Ectoine and hydroxyectoine inhibit aggregation and neurotoxicity of Alzheimer's beta-amyloid

beta-Amyloid peptide (Abeta) is the major constituent of senile plaques, the key pathological feature of Alzheimer's disease. Abeta is physiologically produced as a soluble form, but aggregation of Abeta monomers into oligomers/fibrils causes neurotoxic change of the peptide. In nature, many microorganisms accumulate small molecule chaperones (SMCs) under stressful conditions to prevent the misfolding/denaturation of proteins and to maintain their stability. Hence, it is conceivable that SMCs such as ectoine and hydroxyectoine could be potential inhibitors against the aggregate formation of Alzheimer's Abeta, which has not been studied to date. The current work shows the effectiveness of ectoine and hydroxyectoine on the inhibition of Abeta42 aggregation and toxicity to human neuroblastoma cells. The characterization tools used for this study include thioflavin-T induced fluorescence, atomic force microscopy and cell viability assay. Considering that ectoine and hydroxyectoine are not toxic to cellular environment even at concentrations as high as 100 mM, the results may suggest a basis for the development of ectoines as potential inhibitors associated with neurodegenerative diseases.     

Single chain Fv antibodies against the 25-35 Abeta fragment inhibit aggregation and toxicity of Abeta42

Alzheimer's disease (AD) is characterized by the deposition of amyloid-beta (Abeta) protein in the brain. Immunization studies have demonstrated that anti-Abeta antibodies reduce Abeta deposition and improve clinical symptoms seen in AD. However, conventional antibody-based therapies risk an inflammatory response that can result in meningoencephalitis and cerebral hemorrhage. Here we report on the development of human-based single chain variable domain antibody fragments (scFvs) directed against the Abeta 25-35 region as potential therapeutics for AD that do not risk an inflammatory response. The 25-35 region of Abeta represents a promising therapeutic target since it promotes aggregation and is highly toxic. Two scFvs with differing affinities for Abeta were studied, and both inhibited aggregation of Abeta42 as determined by thioflavin T binding assay and atomic force microscopy analysis and blocked Abeta-induced toxicity toward human neuroblastoma SH-SY5Y cells as determined by MTT and LDH release assays. These results provide additional evidence that scFvs against Abeta provide an attractive alternative to more conventional antibody-based therapeutics for controlling aggregation and toxicity of Abeta.     

Designing peptide inhibitors for oligomerization and toxicity of Alzheimer's beta-amyloid peptide

Convergent biochemical and genetic evidence suggests that the formation of beta-amyloid (Abeta) deposits in the brain is an important and, probably, seminal step in the development of Alzheimer's disease (AD). Recent studies support the hypothesis that Abeta soluble oligomers are the pathogenic species that prompt the disease. Inhibiting Abeta self-oligomerization could, therefore, provide a novel approach to treating the underlying cause of AD. Here, we designed potential peptide-based aggregation inhibitors containing Abeta amino acid sequences (KLVFF) from part of the binding region responsible for Abeta self-association (residues 16-20), with RG-/-GR residues added at their N- and C-terminal ends to aid solubility. Two such peptides (RGKLVFFGR, named OR1, and RGKLVFFGR-NH2, named OR2) were effective inhibitors of Abeta fibril formation, but only one of these peptides (OR2) inhibited Abeta oligomer formation. Interestingly, this same OR2 peptide was the only effective inhibitor of Abeta toxicity toward human neuroblastoma SH-SY5Y cells. Our data support the idea that Abeta oligomers are responsible for the cytotoxic effects of Abeta and identify a potential peptide inhibitor for further development as a novel therapy for AD.     

Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo

Alzheimer's disease (AD) involves amyloid beta (Abeta) accumulation, oxidative damage, and inflammation, and risk is reduced with increased antioxidant and anti-inflammatory consumption. The phenolic yellow curry pigment curcumin has potent anti-inflammatory and antioxidant activities and can suppress oxidative damage, inflammation, cognitive deficits, and amyloid accumulation. Since the molecular structure of curcumin suggested potential Abeta binding, we investigated whether its efficacy in AD models could be explained by effects on Abeta aggregation. Under aggregating conditions in vitro, curcumin inhibited aggregation (IC(50) = 0.8 microM) as well as disaggregated fibrillar Abeta40 (IC(50) = 1 microM), indicating favorable stoichiometry for inhibition. Curcumin was a better Abeta40 aggregation inhibitor than ibuprofen and naproxen, and prevented Abeta42 oligomer formation and toxicity between 0.1 and 1.0 microM. Under EM, curcumin decreased dose dependently Abeta fibril formation beginning with 0.125 microM. The effects of curcumin did not depend on Abeta sequence but on fibril-related conformation. AD and Tg2576 mice brain sections incubated with curcumin revealed preferential labeling of amyloid plaques. In vivo studies showed that curcumin injected peripherally into aged Tg mice crossed the blood-brain barrier and bound plaques. When fed to aged Tg2576 mice with advanced amyloid accumulation, curcumin labeled plaques and reduced amyloid levels and plaque burden. Hence, curcumin directly binds small beta-amyloid species to block aggregation and fibril formation in vitro and in vivo. These data suggest that low dose curcumin effectively disaggregates Abeta as well as prevents fibril and oligomer formation, supporting the rationale for curcumin use in clinical trials preventing or treating AD.     

Inhibition of amyloid fibrillogenesis and toxicity by a peptide chaperone

Aggregation of proteins in tissues is associated with several diseases, including Alzheimer's disease. It is characterized by the accumulation of amyloid beta peptide (Abeta) in the extracellular spaces of the brain cells, resulting in neuronal death and other pathological changes. alpha-Crystallin, a small heat-shock protein in lens, and a peptide chaperone having the functional site sequence DFVIFLDVKHFSPEDLTVK of alphaA-crystallin may inhibit Abeta fibrillogenesis and toxicity. The peptide chaperone (mini-alphaA-crystallin), having an Abeta interacting domain and a complex solubilizing domain, was shown in previous studies to prevent aggregation of several proteins under denaturing conditions. In this in vitro study, using transmission electron microscopy and thioflavin T binding assay, we show that mini-alphaA-crystallin arrests the fibril formation of Abeta peptides. Mini-alphaA-crystallin also suppresses the toxic action of Abeta on rat pheochromocytoma (PC 12) cells. The wide chaperoning capability of the peptide and its ability to inhibit amyloid fibril formation and suppress toxicity suggest that mini-alphaA-crystallin may serve as a universal chaperone in controlling diseases of protein aggregation, including Alzheimer's disease.     

Single chain variable fragments against beta-amyloid (Abeta) can inhibit Abeta aggregation and prevent abeta-induced neurotoxicity

Beta-amyloid (Abeta) is a major pathological determinant of Alzheimer's disease (AD). Both active and passive immunization studies have shown that antibodies against Abeta are effective in decreasing cerebral Abeta levels, reducing Abeta accumulation, and attenuating cognitive deficits in animal models of AD. However, the therapeutic potential of these antibodies in human AD patients is limited because of adverse inflammatory reactions and cerebral hemorrhaging associated with the treatments. Here we show that single chain variable fragments (scFv's) represent an attractive alternative to more conventional antibody-based therapeutics to reduce Abeta toxicity. The binding affinities and binding epitopes of two different scFv's to Abeta were characterized using a surface plasmon resonance (SPR) biosensor. An scFv binding the 17-28 region of Abeta effectively inhibited in vitro aggregation of Abeta as determined by thioflavin T (ThT) fluorescence staining and atomic force microscopy (AFM) analysis, while an scFv binding the carboxyl-terminal region of Abeta (residues 29-40) did not inhibit aggregation. The scFv to the 17-28 region when co-incubated with Abeta not only decreased aggregation but also eliminated any toxic effects of aggregated Abeta on the human neuroblastoma cell line, SH-SY5Y. The ability of scFv's to inhibit both aggregation and cytotoxicity of Abeta indicates that scFv's have potential therapeutic value for treating AD.     

Identification of a mutant amyloid peptide that predominantly forms neurotoxic protofibrillar aggregates

