Neuroprotective effects of PRG on Aß25-35-induced cytotoxicity through activation of the ERK1/2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Phylloporia ribis (Schumach:Fr.)Ryvarden is a genus of needle Phellinus medicinal fungi, parasitic on the living rhizomes of hawthorn and pear trees. As a traditional Chinese medicine, Phylloporia ribis was used in folklore for long-term illness, weakness and memory loss in old age. Previous studies have shown that polysaccharides from Phylloporia ribis (PRG) significantly promoted synaptic growth in PC12 cells in a dose-dependent manner, exhibiting "NGF"-like neurotrophic activity. Aß25-35 damage to PC12 cells produced neurotoxicity and decreased cell survival, and PRG reduced the apoptosis rate, suggesting that PRG has neuroprotective effects. The studies confirmed that PRG had the potential to be a neuroprotective agent, but its neuroprotective mechanism remained unclear. AIM OF THE STUDY: We aimed to elucidate the neuroprotective effects of PRG in an Aß25-35-induced Alzheimer's disease (AD) model. MATERIALS AND METHODS: Highly-differentiated PC12 cells were treated with Aß25-35 (AD model) and PRG, and were assessed for cellular apoptosis, inflammatory factors, oxidative stress, and kinase phosphorylation. RESULTS: The results showed that the PRG groups effectively inhibited the neurotoxicity, mainly manifested by inhibiting mitochondrial oxidative stress, attenuating neuroinflammatory responses, and improving mitochondrial energy metabolism, eventually resulting in higher cell survival. The expression of p-ERK, p-CREB and BDNF proteins was increased in the PRG groups compared to the model group, which confirmed that PRG reversed the inhibition of the ERK pathway. CONCLUSION: We provide evidence for neuroprotection conferred by PRG and its mechanism by inhibiting ERK1/2 hyper-phosphorylation, prevention of mitochondrial stress, and subsequent prevention of apoptosis. The study highlights PRG as a promising candidate with neuroprotective effects, the potential of which can be harnessed for identifying novel therapeutic targets.

Amyloid-ß (25-35) induces the morphological alteration of dendritic spines and decreases NR2B and PSD-95 expression in the hippocampus.

Research on the memory impairment caused by the Amyloid-ß 25-35 (Aß25-35) peptide in animal models has provided an understanding of the causes that occurs in Alzheimer's disease. However, it is uncertain whether this cognitive impairment occurs due to disruption of information encoding and consolidation or impaired retrieval of stored memory. The aim of this study was to determine the effect of the Aß25-35 peptide on the morphology of dendritic spines and the changes in the expression of NR2B and PSD-95 in the hippocampus associated with learning and memory deficit. Vehicle or Aß25-35 peptide (0.1 µg/µL) was bilaterally administered into the CA1 subfield of the rat hippocampus, then tested for spatial learning and memory in the Morris Water Maze. On Day 39, the morphological changes in the CA1 of the hippocampus and dentate gyrus were examined via Golgi-Cox stain. It was observed that the Aß25-35 peptide administered in the CA1 region of the rat hippocampus induced changes to the morphology of dendritic spines and the expression of the NR2B subunit of the NMDA receptor co-localized with both the spatial memory and PSD-95 protein in the hippocampus of learning rats. We conclude that, in soluble form, the Aß25-35 peptide perturbs synaptic plasticity, specifically in the formation of new synapses, thus promoting the progression of memory impairment.

Two polyphenols isolated from Corallodiscus flabellata B. L. Burtt ameliorate amyloid ß-protein induced Alzheimer's disease neuronal injury by improving mitochondrial homeostasis.

