A GPCR/secretase complex regulates β- and γ-secretase specificity for Aβ production and contributes to AD pathogenesis

Dysregulation of β-site APP-cleaving enzyme （BACE） and/or γ-secretase leads to anomalous production of amyloid-β peptide （Aβ） and contributes to the etiology of Alzheimer＇s disease （AD）. Since these secretases mediate proteolytic processing of numerous proteins, little success has been achieved to treat AD by secretase inhibitors because of inevitable undesired side effects. Thus, it is of importance to unravel the regulatory mechanisms of these secretases. Here, we show that δ-opioid receptor （DOR） promotes the processing of Aβ precursor protein （APP） by BACE1 and γ-secretase, but not that of Notch, N-cadherin or APLP. Further investigation reveals that DOR forms a complex with BACE1 and γ-seeretase, and activation of DOR mediates the co-endocytic sorting of the secretases/ receptor complex for APP endoproteolysis. Dysfunction of the receptor retards the endocytosis of BACE1 and γ-secretase and thus the production of Aβ Consistently, knockdown or antagonization of DOR reduces secretase activities and ameliorates Aβ pathology and Aβ-dependent behavioral deficits, but does not affect the processing of Notch, N-cadherin or APLP in AD model mice. Our study not only uncovers a molecular mechanism for the formation of a DOR/secretase complex that regulates the specificity of secretase for Aβ production but also suggests that intervention of either formation or trafficking of the GPCR/secretase complex could lead to a new strategy against AD, potentially with fewer side effects.

γ-Secretase Inhibitor, DAPT Inhibits Self-renewal and Stemness Maintenance of Ovarian Cancer Stem-like Cells In Vitro

Objective: The Notch signaling pathway plays an important role in the stem cell signaling network and contributes to tumorigenesis. However, the functions of Notch signaling in ovarian cancer stem cells (OCSCs) are not well understood. We aimed to investigate the effects of Notch blockade on self-renewal and stemness maintenance of OCSCs. Methods: Ovarian cancer stem-like cells were enriched from ovarian cancer cell lines in serum-free medium. A γ-secretase inhibitor, (DAPT), was used to block Notch signaling. MTT assays were performed to assess self-renewal and proliferation inhibition, flow cytometry was performed to analyze cell surface marker and immunofluorescence, Western Blot and Real-time RT-PCR assays were performed to detect Oct4 and Sox2 protein and mRNA expression of the Ovarian cancer stem-like cells treated with DAPT. Results: Notch blockade markedly inhibits self-renewal and proliferation of ovarian cancer stem-like cells, significantly downregulates the expression of OCSCs-specific surface markers, and reduces protein and mRNA expression of Oct4 and Sox2 in OCSC-like cells. Conclusion: Our results suggest that Notch signaling is not only critical for the self-renewal and proliferation of OCSCs, but also for the stemness maintenance of OCSCs. The γ-secretase inhibitor is a promising treatment targeting OCSCs.

γ-secretase inhibitor DAPT prevents neuronal death and memory impairment in sepsis associated encephalopathy in septic rats

Background Brain dysfunction is a frequent complication of sepsis,usually defined as sepsis-associated encephalopathy （SAE）.Although the Notch signaling pathway has been proven to be involved in both ischemia and neuronal proliferation,its role in SAE is still unknown.Here,the effect of the Notch signaling pathway involved γ-secretase inhibitor DAPT on SAE in septic rats was investigated in a cecal ligation and puncture （CLP） model.Methods Fifty-nine Sprague-Dawley rats were randomly divided into four groups,with the septic group receiving the CLP operation.Twenty-four hours after CLP or sham treatment,rats were sacrificed and their hippocampus was harvested for Western blot analysis.TNF-αexpression was determined using an enzyme-linked immunosorbent assay （ELISA） kit.Neuronal apoptosis was assessed by TUNEL staining,and neuronal cell death was detected by H＆E staining.Finally,a novel object recognition experiment was used to evaluate memory impairment.Results Our data showed that sepsis can increase the expression of hippocampal Notch receptor intracellular domain （NICD） and poly （adenosine diphosphate [ADP]-ribose） polymerase-1 （PARP-1）,as well as the inflammatory response,neuronal apoptosis,neuronal death,and memory dysfunction in rats.The γ-secretase inhibitor N-[N-（3,5-difluorophenacetyl）-1-alanyl]-S-phenylglycine t-butyl ester （DAPT） can significantly decrease the level of NICD and PARP-1,reduce hippocampal neuronal apoptosis and death,attenuate TNF-α release and rescue cognitive impairment caused by CLP.Conclusion The neuroprotective effect of DAPT on neuronal death and memory impairment in septic rats,which could be a new therapeutic approach for treating SAE in the future.

