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The purpose of our study is to determine the protective effects of the newly discovered molecule DDQ (diethyl (3,4-dihydroxyphenethylamino)(quinolin-4-yl) methylphosphonate) against mutant APP and amyloid-beta (Aβ) in Alzheimer's disease (AD). To achieve our objective, we used a well characterized amyloid-beta precursor protein (APP) transgenic mouse model (Tg2576 strain). We administered DDQ, a 20 mg/kg body weight (previously determined in our laboratory) intra-peritoneally 3-times per week for 2 months, starting at the beginning of the 12th month, until the end of the 14th month. Further, using biochemical and molecular methods, we measured the levels of DDQ in the blood, skeletal muscle, and brain. Using Morris Water Maze, Y-maze, open field, and rotarod tests, we assessed cognitive behavior after DDQ treatment. Using q-RT-PCR, immunoblotting, transmission electron microscopy, and Golgi-cox staining methods, we studied mRNA and protein levels of longevity genes SIRTUINS, mitochondrial number & length, and dendritic spine number and length in DDQ-treated APP mice. Our extensive pharmacodynamics analysis revealed high peak levels of DDQ in the skeletal muscle, followed by serum and brain. Our behavioral analysis of rotarod, open field, Y-maze, and Morris Water Maze tests revealed that DDQ ameliorated cognitive decline (Morris Water Maze), improved working memory (Y-Maze), exploratory behavior (open field), and motor coordination (rotarod) in DDQ-treated APP mice. Interestingly, longevity genes SIRTUINS, mitochondrial biogenesis, fusion, mitophagy, autophagy and synaptic genes were upregulated in DDQ-treated APP mice relative to untreated APP mice. Dendritic spines and the quality mitochondria were significantly increased in DDQ treated APP mice. Current study findings, together with our previous study observations, strongly suggest that DDQ has anti-aging, and anti-amyloid-beta effects and a promising molecule to reduce age-and amyloid-beta-induced toxicities in AD., (Copyright © 2021 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

The potential to treat neurodegenerative diseases (NDs) of the major bioactive compound of green tea, epigallocatechin-3-gallate (EGCG), is well documented. Numerous findings now suggest that EGCG targets protein misfolding and aggregation, a common cause and pathological mechanism in many NDs. Several studies have shown that EGCG interacts with misfolded proteins such as amyloid beta-peptide (Aβ), linked to Alzheimer's disease (AD), and α-synuclein, linked to Parkinson's disease (PD). To date, NDs constitute a serious public health problem, causing a financial burden for health care systems worldwide. Although current treatments provide symptomatic relief, they do not stop or even slow the progression of these devastating disorders. Therefore, there is an urgent need to develop effective drugs for these incurable ailments. It is expected that targeting protein misfolding can serve as a therapeutic strategy for many NDs since protein misfolding is a common cause of neurodegeneration. In this context, EGCG may offer great potential opportunities in drug discovery for NDs. Therefore, this review critically discusses the role of EGCG in NDs drug discovery and provides updated information on the scientific evidence that EGCG can potentially be used to treat many of these fatal brain disorders.

Amyloid beta (Aβ) is a major component of amyloid plaques, which are a key pathological hallmark found in the brains of Alzheimer's disease (AD) patients. We show that statins are effective at reducing Aβ in human neurons from nondemented control subjects, as well as subjects with familial AD and sporadic AD. Aβ is derived from amyloid precursor protein (APP) through sequential proteolytic cleavage by BACE1 and γ-secretase. While previous studies have shown that cholesterol metabolism regulates APP processing to Aβ, the mechanism is not well understood. We used iPSC-derived neurons and bimolecular fluorescence complementation assays in transfected cells to elucidate how altering cholesterol metabolism influences APP processing. Altering cholesterol metabolism using statins decreased the generation of sAPPβ and increased levels of full-length APP (flAPP), indicative of reduced processing of APP by BACE1. We further show that statins decrease flAPP interaction with BACE1 and enhance APP dimerization. Additionally, statin-induced changes in APP dimerization and APP-BACE1 are dependent on cholesterol binding to APP. Our data indicate that statins reduce Aβ production by decreasing BACE1 interaction with flAPP and suggest that this process may be regulated through competition between APP dimerization and APP cholesterol binding.

β-Amyloid (Aβ) elevation, tau hyperphosphorylation, and neuroinflammation are major hallmarks of Alzheimer's disease (AD). Glycogen synthase kinase-3β (GSK-3β) is a key protein kinase implicated in the pathogenesis of AD. Blockade of GSK-3β is an attractive therapeutic strategy for AD. Isoorientin, a 6-C-glycosylflavone, was previously shown to be a highly selective inhibitor of GSK-3β, while exerting neuroprotective effects in neuronal models of AD. In the present study, we evaluated the in vivo effects of isoorientin on GSK-3β, tau phosphorylation, Aβ deposition, neuroinflammatory response, long-term potentiation, and spatial memory in amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice using biochemical, electrophysiological, and behavioral tests. Chronic oral administration of isoorientin to APP/PS1 mice at 8 months of age attenuated multiple AD pathogenic hallmarks in the brains, including GSK-3β overactivation, tau hyperphosphorylation, Aβ deposition, and neuroinflammation. For neuroinflammation, isoorientin treatment reduced the number of activated microglia associated with Aβ-positive plaques, and in parallel reduced the levels of pro-inflammatory factors in the brains of APP/PS1 mice. Strikingly, isoorientin reversed deficits in synaptic long-term potentiation and spatial memory relevant to cognitive functions. Together, the findings suggest that isoorientin is a brain neuroprotector and may be a promising drug lead for treatment of AD and related neurodegenerative disorders., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Advanced glycation end products (AGEs) have been reported to cause neurodegeneration, senile plaque formation and spatial learning and memory deficits. There is much evidence describing the beneficial effects of aminoguanidine (AG) on the central nervous system; AG is able to inhibit the receptor for AGEs and beta-amyloid (Aβ) deposition in the brain, thus preventing cognitive decline and neurodegeneration. In this study, we investigated whether AG protects against ovariectomy-induced neuronal deficits and Aβ deposition in rats. Animals in the ovariectomy group (OVX) group, and those in the OVX+AG group were treated with AG (100 mg/kg/day) for 8 weeks. Learning and memory were evaluated using the electric Y maze. AGE and Aβ 1-40 biochemical assessments were performed using enzyme-linked immunosorbent assay (ELISA) kits. Furthermore, evaluations of brain amyloid precursor protein 695 (APP 695 ) mRNA expression by RT-PCR and AGE expression by immunohistochemistry were carried out. Ovariectomized rats exhibited memory impairment and Aβ production disorder with upregulated APP 695 mRNA and AGE expression levels. AG pretreatment relieved the ovariectomy-induced learning and memory disorder and significantly ameliorated the Aβ production disturbance and AGE generation. Additionally, pathological changes in morphology were also significantly recovered. Our data reveal that AG plays a potentially neuroprotective role against ovariectomy-induced learning and cognitive impairment and Aβ production disorder., Advanced glycation end products (AGEs) have been reported to cause neurodegeneration, senile plaque formation and spatial learning and memory deficits. There is much evidence describing the beneficial effects of aminoguanidine (AG) on the central nervous system; AG is able to inhibit the receptor for AGEs and beta-amyloid (Aβ) deposition in the brain, thus preventing cognitive decline and neurodegeneration. In this study, we investigated whether AG protects against ovariectomy-induced neuronal deficits and Aβ deposition in rats. Animals in the ovariectomy group (OVX) group, and those in the OVX+AG group were treated with AG (100 mg/kg/day) for 8 weeks. Learning and memory were evaluated using the electric Y maze. AGE and Aβ 1-40 biochemical assessments were performed using enzyme-linked immunosorbent assay (ELISA) kits. Furthermore, evaluations of brain amyloid precursor protein 695 (APP 695 ) mRNA expression by RT-PCR and AGE expression by immunohistochemistry were carried out. Ovariectomized rats exhibited memory impairment and Aβ production disorder with upregulated APP 695 mRNA and AGE expression levels. AG pretreatment relieved the ovariectomy-induced learning and memory disorder and significantly ameliorated the Aβ production disturbance and AGE generation. Additionally, pathological changes in morphology were also significantly recovered. Our data reveal that AG plays a potentially neuroprotective role against ovariectomy-induced learning and cognitive impairment and Aβ production disorder.