Corallodiscus flabellata B. L. Burtt (CF) is a Chinese folk herb with reported potential for the treatment of Alzheimer's disease (AD). 3,4-Dihydroxyphenylethanol-8-O-[4-O-trans-caffeoyl-ß-D-apiofuranosyl-(1→3)-ß-D-glucopyranosyl (1→6)][1]-ß-D-glucopyranoside (SDC-1-8) and hydroxytyrosol (HT) are two polyphenolic compounds isolated from CF. The aim of this study was to investigate the protective effects of SDC-1-8 and HT on an Aß25-35-induced AD model and to study the underlying mechanism. The AD mouse model was established using a brain injection of amyloid ß-protein 25-35 (Aß25-35, 200 µM), followed by continuous administration of SDC-1-8 and HT for 4 weeks, and found that they improved cognitive dysfunction; ameliorated neuronal damage and apoptosis; decreased oxidative stress, and mitochondrial fission protein levels; and increased mitochondrial fusion protein levels in AD mice. Moreover, SDC-1-8 and HT inhibited mitochondrial membrane depolarization, reduced intracellular stored Ca2+ levels, enhanced mitochondrial respiration, increased mitochondrial fusion, and decreased mitochondrial division in Aß25-35-induced PC12 cells even in the presence of mdivi-1. Furthermore, molecular docking simulations showed that SDC-1-8 and HT interacted with dynamin-related protein 1 with higher affinity than mitofusin 1. Thus, it is summarized that SDC-1-8 and HT may have neuroprotective effects by balancing the abnormalities of mitochondrial fission and fusion, and SDC-1-8 and HT are the components providing the therapeutic basis of CF.

Pyrola incarnata demonstrates neuroprotective effects against ß-amyloid-induced memory impairment in mice.

This study aims to investigate the neuroprotective effects of Pyrola incarnata against ß-amyloid-induced memory impairment in mice. Ethanol extract of Pyrola incarnata (EPI) was obtained and led to eleven phytochemicals successfully by isolation and purification, which were elucidated by spectroscopic analysis (1H NMR, 13C NMR and HR-ESI-MS). Thereinto, ursolic acid was gained as most abundant monomer. C57BL/6 mice were intracerebroventricular injected with aggregated Aß25-35. Open-field test, Barnes maze test and Morris water maze were conducted for evaluating cognition processes of EPI and ursolic acid. EPI significantly improved learning and memory deficits, attenuated the Aß25-35 level of deposition immunohistochemically. Further studies revealed that ursolic acid as bioactive phytochemical of P. incarnata improved spatial memory performance and ameliorated Aß25-35 accumulation by activating microglia cells and up-regulating Iba1 level in the hippocampus. These findings suggest P. incarnata could improve the cognition of mice and be a promising natural source for the treatment of neurodegenerative disease.

Amyloid-ß induced neuropathological actions are suppressed by Padina gymnospora (Phaeophyceae) and its active constituent α-bisabolol in Neuro2a cells and transgenic Caenorhabditis elegans Alzheimer's model.

The inhibition of Aß peptide development and aggregation is a hopeful curative approach for the discovery of disease modifying drugs for Alzheimer's disease (AD) treatment. Recent research mainly focuses on the discovery of drugs from marine setting due to their immense therapeutic potential. The present study aims to evaluate the brown macroalga Padina gymnospora and its active constituent α-bisabolol against Aß25-35 induced neurotoxicity in Neuro2a cells and transgenic Caenorhabditis elegans (CL2006 and CL4176). The results of the in vitro study revealed that the acetone extract of P. gymnospora (ACTPG) and its active constituent α-bisabolol restores the Aß25-35 induced alteration in the oxidation of intracellular protein and lipids. In addition, ACTPG and α-bisabolol inhibited cholinesterase and ß-secretase activity in Neuro2a cells. Moreover, the intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) production was reduced by ACTPG and α-bisabolol in Neuro2a cells. The decrease in the expression level of apoptotic proteins such as Bax and caspase-3 in ACTPG and α-bisabolol treated group indicates that the seaweed and its bioactive compound have anti-apoptotic property. Further, the in vivo study revealed that the ACTPG and α-bisabolol exerts neuroprotective effect against Aß induced proteotoxicity in transgenic C. elegans strains of AD. Moreover it altered the Aß mediated pathways, lifespan, macromolecular damage and down regulated the AD related gene expression of ace-1, hsp-4 and Aß, thereby preventing Aß synthesis. Overall, the outcome of the study signifies the neuroprotective effect of ACTPG and α-bisabolol against Aß mediated AD pathology.

