RS‐4252 Inhibits Amyloid β‐Induced Cytotoxicity in HeLa Cells

Abstract: Progressive deposition of amyloid β peptide in the senile plaques is a principal event in the neurodegenerative process of Alzheimer's disease. Several reports have demonstrated that amyloid β is cytotoxic using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) as an indicator of viability in cells. With the MTT assay, we screened an in‐house library to find compounds which suppress amyloid β‐induced inhibition of MTT reduction. We have previously reported that 6‐ethyl‐N,N'‐bis(3‐hydroxyphenyl)[1,3,5]triazine‐2,4‐diamine (named RS‐0466), found in an in‐house library, was capable of significantly inhibiting amyloid β‐induced cytotoxicity in HeLa cells. From further screening hits, we newly focused on 4‐(7‐hydroxy‐2,2,4‐trimethyl‐chroman‐4‐yl)benzene‐1,3‐diol (named RS‐4252), which show comparable potency to RS‐0466 to ameliorate amyloid β‐induced cytotoxicity. Furthermore, RS‐4252 reversed the decrease in phosphorylated Akt by amyloid β. These results imply that RS‐4252 or one of its derivatives has the potential to be a therapeutic for Alzheimer's disease patients, and that activation of Akt is at least in part involved in the effect.     

6-Ethyl-N,N'-bis(3-hydroxyphenyl)[1,3,5]triazine-2,4-diamine (RS-0466) enhances the protective effect of brain-derived neurotrophic factor on amyloid beta-induced cytotoxicity in cortical neurones

Amyloid beta peptide in the senile plaques of patients with Alzheimer's disease is considered to be responsible for the pathology of Alzheimer's disease. We have previously reported that 6-ethyl-N,N'-bis(3-hydroxyphenyl)[1,3,5]triazine-2,4-diamine, RS-0466, is capable of significantly inhibiting amyloid beta-induced cytotoxicity in HeLa cells. To determine various profiles of RS-0466, we investigated whether RS-0466 would enhance the neuroprotective effect of brain-derived neurotrophic factor on amyloid beta(1-42)-induced cytotoxicity in rat cortical neurones. Consistent with previous observations, brain-derived neurotrophic factor ameliorated amyloid beta(1-42)-induced cytotoxicity. Furthermore, co-application of RS-0466 enhanced the neuroprotective effect of brain-derived neurotrophic factor. RS-0466 also reversed amyloid beta(1-42)-induced decrease of brain-derived neurotrophic factor-triggered phosphorylated Akt. These results raise the possibility that RS-0466 or one of its derivatives has potential to enhance the neuroprotective effect of brain-derived neurotrophic factor, and could serve as a therapeutic agent for patients with Alzheimer's disease.     

Inhibition of β‐amyloid toxicity by short peptides containing N‐methyl amino acids

Abstract: Single N‐methyl amino acid‐containing peptides related to the central hydrophobic region β_16–20 (Lys‐Leu‐Val‐Phe‐Phe) of the β‐amyloid protein are able to reduce the cytotoxicity of natural β_1–42 in PC12 cell cultures. N‐methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t‐test < 0.01%) and are nontoxic in the same assay. These promising results indicate that these peptide molecules could be a starting point for the development of potential therapeutic compounds for the treatment of Alzheimer's disease.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.     

Key mechanisms underlying netrin‐1 prevention of impaired spatial and object memory in Aβ1‐42 CA1‐injected rats

Alzheimer's disease (AD) is a neurodegenerative disorder with an incompletely defined aetiology that is associated with memory and cognitive impairment. Currently available therapeutics only provide temporary assistance with symptoms. In spite of plentiful research in the field and the generation of thousands of studies, much is still to be clarified on precise mechanisms of pathobiology, prevention modalities, disease course and cure. Netrin‐1, a laminin family protein, is said to have anti‐inflammatory and anti‐apoptotic effects and has a key role in neurogenesis and morphogenesis of neural structures. Accordingly, this study was designed to investigate protective effects of bilateral intrahippocampal fissure microinjections of netrin‐1 on memory impairment in rat model of AD. Concomitant administration of netrin‐1 with amyloid beta 1‐42 (Aβ_1‐42) improved cognitive dysfunction in novel object recognition task (NOR), ameliorated impaired spatial memory in Morris water maze (MWM) setting, increased neuronal density and reduced amyloid aggregation in rat AD model. Netrin‐1 was also seen to prevent Aβ_1‐42‐induced caspase‐3, caspase‐7 and NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells) activation. Therefore, based on the data reported here, netrin‐1 may be a promising biologic therapeutic that addresses the memory and neuronal loss associated with AD.     