Huannao Yicong Formula (还脑益聪方) Regulates γ-Secretase Activity through APH-1 and PEN-2 Gene Raguiation Pathways in Hippocampus of APP/PS1 Double Transgenic Mice

Objective To observe the effects of Huannao Yicong Formula (还脑益聪方, HYF) on learning and memory and it’s regulating effect on γ-secretase related anterior pharynx defective 1 (APH-1), presenilin enhancer-2 (PEN-2) signaling pathway, so as to discuss and further clarify the mechanism of HYF on Alzheimer’s disease. Methods Sixty APP/PS1 transgenic mice, randomly allocated into 4 groups, the model group, the donepezil group (0.65 mg/kg), HYF low-dose group (HYF-L, 5.46 g/kg) and HYF high-dose group (HYF-H, 10.92 g/kg), 15 for each group. Another 15 C57BL/6J mice with the same age and same genetic background were allocated into the control group, proper dosage of drugs or distilled water were given by intragastric administration once daily for 12 weeks. After 12 weeks of administration, the learning and memory abilities of mice in each group was evaluated by the morris water maze test, amyloid precursor protein (APP), Aβ1-40 and Aβ1-42 levels in hippocampus were detected by enzyme-linked immunosorbent assay, γ-secretase was detected by dual luciferase assaying, the levels of APH-1a, hypoxia-inducible factor 1α (HIF-1α), cAMP response element-binding protein (CREB) and PEN-2 and their mRNA expression was measured by Western blot and real-time polymerase chain reaction. Results HYF can ameliorate learning and memory deficits in APP/PS1 transgenic mice by decreasing the escape latency, improving the number of platform crossing and swimming speed (P<0.01, P<0.05). HYF can decrease the levels of APP, Aβ1-40, Aβ1-42 and the activity of γ-secretase in hippocampus of Alzheimer’s disease model mice. HYF can down-regulate the levels of CREB and PEN-2 and the expression of their mRNA. Conclusion HYF can improve the learning and memory ability by inhibiting the activity of γ-secretase through the CREB/PEN-2 signaling pathway, and this may be one of the therapeutic mechanisms of HYF in Alzheimer’s disease.

Targeting γ-secretase triggers the selective enrichment of oligomeric APP-CTFs in brain extracellular vesicles from Alzheimer cell and mouse models

Background:We recently demonstrated an endolysosomal accumulation of theβ-secretase-derived APP C-terminal fragment(CTF)C99 in brains of Alzheimer disease(AD)mouse models.Moreover,we showed that the treatment with theγ-secretase inhibitor(D6)led to further increased endolysosomal APP-CTF levels,but also revealed extracellular APP-CTF-associated immunostaining.We here hypothesized that this latter staining could reflect extracellular vesicle(EV)-associated APP-CTFs and aimed to characterize theseγ-secretase inhibitor-induced APPCTFs.Methods:EVs were purified from cell media or mouse brains from vehicle-or D6-treated C99 or APPswedish expressing cells/mice and analyzed for APP-CTFs by immunoblot.Combined pharmacological,immunological and genetic approaches(presenilin invalidation and C99 dimerization mutants(GXXXG))were used to characterize vesicle-containing APP-CTFs.Subcellular APP-CTF localization was determined by immunocytochemistry.Results:Purified EVs from both AD cell or mouse models were enriched in APP-CTFs as compared to EVs from control cells/brains.Surprisingly,EVs from D6-treated cells not only displayed increased C99 and C99-derived C83 levels but also higher molecular weight(HMW)APP-CTF-immunoreactivities that were hardly detectable in whole cell extracts.Accordingly,the intracellular levels of HMW APP-CTFs were amplified by the exosomal inhibitor GW4869.By combined pharmacological,immunological and genetic approaches,we established that these HMW APP-CTFs correspond to oligomeric APP-CTFs composed of C99 and/or C83.Immunocytochemical analysis showed that monomers were localized mainly to the trans-Golgi network,whereas oligomers were confined to endosomes and lysosomes,thus providing an anatomical support for the selective recovery of HMW APP-CTFs in EVs.The D6-induced APP-CTF oligomerization and subcellular mislocalization was indeed due toγ-secretase blockade,since it similarly occurred in presenilin-deficient fibroblasts.Further,our data proposed that besides favoring APP-CTF oligomerization by preventing C99 proteolysis,γ-secretase inhibiton also led to a defective SorLA-mediated retrograde transport of HMW APP-CTFs from endosomal compartments to the TGN.Conclusions:This is the first study to demonstrate the presence of oligomeric APP-CTFs in AD mouse models,the levels of which are selectively enriched in endolysosomal compartments including exosomes and amplified byγ-secretase inhibition.Future studies should evaluate the putative contribution of these exosome-associated APP-CTFs in AD onset,progression and spreading.