Edaravone reduces Aß-induced oxidative damage in SH-SY5Y cells by activating the Nrf2/ARE signaling pathway.

AIMS: Edaravone potentially alleviates cognitive deficits in a mouse model of Alzheimer's disease (AD). However, the mechanism of edaravone in suppressing AD progression remains unclear. We aim to investigate the mechanism of edaravone in suppressing oxidative stress-mediated AD progression in vitro. MAIN METHODS: Human neuroblastoma SH-SY5Y cells were pretreated with different concentrations of edaravone prior to the induction by Aß25-35. Cell viability, apoptosis, reactive oxygen species, and expression of antioxidative response elements (ARE) including Nrf2, SOD, and HO-1 were assessed. KEY FINDINGS: The results showed that apoptosis and reactive oxygen species levels significantly increased in Aß25-35-treated cells, whereas the mRNA and protein levels of Nrf2, SOD and HO-1 decreased. The opposite changes were observed in cells that were pre-treated with edaravone, particularly at a concentration of 40 µM. Aß25-35-treatment suppressed Nrf2 expression and nuclear translocation were rescued by Edaravone. Genetic inhibition of Nrf2 greatly decreased the protective effect of edaravone against cell apoptosis and cytotoxicity induced by Aß25-35, accompanied by decreases in SOD and HO-1 expression. SIGNIFICANCE: Activation of the Nrf2/ARE signaling pathway may underlie the protective effects of edaravone against the oxidative damage associated with Alzheimer's disease.

Asiatic acid protects differentiated PC12 cells from Aß25-35-induced apoptosis and tau hyperphosphorylation via regulating PI3K/Akt/GSK-3ß signaling.

Amyloid ß (Aß) peptide can cause neurotoxicity in Alzheimer's disease (AD). The main purpose of the present study is to investigate the protective role of asiatic acid (AA) against Aß25-35-induced neurotoxicity in neuronally differentiated PC12 cells. Differentiated PC12 cells were pretreated with 5, 10 or 20 µM AA before treatment with 20 µM Aß25-35. The viability and apoptosis of differentiated PC12 cells were determined by MTT assay and Annexin V-FITC/PI double staining, respectively. The mitochondrial membrane potential (MMP) of differentiated PC12 cells was analyzed by JC-1 staining. The expression levels of proteins were detected by western blot analysis. We found that AA significantly increased the viability of differentiated PC12 cells but attenuated the mitochondria-mediated apoptosis dose-dependently when challenging with Aß25-35. Besides, the results of western blot analysis showed that AA prevented IκBα degradation and p65 nuclear translocation, and promoted the phosphorylation of Akt and GSK-3ß in Aß25-35-treated differentiated PC12 cells. Moreover, LY294002, a specific PI3K inhibitor, was found to abolish the beneficial effects of AA on Aß25-35-induced apoptosis and tau protein hyperphosphorylation. Our findings demonstrated that AA protects differentiated PC12 cells from Aß25-35-induced apoptosis and tau protein hyperphosphorylation, which might be partially mediated by the activation of the PI3K/Akt/GSK-3ß signaling pathway.

Neuroprotective effects of cordycepin inhibit Aß-induced apoptosis in hippocampal neurons.