Thiophilic interaction chromatography of Alzheimer's β‐amyloid peptides

Abstract: Alzheimer's disease is characterized by a progressive formation of senile plaques in the brain, the major constituent of which is β‐amyloid (Aβ) peptide, a proteolytic product of the transmembrane β‐amyloid precursor protein (APP). Prior to the measurement of levels of the Aβ peptide for diagnostic purposes, this peptide must be isolated from the myriad of proteins resident in the human serum. Thiophilic interaction chromatography is an effective method for the isolation of proteins and peptides containing clusters of aromatic residues such as tryptophan, phenylalanine and tyrosine. The purpose of the present study was to develop a protocol for binding and recovery of Aβ peptides (1–38), (1–40) and (1–42) to T‐gels by varying T‐gel type and elution conditions such as the salt concentration and type of eluent. We established the minimal salt concentration necessary for the binding of the Aβ(1–40) peptide to the 3S‐gel; binding at that concentration was subsequently compared with that of model proteins, lysozyme and α‐chymotrypsin and this methodology was extended to 2S‐gels and PyS. β‐Amyloid peptide (1–40) showed a remarkably strong affinity for all three types of T‐gels in comparison to lysozyme and α‐chymotrypsin and was found to bind best to 2S‐gel.     

Anti‐Alzheimer's multitarget‐directed ligands with serotonin 5‐HT6 antagonist,butyrylcholinesterase inhibitory,and antioxidant activity

Serotonin 5‐HT₆ receptors, butyrylcholinesterase (BuChE) and oxidative stress are related to the pathophysiology of Alzheimer's disease. Inhibition of BuChE provides symptomatic treatment of the disease and the same effect was demonstrated for 5‐HT ₆ antagonists in clinical trials. Oxidative stress is regarded as a major and primary factor contributing to the development of Alzheimer's disease; therefore, antioxidant agents may provide a disease‐modifying effect. Combining BuChE inhibition, 5‐HT ₆ antagonism, and antioxidant properties may result in multitarget‐directed ligands providing cognition‐enhancing properties with neuroprotective activity. On the basis of the screening of the library of 5‐HT ₆ antagonists against BuChE, we selected two compounds and designed their structural modifications that could lead to improved BuChE inhibitory activity. We synthesized two series of compounds and tested their affinity and functional activity at 5‐HT ₆ receptors, BuChE inhibitory activity and antioxidant properties. Compound 12 with K _i and K _b values against 5‐HT ₆ receptors of 41.8 and 74 nM, respectively, an IC ₅₀ value of 5 µM against BuChE and antioxidant properties exceeding the activity of ascorbic acid is a promising lead structure for further development of anti‐Alzheimer's agents.     

Effects of N‐Methylated Amyloid‐β30–40 Peptides on the Fibrillation of Amyloid‐β1–40

Alzheimer's disease is a neurodegenerative disorder associated with amyloid‐β (Aβ) fibrillation. N‐Methylated amyloid‐β peptides are potent inhibitors of amyloid‐β fibrillation. We investigated the inhibitory effect of N‐Methylated Aβ_30–40 peptides on Aβ_1–40 fibrillation. N‐Methylated Aβ_30–40 peptides affected the fibrillation, and this effect was dependent on the concentration of N‐Methylated peptide and the number and position of N‐Methylated groups. N‐Methylated Aβ_30–40 peptides were co‐aggregated with Aβ_1–40. Spectroscopic technique was adopted to investigate an origin of the observed dependence. Suppression of thioflavin T (ThT) fluorescence count was correlated with the dissociation constant K_d of monomer–dimer equilibrium of each N‐Methylated Aβ_30–40 peptide. Monomeric N‐Methylated peptides decreased ThT fluorescence count during Aβ_1–40 fibrillation. Secondary structure content was not largely different between Aβ_1–40 fibrils and co‐aggregates. These results suggested that N‐Methylated Aβ_30–40 peptides disrupted the regular β‐sheet structure of Aβ_1–40 fibrils and affected the ThT fluorescence count. The monomer–dimer equilibrium of N‐Methylated peptides was (partly) responsible for the observed dependence of their inhibitory effect on the concentration of N‐Methylated peptide and the number and position of N‐Methylated groups. Our study provides a hint to design new N‐Methylated inhibitor peptides of fibrillation.     