Effects of chronic cerebral hypoperfusion of beta- and gamma-secretase on learning and memory in rats

BACKGROUND： Chronic cerebral hypoxia and ischemia have been shown to be related to occurrence of sporadic Alzheimer＇s disease, and β- and y-secretase play an important role in the generation of β-amyloid protein. Early clinical symptoms in Alzheimer＇s disease patients include learning and memory deficits. OBJECTIVE： To measure learning and memory, as well as β- and β-secretase activities in the hippocampus of a cerebral ischemia/hypoxia rat model with chronic cerebral hypoperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Department of Pathology, Capital Medical University from March to December, 2008. MATERIALS： β- and y-secretase activity kits were purchased from R ＆ D Systems, USA. METHODS： Male Sprague Dawiey rats, aged 23 weeks, were randomly assigned to model （n = 56） and sham-surgery （n = 46） groups. Cerebral hypoperfusion rat models were established by bilateral common carotid occlusion. MAIN OUTCOME MEASURES： Morris water maze was used to test changes in escape latency and path length, and β- and y-secretase activities were measured on days 10, 30, 90, and 180 following surgery. RESULTS： Progressive cognitive impairment resulted from 30 days of chronic cerebral hypoperfusion, which lasted for 180 days after cerebral hypoperfusion. β-secretase activity was increased at 10 days after hypoperfusion, which continued until 180 days, with a 14.25% increase compared to the sham-surgery group; y-secretase activity was increased by 10.5%. CONCLUSION： Chronic cerebral hypoperfusion results in impaired spatial memory and upregulated β- and y-secretase activities, which could play an important role in β-amyloid production.

Pharmacological Assessment of <i>γ</i>-Secretase Activity from Rodent and Human Brain

γ-Secretase is involved in the final processing of the amyloid precursor protein into a heterogeneous pool of β-amyloid (Aβ) peptides. Current Alzheimer’s disease drug discovery efforts include targeting γ-secretase activity in brain to attenuate production of the neurotoxic Aβ species. The resulting pharmacology may be affected by species-specific differences in the γ-secretase core complex or its associated proteins. Therefore, we utilized partially purified γ-secretase membranes derived from the brains of different species, including human cortex, to quantitatively assess the de novo production of both Aβ42 and Aβ40 following treatment with known γ-secretase inhibitors and modulators. We determined that the inhibitory activity of a Notch-1 sparing γ-secretase inhibitor and the modulatory activity of two classes of γ-secretase modulators were equipotent at affecting the production of Aβ across rodent and human brain membrane preparations. Additionally, the observed modulator-specific Aβ profile in isolated brain membranes across species was similar to that observed in HeLa cell membranes, and the brain and CSF of guinea pigs following oral administration. By utilizing rapidly purified γ-secretase, we were able to probe and compare the complex pharmacology of γ-secretase in the brain across common rodent species and human cortex.