Alzheimer's disease (AD) is the most common kind of dementia in the aging population leading to great social and financial burdens in many countries around the world. For decades, disease-modifying drug developed using the "one target, one drug" strategy failed to conquer this disease. Recently, we have designed and synthesized 9R, which exhibited dual inhibition of cholinesterase and amyloid beta (Aβ) aggregation in vitro. Herein, we evaluated the in vivo efficacy of 9R in a triple transgenic AD (3xTg-AD) mouse model. 3xTg-AD mice (10-month-old) were dosed intraperitoneally with 9R (daily 3, 10 or 30 mg/kg) for a month. Known cholinesterase inhibitor donepezil (0.3 mg/kg) and Aβ aggregation inhibitor tramiprosate (30 mg/kg) were used as positive controls. Cognitive performance of the mice was then evaluated by using Morris Water Maze (MWM), Y-maze tasks and Open Field test. The acetylcholine level, degree of Aβ deposition, amyloid precursor protein (APP) processing, neuroinflammation, tau deposition and tau hyperphosphorylation in the brains of the 3xTg-AD mice were examined. We have observed that one-month treatment with 9R significantly improved cognitive deficits in 3xTg-AD mice. Moreover, 9R treatment enhanced the brain acetylcholine level and mitigated the amyloid burden, tau hyperphosphorylation and neuroinflammation in the mouse brains. The effects of 9R on APP processing, neuroinflammation, tau hyperphosphorylation and Cdk-p25 action demonstrated its multifunctional role in 3xTg-AD mouse model. Our results suggested that the use of multi-target compound could be a potential approach to treat AD., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: A disease-modifying therapy for Alzheimer's disease (AD) is still an unmet clinical need. The formation of amyloid-β (Aβ) requires the initial cleavage of the amyloid-β protein precursor (AβPP) by BACE1 (beta-site AβPP cleaving enzyme 1), which is a prime therapeutic target for AD., Objective: We aimed to design and develop a selective BACE1 inhibitor suitable to AD treatment., Methods: The new BACE1 inhibitors consist on a chimeric peptide including a sequence related to the human Swedish mutant form of AβPP (AβPPswe) conjugated with the TAT carrier that facilitates cell membrane permeation and the crossing of the blood-brain barrier. Additionally to the chimeric peptide in the L-form, we developed a D-retroinverso chimeric peptide. The latter strategy, never used with BACE1 inhibitors, is considered to favor a significantly higher half-life and lower immunogenicity., Results: We found that both chimeric peptides inhibit recombinant BACE1 activity and decrease Aβ40/42 production in Neuro-2a (N2A) cells expressing AβPPswe without inducing cytotoxicity. The intraperitoneal administration of these peptides to 3xTg-AD mice decreased plasma and brain Aβ40/42 levels, as well as brain soluble AβPPβ production. Also, a reduction of insoluble Aβ was observed in the brain after chronic treatment. Noteworthy, the chimeric peptides selectively inhibited the AβPP-β cleavage relatively to the proteolysis of other BACE1 substrates such as close homologue of L1 (CHL1) and seizure-related gene 6 (SEZ6)., Conclusions: Overall these new BACE1 chimeric peptideshold promising potential as a selective disease-modifying therapy for AD.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder without effective treatment. Accumulating evidence demonstrates the production and deposition of amyloid-β peptides (Aβ) in the pathological mechanism of this disease. In our study, we investigated the effect of an ozone intraperitoneal injection on AD pathology in APP/PS1 transgenic mouse model. The male mice (5-months-old) received either ozone intraperitoneal injection (at 30 μg/ml or 50 μg/ml) or abdominocentesis administration daily for 25 days, and they were evaluated in the Morris water maze and the open field test for improvements in spatial learning-memory and working memory and anxious. Prefrontal cortex and hippocampus amyloid-β precursor protein (APP), along with other relevant biomarkers for AD, were measured through ELISA, western blot and immunohistochemistry. Results showed that ozone ameliorated the behavioral and pathological deterioration of APP/PS1 transgenic mice, and reduced the level of APP, which supports the therapeutic potential of administration of ozone in APP/PS1 mice., (Copyright © 2019. Published by Elsevier Ltd.)

The neurotoxin β-N-methylamino-l-alanine replaces l-serine in proteins and produces Alzheimer-like pathology. In proteoglycans, e.g. glypican-1, this should preclude substitution with heparan sulfate chains. Reduced release of heparan sulfate should increase β-secretase activity and processing of amyloid precursor protein. Cultured cells were treated with β-N-methylamino-l-alanine during the growth-phase and the effect on heparan sulfate substitution and amyloid precursor protein processing was evaluated using antibodies specific for heparan sulfate, the N- and C-termini of the C-terminal fragment of β-cleaved amyloid precursor protein, and amyloid beta followed by immunofluorescence microscopy, flow cytometry or SDS-PAGE. Mouse fibroblasts, N2a neuroblastoma cells and human neural stem cells released less heparan sulfate when grown in the presence of β-N-methylamino-l-alanine. Cells expressing a recombinant, anchor-less glypican-1 secreted heparan sulfate-deficient glypican-1. There was increased processing of amyloid precursor protein in N2a cells when grown in the presence of the neurotoxin. The degradation products accumulated in cytoplasmic clusters. Secretion of amyloid beta increased approx. 3-fold. Human neural stem cells also developed cytoplasmic clusters containing degradation products of amyloid precursor protein. When non-dividing mouse N2a cells or cortical neurons were exposed to β-N-methylamino-l-alanine there was no effect on heparan sulfate substitution in glypican-1 or on amyloid precursor protein processing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The abnormal self-assembly of amyloid-β (Aβ) peptides into toxic fibrillar aggregates is associated with the pathogenesis of Alzheimer's disease (AD). The inhibition of β-sheet-rich oligomer formation is considered as the primary therapeutic strategy for AD. Previous experimental studies reported that norepinephrine (NE), one of the neurotransmitters, is able to inhibit Aβ aggregation and disaggregate the preformed fibrils. Moreover, exercise can markedly increase the level of NE. However, the underlying inhibitory and disruptive mechanisms remain elusive. In this work, we performed extensive replica-exchange molecular dynamic (REMD) simulations to investigate the conformational ensemble of Aβ 1-42 dimer with and without NE molecules. Our results show that without NE molecules, Aβ 1-42 dimer transiently adopts a β-hairpin-containing structure, and the β-strand regions of this β-hairpin (residues 15QKLVFFA21 and 33GLMVGGVV40) strongly resemble those of the Aβ fibril structure (residues 15QKLVFFA21 and 30AIIGLMVG37) reported in an electron paramagnetic resonance spectroscopy study. NE molecules greatly reduce the interpeptide β-sheet content and suppress the formation of the above-mentioned β-hairpin, leading to a more disordered coil-rich Aβ dimer. Five dominant binding sites are identified, and the central hydrophobic core 16KLVFFA21 site and C-terminal 31IIGLMV36 hydrophobic site are the two most favorable ones. Our data reveal that hydrophobic, aromatic stacking, hydrogen-bonding and cation-π interactions synergistically contribute to the binding of NE molecules to Aβ peptides. MD simulations of Aβ 1-42 protofibril show that NE molecules destabilize Aβ protofibril by forming H-bonds with residues D1, A2, D23, and A42. This work reveals the molecular mechanism by which NE molecules inhibit Aβ 1-42 aggregation and disaggregate Aβ protofibrils, providing valuable information for developing new drug candidates and exercise therapy against AD.

Alzheimer's disease (AD) is characterized by the accumulation of neurotoxic amyloid-β (Aβ) peptides consisting of 39-43 amino acids, proteolytically derived fragments of the amyloid-β protein precursor (AβPP), and the accumulation of the hyperphosphorylated microtubule-associated protein tau. Inhibiting Aβ production may reduce neurodegeneration and cognitive dysfunction associated with AD. We have previously used an AβPP-firefly luciferase enzyme complementation assay to conduct a high throughput screen of a compound library for inhibitors of AβPP dimerization, and identified a compound that reduces Aβ levels. In the present study, we have identified an analog, compound Y10, which also reduced Aβ. Initial kinase profiling assays identified the receptor tyrosine kinase cKit as a putative Y10 target. To elucidate the precise mechanism involved, AβPP phosphorylation was examined by IP-western blotting. We found that Y10 inhibits cKit phosphorylation and increases AβPP phosphorylation mainly on tyrosine residue Y743, according to AβPP751 numbering. A known cKit inhibitor and siRNA specific to cKit were also found to increase AβPP phosphorylation and lower Aβ levels. We also investigated a cKit downstream signaling molecule, the Shp2 phosphatase, and found that known Shp2 inhibitors and siRNA specific to Shp2 also increase AβPP phosphorylation, suggesting that the cKit signaling pathway is also involved in AβPP phosphorylation and Aβ production. We further found that inhibitors of both cKit and Shp2 enhance AβPP surface localization. Thus, regulation of AβPP phosphorylation by small molecules should be considered as a novel therapeutic intervention for AD.