The amyloid peptide (Abeta), derived from the proteolytic cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases, undergoes multistage assemblies to fibrillar depositions in the Alzheimer's brains. Abeta protofibrils were previously identified as an intermediate preceding insoluble fibrils. While characterizing a synthetic Abeta variant named EV40 that has mutations in the first two amino acids (D1E/A2V), we discerned unusual aggregation profiles of this variant. In comparison of the fibrillogenesis and cellular toxicity of EV40 to the wild-type Abeta peptide (Abeta40), we found that Abeta40 formed long fibrillar aggregates while EV40 formed only protofibrillar aggregates under the same in vitro incubation conditions. Cellular toxicity assays indicated that EV40 was slightly more toxic than Abeta40 to human neuroblastoma SHEP cells, rat primary cortical, and hippocampal neurons. Like Abeta40, the neurotoxicity of the protofibrillar EV40 could be partially attributed to apoptosis since multiple caspases such as caspase-9 were activated after SHEP cells were challenged with toxic concentrations of EV40. This suggested that apoptosis-induced neuronal loss might occur before extensive depositions of long amyloid fibrils in AD brains. This study has been the first to show that a mutated Abeta peptide formed only protofibrillar species and mutations of the amyloid peptide at the N-terminal side affect the dynamic amyloid fibrillogenesis. Thus, the identification of EV40 may lead to further understanding of the structural perturbation of Abeta to its fibrillation.     

Compensatory function of transthyretin in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of age-related primary neurodegenerative diseases characterized by progressive memory loss, aphasia, and intellectual and mental breakdown. Pathogenesis of AD is based on the early synaptic dysfunction following neurodegeneration and neuronal death. According to modern concepts, the development of neuropathological processes is due to progressively deposited intermediates of amyloid fibrils that represent oligomers consisting of short peptide named amyloid beta protein (Abeta). In this context, it is reasonable to propose that one of the compensatory mechanisms of AD might be inhibition of Abeta oligomerization by sequestration or clearance of Abeta. Experiments with transgenic animals and epidemiological studies demonstrate that major protein of cerebrospinal fluid, transthyretin, is a natural neuroprotector that inhibits Abeta amyloid formation and restore cognitive functions. The study of Abeta-transthyretin complexes allowed to create peptides that are mimetics of transthyretin. These mimetics inhibit amyloid formation in vitro and, therefore, could be used in therapeutic treatment of AD.     

Role of glutathione in intracellular amyloid-alpha precursor protein/carboxy-terminal fragment aggregation and associated cytotoxicity

Abstract Alterations in glutathione (GSH) metabolism are associated with neurodegeneration in Alzheimer's disease (AD), and GSH depletion follows application of exogenous fibrillar amyloid beta (Abeta) peptides in experimental systems; these results are commonly cited as evidence of oxidative damage in AD. We used MC65 human neuroblastoma cells that conditionally express carboxy-terminal fragments of the Abeta precursor protein (Abeta/CTFs) to directly test the hypothesis that GSH is part of the cellular response to stressors associated with Abeta/CTF accumulation and not simply a marker of oxidative damage. Our data showed that Abeta/CTFs accumulated by post-translational processes and were associated with progressive increases in oxidative damage and cytotoxicity. Ethycrinic acid (EA) or diethyl maleate (DEM), reagents that deplete GSH through non-specific thiol adduction, gave rise to dose-dependent cytotoxicity that was independent of Abeta/CTF expression and minimally responsive to alpha-tocopherol (AT). In contrast, buthionine sulfoximine (BSO), a selective inhibitor of GSH synthase, not only augmented Abeta/CTF-associated cell death but unexpectedly potentiated Abeta/CTF accumulation; both outcomes were completely suppressed by AT. These data suggest that antioxidants may serve as 'Abeta targeting' therapies that suppress toxic protein aggregation rather than simply acting as downstream radical scavengers.     

Dimerisation of N-acetyl-L-tyrosine ethyl ester and Abeta peptides via formation of dityrosine

Alzheimer's disease (AD) is characterised by the formation of amyloid deposits composed primarily of the amyloid beta-peptide (Abeta). This peptide has been shown to bind redox active metals ions such as copper and iron, leading to the production of reactive oxygen species (ROS) and formation of hydrogen peroxide (H(2)O(2)). The generation of H(2)O(2) has been linked with Abeta neurotoxicity and neurodegeneration in AD. Because of the relative stability of a tyrosyl radical, the tyrosine residue (Tyr-10) is believed to be critical to the neurotoxicity of Abeta. This residue has also been shown to be important to Abeta aggregation and amyloid formation. It is possible that the formation of an Abeta tyrosyl radical leads to increased aggregation via the formation of dityrosine as an early aggregation step, which is supported by the identification of dityrosine in amyloid plaque. The role of dityrosine formation in Abeta aggregation and neurotoxicity is as yet undetermined, partly because there are no facile methods for the synthesis of Abeta dimers containing dityrosine. Here we report the use of horseradish peroxidase and H(2)O(2) to dimerise N-acetyl-L-tyrosine ethyl ester and apply the optimised conditions for dityrosine formation to fully unprotected Abeta peptides. We also report a simple fluorescent plate reader method for monitoring Abeta dimerisation via dityrosine formation.     