Synthesis and activity of hydroxyethylene peptidomimetic inhibitors of humanβ-secretase

A series of β-secretase peptidomimetic inhibitors with Leu＊Ala hydroxyethylene dipeptide isostere were synthesized and their β-secretase inhibitory activities were measured. The most potent compound N9 showed an inhibitory rate of 59.66% （10 mg/mL）. Compound N9 might be further modified by means of computational chemical methodology.

Synthesis of Hydroxyethylene-based β-Secretase Inhibitors

Aim To discuss in depth the synthesis of hydroxyethylene dipeptide-based β-secretase inhibitors; Methods Organic reactions such as nucleophilic addition and substitution assisted by organometallic agents, catalytic hydrogenation, and classic peptide coupling were used to synthesize peptidomimetic β-secretase inhibitors. Results Ideal reaction conditions and potential problems were investigated, and one of the designed β-secretase inhibitors 13 （as a model） was synthesized successfully; Conclusion This approach might be used to build up the β-secretase inhibitor library and to search for new molecular candidates.

Influence of Ginkgo Biloba extract on beta-secretase in rat hippocampal neuronal cultures following chronic hypoxic and hypoglycemic conditions

BACKGROUND： Preparation of Ginkgo leaf has been widely used to improve cognitive deficits and dementia, in particular in Alzheirner＇s disease patients. However, the precise mechanism of action of Ginkgo leaf remains unclear. OBJECTIVE： To explore the effect of Ginkgo Biloba extract （Egb761）, Ginaton, on β -secretase expression in rat hippocampal neuronal cultures following chronic hypoxic and hypoglycemic conditions. DESIGN, TIME AND SETTNG： Completely by randomized, grouping study. The experiment was performed at the Laboratory of Molecular Imaging, Southeast University between August 2006 and August 2007. MATERIALS： A total of 128 Wistar rats aged 24 hours were selected, and hippocampal neurons were harvested for primary cultures. METHODS： On day 7, primary hippocampal neuronal cultures were treated with Egb761 （0, 25, 50, 100, 150, and 200μg/mL） under hypoxic/hypoglycemic or hypoglycemic culture conditions for 12, 24, and 36 hours, respectively. Hippocampal neurons cultured in primary culture medium served as control. MAIN OUTCOME MEASURES： Cell viability was assayed using 3-（4,5-Dimethylthiazol-2-yl）- 2,5-diphenyltetrazolium bromide （MTT）; fluorescence detection of β -secretase activity was performed; Western Blot was used to measure β -secretase expression. RESULTS： Cell viability under hypoxic/hypoglycemic or hypoglycemic culture conditions was significantly less than control cells （P 〈 0.05）. Under hypoxic/hypoglycemic or hypoglycemic culture conditions, treatment with 25 μg/mL Egb761 did not alter cell viability. However, 〉 25 μg/mL Egb761 induced greater cell viability （P 〈 0.05）. No differences were observed between hypoxic/hypoglycemic or hypoglycemic cells （P 〉 0.05）. α -secretase activity was increased after 12 hours in hypoxic/hypoglycemic culture （P 〈 0.01）. There were no significant differences between the 12-, 24-, or 36-hour Egb761 groups and the hypoxic/hypoglycemic groups （P 〉 0.05）. β -secretase activity was greater after 12, 24, and 36 hours in hypoxic/hypoglycemic culture conditions, compared with control conditions （P 〈 0.05）. β-secretase activity was significantly decreased in neurons treated with Egb761 for 12, 24, or 36 hours, compared with the hypoxichaypoglycemic group （P 〈 0.05）. β -secretase protein expression was significantly up-regulated in neurons cultured in hypoxic/hypoglycemic conditions for 12, 24, or 36 hours, compared to control cells （P 〈 0.05）, and was decreased compared to neurons treated with Egb761 （P 〈 0.05）. CONCLUSION： β -secretase expression and activity in rat neonatal hippocampal neurons were influenced by hypoxic and hypoglycemic culture. Egb761 played a protective role in hippocampal neurons damaged by chronic hypoxic and hypoglycemic culture conditions, possibly through its effect on β -secretase expression and activity.