In Alzheimer's disease (AD), ß-amyloid (Aß) protein toxicity increases the formation of reactive oxygen species (ROS) and intracellular calcium levels, resulting in neuronal dysfunction, neurodegenerative disorders, and cell death. Cordycepin is a derivative of the nucleoside adenosine; also, it is speculated to exert neuroprotective effects against Aß-induced oxidative toxicity in hippocampal neurons. In the present study, the fluorescence detection method and whole-cell patch-clamp recordings were used to study the neuroprotective effects against Aß-induced toxicity in the primary hippocampal cultured neurons. The results revealed that Aß25-35 toxicity causes increased cellular ROS production and abnormal calcium homeostasis in hippocampal neurons. Moreover, Aß25-35-induced cytotoxicity led to a series of downstream events, including the activation of acetylcholinesterase, increased p-Tau expression, and increased apoptosis. Cordycepin inhibits ROS production, elevated levels of Ca2+ induced by Aß25-35, and the activation of acetylcholinesterase; all these are involved in oxidative-induced apoptosis. In addition, it decreases the increased p-Tau expression that plays a key role in the initiation of the AD. Results showed that the anti-apoptotic effects of cordycepin are partially dependent on the activation of adenosine A1 receptor, whereas an antagonist selectively attenuated the neuroprotective effects of cordycepin. Collectively, these results suggest that cordycepin could be a potential future therapeutic agent for neuronal disorders, such as AD.

Anti-amyloidogenic and anti-apoptotic effect of α-bisabolol against Aß induced neurotoxicity in PC12 cells.

Alzheimer's disease (AD) is a life-threatening neurodegenerative disorder leading to dementia, with a progressive decline in memory and other thinking skills of elderly populace. Of the multiple etiological factors of AD, the accumulation of senile plaques (SPs) particularly as Aß oligomers correlates with the relentlessness cognitive impairment in AD patients and play a vital role in AD pathology. Since natural essential oil constituents have successfully served as a source of drugs for AD treatment, the present study aims at the in vitro and in silico investigation of anti-amyloidogenic potential and anti-apoptotic property of the α-bisabolol against Aß25-35 induced neurotoxicity in PC12 cells. Treatment with α-bisabolol (5 µg/ml) after 24 h incubation with Aß25-35 reduced the aggregation propensity of Aß (p < 0.05), as observed by the reduced fluorescence intensity of thioflavin T (ThT). Confocal laser scanning microscopy (CLSM) analysis, Transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopic analysis and molecular dynamics simulation study also substantiated the Aß fibril formation hampering ability of α-bisabolol even after 9 days of incubations. The results of antiaggregation and disaggregation assay showed an increase in fluorescence intensity in Aß treated group, whereas the co-treatment of α-bisabolol (5 µg/ml) with Aß25-35 showed an extensive decrease in the fluorescence intensity, which suggests that α-bisabolol prevents the oligomers formation as well as disaggregates the matured fibrils. FACS analysis of the cells revealed the competency of α-bisabolol in rescuing the PC12 cells from Aß induced neurotoxicity and chromosomal damage and clonogenic assay proved its ability to retain the colony survival of cells. Overall, the anti-amyloidogenic and anti-apoptotic effect of α-bisabolol proves that it could be used as an excellent therapeutic drug to combat AD.

DHA, EPA and their combination at various ratios differently modulated Aß25-35-induced neurotoxicity in SH-SY5Y cells.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), have been reported to prevent neurodegenerative diseases such as Alzheimer's disease (AD) in both experimental and clinical/epidemiological studies. However, whether DHA and EPA from natural products exert similar or different neuroprotective effects and how these n-3 PUFAs target cellular and molecular mechanisms associated with neurodegenerative disease pathogenesis are unknown. In the present study, we used amyloid-ß (Aß)25-35-treated differentiated SH-SY5Y cells as a model of AD to compare the neuroprotective effect of DHA, EPA and their combination at various ratios. Administration of 20µM Aß25-35 significantly decreased SH-SY5Y cell viability, the expression of nerve growth factor (NGF), its TrkA receptor, and the level of glutathione (GSH) and increased reactive oxygen species (ROS), nitric oxide, tumor necrosis factor (TNF)-α, brain derived neurotrophic factor (BDNF) and its TrkB receptor. Aß25-35 also increased the Bax/Bcl-2 ratio and the expression of Caspase-3 in these cells. Compared with the Aß group, pretreatment with DHA/EPA significantly reduced cell death, especially at ratio of 1:1 and 2:1 DHA/EPA or pure DHA. However, the most efficient ratio for reducing changes in ROS and GSH and for decreasing TNF-α appeared at ratio of 1:2 and 1:1, respectively. The ratio of 1:1, 2:1 and pure DHA resulted in significant increase in the level of NGF. Furthermore, pure DHA was the most efficient for reducing Bax/Bcl ratio and Caspase-3 expression. In conclusion, DHA, EPA and their combination differently modulated Aß25-35-induced neurotoxicity in SH-SY5Y cells by exerting anti-oxidative, anti-inflammatory and neurotrophic effects.