Salvianolic acid B inhibits Abeta fibril formation and disaggregates preformed fibrils and protects against Abeta-induced cytotoxicty

One of the major pathological features of Alzheimer's disease (AD) is the appearance of senile plaques characterized by extracellular aggregation of amyloid beta-peptide (Abeta) fibrils. Inhibition of Abeta fibril aggregation is therefore viewed as one possible method to halt the progression of AD. Salvianolic acid B (Sal B) is an active ingredient isolated from Salvia miltiorrhiza, a Chinese herbal medicine commonly used for the treatment of cardiovascular and cerebrovascular disorders. Recent findings show that Sal B prevents Abeta-induced cytotoxicity in a rat neural cell line. To understand the mechanism of Sal B-mediated neuroprotection, its effects on the inhibition of Abeta1-40 fibril formation and destabilization of the preformed Abeta1-40 fibrils were studied. The results were obtained using Thioflavin T fluorescence assay and Abeta aggregating immunoassay. We found that Sal B can inhibit fibril aggregation (IC(50): 1.54-5.37 microM) as well as destabilize preformed Abeta fibril (IC(50): 5.00-5.19 microM) in a dose- and time-dependent manner. Sal B is a better aggregation inhibitor than ferulic acid but less active than curcumin in the inhibition of Abeta1-40 aggregation. In electron microscope study, Sal B-treated Abeta1-40 fibrils are seen in various stages of shortening or wrinkling with numerous deformed aggregates of amorphous structure. Circular dichroism data indicate that Sal B dose dependently prevents the formation of beta-structured aggregates of Abeta1-40. Addition of preincubated Sal B with Abeta1-42 significantly reduces its cytotoxic effects on human neuroblastoma SH-SY5Y cells. These results suggest that Sal B has therapeutic potential in the treatment of AD, and warrant its study in animal models.     

Influence of fluorinated and hydrogenated nanoparticles on the structure and fibrillogenesis of amyloid beta-peptide

Peptide aggregation in amyloid fibrils is implicated in the pathogenesis of several diseases such as Alzheimer's disease. There is a strong correlation between amyloid fibril formation and a decrease in conformational stability of the native state. Amyloid-beta peptide (Abeta), the aggregating peptide in Alzheimer's disease, is natively unfolded. The deposits found in Alzheimer's disease are composed of Abeta fibrillar aggregates rich in beta-sheet structure. The influence of fluorinated complexes on the secondary structure and fibrillogenesis of Abeta peptide was studied by circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). CD spectra show that complexes of polyampholyte and fluorinated dodecanoic acid induce alpha-helix structure in Abeta, but their hydrogenated analogous lead to beta-sheet formation and aggregation. The fluorinated nanoparticles with highly negative zeta potential and hydrophobic fluorinated core have the fundamental characteristics to prevent Abeta fibrillogenesis.     

The toxicity of beta-amyloid is attenuated by interaction with its specific human scFv E3 in vitro

Beta-amyloid (Abeta) has been suggested as a potent neurotoxic agent. The Abeta-targeted immunotherapy aims to clear diffuse amyloid deposits and reverse memory deficits in Alzheimer's disease. We generated a human single chain variable domain antibody fragment (scFv) against Abeta40, termed E3, by screening a phage antibody library. E3 scFv could recognize Abeta in human cerebral cortex. It was able not only to prevent the aggregation of Abeta but also to disrupt the Abeta preexisting fibrils. Moreover, the Abeta toxicity to SK-N-SH cells was attenuated by addition of E3 scFv. Our results indicate that site-directed human scFv might be a potential therapeutic agent for Alzheimer's disease.     

Oxidative metabolites accelerate Alzheimer's amyloidogenesis by a two-step mechanism, eliminating the requirement for nucleation