Improving cognitive impairment by Tongxinluo via inhibiting expression of beta-secretase 1/beta-amyloid peptide in experimental vascular dementia

BACKGROUND： Tongxinluo has been clinically proven to be effective in improving memory and cognitive function in patients with post-stroke vascular dementia. Is the mechanism related to the deposition of beta-amyloid peptide （A β ） in hippocampus？ OBJECTIVE： To observe the effect of Tongxinluo on cognitive impairment in a mouse model with vascular dementia and the changes of A β deposition and β -secretase 1 （BACE1） expression. DESIGN： Randomized controlled study. SETTING： State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University and Affiliated Drum Tower Hospital of Nanjing University Medical School. MATERIALS： The experiment was carried out in the State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University and Affiliated Drum Tower Hospital of Nanjing University Medical School from March 2006 to January 2007. A total of 36 healthy Kunming mice, 18 of each gender, were chosen. The study was conducted in accordance with the National Regulations of Experimental Animal Administration, and all animal experiments were approved by the Committee of Experimental Animal Administration of Nanjing University. Tongxinluo was provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd. METHODS： All mice were randomly divided into 6 groups, including naive control （n=6）, sham-operated control （n=6） and experimental groups treated with different doses of Tongxinluo （0.2, 0.4, and 0.6 g/kg/d; n=6 for each group） or vehicle （n=6）. Five groups were subjected to bilateral common carotid arteries （2-VO） occlusion to produce a vascular dementia model （no occlusion was performed in sham-operated group）. The mice in the Tongxinluo treatment groups were intragastricly administered daily with a Tongxinluo suspension （40 g/L in distilled water） at doses of 0.2, 0.4 or 0.6 g/kg/d from day 1 to day 30 post-surgery. The animals in vehicle, sham-operated and naive groups were administered an equal volume of distilled water. MAIN OUTCOME MEASURES： ①Escape latency time determined in all groups of mice before and after 2-VO occlusion by Morris water maze. ②Changes in BACEI mRNA expression in the hippocampi of mice among the six groups by RT-PCR assay, and BACEI and A β protein expression in the hippocampi of mice by Western blot. RESULTS： All 36 mice were involved in the final analysis.① No difference was detected in escape latency time to a hidden platform among all groups in water maze test before surgery （P 〉 0.05） At 30 days after 2-VO occlusion, the vehicle animals exhibited a significantly longer latency in finding the hidden platform compared to that of sham-operated and naive animals （P 〈 0.01）. The prolonged escape latency was significantly reduced by oral administration of 0.4 g or 0.6 kg/day （P 〈 0.01, P 〈 0.05）. BACEI mRNA and protein expression in vehicle animals were much higher than in sham-operated and naive animals （P 〈 0.01）. The ischemia-induced increases in BACE1 mRNA and protein level were attenuated by all three doses of Tongxinluo treatment （P 〈 0.01）, and the 0.4 g/kg/d treatment was the most effective. A β protein expression in vehicle animals after 2-VO occlusion were much higher than in sham-operated and naive animals （P 〈 0.01）. 2-VO occlusion-induced A β generation was significantly attenuated by all doses of Tongxinluo treatment, with the most effective dose being 0.4 g/kg/d （P 〈 0.01）. CONCLUSION： BACE1 mRNA levels and protein levels of BACEI and A β are reduced in the hippocampi of vascular dementia model mice by all three doses of Tongxinluo treatment, with the most effective dose being 0.4 g/kg/d. The results suggest that inhibition of post-ischemia BACEI expression and A β generation in brain might underlie Tongxinluo＇s effects in improving cognitive impairment.

Divalent cation tolerance protein binds to β-secretase and inhibits the processing of amyloid precursor protein

The deposition of amyloid-beta is a pathological hallmark of Alzheimer＇s disease, Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase （beta-site amyloid precursor protein-cleaving enzyme 1） and r-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of AIzheimer＇s disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1- interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer＇s disease.

