection.Various genetic variants have also been identified and as CCA develops on the background of biliary inflammation,diverse range of molecular mechanisms are involved in its progression.Among these,the Notch signalling pathway acts as a major driver of cholangiocarcinogenesis and its components(receptors,ligands and downstream signalling molecules)represent a promising therapeutic targets.Gamma-Secretase Inhibitors have been recognized in inhibiting the Notch pathway efficiently.A comprehensive knowledge of the molecular pathways activated by the Notch signalling cascade as well as its functional crosstalk with other signalling pathways provide better approach in developing innovative therapies against CCA.

Targeting β-secretase with RNAi in neural stem cells for Alzheimer's disease therapy

There are several major pathological changes in Alzheimer＇s disease, including apoptosis of cho- linergic neurons, overactivity or overexpression of 13-site amyloid precursor protein cleaving enzyme 1 （BACE1） and inflammation. In this study, we synthesized a 19-nt oligonucleotide targeting BACE1, the key enzyme in amyloid beta protein （AI3） production, and introduced it into the pSilenCircle vector to construct a short hairpin （shRNA） expression plasmid against the BACE1 gene. We transfected this vector into C17.2 neural stem cells and primary neural stem cells, resulting in downregulation of the BACE1 gene, which in turn induced a considerable reduction in reducing AI3 protein production. We anticipate that this technique combining cell transplantation and gene ther- apy will open up novel therapeutic avenues for Alzheimer＇s disease, particularly because it can be used to simultaneously target several pathogenetic changes in the disease.

Platelets and Alzheimer's disease:Potential of APP as a biomarker

Platelets are the first peripheral source of amyloid precursor protein(APP). They possess the proteolytic machinery to produce Aβ and fragments similar to those produced in neurons, and thus offer an ex-vivo model to study APP processing and changes associated with Alzheimer's disease(AD). Platelet process APP mostly through the α-secretase pathway to release soluble APP(s APP). They produce small amounts of Aβ, predominantly Aβ40 over Aβ42. s APP and Aβ are stored inα-granules and are released upon platelet activation by thrombin and collagen, and agents inducing platelet degranulation. A small proportion of full-length APP is present at the platelet surface and this increases by 3-fold upon platelet activation. Immunoblotting of platelet lysates detects APP as isoforms of 130 kD a and106-110 kD a. The ratio of these of APP isoforms is significantly lower in patients with AD and mild cognitive impairment(MCI) than in healthy controls. This ratio follows a decrease that parallels cognitive decline andcan predict conversion from MCI to AD. Alterations in the levels of α-secretase ADAM10 and in the enzymatic activities of α- and β-secretase observed in platelets of patients with AD are consistent with increased processing through the amyloidogenic pathway. β-APP cleaving enzyme activity is increased by 24% in platelet membranes of patients with MCI and by 17% in those with AD. Reports of changes in platelet APP expression with MCI and AD have been promising so far and merit further investigation as the search for blood biomarkers in AD, in particular at the prodromal stage, remains a priority and a challenge.

Advances in the study of Alzheimer's disease

Alzheimer＇ s disease （AD） is the most common cause of dementia, and the only treatment currently available for the disease is acetylcholinesterase inhibitors. Recent progress in understanding the molecular and cellular pathophysiology of Alzheimer＇s disease has suggested possible pharmacological interventions, including acetylcholineseterase inhibitors; secretase inhibitors; cholesterol lowering drugs; metal chelators and amyloid immunization. The objective of this paper is to review the main drugs possibly used for AD and their future therapeutic effects.

Regulation of β cleavage of amyloid precursor protein

Alzheimer’s disease ranks the first cause for senile dementia.The amyloid cascade is proposed to contribute to the pathogenesis of this disease.In this cascade,amyloid β peptide（Aβ）is produced through a sequential cleavage of amyloid precursor protein（APP）by β and γ secretases,while its cleavage by α secretase precludes Aβ production and generates neurotrophic sAPPα.Thus,enhancing α secretase activity or suppressing β and γ cleavage may reduce Aβ formation and ameliorate the pathological process of the disease.Several regulatory mechanisms of APP cleavage have been established. The present review mainly summarizes the signaling pathways pertinent to the regulation of APP β cleavage.