Background: Various methodologies have been employed for the therapeutic interpolation of the progressive brain disorder Alzheimer's disease. Thus, β-secretase inhibition is significant to prevent disease progression in the early stages., Objective: This study seeks to purify and characterize a novel β-secretase inhibitory peptide from Pacific hake enzymatic hydrolysate., Methods: A potent β-secretase inhibitory peptide was isolated by sequential purifications using Sephadex G-25 column chromatography and octadecylsilane (ODS) C18 reversed-phase HPLC. A total of seven peptides were synthesized using the isolated peptide sequences. SH-SY5Y cells stably transfected with the human ''Swedish'' amyloid precursor protein (APP) mutation APP695 (SH-SY5YAPP695swe) were used as an in-vitro model system to investigate the effect of Leu-Asn peptide on APP processing., Results: The β-secretase inhibitory activity (IC50) of the purified peptide (Ser-Leu-Ala-Phe-Val-Asp- Asp-Val-Leu-Asn) from fish protein hydrolysate was 18.65 μM and dipeptide Leu-Asn was the most potent β-secretase inhibitor (IC50 value = 8.82 µM). When comparing all the seven peptides, the inhibition pattern of Leu-Asn dipeptide was found to be competitive by Lineweaver-Burk plot and Dixon plot (Ki value = 4.24 µM). The 24 h treatment with Leu-Asn peptide in SH-SY5Y cells resulted in reducing the β-amyloid (Aβ) production in a dose-dependent manner., Conclusion: Therefore, the results of this study suggest that β-secretase inhibitory peptides derived from marine organisms could be potential candidates to develop nutraceuticals or pharmaceuticals as antidementia agents., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

As the most common cause of progressive intellectual failure in elderly humans, Alzheimer's disease (AD) is pathologically featured by amyloid plaques, synaptic loss, and neurofibrillary tangles. The amyloid plaques are mainly aggregates of amyloid β-peptide (Aβ), a primary factor contributing to the pathogenesis of AD. Elimination or reduction of the level of Aβ is considered an important strategy in AD treatment. The pharmacotherapeutic efficacy of selenium (Se), an essential biological trace element for mammalian species, has been confirmed in a number of experimental models of neurodegenerative diseases. Selenium-enriched yeast (Se-yeast) is commonly used as a nutritional supplement for Se. In this study, we investigated the effects and underlying mechanisms of Se-yeast on Aβ pathology in a 4-month-old triple transgenic mouse model of AD (3×Tg-AD mice). The administration of Se-yeast attenuated the deposition of Aβ in the brains of AD mice, which was concomitant with decreased levels of LC3II. The Se-yeast treatment decreased the level of amyloid-protein precursor (APP), downregulated the activity of AMP-activated protein kinase (AMPK) and upregulated the activity of AKT/mTOR/p70S6K. Furthermore, the levels of p62 also significantly decreased, and the cathepsin D levels increased, accompanied by increased turnover of Aβ and APP in Se-yeast-treated AD mice. In addition to decreasing the generation of Aβ, Se-yeast also inhibited the initiation of autophagy by modulating the AMPK/AKT/mTOR/p70S6K signaling pathway and enhanced autophagic clearance, thus reducing the burden of Aβ accumulation in the brains of AD mice. Our results further highlight the potential therapeutic effects of Se-yeast on AD.

Altered proteostasis induced by amyloid peptide aggregation and hyperphosphorylation of tau protein, is a prominent feature of Alzheimer's disease, which highlights the occurrence of endoplasmic reticulum stress and triggers the activation of the unfolded protein response (UPR), a signaling pathway that enforces adaptive programs to sustain proteostasis. In this study, we investigated the role of geniposide in the activation of UPR induced by high glucose in primary cortical neurons. We found that high glucose induced a significant activation of UPR, and geniposide enhanced the effect of high glucose on the phosphorylation of IRE1α, the most conserved UPR signaling branch. We observed that geniposide induced the expression of HRD1, an ubiquitin-ligase E3 in a time dependent manner, and amplified the expression of HRD1 induced by high glucose in primary cortical neurons. Suppression of IRE1α activity with STF-083010, an inhibitor of IRE1 phosphorylation, prevented the roles of geniposide on the expression of HRD1 and APP degradation in high glucose-treated cortical neurons. In addition, the results from RNA interfere on HRD1 revealed that HRD1 was involved in geniposide regulating APP degradation in cortical neurons. These data suggest that geniposide might be benefit to re-establish proteostasis by enhancing the UPR to decrease the load of APP in neurons challenged by high glucose.

Amyloid-β peptide (Aβ) initiates a cascade of pathological events, including activation of microglial cells, oxidative stress, and inflammation, leading to neuronal death and the typical pathological changes in Alzheimer's disease (AD). Flavonoids have been reported to exert neuroprotective activities, not only through their generally accepted antioxidant effects, but also through their ability to protect against neurotoxin-induced injury. Flavonoids reduce Aβ production, inhibit neuroinflammation, increase cerebrovascular function, and improve cognitive performance. Here, we analyzed the effects of a flavonoid-rich ethanol extract from the leaves of Diospyros kaki (FLDK) in APP/PS1 transgenic mice. We found that oral treatment with FLDK reversed learning and memory impairment, reduced Aβ burden and expression of β-site amyloid precursor protein cleavage enzyme 1 (BACE1), and decreased microglial activation in senile plaques. FLDK restored antioxidant enzyme activities, as well as reduced the lipid peroxidation product, malondialdehyde, and inflammatory mediators. These results demonstrate that FLDK alleviates cognitive decline and reduces Aβ burden, microglial activation, oxidative stress, and inflammation responses. Thus, FLDK treatment may be a potential therapeutic strategy for preventing and treating AD, at least in part via its anti-oxidant and anti-inflammatory biological activities and its effect on the Aβ producing enzyme BACE1., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The excess accumulation of amyloid-β (Aβ) peptides derived from the sequential cleavage of amyloid precursor protein (APP) by secretases, is one of the toxic key events leading to neuronal loss in Alzheimer's disease (AD). Studies have shown that cholinergic activity may also be involved in the regulation of APP metabolism. In the current study, we have investigated the roles of toluidine blue O (TBO) and thionine (TH), newly recognized phenothiazine-derived cholinesterase inhibitors, on the metabolism of APP in Chinese hamster ovary cells stably expressing human APP751 and presenilin 1 (PS70 cells). We assessed the effects of both compounds on the levels of Aβ, soluble APP-α (sAPPα), intracellular APP and β-site APP-cleaving enzyme 1 (BACE1). After treatment of PS70 cells with TBO or TH without any side effect on cell viability, the levels of secreted Aβ40, Aβ42 and sAPPα were assayed by specific sandwich ELISAs while APP and BACE1 in cell lysates were analyzed using Western blot. The secreted Aβ40, Aβ42 and sAPPα in TBO- and TH-treated cells were found to be reduced in a dose-dependent manner compared to vehicle-treated cells. Results suggest that TH mitigated the Aβ pathology by lowering APP levels whereas reduced Aβ caused by TBO treatment seems to be the outcome of both less substrate availability and amyloidogenic APP processing. Taken together, our results represent the first report demonstrating that TBO and TH can affect amyloid metabolism in vitro., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Objective: To investigate the effect of β-sheet breaker peptide H102 on APP associated secretase in the hippocampus brain regions of APP/PS1 double transgenic mice(AD mice)., Methods: Thirty 6-month-old APP/PS1 double transgenic mice were randomly divided into AD group and H102 group, a group of C57BL/6J mice with the same age, number and background was set as controls( n =15). H102 (5.8 mg/kg) 5 μl was infused by intranasal administration to mice in H102 treatment group, and equal volume of blank solution of H102 was given to mice in control group and AD group. The ability of spatial reference memory was tested by Morris water maze after 30 days of treatment. And then immunohistochemistry tests and Western blot were used to detect the content of α-secretase (ADAM10, ADAM17), β-secretase (BACE1), γ-secretase (PS1, APH1a, PEN2) in the hippocampus brain regions., Results: Compared with the control group, the expression of BACE1, PS1, PEN-2 and APH1-a protein in the hippocampus of AD group were significantly increased, ADAM10, ADAM17 protein expression were significantly reduced ( P <0.05); Compared with the model group, H102 could significantly improve the spatial learning and memory ability of AD mice, significantly decreased the expression of BACE1, PS1, PEN-2 and APH1-a protein in the hippocampus, significantly increased the expression of ADAM10 and ADAM17 protein( P <0.05)., Conclusions: β sheet peptides blocked H102 can reduce the formation of Aβ in the hippocampus brain area, improve the activity of α-secretase in the hippocampus brain region, decrease the activity of β-and γ-secretase, improve the learning and memory ability of AD mice.