Precise location of proton of beta-secretase for catalytic aspartates(Asp 32 and Asp 228)in Alzheimer’s patients

BACKGROUND：β-secretase （β-site APP cleavage rate-limiting enzyme, BACE） has been proposed as a promising therapeutic target for Alzheimer＇s disease （AD）. BACE inhibition reduces production of β-amyloid peptide （Aβ） and promotes neural regeneration. Two catalytic aspartates （Asp 32 and Asp 228） exist in a monoprotonated state in the active BACE site, but the precise proton location remains unclear.OBJECTIVE：To explore the entire process of BACE enzymatic hydrolysis using quantum chemistry calculations, and to identify the precise proton location for Asp 32 and Asp 228 during the enzymatic process.DESIGN, TIME AND SETTING：According to protonation state of BACE, four tautomers were designed and quantum chemistry calculations were performed at the Department of Human Anatomy, Zhongshan School of Medicine, Sun Yat-sen University, China between October 2008 and March 2009.MATERIALS：Hardware：linux workstation （Department of Equipment, Sun Yat-sen University, China）; software：QSITE, Glide, Maestro （Schrodinger LLC, USA）, MOPAC 2007 （CAChe Research LLC, USA）, Triton 4.0 （National Centre for Biomolecular Research, Czech Republic） were used.METHODS：Using crystal structures of BACE to build a catalytic model （enzyme, catalytic water, and substrate peptide EVNLAAEF） on the computer and superimposition, four BACE tautomers （32i, 320, 228i, and 2280） in the monoprotonated state were developed with Schrodinger package. Hybrid quantum mechanical/molecular mechanic （QM/MM） calculations were performed at the B3LYP density functional theory level to identify the precise proton location for the dyad aspartic residues （Asp 32 and Asp 228）. Using the most possible tautomer as the reactant, the entire enzymatic hydrolysis of substrate EVNL/AAEF was simulated at the semiempirical level.MAIN OUTCOME MEASURES：The precise proton location of was measured by analyzing co-planarities of 4 BACE tautorners （32i, 32o, 228i, and 2280） in the monoprotonated state, because the dihedral formed by the carboxyl oxygen atoms of the dyad aspartic residues. The transition state and the production state, as well as activation energies and reaction enthalpies, were measured by calculating geometric and energy changes during catalytic reaction of the system.RESULTS：In the 2280 BACE tautomer, the dihedral angle of the four oxygen atoms in the catalytic aspartates was 8.7°, which was the lowest of four tautomers. The lowest activation energy and highest reaction enthalpy （Ea = 216.30 kJ/mol, AH = 30.98 kJ/mol） were also found in 2280, among the four tautomers during the catalytic reaction. In addition, when the reaction proceeded to the transition state, followed by product generation, the proton location was reversed to the inner oxygen of Asp 32 （32i） from the outer oxygen of Asp 228 （228o）.CONCLUSION：Results demonstrated the mechanism of Aβ generation. At beginning of BACE catalytic reaction, the precise proton location was preferred on the outer oxygen of Asp 228 （228o）. In this protonation state, catalytic reaction can proceed smoothly, with reduced active energy and heat release. When the reaction proceeded to the transition state and product generation, the proton location was reversed to the inner oxygen of Asp 32 （32i）. These results provide theoretical guidance for designing new drugs to protect neural cells and promote neural regeneration in Alzheimer＇s patients.

Purification of Full-Length <i>β</i>-Secretase Involved in Alzheimer’s Disease, and Proteomic Identification of Binding Partners

β-Secretase (BACE1 or β-site APP cleaving enzyme) is an acid protease that releases the neurotoxic 40 - 42 residue peptides (β-amyloid or A-β) from its glycoprotein precursor, (APP or amyloid precursor protein) which when released in brain is thought to give rise to cognitive decline in patients with Alzheimer’s Disease. Most structural studies on β-secretase have previously been performed with recombinant forms of the protease, in which the transmembrane coding region has been deleted. However, interactions with proteins of the same species are best studied using the full-length β-secretase as interactions are likely to be influenced by the hydrophobic nature and localization of its transmembrane regions. Here we develop a multi-step purification procedure that isolates a complex containing BACE1 from recombinant human cells using mild detergents in a procedure that retains other proteins within the complex and remains active in its β-site APP cleaving activity. Some of these proteins, eg reticulon 4, are identified by proteomics, and are known by previous studies performed by others to regulate the activity of BACE1 against APP. These interactions may aid the development of small proteins and peptides that could inhibit the release of aggregated forms of β-amyloid, and thus be useful therapeutically.