Ginsenoside Rg3 promotes beta‐amyloid peptide degradation by enhancing gene expression of neprilysin

Objectives It has been hypothesized that the accumulation of beta‐amyloid peptide (Aβ) in the brain is a triggering event leading to the pathological cascade of Alzheimer's disease. The steady‐state levels of Aβ are determined by the metabolic balance between anabolic and catabolic activity and the dysregulation of this activity leads to Alzheimer's disease. Recent evidence has shown that neprilysin (NEP) is the rate‐limiting enzyme in the Aβ degradation in the brain. Ginseng, the root of Panax ginseng C.A. Meyer, is widely used as a tonic for the prevention and treatment of age‐related disorders in China. We aimed to investigate the basis of this use. Methods In this study, we investigated the effect of ginsenoside Rg3, one of the major active components of ginseng, on the metabolism of Aβ40 and Aβ42 in SK‐N‐SH cells transfected with Swedish mutant β‐amyloid precursor protein (SweAPP). Results The ELISA result showed that Rg3 significantly reduced the levels of Aβ40 and Aβ42, 19.65 ± 6.05%, 23.61 ± 6.74%, respectively (P < 0.01). The Western blot analysis showed that Rg3 reduced the levels of Aβ40 and Aβ42 through enhancing NEP gene expression, and real‐time PCR assay showed that 50 μM Rg3 could significantly enhance NEP gene expression (2.9 fold at 48 h). Conclusions Our findings suggest that the Rg3 compound of ginseng may be useful for treating patients suffering with Alzheimer's disease.     

Synthesis,Biological Evaluation,and Molecular Docking of 8‐imino‐2‐oxo‐2H,8H‐pyrano[2,3‐f]chromene Analogs: New Dual AChE Inhibitors as Potential Drugs for the Treatment of Alzheimer's Disease

Alzheimer's disease onset and progression are associated with the dysregulation of multiple and complex physiological processes, and a successful therapeutic approach should therefore address more than one target. In line with this modern paradigm, a series of 8‐imino‐2‐oxo‐2H,8H‐pyrano[2,3‐f]chromene analogs ( 4a – q ) were synthesized and evaluated for their multitarget‐directed activity on acetylcholinesterase, butyrylcholinesterase (BuChE), 2,2’‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) (ABTS) radical, and amyloid‐β peptide (Aβ) specific targets for Alzheimer's disease therapy. Most of the synthesized compounds showed remarkable acetylcholinesterase inhibitory activities in low nm concentrations and good ABTS radical scavenging activity, however, no evidence of BuChE inhibitory activity. Among them, 3‐bromobenzylamide derivative 4m exhibited the best acetylcholinesterase inhibitory activity with IC₅₀ value of 13 ± 1.4 nm which is 51‐fold superior to galantamine, a reference drug. Kinetic and molecular docking studies indicated 4m as mixed‐type inhibitor, binding simultaneously to catalytic active and peripheral anionic sites of acetylcholinesterase. Five compounds 4e , 4f , 4g , 4j , and 4k have shown 1.4‐ to 2.5‐fold of higher antioxidant activities than trolox. Interestingly, the most active compound 4m demonstrated dosage‐dependent acceleration of Aβ_1?42 aggregation, which may reduce toxicity of oligomers. Overall, these results lead to discovery of fused tricyclic coumarins as promising dual binding site inhibitors of acetylcholinesterase and afford multifunctional compounds with potential impact for further pharmacological development in Alzheimer's therapy.     