The deposition of amyloid-β (Aβ) oligomers in brain parenchyma has been implicated in the pathophysiology of Alzheimer's disease. Here we present a systems pharmacology model describing the changes in the amyloid precursor protein (APP) pathway after administration of three different doses (10, 30, and 125 mg/kg) of the β-secretase 1 (BACE1) inhibitor MBi-5 in cisterna magna ported rhesus monkeys. The time course of the MBi-5 concentration in plasma and cerebrospinal fluid (CSF) was analyzed in conjunction with the effect on the concentrations of the APP metabolites Aβ42, Aβ40, soluble β-amyloid precursor protein (sAPP) α, and sAPPβ in CSF. The systems pharmacology model contained expressions to describe the production, elimination, and brain-to-CSF transport for the APP metabolites. Upon administration of MBi-5, a dose-dependent increase of the metabolite sAPPα and dose-dependent decreases of sAPPβ and Aβ were observed. Maximal inhibition of BACE1 was close to 100% and the IC50 value was 0.0256 μM (95% confidence interval, 0.0137-0.0375). A differential effect of BACE1 inhibition on Aβ40 and Aβ42 was observed, with the Aβ40 response being larger than the Aβ42 response. This enabled the identification of an Aβ42 oligomer pool in the systems pharmacology model. These findings indicate that decreases in monomeric Aβ responses resulting from BACE1 inhibition are partially compensated by dissociation of Aβ oligomers and suggest that BACE1 inhibition may also reduce the putatively neurotoxic oligomer pool., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Tropisetron was identified in a screen for candidates that increase the ratio of the trophic, neurite-extending peptide sAPPα to the anti-trophic, neurite-retractive peptide Aβ, thus reversing this imbalance in Alzheimer's disease (AD). We describe here a hierarchical screening approach to identify such drug candidates, moving from cell lines to primary mouse hippocampal neuronal cultures to in vivo studies. By screening a clinical compound library in the primary assay using CHO-7W cells stably transfected with human APPwt, we identified tropisetron as a candidate that consistently increased sAPPα. Secondary assay testing in neuronal cultures from J20 (PDAPP, huAPP(Swe/Ind)) mice showed that tropisetron consistently increased the sAPPα/Aβ 1-42 ratio. In in vivo studies in J20 mice, tropisetron improved the sAPPα/Aβ ratio along with spatial and working memory in mice, and was effective both during the symptomatic, pre-plaque phase (5-6 months) and in the late plaque phase (14 months). This ameliorative effect occurred at a dose of 0.5mg/kg/d (mkd), translating to a human-equivalent dose of 5mg/day, the current dose for treatment of postoperative nausea and vomiting (PONV). Although tropisetron is a 5-HT3 receptor antagonist and an α7nAChR partial agonist, we found that it also binds to the ectodomain of APP. Direct comparison of tropisetron to the current AD therapeutics memantine (Namenda) and donepezil (Aricept), using similar doses for each, revealed that tropisetron induced greater improvements in memory and the sAPPα/Aβ1-42 ratio. The improvements observed with tropisetron in the J20 AD mouse model, and its known safety profile, suggest that it may be suitable for transition to human trials as a candidate therapeutic for mild cognitive impairment (MCI) and AD, and therefore it has been approved for testing in clinical trials beginning in 2014., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Accumulation of β-amyloid peptides (Aβ) and its progressive deposition into amyloid plaques are key events in the aetiology of Alzheimer's disease (AD). To date, AD treatment is symptomatic and consists of drugs treating the cognitive decline., Objective: Identifying molecules specifically targeting Aβ production or aggregation represents a huge challenge in the development of specific AD treatments. Several molecules reported as γ-secretase inhibitors or modulators have been evaluated, but so far none of them have proven to be selective or fully efficient. We have previously investigated the potential interest of plant extracts and we reported that Pterocarpus erinaceus stem-bark extract was active on Aβ release. Our aim here was to characterize the mechanisms by which this extract reduces Aβ levels., Methods: We tested P. erinaceus extract at non-toxic concentrations on cells expressing the human amyloid precursor protein (APP695) or its amyloidogenic β-cleaved C-terminal fragment (C99), as well as on neuronal cell lines. P. erinaceus extract was found to inhibit Aβ release. We further showed that this extract inhibited γ-secretase activity in cell-free and in vitro assays, strongly suggesting that P. erinaceus extract is a natural γ-secretase inhibitor. Importantly, this extract did not inhibit γ-secretase-dependent Notch intracellular domain release., Conclusion: P. erinaceus extract appears as a new potent γ-secretase inhibitor selective towards APP processing., (2013 S. Karger AG, Basel)

A role of amyloid β (Aβ) peptide aggregation and deposition in Alzheimer's disease (AD) pathogenesis is widely accepted. Significantly, abnormalities induced by aggregated Aβ have been linked to synaptic and neuritic degeneration, consistent with the "dying-back" pattern of degeneration that characterizes neurons affected in AD. However, molecular mechanisms underlying the toxic effect of aggregated Aβ remain elusive. In the last 2 decades, a variety of aggregated Aβ species have been identified and their toxic properties demonstrated in diverse experimental systems. Concurrently, specific Aβ assemblies have been shown to interact and misregulate a growing number of molecular effectors with diverse physiological functions. Such pleiotropic effects of aggregated Aβ posit a mayor challenge for the identification of the most cardinal Aβ effectors relevant to AD pathology. In this review, we discuss recent experimental evidence implicating amyloid β precursor protein (APP) as a molecular target for toxic Aβ assemblies. Based on a significant body of pathologic observations and experimental evidence, we propose a novel pathologic feed-forward mechanism linking Aβ aggregation to abnormalities in APP processing and function, which in turn would trigger the progressive loss of neuronal connectivity observed early in AD., (Copyright © 2013 Elsevier Inc. All rights reserved.)

γ-Secretase modulators (GSM), which reduce amyloidogenic Aβ(42) production while maintaining total Aβ levels, and Notch-sparing γ-secretase inhibitors (GSIs) are promising therapies for the treatment of Alzheimer's Disease (AD). To have a safety margin for therapeutic use, GSMs and GSIs need to allow Notch intracellular domain (NICD) production, while preventing neurotoxic Aβ peptide production. Typically, GSI and GSM effects on these substrates are determined using two different cell lines, one for the measurement of enzyme activity against each substrate. However, predicting selectivity for different substrates across cell systems may reduce the reliability of such ratios such that the in vitro data are not useful for predicting in vivo safety margins. This is especially concerning since the IC(50)'s of some GSIs vary depending upon the level of APP expression in a cell line. To circumvent this problem, we utilized the SUP-T1 cell line which expresses a truncated Notch receptor fragment that does not need sheddase cleavage to be a γ-secretase substrate. When combined with a sensitive method of measuring Aβ production, this assay system allows both substrates to be measured simultaneously, reducing the potential to calculate imprecise selectivity margins. To demonstrate the value of this system, known GSIs and GSMs were examined in the SUP-T1 dual substrate assay. IC(50)'s were determined for both substrates and the in vitro selectivity margin was calculated. These data suggest using a single cell line is a more accurate prediction of the fold difference between NICD inhibition and Aβ(42) lowering for therapeutically promising GSIs and GSMs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Stress, depending on its level and quality, may cause adaptive and maladaptive alterations in brain functioning. As one of its multiple effects, elevated blood cortisol levels decrease the synthesis of the neuroprotective BDNF, thus leading to hippocampal atrophy and synapse loss, and rendering it a possible cause for the Alzheimer's disease (AD) related neuropathological and cognitive changes. As a result of the stress response, intraneuronal alterations--also affecting the metabolism of beta-actin--can develop. These have a role in the regulation of memory formation (LTP), but in pathological conditions (AD) they could lead to the accumulation of Hirano bodies (actin-cofilin rods). According to the dementia treatment guidelines, the behavioural and psychological symptoms of AD can be treated with certain antipsychotics. Therefore, the aim of our study was to examine the effects of sertindole (currently not used in the standard management of AD) on the transcription of some AD associated genes (amyloid precursor protein [APP], mitogen activated protein kinase-1 [MAPK-1], beta-actin) in the brain of rats exposed to chronic immobilization stress (CIS). Male Wistar rats were exposed to CIS for three weeks. The four groups were: control (n = 16), CIS (n = 10), 10 mg/kg sertindole (n = 5) and 10 mg/kg sertindole + CIS (n = 4). Following transcardial perfusion, the relative levels of hippocampal and cortical mRNA of the previously mentioned genes were measured with real-time PCR. CIS induced hippocampal beta-actin (p < 0.01), MAPK-1 and APP (p < 0.05) mRNA overexpression. The simultaneous administration of sertindole suppressed this increase in beta-actin, MAPK-1 and APP expression (p < 0.05). Ours is the first report about CIS induced beta-actin gene overexpression. This finding, in accordance with the similar results in APP and MAPK-1 expression, underlines the significance of cytoskeletal alterations in AD pathogenesis. The gene expression reducing effect of sertindole suggests that antipsychotic drugs may have a neuroprotective effect.