Design, Synthesize and Bio-Evaluate 1,2-Dihydroisoquinolin-3(4H)-One Derivates as Acetylcholinesterase and β-Secretase Dual Inhibitors in Treatment with Alzheimer’s Disease

With the recent research advances in molecular biology and technology, many credible hypothe-ses about the progress of Alzheimer’s disease (AD) have been proposed, among which the amyloid and cholinergic hypotheses are commonly used to develop reliable therapeutic agents. The multitarget-directed ligand (MTDL) approach was taken in this work to develop multi-functional agents, which can mainly serve as dual BACE 1 and AChE inhibitors. Depending on the scaffolds of (+)-(S)- dihydro-ar-tumerone and (-)-gallocatechin gallate, 3 series of new compounds have been designed, synthesized and evaluated, from which we have identified 2-(2-(3-methylbenzoyl)-3-oxo-1,2,3,4- tetrahydroisoquinolin-6-yl) isoindoline-1,3-dione (3d) as a new cholinesterase and β-secretase dual inhibitor without toxicity. Furthermore, 3d also exhibits hydrogen peroxide scavenging activity which could help to reduce the reactive oxygen species (ROS) in the brain of AD patients.

BACE1 inhibitors:A promising therapeutic approach for the management of Alzheimer’s disease

Alzheimer’s disease is a neurological disorder marked by the accumulation of amyloid beta(Aβ)aggregates,resulting from mutations in the amyloid precursor protein.The enzymeβ-secretase,also known asβ-site amyloid precursor protein cleaving enzyme 1(BACE1),plays a crucial role in generating Aβpeptides.With no targeted therapy available for Alzheimer’s disease,inhibiting BACE1 aspartic protease has emerged as a primary treatment target.Since 1999,compounds demonstrating potential binding to the BACE1 receptor have advanced to human trials.Structural optimization of synthetically derived compounds,coupled with computational approaches,has offered valuable insights for developing highly selective leads with drug-like properties.This review highlights pivotal studies on the design and development of BACE1 inhibitors as anti-Alzheimer’s disease agents.It summarizes computational methods employed in facilitating drug discovery for potential BACE1 inhibitors and provides an update on their clinical status,indicating future directions for novel BACE1 inhibitors.The promising clinical results of Elenbecestat(E-2609)catalyze the development of effective,selective BACE1 inhibitors in the future.

Notch1 downregulation combined with interleukin-24 inhibits invasion and migration of hepatocellular carcinoma cells

AIM: To confirm the anti-invasion and anti-migration effects of down-regulation of Notch1 combined with interleukin(IL)-24 in hepatocellular carcinoma(HCC) cells.METHODS: γ-secretase inhibitors(GSIs) were used to down-regulate Notch1.Hep G2 and SMMC7721 cells were seeded in 96-well plates and treated with GSI-I or/and IL-24 for 48 h.Cell viability was measured by MTT assay.The cellular and nuclear morphology was observed under a fluorescence microscope.To further verify the apoptotic phenotype,cell cultures were also analyzed by flow cytometry with Annexin V-FITC/propidium iodide staining.The expression of Notch1,SNAIL1,SNAIL2,E-cadherin,IL-24,XIAP and VEGF was detected by Western blot.The invasion and migration capacities of HCC cells were detected by wound healing assays.Notch1 and Snail were downregulated by RNA interference,and the target proteins were analyzed by Western blot.To investigate the mechanism of apoptosis,we analyzed Hep G2 cells treated with si Notch1 or si CON plus IL-24 or not for 48h by caspase-3/7 activity luminescent assay.RESULTS: GSI-I at a dose of 2.5 μmol/L for 24 h caused a reduction in cell viability of about 38% in Hep G2 cells.The addition of 50 ng/m L IL-24 in combination with 1 or 2.5 μmol/L GSI-I reduced cell viability of about 30% and 15%,respectively.Treatment with IL-24 alone did not induce any cytotoxic effect.In SMMC7721 cells with the addition of IL-24 to GSI-I(2.5 μmol/L),the reduction of cell viability was only about 25%.Following GSI-I/IL-24 combined treatment for 6 h,the apoptotic rate of Hep G2 cells was 47.2%,while no significant effect was observed in cells treated with the compounds employed separately.Decreased expression of Notch1 and its associated proteins SNAIL1 and SNAIL2 was detected in Hep G2 cells.Increased E-cadherin protein expression was noted in the presence of IL-24 and GSI-I.Furthermore,the increased GSI-I and IL-24 in Hep G2 cell was associated with downregulation of MMP-2,XIAP and VEGF.In the absence of treatment,Hep G2 cells could migrate into the scratched space in 24 h.With IL-24 or GSI-I treatment,the wound was still open after 24 h.And the distance of the wound closure strongly correlated with the concentrations of IL-24 and GSI-I.Treatment of Notch-1 silenced Hep G2 cells with 50 ng/m L IL-24 alone for 48 h induced cytotoxic effects very similar to those observed in non-silenced cells treated with GSI-I/IL-24 combination.Caspase-3/7 activity was increased in the presence of si Notch1 plus IL-24 treatment.CONCLUSION: Down-regulation of Notch1 by GSI-I or si RNA combined with IL-24 can sensitize apoptosis and decrease the invasion and migration capabilities of Hep G2 cells.