Reduction of Aβ42 in brains of transgenic APPswe mice by 2-3-chlorophenylaminophenylacetate

• 1 Imbalanced generation of the Aβ42 peptide from the amyloid β protein precursor (APP) is implicated in the pathogenesis of Alzheimer's disease.
• 2 The present study is the first to evaluate the ability of 2‐[3‐chlorophenylamino]phenylacetic acid (GLY‐230), a new drug in clinical development for the treatment of vascular complications of diabetes, to modulate Aβ42 levels in transgenic mice expressing APP.
• 3 Oral administration of 7.5 mg/kg GLY‐230 twice a day for 14 days to APPswe transgenic mice aged 3 months significantly reduced brain Aβ42 and increased plasma Aβ42 levels by 50 and 20%, respectively.
• 4 GLY‐230 readily entered the brain after administration of a dose (7.5 mg/kg) that decreased brain Aβ42.
• 5 These results are the first to demonstrate that GLY‐230, which exhibits antiglycation but no cyclo‐oxygenase inhibitory properties, lowers brain Aβ42 levels in this experimental model of Alzheimer's disease.

     

In Silico Design of Beta‐Secretase Inhibitors in Alzheimer's Disease

Alzheimer's disease is the major cause of senile dementia, flewing out 10% of 65 years old and 50% of 85 years old global population. The major fisiopathologic characteristics of Alzheimer's disease are the deposition of extracellular neuritic plaques and the presence of intracellular neurofibrillary tangles in memory‐related areas of the brain. The plaques are composed by the β‐amyloid peptide with 40 or 42 residues, result from hydrolysis of the amyloid precursor protein by the β‐secretase 1 (BACE‐1) on the amyloidogenic pathway, that begins with the BACE‐1 and which inhibition is considered one of the most promising treatments available of Alzheimer's disease. In this work, molecular modeling techniques such as virtual screening of compound libraries, pharmacophore‐based virtual screening, molecular interaction fields, ADME/Tox predictions, and similarity search were used to design novel inhibitors of BACE‐1, starting from structures available in the Protein Data Bank. The results obtained from all virtual screening techniques leaded to 10 promising compounds, which were then evaluated by enzymatic assays, and, three of them showed inhibitory activity of BACE‐1 at a 1 μm range.     

Structure‐Activity Relationships for a Series of Compounds that Inhibit Aggregation of the Alzheimer's Peptide,Aβ42

Inhibiting aggregation of the amyloid‐beta (Aβ) peptide may be an effective strategy for combating Alzheimer's disease. As the high‐resolution structure of the toxic Aβ aggregate is unknown, rational design of small molecule inhibitors is not possible, and inhibitors are best isolated by high‐throughput screening. We applied high‐throughput screening to a collection of 65 000 compounds to identify compound D737 as an inhibitor of Aβ aggregation. D737 diminished the formation of oligomers and fibrils, and reduced Aβ42‐induced cytotoxicity. Most importantly, D737 increased the life span and locomotive ability of transgenic flies in a Drosophila melanogaster model of Alzheimer's disease (J Biol Chem, 287, 2012, 38992). To explore the chemical features that make D737 an effective inhibitor of Aβ42 aggregation and toxicity, we tested a small collection of eleven analogues of D737 . Overall, the ability of a compound to inhibit Aβ aggregation was a good predictor of its efficacy in prolonging the life span and locomotive ability of transgenic flies expressing human Aβ42 in the central nervous system. Two compounds ( D744 and D830 ) with fluorine substitutions on an aromatic ring were effective inhibitors of Aβ42 aggregation and increased the longevity of transgenic flies beyond that observed for the parent compound, D737 .     

Functional identification of β3‐adrenoceptors in the guinea‐pig ileum using the non‐selective β‐adrenoceptor antagonist (±)‐bupranolol