The process of amyloid formation by the amyloid beta peptide (Abeta), i.e., the misassembly of Abetapeptides into soluble quaternary structures and, ultimately, amyloid fibrils, appears to be at the center of Alzheimer's disease (AD) pathology. We have shown that abnormal oxidative metabolites, including cholesterol-derived aldehydes, modify Abeta and accelerate the early stages of amyloidogenesis (the formation of spherical aggregates). This process, which we have termed metabolite-initiated protein misfolding, could explain why hypercholesterolemia and inflammation are risk factors for sporadic AD. Herein, the mechanism by which cholesterol metabolites hasten Abeta 1-40 amyloidogenesis is explored, revealing a process that has at least two steps. In the first step, metabolites modify Abeta peptides by Schiff base formation. The Abeta-metabolite adducts form spherical aggregates by a downhill polymerization that does not require a nucleation step, dramatically accelerating Abeta aggregation. In agitated samples, a second step occurs in which fibrillar aggregates form, a step also accelerated by cholesterol metabolites. However, the metabolites do not affect the rate of fibril growth in seeded aggregation assays; their role appears to be in initiating amyloidogenesis by lowering the critical concentration for aggregation into the nanomolar range. Small molecules that block Schiff base formation inhibit the metabolite effect, demonstrating the importance of the covalent adduct. Metabolite-initiated amyloidogenesis offers an explanation for how Abeta aggregation could occur at physiological nanomolar concentrations.     

Internalized antibodies to the Abeta domain of APP reduce neuronal Abeta and protect against synaptic alterations

Immunotherapy against beta-amyloid peptide (Abeta) is a leading therapeutic direction for Alzheimer disease (AD). Experimental studies in transgenic mouse models of AD have demonstrated that Abeta immunization reduces Abeta plaque pathology and improves cognitive function. However, the biological mechanisms by which Abeta antibodies reduce amyloid accumulation in the brain remain unclear. We provide evidence that treatment of AD mutant neuroblastoma cells or primary neurons with Abeta antibodies decreases levels of intracellular Abeta. Antibody-mediated reduction in cellular Abeta appears to require that the antibody binds to the extracellular Abeta domain of the amyloid precursor protein (APP) and be internalized. In addition, treatment with Abeta antibodies protects against synaptic alterations that occur in APP mutant neurons.     

Phosphorylated PP2A (tyrosine 307) is associated with Alzheimer neurofibrillary pathology

Down-regulation of protein phosphatase 2A (PP2A) is thought to play a critical role in tau hyperphosphorylation in Alzheimer's disease (AD). In vitro phosphorylation of PP2A catalytic subunit at Y307 efficiently inactivates PP2A. A specific antibody against phosphorylated (p) PP2A (Y307) (PP2Ac-Yp307) was used to investigate possible PP2A down-regulation by known pathophysiological changes associated with AD, such as Abeta accumulation and oestrogen deficiency. Immunohistochemistry and immunofluorescence confocal microscopy showed an aberrant accumulation of PP2Ac-Yp307 in neurons that bear pretangles or tangles in the susceptible brain regions, such as the entorhinal cortical cortex and the hippocampus. Experimentally, increased PP2Ac-Yp307 was observed in mouse N2a neuroblastoma cells that stably express the human amyloid precursor protein with Swedish mutation (APPswe) compared with wild-type, and in the brains of transgenic APPswe/ presenilin (PS1, A246E) mice, which corresponded to the increased tau phosphorylation. Treating N2a cells with Abeta25-35 mimicked the changes of PP2Ac-Yp307 and tau phosphorylation in N2a APPswe cells. Knockout of oestrogen receptor (ER) alpha or ERbeta gave similar changes of PP2Ac-Yp307 level and tau phosphorylation in the mouse brain. Taken together, these findings suggest that increased PP2A phosphorylation (Y307) can be mediated by Abeta deposition or oestrogen deficiency in the AD brain, and consequently compromise dephosphorylation of abnormally hyperphosphorylated tau, and lead to neurofibrillary tangle formation.     

Acyl peptide hydrolase, a serine proteinase isolated from conditioned medium of neuroblastoma cells, degrades the amyloid-beta peptide

Considerable evidence indicates that the amyloid-beta (Abeta) peptide, a proteolytic fragment of the amyloid precursor protein, is the pathogenic agent in Alzheimer's disease (AD). A number of proteases have been reported as capable of degrading Abeta, among them: neprilysin, insulin-degrading enzyme, endothelin-converting enzyme-1 and -2, angiotensin-converting enzyme and plasmin. These proteases, originating from a variety of cell types, degrade Abeta of various conformational states and in different cellular locations. We report here the isolation of a serine protease from serum-free conditioned medium of human neuroblastoma cells. Tandem mass spectrometry (MS/MS)-based sequencing of the isolated protein identified acyl peptide hydrolase (APH; EC3.4.19.1) as the active peptidase. APH is one of four members of the prolyl oligopeptidase family of serine proteases expressed in a variety of cells and tissues, including erythrocytes, liver and brain, but its precise biological activity is unknown. Here, we describe the identification of APH as an Abeta-degrading enzyme, and we show that the degradation of Abeta by APH isolated from transfected cells is inhibited by APH-specific inhibitors, as well as by synthetic Abeta peptide. In addition, we cloned APH from human brain and from neuroblastoma cells. Most importantly, our results indicate that APH expression in AD brain is lower than in age-matched controls.     