A screen of a library of synthetic drugs and natural product extracts identified a botanical extract that modulates the processing of amyloid precursor protein (APP) in cultured cells to produce a lowered ratio of amyloid-beta peptide (1-42) (Aβ42) relative to Aβ40. This profile is of interest as a potential treatment for Alzheimer's disease. The extract, from the black cohosh plant (Actaea racemosa), was subjected to bioassay guided fractionation to isolate active components. Using a combination of normal-phase and reverse-phase chromatography, a novel triterpene monoglycoside, 1, was isolated. This compound was found to have an IC(50) of 100 nM for selectively reducing the production of amyloidogenic Aβ42 while having a much smaller effect on the production of Aβ40 (IC(50) 6.3 μM) in cultured cells overexpressing APP. Using IP-MS methods, this compound was found to modulate the pool of total Aβ produced by reducing the proportion of Aβ42 while increasing the relative amounts of shorter and less amyloidogenic Aβ37 and Aβ39. Concentrations of 1 sufficient to lower levels of Aβ42 substantially (up to 10 μM) did not significantly affect the processing of Notch or other aspects of APP processing. When 1 (10 μg) was administered to CD-1 normal mice intracerebroventricularly, the level of Aβ42 in brain was reduced. Assays for off-target pharmacology and the absence of overt signs of toxicity in mice dosed with compound 1 suggest a comparatively selective pharmacology for this triterpenoid. Compound 1 represents a new lead for the development of potential treatments for Alzheimer's disease via modulation of gamma-secretase.

Purpose: To examine the changes in and localization of phosphorylated presenilin1 (p-PS1) and amyloid precursor protein (APP) in the optic nerve after intravitreal injection of TNF and to investigate the role of γ-secretase in the cleavage of APP in optic nerve degeneration., Methods: Groups of rats were euthanatized at 1 or 2 weeks after intravitreal injection of TNF. Levels of p-PS1 protein in the optic nerve were determined by immunoblotting and immunohistochemistry. The localization of APP was determined by immunohistochemistry, and its downstream cleavage was determined by immunoprecipitation using 6E10 antibody followed by immunoblotting with an APP intracellular domain (AICD) antibody. The effect of a γ-secretase inhibitor on TNF-induced optic nerve degeneration was determined by counting the number of axons., Results: p-PS1 was increased in the optic nerve after TNF injection and was found to colocalize with vimentin and glial fibrillary acidic protein, markers of astrocytes. Immunoprecipitation using 6E10 antibody followed by immunoblotting with AICD antibody revealed an increase in γ-secretase activation in the optic nerve after TNF injection, which was inhibited by treatment with the γ-secretase inhibitor. Moreover, γ-secretase inhibition significantly prevented the loss of axons in the optic nerve after TNF injection., Conclusions: The increase in p-PS1 and activation of γ-secretase in the optic nerve may be associated with TNF-induced axonal degeneration. Modulation of γ-secretase activity may be useful for the treatment of TNF-related optic neuropathy.

Amyloid precursor protein (APP) can be proteolytically processed along two pathways, the amyloidogenic that leads to the formation of the 40-42 amino acid long Alzheimer-associated amyloid β (Aβ) peptide and the non-amyloidogenic in which APP is cut in the middle of the Aβ domain thus precluding Aβ formation. Using immunoprecipitation and mass spectrometry we have shown that Aβ is present in cerebrospinal fluid (CSF) as several shorter isoforms in addition to Aβ1-40 and Aβ1-42. To address the question by which processing pathways these shorter isoforms arise, we have developed a cell model that accurately reflects the Aβ isoform pattern in CSF. Using this model, we determined changes in the Aβ isoform pattern induced by α-, β-, and γ-secretase inhibitor treatment. All isoforms longer than and including Aβ1-17 were γ-secretase dependent whereas shorter isoforms were γ-secretase independent. These shorter isoforms, including Aβ1-14 and Aβ1-15, were reduced by treatment with α- and β-secretase inhibitors, which suggests the existence of a third and previously unknown APP processing pathway involving concerted cleavages of APP by α- and β-secretase., (Copyright © 2009 Elsevier Inc. All rights reserved.)

Several lines of evidence support protective as well as deleterious effects of oleic acid (OA) on Alzheimer's disease (AD) and other neurological disorders; however, the bases of these effects are unclear. Our investigation demonstrates that amyloid precursor protein (APP) 695 transfected Cos-7 cells supplemented with OA have reduced secreted amyloid-beta (Aβ) levels. An early-onset AD transgenic mouse model expressing the double-mutant form of human APP, Swedish (K670N/M671L) and Indiana (V717F), corroborated our in vitro findings when they were fed a high-protein, low-fat (18% reduction), cholesterol-free diet enriched with OA. These mice exhibited an increase in Aβ40/Aβ42 ratio, reduced levels of beta-site APP cleaving enzyme (BACE) and reduced presenilin levels along with reduced amyloid plaques in the brain. The decrease in BACE levels was accompanied by increased levels of a non-amyloidogenic soluble form of APP (sAPPα). Furthermore, the low-fat/+OA diet resulted in an augmentation of insulin-degrading enzyme and insulin-like growth factor-II. These results suggest that OA supplementation and cholesterol intake restriction in a mouse model of AD reduce AD-type neuropathology., (© 2010 The Authors; Brain Pathology © 2010 International Society of Neuropathology.)

Sequential processing of the β-amyloid precursor protein by β- and γ-secretase generates the amyloid β-peptide (Aβ), which is widely believed to play a causative role in Alzheimer disease. Selective lowering of the pathogenic 42-amino acid variant of Aβ by γ-secretase modulators (GSMs) is a promising therapeutic strategy. Here we report that mutations in presenilin (PS), the catalytic subunit of γ-secretase, display differential responses to non-steroidal anti-inflammatory drug (NSAID)-type GSMs and more potent second-generation compounds. Although many pathogenic PS mutations resisted lowering of Aβ(42) generation by the NSAID sulindac sulfide, the potent NSAID-like second-generation compound GSM-1 was capable of lowering Aβ(42) for many but not all mutants. We further found that mutations at homologous positions in PS1 and PS2 can elicit differential Aβ(42) responses to GSM-1, suggesting that a positive GSM-1 response depends on the spatial environment in γ-secretase. The aggressive pathogenic PS1 L166P mutation was one of the few pathogenic mutations that resisted GSM-1, and Leu-166 was identified as a critical residue with respect to the Aβ(42)-lowering response of GSM-1. Finally, we found that GSM-1-responsive and -resistant PS mutants behave very similarly toward other potent second-generation compounds of different structural classes than GSM-1. Taken together, our data show that a positive Aβ(42) response for PS mutants depends both on the particular mutation and the GSM used and that attenuated Aβ(42) responses to low potency GSMs can be overcome for many PS mutants by second generation GSMs.