1 To clarify whether there is a species difference or a tissue difference in β₃‐adrenoceptors, the β₃‐adrenoceptors mediating relaxations to catecholamines ((–)‐isoprenaline, (–)‐noradrenaline and (–)‐adrenaline), a selective β₃‐adrenoceptor agonist BRL37344 and a non‐conventional partial β₃‐adrenoceptor agonist (±)‐CGP12177A (a potent β₁‐ and β₂‐adrenoceptor antagonist with a partial β₃‐adrenoceptor agonist property) were investigated in the guinea‐pig ileum. 2 Catecholamines and β₃‐adrenoceptor agonists induced concentration‐dependent relaxations of pre‐contracted strips of the guinea‐pig ileum. The rank order for their relaxing potency was (–)‐isoprenaline (pD₂: 7.60) > BRL37344 (7.05) > (–)‐noradrenaline (6.38) > (±)‐CGP12177A (6.25) > (–)‐adrenaline (6.07). 3 In the presence of the non‐selective β₁‐ and β₂‐adrenoceptor antagonist (±)‐propranolol (1 μM ), only small rightward shifts of the concentration–response curves (CRCs) to these agonists were observed and the rank order of potency of agonists was BRL37344 (pD₂: 7.00) > (±)‐CGP12177A (6.17) > (–)‐isoprenaline (6.01) > (–)‐noradrenaline (5.69) > (–)‐adrenaline (5.41). 4 In the presence of (±)‐propranolol (1 μM ), the additional presence of (±)‐bupranolol (3–30 μM ), a non‐selective β₁‐, β₂‐ and β₃‐adrenoceptor antagonist, caused a concentration‐dependent rightward shift of the CRCs to catecholamines and β₃‐adrenoceptor agonists. Schild plot analyses of (±)‐bupranolol against these agonists gave pA₂ values of 6.02 ((–)‐isoprenaline), 6.03 ((–)‐noradrenaline), 6.01 ((–)‐adrenaline), 6.56 (BRL37344) and 5.74 ((±)‐CGP12177A), respectively. All Schild plot slopes were not significantly different from unity. The pA₂ values of (±)‐bupranolol obtained for the guinea‐pig β₃‐adrenoceptors were about one log unit less than the values obtained for the rat β₃‐adrenoceptors and about two log units less than the values obtained for dog β₃‐adrenoceptors. 5 These results confirm that functional β₃‐adrenoceptors are present in the guinea‐pig ileum and that the relaxations of these agonists are mainly mediated via β₃‐adrenoceptors in this tissue. The differential antagonistic potency of (±)‐bupranolol may suggest that there is a species difference between the three species (guinea‐pig, dog and rat) in their β₃‐adrenoceptors.     

Protective effects of 1,3‐diaryl‐2‐propen‐1‐one derivatives against H2O2‐induced damage in SK‐N‐MC cells

Alzheimer's disease (AD) is an age‐related neurodegenerative disorder of the central nervous system resulting in memory loss and dementia. Some of the associated pathogenic changes are amyloid peptide aggregation, excitotoxicity, oxidative stress and inflammation. Oxidative stress plays an indispensable role in the pathophysiology of AD. Therefore, antioxidant therapies appear to be promising approaches in dealing with AD patients. In that line, we evaluated the free radical scavenging capabilities of 13 different chalcones (1,3‐diphenyl‐2‐propen‐1‐one) derivatives against the free‐radical damaging effects of hydrogen peroxide (H₂O₂) on the SK‐N‐MC neuroblastoma cell line. Pretreatment of the cells for 3 h with 20 µ m of each of these derivatives (compounds 8 – 20 ) followed by exposure to 300 µ m H₂O₂ for 24 h indicated that all compounds, except compound 20 , were capable of restoring the viabilities of cells relative to the control (H₂O₂–treated) cells. The destructive effect of H₂O₂ on the adhesive behavior of the cells was almost totally restored by each of the derivatives. In addition, each of the derivatives except compounds 20 and 14 significantly reduced the extent of lipofuscin formation among the cells time‐dependently. Despite these activities, some of the derivatives, such as compounds 12 and 19 , did not reduce the H₂O₂‐induced intracellular ROS (reactive oxygen species) levels, meaning that these two derivatives act through a different mechanism other than free‐radical scavenging activity. On the other hand, for those derivatives acting as anti‐oxidants, structure–activity evaluation clearly revealed that the hydroxyl group of vanillin ring is required for their free‐radical scavenging activities. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis and 11C‐labelling of (E,E)‐1‐(3′,4′‐dihydroxystyryl)‐4‐(3′‐methoxy‐4′‐hydroxystyryl) benzene for PET imaging of amyloid deposits†