Rapamycin suppresses Aß25-35- or LPS-induced neuronal inflammation via modulation of NF-κB signaling.

Rapamycin (RAPA), an inhibitor of mammalian target of rapamycin (mTOR), exhibits a high neuroprotective action against neurodegenerative diseases in mouse models. Since neuroinflammation has been shown to be involved in Alzheimer's disease (AD) development and progression, the aim of this study was to examine the anti-inflammatory role of RAPA in AD in vivo and in vitro, and investigate the underlying mechanisms. We found that amyloid-ß (Aß) induced neuronal inflammation and a remarkable increase in mTOR activity in in-vivo and in-vitro models of inflammation, suggesting the critical role of mTOR signaling in neuronal inflammation. In addition, administration of RAPA was found to down-regulate mTOR, p-mTOR, Nuclear factor kappa B (NF-κB) p65, p-p65, TNF-α, IL-1ß and Bax protein expression in Aß25-35- or lipopolysaccharides (LPS)-treated mice and cultured Neuro-2a (N2a) cells. Moreover, RAPA disrupted Aß25-35-induced nuclear translocation of mTOR and NF-κB. Our findings indicate that RAPA inhibits Aß25-35- or LPS-induced neuronal inflammation through suppressing mTOR signaling and reducing nuclear import of NF-κB.

Riluzole ameliorates learning and memory deficits in Aß25-35-induced rat model of Alzheimer's disease and is independent of cholinoceptor activation.

Alzheimer's disease (AD) is a major global public health concern and social care problem that is associated with learning, memory, and cognitive deficits. Riluzole is a glutamate modulator which has shown to improve memory performance in aged rats and may be of benefit in Alzheimer's disease. In the present study, its beneficial effect on attenuation of learning and memory deficits in Aß25-35-induced rat model of AD was assessed. Riluzole administration at a dose of 10mg/kg/day p.o. improved spatial memory in Morris water maze and retention and recall in passive avoidance task and its protective effect was not neutralized following intracerebroventricular microinjection of muscarinic or nicotinic receptor antagonists. Further biochemical analysis showed that riluzole pretreatment of intrahippocampal Aß-microinjected rats is able to attenuate hippocampal AChE activity and lower some oxidative stress markers, i.e. MDA and nitrite, with no significant change of the defensive enzyme catalase. Furthermore, riluzole prevented hippocampal CA1 neuronal loss and reduced 3-nitrotyrosine immunoreactivity. It is concluded that riluzole could exert a protective effect against memory decline induced by intrahippocampal Aß25-35 through anti-oxidative, anti-cholinesterase, and neuroprotective potential and its beneficial effect is possibly independent of cholinoceptor activation.

Design, synthesis of allosteric peptide activator for human SIRT1 and its biological evaluation in cellular model of Alzheimer's disease.