Alzheimer's disease (AD) is the most common form of dementia in the United States and is increasing in prevalence every year throughout the world. Recent clinical trial failures highlight the need for further insights into the molecular events that underlie the neurobiology of AD. Pathological aberrations in AD are believed to result, in part, from excess accumulation of amyloid-beta peptide (Aβ), a product of Aβ precursor protein (APP). Targeting APP levels would then be expected to reduce Aβ production in all forms of AD. Therefore, clarifying the regulatory network that governs APP expression is likely to reveal molecular players that could serve as novel drug targets. This review highlights recent work demonstrating the involvement of microRNA (miRNA) in this regulatory network. MiRNA are small, non-coding RNA that interact with target mRNA at sites of imperfect complementarity and mediate translational inhibition or transcript destabilization. We first review the neurobiology of AD and describe current therapeutic strategies. We then review transcriptional and post-transcriptional mechanisms utilized by cells to control APP expression. We conclude by highlighting recent work, including our own, which suggests miRNA are integral components of this regulatory framework and potential targets for future AD therapeutics.

Alzheimer's disease-associated amyloid-β (Aβ) peptide is neurotoxic as an oligomer, but not as a monomer, by an unknown mechanism. We showed previously that Aβ interacts with the amyloid-β precursor protein (AβPP), leading to caspase cleavage and cell death induction. To characterize this structure and interaction further, we purified the extracellular domain of AβPP695 (eAβPP) and its complex with Aβ oligomers (AβOs) of varying sizes, and then performed small angle X-ray scattering (SAXS). In the absence of any Aβ, eAβPP was a compact homodimer with a tight association between the E1 and E2 domains. Dimeric Aβ oligomers induced monomerization of eAβPP while larger oligomers also bound eAβPP but preserved the homodimer. Efficient binding of the larger oligomers correlated with the presence of prefibrillar oligomers, suggesting that the eAβPP binding is limited to a conformational subset of Aβ oligomers. Both forms of Aβ bound to eAβPP at the Aβ-cognate region and induced dissociation of the E1 and E2 domains. Our data provide the first structural evidence for Aβ-AβPP binding and suggest a mechanism for differential modulation of AβPP processing and cell death signaling by Aβ dimers versus conformationally-specific larger oligomers.

Hypoxia which is mainly mediated by hypoxia-inducible factor 1 (HIF-1), can greatly contribute to the occurrence of Alzheimer's disease (AD) by increasing beta-site APP cleaving enzyme (BACE1) gene expression, protein level and beta-secretase activity, resulting in a significant generation of amyloid-beta (Abeta). Salidroside has been reported to have great neuroprotective effects. The aim of this study was to investigate the effects of salidroside on hypoxia-induced abnormal processing of the amyloid precursor protein (APP) in SH-SY5Y cells and its possible mechanism. Western blot analysis showed that 200muM of salidroside pretreatment significantly decreased BACE1 protein level and promoted the secretion of sAPPalpha in hypoxic condition. Salidroside had no effect on the level of APP, ADAM10 and ADAM17. ELISA analysis revealed that salidroside was able to inhibit the increase of beta-secretase activity and Abeta generation induced by hypoxia, with no effect on gamma-secretase activity. Notably, under hypoxia condition, mRNA of BACE1 and protein level of HIF-1alpha were decreased by salidroside pretreatment. These results demonstrated for the first time that salidroside was able to attenuate abnormal processing of amyloid precursor protein induced by hypoxia in SH-SY5Y cells, providing a new insight into prevention and treatment of Alzheimer's disease., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

A critical requirement in the development of Alzheimer's disease (AD) therapeutics is a demonstration of the in vivo efficacy of compounds in pre-clinical disease relevant models. One of the most frequently used models in AD research are transgenic mice overexpressing mutant forms of human amyloid precursor protein (APP) that are associated with early-onset familial AD. These mice exhibit an age-dependent accumulation and deposition of amyloid -peptide (A) as extracellular plaques in the brain, and thereby depict one of the key pathologies observed in the brains of AD patients. Although these mouse models do not recapitulate all the pathological features of AD, they have been invaluable in the development of therapeutic agents aimed at lowering A production, inhibiting A deposition or facilitating A clearance. Further development of these APP transgenic models led to the incorporation of transgenes for human mutant presenilins, resulting in an accelerated A deposition rate and human mutant tau protein leading to neurofibrillary tangle-like pathology. The latter was a major advance in the development of AD models, as it allowed researchers to investigate the interplay between the two key pathologies of AD. This review highlights how APP transgenic mouse models have successfully been used in drug discovery to support the progression of A lowering therapeutics to clinical trials to ultimately test the 'amyloid hypothesis' of AD.

Alzheimer's disease (AD) is a progressive, neurodegenerative disease histochemically characterized by extracellular deposits of amyloid beta (Abeta) protein and intracellular neurofibrillary tangles of hyperphosphorylated tau protein. AD is considered to be a complex, multifactorial syndrome, with numerous causal factors contributing to its pathogenesis. Thus, for any novel therapeutic molecule to have a "disease-modifying" effect on AD, it must be able to modulate multiple, synergistic targets simultaneously. In this context, we have studied two compounds of plant origin [withanolide A (1) and asiatic acid (2)] for their potential activities against multiple targets associated with Abeta pathways (BACE1, ADAM10, IDE, and NEP). BACE1 is a rate-limiting enzyme in the production of Abeta from amyloid-beta precursor protein (AbetaPP), while ADAM10 is involved in non-amyloidogenic processing of AbetaPP. IDE and NEP are two of the prominent enzymes involved in effectively degrading Abeta. It was found that both 1 and 2 significantly down-regulated BACE1 and also up-regulated ADAM10 in primary rat cortical neurons. In addition, 1 significantly up-regulated IDE levels, which may help in degrading excess Abeta from the AD brain. On the basis of the data obtained, the two multifunctional compounds may prove valuable in developing novel, effective therapeutics for the prevention and treatment of AD-associated amyloid pathology.

MicroRNAs (miRNAs) are emerging as a class of small regulated RNAs, and the alterations of miRNAs are implicated in the initiation and progression of human cancers. Our study shows that inhibition of miR-20a in OVCAR3 ovarian cancer cell line could suppress, whereas overexpression of miR-20a could enhance cell long-term proliferation and invasion. We also confirmed amyloid precursor protein (APP) as a direct target gene of miR- 20a. Furthermore, suppression of APP expression could also promote ovarian cancer cell proliferation and invasion, which is consistent with the results of miR-20a overexpression. Therefore, we concluded that the regulation of APP is an important mechanism for miR-20a to promote proliferation and invasion in ovarian cancer cells.

Mounting evidence suggests that Alzheimer's disease (AD) is caused by the accumulation of the small peptide, amyloid-β (Aβ), a proteolytic cleavage product of amyloid-β protein precursor (AβPP). Aβ is generated through a serial cleavage of AβPP by β- and γ-secretase. Aβ40 and Aβ42 are the two main components of amyloid plaques in AD brains, with Aβ42 being more prone to aggregation. AβPP can also be processed by α-secretase, which cleaves AβPP within the Aβ sequence, thereby preventing the generation of Aβ. Little is currently known regarding the effects of cell density on AβPP processing and Aβ generation. Here we assessed the effects of cell density on AβPP processing in neuronal and non-neuronal cell lines, as well as mouse primary cortical neurons. We found that decreased cell density significantly increases levels of Aβ40, Aβ42, total Aβ, and the ratio of Aβ42: Aβ40. These results also indicate that cell density is a significant modulator of AβPP processing. Overall, these findings carry profound implications for both previous and forthcoming studies aiming to assess the effects of various conditions and genetic/chemical factors, e.g., novel drugs on AβPP processing and Aβ generation in cell-based systems. Moreover, it is interesting to speculate whether cell density changes in vivo may also affect AβPP processing and Aβ levels in the AD brain.

Alzheimer's disease (AD) is a devastating age-related neurodegenerative disorder characterized by progressive impairment of cognition and short-term memory loss. The deposition of amyloid-beta (Abeta) 1-42 into senile plaques is an established feature of AD neuropathology. Controversy still exists about the amyloid pathway as the initiating mechanism or a mere consequence of the events leading to AD. Nevertheless, Abeta toxicity has been probed in vitro and in vivo and increased production or decreased clearance of Abeta peptides are reported to play a major role in the development of AD. Treatment of neural stem cells with Abeta in vitro induces neuronal differentiation. Increased neurogenesis has been also described in AD patients as well as in amyloid-beta protein precursor (AbetaPP) transgenic mice. Adult neurogenesis is greatly enhanced in young AbetaPP transgenic mice, before other AD-liked pathologies, and reduced in older animals. This increased neurogenesis at young ages might be the first pathology related to AD, which is detectable long before other harmful manifestation of the disease. Therefore, understanding the mechanisms of Abeta-induced neurogenesis will reveal insights into the pathogenesis of AD and may prove useful as an early AD biomarker.