Stepping closer to treating Alzheimer’s disease patients with BACE1 inhibitor drugs

Alzheimer’s disease(AD)is the most common age-dependent neurodegenerative disease which impairs cognitive function and gradually causes patients to be unable to lead normal daily lives.While the etiology of AD remains an enigma,excessive accumulation ofβ-amyloid peptide(Aβ)is widely believed to induce pathological changes and cause dementia in brains of AD patients.BACE1 was discovered to initiate the cleavage of amyloid precursor protein(APP)at theβ-secretase site.Only after this cleavage doesγ-secretase further cleave the BACE1-cleaved C-terminal APP fragment to release Aβ.Hence,blocking BACE1 proteolytic activity will suppress Aβgeneration.Due to the linkage of Aβto the potential cause of AD,extensive discovery and development efforts have been directed towards potent BACE1 inhibitors for AD therapy.With the recent breakthrough in developing brain-penetrable BACE1 inhibitors,targeting amyloid deposition-mediated pathology for AD therapy has now become more practical.This review will summarize various strategies that have successfully led to the discovery of BACE1 drugs,such as MK8931,AZD-3293,JNJ-54861911,E2609 and CNP520.These drugs are currently in clinical trials and their updated states will be discussed.With the promise of reducing Aβgeneration and deposition with no alarming safety concerns,the amyloid cascade hypothesis in AD therapy may finally become validated.

α-secretase ADAM IO physically interacts with β-secretase BACE1 in neurons and regulates CHL1 proteolysis

α-secretase and β-secretase are known to compete for amyloid precursor protein （APP） processing and thus play a vital role in Alzheimer＇s disease pathogenesis. A disintegrin and metaUoproteinase 10 （ADAM10） and β-site APP cleaving enzyme 1 （BACE1） mediate the major activities of α-secretase and β-secretase in brain and share various common substrates. However, whether they function separately or together is poorly understood. Here, we show that ADAM10 and BACE1 co-localize in the neurites of mouse primary neurons. Co-immunoprecipitation and fluorescence resonance energy transfer analysis revealed that ADAM10 and BACE1 interact with each other under both endogenous and exogenous conditions. In addition, we found that ADAMIO enhances the proteolysis of neural cell adhesion molecule close homolog of L1 （CHL1） by BACE1. Further studies found that ADAM10-BACE1 interaction interfering peptide LT52 attenuates the regulation of ADAM10 on BACEl-mediated cleavage of CHL1. Our data indicate that ADAM10-BACE1 interaction regulates the proteolysis of some specific substrates and may play a potential role in brain function.

Biochemical and behavioral characterization of the double transgenic mouse model (APPswe/PS1dE9) of Alzheimer’s disease

Objective The double transgenic mouse model （APPswe/PSldE9） of Alzheimer＇s disease （AD） has been widely used in experimental studies. β-Amyloid （Aβ） peptide is excessively produced in AD mouse brain, which affects synaptic function and the development of central nervous system. However, little has been reported on characterization of this model. The present study aimed to characterize this mouse AD model and its wild-type counterparts by biochemical and functional approaches. Methods Blood samples were collected from the transgenic and the wild-type mice, and radial arm water maze behavioral test was conducted at the ages of 6 and 12 months. The mice were sacrificed at 12-month age. One hemisphere of the brain was frozen-sectioned for immunohistochemistry and the other hemisphere was dissected into 7 regions. The levels ofAβ1-40, Aβ1-42 and 8-hydroxydeoxyguanosine （8-OHdG） in blood or/and brain samples were analyzed by ELISA. Secretase activities in brain regions were analyzed by in vitro assays. Results The pre-mature death rate of transgenic mice was approximately 35% before 6-month age, and high levels of Aβ1-40 and Aβ1-42 were detected in these dead mice brains with a ratio of 1 ： 1 0. The level of blood-borne Aβ at 6-month age was similar with that at 12-month age. Besides, Aβ1-40 level in the blood was significantly higher than Aβ1-42 level at the ages of 6 and 12 months （ratio 2.37：1）. In contrast, the level of Aβ1-42 in the brain （160.6 ng/mg protein） was higher than that of Aβ1-40 （74 ng/mg protein） （ratio 2.17：1）. In addition, the levels of Aβ1-40 and Aβ1-42 varied markedly among different brain regions. Aβ1-42 level was significantly higher than Aβ1-40 level in cerebellum, frontal and posterior cortex, and hippocampus. Secretase activity assays did not reveal major differences among different brain regions or between wild-type and transgenic mice, suggesting that the transgene PS1 did not lead to higher 7-secretase activity but was more efficient in producing Aβ1-42 peptides. 8-OHdG, the biomarker of DNA oxidative damage, showed a trend of increase in the blood of transgenic mice, but with no significant difference, as compared with the wild-type mice. Behavioral tests showed that transgenic mice had significant memory deficits at 6-month age compared to wild-type controls, and the deficits were exacerbated at 12-month age with more errors. Conclusion These results suggest that this mouse model mimics the early-onset human AD and may represent full-blown disease at as early as 6-month age for experimental studies.