Sirtuin 1 (SIRT1) is one of the member of the mammalian proteins of the Sirtuin family of NAD+ dependent deacetylases, has recently been shown to attenuate amyloidogenic processing of amyloid protein precursor (APP) in in-vitro cell culture studies and transgenic mouse models of Alzheimer's disease (AD). SIRT1 has been shown to have a protective role against (AD). It has been reported earlier that increasing SIRT1 activity can prevent AD in mice model. Tripeptide as an activator of SIRT1 were screened on the basis of structural information by molecular docking and synthesized by solid phase method. The enhancement of biochemical activity of pure recombinant SIRT1 as well as SIRT1 in serum of AD patients in presence of tripeptide was done by Fluorescent Activity Assay. The activity of SIRT1 by peptide was assessed in IMR-32 cell line by measuring acetylated p53 level. Further the protective effect of SIRT1 activator in cellular model of AD was analyzed by MTT assay. We find CWR tripeptide as a SIRT1 activator by molecular docking, enhanced the activity of SIRT1 protein by lowering the Michaelis constant, Km by allosteric mechanism. The activity of serum SIRT1 of AD was also increases by CWR. It also decreased the acetylation of p53 in IMR32 neuroblastoma cells and protected the cell death caused by Aß amyloid fragments in cell line model of AD. Thus, it can be concluded that CWR may serve as platform to elucidate further small molecule activator as a therapeutic agent for AD targeting SIRT1.

New selective glucocorticoid receptor modulators reverse amyloid-ß peptide-induced hippocampus toxicity.

In Alzheimer's disease (AD), cognitive deficits and psychological symptoms are associated with an early deregulation of the hypothalamic-pituitary-adrenal axis. Here, in an acute model of AD, we investigated if antiglucocorticoid strategies with selective glucocorticoid receptor (GR) modulators (CORT108297 and CORT113176) that combine antagonistic and agonistic GR properties could offer an interesting therapeutic approach in the future. We confirm the expected properties of the nonselective GR antagonist (mifepristone) because in addition to restoring basal circulating glucocorticoids levels, mifepristone totally reverses synaptic deficits and hippocampal apoptosis processes. However, mifepristone only partially reverses cognitive deficit, effects of the hippocampal amyloidogenic pathway, and neuroinflammatory processes, suggesting limits in its efficacy. By contrast, selective GR modulators CORT108297 and CORT113176 at a dose of 20 and 10 mg/kg, respectively, reverse hippocampal amyloid-ß peptide generation, neuroinflammation, and apoptotic processes, restore the hippocampal levels of synaptic markers, re-establish basal plasma levels of glucocorticoids, and improve cognitive function. In conclusion, selective GR modulators are particularly attractive and may pave the way to new strategies for AD treatment.

ß-Amyloid-acetylcholine molecular interaction: new role of cholinergic mediators in anti-Alzheimer therapy?

BACKGROUND: For long time Alzheimer's disease has been attributed to a cholinergic deficit. More recently, it has been considered dependent on the accumulation of the amyloid beta peptide (Aß), which promotes neuronal loss and impairs neuronal function. Results/methodology: In the present study, using biophysical and biochemical experiments we tested the hypothesis that in addition to its role as a neurotransmitter, acetylcholine may exert its action as an anti-Alzheimer agent through a direct interaction with Aß. CONCLUSION: Our data provide evidence that acetylcholine favors the soluble peptide conformation and exerts a neuroprotective effect against the neuroinflammatory and toxic effects of Aß. The present paper paves the way toward the development of new polyfunctional anti-Alzheimer therapeutics capable of intervening on both the cholinergic transmission and the Aß aggregation.

Cyanidin 3-O-ß-glucopyranoside activates peroxisome proliferator-activated receptor-γ and alleviates cognitive impairment in the APP(swe)/PS1(ΔE9) mouse model.