Amyloid precursor-like protein 2 C-terminal fragments upregulate S100A9 gene and protein expression in BV2 cells

The murine microglial cell line BV2 has neuroprotective effects, but is toxic to neurons by secret-ing inlfammatory cytokines, and is an important target in the treatment of nerve inlfammation and neurodegenerative diseases. In the present study, we observed the effects of transfecting three amyloid precursor-like protein 2 （APLP2） C-terminal fragments （CTFs; C57, C50 and C31） in the pEGFP-N1 vector on S100A9 expression in BV2 cells. Reverse transcription-PCR, western blot assay and immunocytochemistry revealed that S100A9 protein and mRNA expression was greater in BV2 cells after CTF transfection than after mock transfection with an empty vector. Furthermore, transfection of full-length APLP2-751 resulted in low levels of S100A9 protein ex-pression. Our results show that APLP2-CTFs upregulate S100A9 protein and mRNA expression in BV2 cells, and identify a novel pathway involved in neuronal injury and apoptosis, and repair and protection in Alzheimer’s disease.

Presenilin mutations and their impact on neuronal differentiation in Alzheimer’s disease

The presenilin genes(PSEN1 and PSEN2)are mainly responsible for causing early-onset familial Alzheimer’s disease,harboring~300 causative mutations,and representing~90%of all mutations associated with a very aggressive disease form.Presenilin 1 is the catalytic core of theγ-secretase complex that conducts the intramembranous proteolytic excision of multiple transmembrane proteins like the amyloid precursor protein,Notch-1,N-and E-cadherin,LRP,Syndecan,Delta,Jagged,CD44,ErbB4,and Nectin1a.Presenilin 1 plays an essential role in neural progenitor maintenance,neurogenesis,neurite outgrowth,synaptic function,neuronal function,myelination,and plasticity.Therefore,an imbalance caused by mutations in presenilin 1/γ-secretase might cause aberrant signaling,synaptic dysfunction,memory impairment,and increased Aβ42/Aβ40 ratio,contributing to neurodegeneration during the initial stages of Alzheimer’s disease pathogenesis.This review focuses on the neuronal differentiation dysregulation mediated by PSEN1 mutations in Alzheimer’s disease.Furthermore,we emphasize the importance of Alzheimer’s disease-induced pluripotent stem cells models in analyzing PSEN1 mutations implication over the early stages of the Alzheimer’s disease pathogenesis throughout neuronal differentiation impairment.

Sex effect on presenilins expression in post-natal rat brain

Presenilin 1 and presenilin 2 are widely expressed during brain development. Several mutations in these proteins have been associated with autosomal-dominant inherited forms of Alzheimer disease. Their expression is regulated by various cellular and extracellular factors, which change with age and sex. Both age and sex are key risk factors for Alzheimer’s disease, but the issue of whether the expression of presenilins is influenced by the sex during early postnatal development of the brain has been poorly investigated so far. In this study, we report that transcript levels of presenilins, and the subset of neurons expressing these proteins in various brain areas of the developing post-natal brain are different in male and female rats, suggesting that their function(s) may contribute to sexual dimorphism in the brain, both at morphological and functional levels.