Novel aspects of the apolipoprotein-E receptor family： regulation and functional role of their proteolytic processing

Studies related to the functional and regulatory aspects of proteolytic processing are of interest to cell biologists, developmental biologists and investigators who work on human diseases. Much of what is known about this topic derives from the study of the proteolytic processing of the amyloid precursor protein （APP）, which is involved in the pathology of Alzheimer＇s disease, and of the Notch protein and its Delta ligand, which play roles during embryonic development and in biologic processes in the adult. The proteolytic processing of plasma membrane receptor proteins is under the control of different enzymes that are responsible for releasing the ectodomain into the extracellular environment, where it has the potential to function as a signaling molecule and/or regulate the availability of the receptor＇s ligand. Following shedding of the ectodomain, the y-secretase enzymatic complex cleaves the transmembrane domain and releases the cytoplasmic domain （ICD） of the receptor. The ICD can function in the cytoplasm and/or at the nucleus. Members of the low-density lipoprotein receptor （LDLR） family are cndocytic-signaling proteins that perform a wide variety of physiologic functions during development and in the adult life. In addition these receptors have been implicated in a variety of diseases in adults. The prototypic receptor for this family of proteins is the LDLR itself. Besides their binding to apolipoproteins, these receptors bind many ligands that are destined for internalization and degradation. Some ligands have signaling properties. The proteolytic processing of certain members of the LDLR family not only controls receptor availability at the cell surface but also has functional consequences that amplify the spectrum of roles that these receptors perform. In addition, many complex regulatory mechanisms control the proteolytie processing of these receptors.

Role of BACE1 in Alzheimer’s synaptic function

Alzheimer’s disease(AD)is the most common age-dependent disease of dementia,and there is currently no cure available.This hallmark pathologies of AD are the presence of amyloid plaques and neurofibrillary tangles.Although the exact etiology of AD remains a mystery,studies over the past 30 have shown that abnormal generation or accumulation of β-amyloid peptides(Aβ)is likely to be a predominant early event in AD pathological development.Aβ is generated from amyloid precursor protein(APP)via proteolytic cleavage by β-site APP cleaving enzyme 1(BACE1).Chemical inhibition of BACE1 has been shown to reduce Aβ in animal studies and in human trials.While BACE1 inhibitors are currently being tested in clinical trials to treat AD patients,it is highly important to understand whether BACE1 inhibition will significantly impact cognitive functions in AD patients.This review summarizes the recent studies on BACE1 synaptic functions.This knowledge will help to guide the proper use of BACE1 inhibitors in AD therapy.

GPCR, a rider of Alzheimer’s disease

Alzheimer’s disease(AD)is the most common type of dementia that affects thinking,learning,memory and behavior of older people.Based on the previous studies,three pathogenic pathways are now commonly accepted as the culprits of this disease namely,amyloid-β pathway,tauopathology and cholinergic dysfunction.This review focuses on the current findings on the regulatory roles of G protein-coupled receptors(GPCRs)in the pathological progression of AD and discusses the potential of the GPCRs as novel therapeutic targets for AD.

Effects of lipids on the activity and structural dynamics of gamma secretase: A study using coarse-grained molecular dynamics simulations

Gamma secretase(GS)is an intramembranous enzyme that acts on the amyloid precursor protein and Notch inside lipid membranes.The enzyme is responsible for amyloid-β propagation,one of the well-known causes of Alzheimer’s disease.However,the effects of lipids on GS activity and structural dynamics are unknown.Therefore,in this study,we performed coarse-grained molecular dynamics simulations to probe the effects of five individual lipids on GS.These lipids included 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine(POPE),1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine(POPC),1,2-dipalmitoyl-sn-phosphatidylcholine(DOPC),2-dimyristoyl-snglycero-3-phosphocholine(DMPC),and 1,2-dilauroyl-sn-glycero-3-phosphocholine(DLPC).These lipids are structurally characterized by different heads(i.e.,NH_(3)[PE]for POPE vs.NC_(3)[PC]for POPC),number of double bonds(one for POPC vs.two for DOPC),and alkyl tail chain lengths(16:1/18:1 for DOPC vs.14:0/14:0 for DMPC vs.12:0/12:0 for DLPC).This indicates distinct microenvironments and adjustable structural elements for catalytic function when GS is embedded.Our results revealed that the presence of more unsaturated bonds in DOPC than in POPC resulted in greater GS stability.Moreover,lipids with short alkyl tail chains or with PC heads instead of PE heads had improved mobility of the sixth transmembrane helix of GS,which is responsible for the considerable active site flexibility and presenilin 1 subunit plasticity.The length of the DMPC alkyl tail chain was between that of DOPC and DLPC because the up-down and cross-correlation motions of GS in DMPC was the lowest among the three lipids,and GS mobility in DMPC was the lowest among all five lipids.This may be because the alkyl tail chain length(i.e.,3.8 nm thickness of the DMPC bilayer)was suitable for GS embedding,thereby restraining more GS motions than that of the long(DOPC)or short lipids(DLPC).Collectively,these results indicated that GS activity can be modulated through changes in conformational fluctuations,structural perturbations,molecular motion,and cross-correlation motion when embedded in different lipids.Exploration of such fundamental information can reveal the possible mechanisms by which GS is affected by individual lipid species.