Alzheimer's disease (AD) is currently one of the most common neurodegenerative disorders worldwide. To date, no cure has been developed for AD, and some disease-modifying treatments show side effects and low efficacy. Increasing evidence shows that cyanidin 3-O-ß-glucopyranoside (Cy3G), which is naturally derived from many plants, may provide protection against neurodegenerative diseases including AD; however, its exact role is still unclear. Therefore, we investigated the mechanisms of the effects of Cy3G on beta-amyloid 25-35 (Aß25-35)-induced SH-SY5Y cell injury and cognitive impairment in the APP(swe)/PS1(ΔE9) (PAP) mouse model of AD. Furthermore, we aimed to determine the molecular target initiated by Cy3G. The data indicated that Cy3G-mediated neuroprotection involved the inhibition of Aß25-35 binding to the cell surface and spontaneous aggregation of Aß25-35 fibrils at the molecular level. Furthermore, in an in vitro study, Aß25-35-mediated cytotoxicity, which was caused by inducing apoptotic cell death and ROS formation, was also ameliorated by Cy3G intervention. In addition, upregulation of peroxisome proliferator-activated receptor-γ (PPARγ) protein involved in glucose/lipid metabolism by Cy3G treatment verified that the initiated molecule was Cy3G. In an in vivo study, Cy3G was shown to alleviate cognitive impairment, improve cerebral glucose uptake and decrease fasting blood glucose levels. In conclusion, Cy3G ameliorates amyloid ß peptide-induced injury both in vitro and in vivo through the PPARγ pathway. Thus, Cy3G has a good safety profile as a potential natural PPARγ agonist and may be used as an ideal alternative to traditional disease-modifying treatments against AD.

Interaction of the Alzheimer Aß(25-35) peptide segment with model membranes.

Alzheimer's disease is characterized by the presence of amyloid plaques in the brain. The main components of these plaques are the Aß(1-40) and Aß(1-42) peptides but the Aß(25-35) sequence is the most frequently studied fragment because it represents a biologically active region of the longer Aß peptides. In the present work, the interactions of Aß(25-35) peptide with model membranes were investigated, taking into consideration the aggregation state of the peptide. Monolayers and liposomes were taken as model membranes with two lipid compositions: the equimolar ternary mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM), and cholesterol (Chol) and the equimolar POPC/SM binary mixture. The interaction of Aß(25-35) with the monolayers, investigated at low concentrations (0.25-4µM), suggested a three step mechanism: adsorption-monomers or dimers adsorb at the polar region of the lipid monolayer; nucleation-adsorbed peptides act as nucleation sites for higher aggregates; and penetration-these aggregates insert in the hydrophobic region of the monolayer. Chol slightly enhances the peptide-lipid monolayer interaction. The large aggregates nucleated in the bulk solution evidenced a weak interaction with monolayers. The interaction of Aß(25-35) with liposomes, followed by a Quartz Crystal Microbalance with Dissipation (QCM-D) in a large range of peptide concentrations (10-80µM), was very small, independently of the peptide concentration.

Amyloid ß25-35 induced ROS-burst through NADPH oxidase is sensitive to iron chelation in microglial Bv2 cells.

Iron chelation therapy and inhibition of glial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can both represent possible routes for Alzheimer's disease modifying therapies. The metal hypothesis is largely focused on direct binding of metals to the N-terminal hydrophilic 1-16 domain peptides of Amyloid beta (Aß) and how they jointly give rise to reactive oxygen species (ROS) production. The cytotoxic effects of Aß through ROS and metals are mainly studied in neuronal cells using full-length Aß1-40/42 peptides. Here we study cellularly-derived ROS during 2-60min in response to non-metal associated mid domain Aß25-35 in microglial Bv2 cells by fluorescence based spectroscopy. We analyze if Aß25-35 induce ROS production through NADPH oxidase and if the production is sensitive to iron chelation. NADPH oxidase inhibitor diphenyliodonium (DPI) is used to confirm the production of ROS through NADPH oxidase. We modulate cellular iron homeostasis by applying cell permeable iron chelators desferrioxamine (DFO) and deferiprone (DFP). NADPH oxidase subunit gp91-phox level was analyzed by Western blotting. Our results show that Aß25-35 induces strong ROS production through NADPH oxidase in Bv2 microglial cells. Intracellular iron depletion resulted in restrained Aß25-35 induced ROS.