Carboxylic acid derivatives of the amyloid‐binding dye Congo red do not enter the brain well and are thus unable to serve as in vivo amyloid‐imaging agents. A neutral amyloid probe, (E,E)‐1‐(3′,4′‐dihydroxystyryl)‐4‐(3′‐methoxy‐4′‐hydroxystyryl)benzene ( 3 ), devoid of any carboxylate groups has been designed and synthesized via a 12‐step reaction sequence with a total yield of 30%. The unsymmetric compound 3 has also been labelled with C‐11 via [¹¹C]methyl iodide ([¹¹C]CH₃I) methylation of a symmetric 4,4′‐dimesyl protected precursor followed by deprotection. Preliminary evaluation indicated that compound 3 selectively stained plaques and neurofibrillary tangles in post‐mortem AD brain, and exhibited good binding affinity (K_i=38±8 nM) for Aβ(1–40) fibrils in vitro. In vivo pharmacokinetic studies indicated that [¹¹C] 3 exhibited higher brain uptake than its carboxylic acid analogs and good clearance from normal control mouse brain. [¹¹C] 3 also exhibited specific in vivo binding to pancreatic amyloid deposits in the NOR‐beta transgenic mouse model. These results justify further investigation of 3 and similar derivatives as surrogate markers for in vivo quantitation of amyloid deposits. Copyright © 2002 John Wiley & Sons, Ltd.     

Design and characterization of a membrane permeable N‐methyl amino acid‐containing peptide that inhibits Aβ1–40 fibrillogenesis

Abstract: Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of β‐sheet containing fibrils. In a previous publication, we demonstrated that the self‐association of the Alzheimer's β‐amyloid (Aβ) peptide is inhibited by peptides homologous to the central core domain of Aβ, but containing N‐methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, β‐strand conformation, the alternating position of N‐methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen‐bonding capabilities due to the replacement of the amide protons by N‐methyl groups. Here, we demonstrate, through two‐dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N‐methyl amino acids, Aβ16–20m or Ac‐K(Me)LV(Me)FF‐NH₂, does indeed have the intended structure of an extended β‐strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine‐HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm , in contrast to the nonmethylated congener, Aβ16–20 (Ac‐KLVFF‐NH₂). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Aβ16–20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through β‐strand domains.     

3‐butyl‐6‐fluoro‐1(3H)‐isobenzofuranone (6‐F‐NBP), a derivative of dl‐n‐butylphthalide,inhibits glutamate‐induced cytotoxicity in PC12 cells

It is widely recognized that glutamate (Glu)‐induced cytotoxicity, intracellular calcium overload and the excessive free radical production are key events in the development and progression of ischemic brain injury. dl‐3‐n‐butylphthalide (NBP), an anti‐ischemic agent, has therapeutic effects in animal models of vascular dementia. The aim of the present study was to investigate the protective effect of 3‐butyl‐6‐fluoro‐1(3H)‐isobenzofuranone (6‐F‐NBP), a derivative of NBP on Glu‐induced cytotoxicity in rat pheochromocytoma (PC12) cells, and to compare this action with NBP. The results showed that after 24‐h incubation with Glu (5 mM), cell viability and mitochondrial membrane potential (MMP) were decreased. In contrast, the content of reactive oxygen species (ROS), activity of nitric oxide synthase (NOS), and apoptosis rate, as well as intracellular accumulation of [Ca²⁺]_i, were increased, 6‐F‐NBP inhibited the damage induced by Glu in a dose‐dependent manner and exerted a more potent effect than NBP, indicating that 6‐F‐NBP exhibited a protective effect against Glu‐induced cytotoxicity in cultured PC12 cells. Drug Dev Res 73: 11–17, 2012. © 2011 Wiley Periodicals, Inc.     

Metformin inhibits Aβ25‐35‐induced apoptotic cell death in SH‐SY5Y cells

Metformin, a first‐line drug for type‐2 diabetes, plays a potentially protective role in preventing Alzheimer's disease (AD), but its underlying mechanism is unclear. In this study, Aβ_25‐35‐treated SH‐SY5Y cells were used as a cell model of AD to investigate the neuroprotective effect of metformin, as well as its underlying mechanisms. We found that metformin decreased the cell apoptosis rate and death, ratio of Bcl‐2/Bax, and expression of NR2A and NR2B, and increased the expression of LC3 in Aβ_25‐35‐treated SH‐SY5Y cells. Metformin also reduced intracellular and extracellular Glu concentrations, as well as the intracellular concentration of Ca²⁺ and ROS in Aβ_25‐35‐treated SH‐SY5Y cells. These findings suggest that metformin inhibits Aβ_25‐35‐treated SH‐SY5Y cell death by inhibiting apoptosis, decreasing intracellular Ca²⁺ and ROS by reducing neurotoxicity of excitatory amino acids, and by possibly reversing autophagy disorder via regulating autophagy process.