The up-regulation of the angiogenic vascular endothelial growth factor (VEGF) in brains of Alzheimer patients in close relationship to beta-amyloid (Abeta) plaques, suggests a link of VEGF action and processing of the amyloid precursor protein (APP). To reveal whether VEGF may affect APP processing, brain slices derived from 17-month-old transgenic Tg2576 mice were exposed with 1ng/ml VEGF for 6, 24, and 72h, followed by assessing cytosolic and membrane-bound APP expression, level of both soluble and fibrillar Abeta-peptides, as well as activities of alpha- and beta-secretases in brain slice tissue preparations. Treatment of brain slices with VEGF did not significantly affect the expression level of APP, regardless of the exposure time studied. In contrast, VEGF exposure of brain slices for 6h reduced the formation of soluble, SDS extractable Abeta(1-40) and Abeta(1-42) as compared to brain slice cultures incubated in the absence of any drug, while the fibrillar Abeta peptides did not change significantly. This effect was less pronounced 24h after VEGF exposure, but was no longer detectable when brain slices were exposed by VEGF for 72h, which indicates an adaptive response to chronic VEGF exposure. The VEGF-mediated reduction in Abeta formation was accompanied by a transient decrease in beta-secretase activity peaking 6h after VEGF exposure. To reveal whether the VEGF-induced changes in soluble Abeta-level may be due to actions of VEGF on Abeta fibrillogenesis, the fibrillar status of Abeta was examined using the thioflavin-T binding assay. Incubation of Abeta preparations obtained from Tg2576 mouse brain cortex, in the presence of VEGF slightly decreased the fibrillar content with increasing incubation time up to 72h. The data demonstrate that VEGF may affect APP processing, at least in vitro, suggesting a role of VEGF in the pathogenesis of Alzheimer's disease.

The amyloid precursor protein (APP) is cleaved enzymatically by non-amyloidogenic and amyloidogenic pathways. alpha-Secretase cleaves APP within beta-amyloid protein (Abeta) sequence, resulting in the release of a secreted fragment of APP (sAPPalpha) and precluding Abeta generation. Cryptotanshinone (CTS), an active component of the medicinal herb Salvia miltiorrhiza, has been shown to improve learning and memory in several pharmacological models of Alzheimer's disease (AD). However, the effects of CTS on the Abeta plaque pathology and the APP processing in AD are unclear. Here we reported that CTS strongly attenuated amyloid plaque deposition in the brain of APP/PS1 transgenic mice. In addition, CTS significantly improved spatial learning and memory in APP/PS1 mice assessed by the Morris water maze testing. To define the exact molecular mechanisms involved in the beneficial effects of CTS, we investigated the effects of the CTS on APP processing in rat cortical neuronal cells overexpressing Swedish mutant human APP695. CTS was found to decrease Abeta generation in concentration-dependent (0-10muM) manner. Interestingly, the N-terminal APP cleavage product, sAPPalpha was markedly increased by CTS. Further study showed that alpha-secretase activity was increased by CTS. Taken together, our results suggested CTS improved the cognitive ability in AD transgenic mice and promoted APP metabolism toward the non-amyloidogenic products pathway in rat cortical neuronal cells. CTS shows a promising novel way for the therapy of AD.

Background: Epidemiological studies have suggested that long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced incidence of Alzheimer's disease (AD). Several mechanisms have been proposed to explain these findings including increased shedding of the soluble ectodomain of the amyloid precursor protein (sAPP), which functions as a neurotrophic and neuroprotective factor in vitroand in vivo., Objective: To clarify whether NSAIDs consistently stimulate sAPP secretion., Methods: 293-EBNA cells with stable overexpression of an APP-alkaline phosphatase fusion protein (APP-AP), SH-SY5Y and PC12 cells or primary telencephalic chicken neurons were treated with ibuprofen or indomethacin. APP shedding was then determined by measuring AP activity in conditioned media, Western blot analysis with antibodies against total sAPP or specific for sAPP-alpha, or in a pulse-chase paradigm., Results: AP activity in conditioned media was not increased after NSAID treatment of 293-EBNA cells whereas it was elevated by phorbol ester. Surprisingly, ibuprofen or indomethacin treatment of SH-SY5Y and PC12 cells expressing endogenous APP did not cause changes in sAPP or sAPP-alpha secretion or downregulation of cellular APP. These findings were further corroborated in primary chicken neuronal cultures., Conclusions: Using various experimental settings, we were unable to confirm sAPP or sAPP-alpha stimulation with the NSAIDs ibuprofen and indomethacin in transfected and nontransfected cells of neuronal and nonneuronal origin. Importantly, these findings seem to rule out chronic sAPP stimulation as an alternative mechanism of NSAID action in AD., (Copyright 2008 S. Karger AG, Basel.)

The effect of testosterone on the levels of the Alzheimer's disease amyloid-beta peptide (Abeta) was investigated in guinea pigs. Castrated guinea pigs (GPX) were administered testosterone at two different dosages, following which plasma and cerebrospinal fluid (CSF) Abeta&#95;{40} levels were measured. Plasma Abeta&#95;{40} levels were reduced in GPX in the early stages of low-dose testosterone treatment, whereas CSF Abeta&#95;{40} levels were only reduced by the time circulating testosterone had returned to untreated GPX levels. The supraphysiological testosterone dose did not reduce CSF Abeta&#95;{40} levels significantly until circulating testosterone was back to uncastrated levels, whereas plasma Abeta&#95;{40} levels significantly increased over time in these animals. These results indicate that the extent of testosterone-induced changes to Abeta&#95;{40} levels and their response rates depend on both the tissue examined and testosterone dosage.

Advanced age and mutations in the genes encoding amyloid precursor protein (APP) and presenilin (PS1) are two serious risk factors for Alzheimer's disease (AD). Finding common pathogenic changes originating from these risks may lead to a new therapeutic strategy. We observed a decline in memory performance and reduction in hippocampal long-term potentiation (LTP) in both mature adult (9-15 months) transgenic APP/PS1 mice and old (19-25 months) non-transgenic (nonTg) mice. By contrast, in the presence of bicuculline, a GABA(A) receptor antagonist, LTP in adult APP/PS1 mice and old nonTg mice was larger than that in adult nonTg mice. The increased LTP levels in bicuculline-treated slices suggested that GABA(A) receptor-mediated inhibition in adult APP/PS1 and old nonTg mice was upregulated. Assuming that enhanced inhibition of LTP mediates memory decline in APP/PS1 mice, we rescued memory deficits in adult APP/PS1 mice by treating them with another GABA(A) receptor antagonist, picrotoxin (PTX), at a non-epileptic dose for 10 days. Among the saline vehicle-treated groups, substantially higher levels of synaptic proteins such as GABA(A) receptor alpha1 subunit, PSD95, and NR2B were observed in APP/PS1 mice than in nonTg control mice. This difference was insignificant among PTX-treated groups, suggesting that memory decline in APP/PS1 mice may result from changes in synaptic protein levels through homeostatic mechanisms. Several independent studies reported previously in aged rodents both an increased level of GABA(A) receptor alpha1 subunit and improvement of cognitive functions by long term GABA(A) receptor antagonist treatment. Therefore, reduced LTP linked to enhanced GABA(A) receptor-mediated inhibition may be triggered by aging and may be accelerated by familial AD-linked gene products like Abeta and mutant PS1, leading to cognitive decline that is pharmacologically treatable at least at this stage of disease progression in mice.