Leucettine L41, a DYRK1A-preferential DYRKs/CLKs inhibitor, prevents memory impairments and neurotoxicity induced by oligomeric Aß25-35 peptide administration in mice.

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) and cdc2-like kinases (CLKs) are implicated in the onset and progression of Down syndrome (DS) and Alzheimer's disease (AD). DYRK1A has emerged as a possible link between amyloid-ß (Aß) and Tau, the major pathological proteins in AD. We here assessed the neuroprotective potential of a novel inhibitor of DYRKs/CLKs. The Leucettine L41, acting preferentially on DYRK1A, was tested in Aß25-35-treated mice, a nontransgenic model of AD-like toxicity. We co-injected intracerebroventricularly oligomeric Aß25-35 peptide and L41 in Swiss male mice. After 7 days, they were submitted to behavioral tests addressing spatial and non-spatial, short- and long-term memories. The oxidative stress, apoptotic markers, kinases involved in Tau phosphorylation, and synaptic integrity were analyzed by Western blot and ELISA in the hippocampus. L41, tested at 0.4, 1.2, 4 µg, prevented the Aß25-35-induced memory deficits in the Y-maze, passive avoidance and water-maze tests, with the most active dose being 4 µg. The inhibitor prevented the Aß25-35-induced oxidative stress, as revealed by measures of lipid peroxidation levels and reactive oxygen species accumulation, and abolished Aß25-35-induced expression of pro-apoptotic markers. L41 prevented the Aß25-35-induced decrease of AKT activation and increase of glycogen synthase kinase-3ß (GSK-3ß) activation, resulting in a decrease of Tau phosphorylation. Finally, L41 restored Aß25-35-reduced levels of synaptic markers. The novel DYRK1A-preferential inhibitor L41 therefore prevented Aß25-35-induced memory impairments and neurotoxicity in the mouse hippocampus. These in vivo data highlighted particularly DYRK1A as a major kinase involved in Aß pathology and suggested therapeutic developments for DYRK1A inhibitors in AD.

Selective activation of α7 nicotinic acetylcholine receptor by PHA-543613 improves Aß25-35-mediated cognitive deficits in mice.

Agonists of α7 nicotinic acetylcholine receptors (nAChRs) are currently being considered as therapeutic approaches for managing cognitive deficits in Alzheimer's disease (AD). Present study was designed to evaluate the effect of α7 nAChR selective activation by PHA-543613 (PHA) on beta-amyloid (Aß)25-35-mediated cognitive deficits in mice. For this purpose, PHA (1mg/kg, i.p.), a selective α7 nAChR agonist, and galantamine (Gal) (3mg/kg, s.c.), an acetylcholine-esterase inhibitor (AChEI) effects on α7 nAChR were tested in Aß25-35-received (intracerebroventricular, 10 nmol) mice model of AD. Methyllycaconitine (MLA) (1mg/kg, i.p.), a α7 nAChR antagonist, was used for receptor blockage effects evaluation. Working and reference memory in animals was assessed by the Morris water maze (MWM) task. The mRNA and protein levels of α7 subunit were analyzed by real-time PCR and Western blotting, respectively. PHA and Gal, ameliorate Aß-impaired working and reference memory. However, Gal had less effect than PHA in this regard. Pretreatment with MLA reverses both Gal and PHA effects in MWM. PHA and Gal treatment prevent Aß-induced α7 subunit protein reduction, but Gal has lesser effect than PHA. This effect blocked by pretreatment with MLA. In neither the pretreatment nor treatment group, the mRNA levels of nAChR α7 subunit were significantly changed. Therefore, α7 nAChR activation, reduces Aß-induced cognitive deficits and increases the α7 protein level and subsequent neuron survival. However, blockage of receptor, increases Aß toxicity and cognitive impairment and reduces the α7 nAChR protein level and flowing neuroprotection.