Induction of NADPH cytochrome P450 reductase by the Alzheimer β-protein. Amyloid as a 'foreign body'

A large body of data suggests that the Alzheimer's amyloid peptide (Abeta) causes degeneration and death of neurons by mechanisms that involve reactive oxygen species. The pathways involved in Abeta-mediated oxidative injury are only partially understood. We theorized that abnormal microaggregates and/or pathological conformations of Abeta peptides may behave as xenobiotics and trigger the induction of NADPH cytochrome P450 reductase (CP450r), an enzyme which, if induced by non-physiological substrates (such as xenobiotics like drugs or other 'foreign molecules'), is known to cause oxidative stress. In order to test this hypothesis, i.e. that Abeta can increase the expression of CP450r, SK-N-SH human neuroblastoma cells were exposed to Abeta25-35 and Abeta1-42 and then examined for induction of this enzyme in immunoblots, using specific antibodies. Following exposure to Abeta peptides, neuroblastoma cells showed a clear-cut induction of CP450r. To determine whether this mechanism is operational in vivo, we investigated the expression of CP450r in a transgenic mouse model of Alzheimer's disease (AD) and in brains from patients afflicted with AD, using an immunocytochemical approach. Tissue sections from brains of transgenic mice exhibited strong immunoreactivity for CP450r, surrounding amyloid deposits. The pattern of expression of CP450r was similar to that exhibited by neuritic and oxidative stress markers. Sections from non-transgenic mice showed no detectable immunoreactivity. Immunostaining of sections from four brains with neuropathologically confirmed AD showed a pattern of abnormality different from transgenic mice that was characterized by abnormal immunoreactivity for CP450r within the cytoplasm of cortical neurons. No labeling was seen in sections from aged-matched control brains. The data showed that CP450r is induced by Alzheimer amyloid peptide and that such a response must be considered as one possible mechanism whereby Abeta causes oxidative stress.     

Different tau epitopes define Abeta42-mediated tau insolubility

Alzheimer's disease (AD) is characterized by extracellular beta-amyloid (Abeta(42))-containing plaques and intracellular neurofibrillary tangles. The latter are composed of hyperphosphorylated filamentous aggregates of the microtubule-associated protein tau. Previously we demonstrated pathological interactions between these two histopathological hallmarks using human SH-SY5Y neuroblastoma cells overexpressing wild-type and mutant forms of human tau. Exposure to pre-aggregated forms of Abeta(42) caused both the formation of AD-like tau-containing filaments and a decreased solubility of tau, both of which were prevented by mutating the S422 phospho-epitope of tau. Here, we expressed additional tau mutants in SH-SY5Y cells to assess the role of phosphorylation and cleavage sites of tau in tau aggregation. We found that the Abeta(42)-mediated decrease in tau solubility depends on the interplay of distinct phospho-epitopes of tau and not only on phosphorylation of the S422 epitope.     

Determination of hypoxic effect on neprilysin activity in human neuroblastoma SH-SY5Y cells using a novel HPLC method

Alzheimer's disease (AD) is characterized by extracellular deposition of amyloid-beta-peptide (Abeta), which is closely associated with the metabolic balance between Abeta production and clearance activities. Neprilysin is one of the important enzymes to degrade Abeta in the brain and alternation of its activity would contribute to the AD neuropathology. However, measurement of neprilysin activity in neuronal cells, especially the extracellular activity, is very difficult because of its weak activities. In the present study, we established a sensitive method enough to estimate extracellular neprilysin activity of living cell cultivated in a 96-well plate using HPLC-fluorometric system, and investigated the effect of hypoxia, a closely associated event with neurodegenerative diseases, on neprilysin activity of human neuroblastoma SH-SY5Y cells. We demonstrated that chronic but not acute hypoxia significantly attenuated neprilysin activity without any alterations of neprilysin gene expression. The present study suggests that chronic hypoxia may down-regulate extracellular neprilysin activity of neuronal cells to impair Abeta degradation and associate with the development of amyloid pathology.