It is known that 5-HT(4) receptor agonists increase sAPPalpha levels in the cortex and hippocampus of mice as well as in a model of Alzheimer's disease (AD). As sAPPalpha is thought to have pro-mnesic properties, we assessed whether its increase induces cognitive improvement in a spatial memory task and whether it reverses a scopolamine-induced memory deficit. Mice treated or not treated with scopolamine were trained in the Morris water maze for 3 days. Before the probe test, they received an injection of either a 5-HT(4) receptor agonist (prucalopride or RS 67333), or an acetylcholinesterase inhibitor (donepezil), or both drugs. As expected, scopolamine decreased performance, an effect that was not reversed by the drugs tested when injected alone. However, prucalopride (5 mg kg(-1), s.c.) acted synergistically with donepezil (0.75 mg kg(-1), s.c.) to counteract completely scopolamine-induced amnesia. Western blot analysis of tissue homogenates in the cortex and hippocampus shows that sAPPalpha levels did not differ between saline- and scopolamine-treated mice. Furthermore, a region-dependent drug action was observed since the scopolamine-treated mice display a tendency to increase sAPPalpha levels in the hippocampus after donepezil or in the cortex after prucalopride. Our results suggest that a combined treatment with a 5-HT(4) receptor agonist with an acetylcholinesterase inhibitor has beneficial effects on memory in mice. Moreover, it seems to enhance sAPPalpha levels in two brain regions highly affected in AD. Thus, a drug polytherapy could be interesting not only to enhance cognitive performance and decrease drawbacks but also to get the best action in each brain region.

Sequential cleavages of the beta-amyloid precursor protein cleaving enzyme 1 (BACE1) by beta-secretase and gamma-secretase generate the amyloid beta-peptides, believed to be responsible of synaptic dysfunction and neuronal cell death in Alzheimer's disease (AD). Levels of BACE1 are increased in vulnerable regions of the AD brain, but the underlying mechanism is unknown. Here we show that oxidative stress (OS) stimulates BACE1 expression by a mechanism requiring gamma-secretase activity involving the c-jun N-terminal kinase (JNK)/c-jun pathway. BACE1 levels are increased in response to OS in normal cells, but not in cells lacking presenilins or amyloid precursor protein. Moreover, BACE1 is induced in association with OS in the brains of mice subjected to cerebral ischaemia/reperfusion. The OS-induced BACE1 expression correlates with an activation of JNK and c-jun, but is absent in cultured cells or mice lacking JNK. Our findings suggest a mechanism by which OS induces BACE1 transcription, thereby promoting production of pathological levels of amyloid beta in AD.

Alpha-secretase (alpha-secretase), cleaves the amyloid precursor protein (APP) within the amyloid-beta (Abeta) sequence, resulting in the release of a secreted fragment of APP (alphaAPPs) and precluding Abeta generation. We investigated the effects of the acetylcholinesterase inhibitor, huperzine A (Hup A), on APP processing and Abeta generation in human neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. Hup A dose-dependently (0-10 microM) increased alphaAPPs release. Therefore, we evaluated two alpha-secretase candidates, a disintegrin and metalloprotease (ADAM) 10 and ADAM17 in Hup A-induced non-amyloidogenic APP metabolism. Hup A enhanced the level of ADAM10, and the inhibitor of tumor necrosis factor-alpha converting enzyme (TACE)/ADAM17 inhibited the Hup A-induced rise in alphaAPPs levels, further suggesting Hup A directed APP metabolism toward the non-amyloidogenic alpha-secretase pathway. Hup A had no effect on Abeta generation in this cell line. The steady-state levels of full-length APP and cell viability were unaffected by Hup A. Alpha-APPs release induced by Hup A treatment was significantly reduced by muscarinic acetylcholine receptor antagonists (particularly by an M1 antagonist), protein kinase C (PKC) inhibitors, GF109203X and calphostin C, and the mitogen-activated kinase kinase (MEK) inhibitors, U0126 and PD98059. Furthermore, Hup A markedly increased the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, which was blocked by treatment with U0126 and PD98059. In addition, Hup A inhibited acetylcholinesterase activity by 20% in neuroblastoma cells. Our results indicate that the activation of muscarinic acetylcholine receptors, PKC and MAP kinase may be involved in Hup A-induced alphaAPPs secretion in neuroblastoma cells and suggest multiple pharmacological mechanisms of Hup A regarding the treatment of Alzheimer's disease (AD).

While much of the focus on Alzheimer's disease therapeutics has been directed at beta-amyloid peptide or at cholinergic synaptic transmission, recent data suggest that targeting signal transduction by the amyloid precursor protein (APP) itself may be an alternative approach with significant potential [1]. Here we discuss the possibility that APP-mediated signal transduction, downstream from amyloid-beta peptide production itself, may be an appropriate therapeutic target in Alzheimer's disease.

One of the main neuropathological lesions observed at brain autopsy of Alzheimer's disease (AD) patients are the extracellular senile plaques mainly composed of amyloid-beta (Abeta) peptides. Abeta is generated by proteolytic processing of amyloid precursor protein (APP) via beta and gamma-secretases. The beta-secretase APP cleaving enzyme 1 (BACE1) has become a target of intense research aimed at blocking the enzyme activity. Recent studies showed that BACE1 is involved in processing other non-APP substrates, and that other proteases are involved in APP processing. We have recently established a novel approach to inhibit Abeta production via antibodies against the beta-secretase cleavage site of APP. These antibodies bind wild type and Swedish mutated APP expressed in transgenic mice brain tissues. The isolated antibodies do not bind any form of Abeta peptides. Antibody up-take experiments, using Chinese hamster ovary cells expressing wild-type APP, suggest that antibody internalization and trafficking are mediated via the endocytic pathway. Administration of antibodies to the cells growing media resulted in a considerable decrease in intracellular Abeta levels, as well as in the levels of the corresponding C-terminal fragment (C99). The relevance of intra-neuronal accumulation of mainly Abeta42 as an early event in AD pathogenesis suggests that this approach may be applicable as a novel therapeutic strategy in AD treatment.

Amyloid precursor protein generates the secreted amyloid precursor protein alpha, which protects hippocampal neurons from ischemic injury and facilitates neuronal survival and synaptogenesis in the developing nervous system. Here, we examined whether platelet-derived growth factor regulates the generation of secreted amyloid precursor protein alpha during the neuronal differentiation of hippocampal precursor cells, HiB5. We showed that platelet-derived growth factor promoted amyloid precursor protein production and secreted amyloid precursor protein alpha secretion. These effects of platelet-derived growth factor were diminished by the PI3K-specific inhibitor wortmannin and the protein kinase C-specific inhibitor GF109203X, suggesting the involvement of the PI3K and protein kinase C-signaling pathway. Furthermore, the conditioned media enriched with secreted amyloid precursor protein alpha promoted the survival of HiB5 cells during neuronal differentiation. These results suggest that the neurotrophic effect of platelet-derived growth factor is mediated in part via upregulation of the expression and release of secreted amyloid precursor protein alpha.

The Alzheimer's beta-amyloid peptides derive from the proteolytic processing of the beta-amyloid precursor protein, APP, by beta- and gamma-secretases. The regulation of this processing is not fully understood. Experimental evidence suggests that the activation of pathways involving protein tyrosine kinases, such as PDGFR and Src, could induce the cleavage of APP and in turn the generation of amyloid peptides. In this paper we addressed the effect of receptor and nonreceptor protein tyrosine kinases on the cleavage of APP and the mechanisms of their action. To this aim, we developed an in vitro system based on the APP-Gal4 fusion protein stably transfected in SHSY5Y neuroblastoma cell line. The cleavage of this molecule, induced by various stimuli, results in the activation of the transcription of the luciferase gene under the control of Gal4 cis-elements. By using this experimental system we demonstrated that, similarly to Src, three tyrosine kinases, TrkA, Ret and EGFR, induced the cleavage of APP-Gal4. We excluded that this effect was mediated by the activation of Ras-MAPK, PI3K-Akt and PLC-gamma pathways. Furthermore, the direct phosphorylation of the APP cytosolic domain does not affect Abeta peptide generation. On the contrary, experiments in cells lacking the LDL-receptor related protein LRP support the hypothesis that the interaction of APP with LRP is required for the induction of APP cleavage by tyrosine kinases., (2007 S. Karger AG, Basel)

In recent years, the most common pharmacological treatment for Alzheimer's disease (AD) has been acetylcholinesterase (AChE) inhibition. However, this single-target approach has limited effectiveness and there is evidence that a multitarget approach might be more effective. Huperzine A (HupA), a novel alkaloid isolated from a Chinese herb, has neuroprotective effects that go beyond the inhibition of AChE. Recent data have demonstrated that HupA can ameliorate the learning and memory deficiency in animal models and AD patients. Its potentially beneficial actions include modification of beta-amyloid peptide processing, reduction of oxidative stress, neuronal protection against apoptosis, and regulation of the expression and secretion of nerve growth factor (NGF) and NGF signaling.