Association of β-amyloid peptide fragments with neuronal nitric oxide synthase: Implications in the etiology of Alzheimers disease

Neuronal nitric oxide synthase (nNOS) was purified on DEAE-Sepharose anion-exchange in a 38% yield, with 3-fold recovery and specific activity of 5 µmol.min^?1.mg^?1. The enzyme was a heterogeneous dimer of molecular mass 225?kDa having a temperature and pH optima of 40°C and 6.5, K_m and V_max of 2.6 μM and 996 nmol.min^?1.ml^?1, respectively and was relatively stable at the optimum conditions (t_½?=?3?h). β-Amyloid peptide fragments Aβ_17–28 was the better inhibitor for nNOS (K_i?=?0.81 µM). After extended incubation of nNOS (96?h) with each of the peptide fragments, Congo Red, turbidity and thioflavin-T assays detected the presence of soluble and insoluble fibrils that had formed at a rate of 5?nM.min^?1. A hydrophobic fragment Aβ_17–21 [Leu₁₇ – Val₁₈ – Phe₁₉ – Phe₂₀ – Ala₂₁] and glycine zipper motifs within the peptide fragment Aβ_17–35 were critical in binding and in fibrillogenesis confirming that nNOS was amyloidogenic catalyst.     

Methionine oxidation: Implications for the mechanism of toxicity of the β-amyloid peptide from Alzheimer's disease

Summary The amyloid β-peptide, Aβ is toxic to neurons and this toxicity plays a central role in the progression of Alzheimer's disease. The mechanism(s) by which Aβ exerts its toxicity has been hotly debated with several theories postulated. Here we discuss the role of oxidation of the sulfur atom of Met35 in Aβ42 (Met(O)Aβ), a modification that has significant implications for the mechanism of Aβ toxicity. Both Met(O)Aβ and its native form display toxicity to primary neuronal cells in culture which can be rescued by catalase, a H₂O₂ inhibitor and clioquinol a mild copper chelator. However both native Aβ and Met(O)Aβ differ substantially in primary and secondary structures, solubility, ability to penetrate lipid membranes, and oligomerization profiles. It is clearly evident that metals play an important role in the oxidation of Aβ to Met(O)Aβ via Fenton chemistry and that regulation of this pathway has a potential therapeutic application for the regulation of Alzheimer's disease.     

Cross interaction of β-amyloid peptide and prion protein fragments

Summary This article presents kinetic studies of cross interaction of β-amyloid peptide and prion protein fragments. Syntheses of three peptides (β25-35, β22-35 and PrP 109–126) were performed. Those peptides were used for aggregation studies in PBS and TRIS buffers using HPLC with DAD detector. Comparison of aggregation of peptides alone and in combination with other fragments was investigated. In all cases aggregation was faster in PBS than in TRIS solution. Obtained results suggest that β-amyloid peptide and prion protein may interact to form macromolecular complexes with different ability for aggregation.     

Investigating the binding interactions of galantamine with β-amyloid peptide

The anti-Alzheimer’s agent galantamine is known to possess anti-amyloid properties. However the exact mechanisms are not clear. We studied the binding interactions of galantamine with amyloid peptide dimer (Aβ_1–40) through molecular docking and molecular dynamics simulations. Galantamine’s binding site within the amyloid peptide dimer was identified by docking experiments and the most stable complex was analyzed by molecular dynamics simulation. These studies show that galantamine was interacting with the central region of the amyloid dimer (Lys16–Ala21) and the C-terminal region (Ile31–Val36) with minimum structural drift of Cα atom in those regions. Strikingly, a significant drift was observed at the turn region from Asp23-Gly29 (Cα atom RMSD = 9.2 Å and 11.6 Å at 50 fs and 100 fs respectively). Furthermore, galantamine’s binding mode disrupts the key pi–pi stacking interaction between aromatic rings of Phe19 (chain A) and Phe19 (chain B) and intermolecular hydrogen bonds seen in unbound peptide dimer. Noticeably, the azepine tertiary nitrogen of galantamine was in close proximity to backbone CO of Leu34 (distance <3.5 Å) to stabilize the dimer conformation. In summary, the results indicate that galantamine binding to amyloid peptide dimer leads to a significant conformational change at the turn region (Asp23–Gly29) that disrupts interactions between individual β-strands and promotes a nontoxic conformation of Aβ_1–40 to prevent the formation of neurotoxic oligomers.     

Nitric oxide control of cardiac function: is neuronal nitric oxide synthase a key component?

Nitric oxide (NO) has been shown to regulate cardiac function, both in physiological conditions and in disease states. However, several aspects of NO signalling in the myocardium remain poorly understood. It is becoming increasingly apparent that the disparate functions ascribed to NO result from its generation by different isoforms of the NO synthase (NOS) enzyme, the varying subcellular localization and regulation of NOS isoforms and their effector proteins. Some apparently contrasting findings may have arisen from the use of non-isoform-specific inhibitors of NOS, and from the assumption that NO donors may be able to mimic the actions of endogenously produced NO. In recent years an at least partial explanation for some of the disagreements, although by no means all, may be found from studies that have focused on the role of the neuronal NOS (nNOS) isoform. These data have shown a key role for nNOS in the control of basal and adrenergically stimulated cardiac contractility and in the autonomic control of heart rate. Whether or not the role of nNOS carries implications for cardiovascular disease remains an intriguing possibility requiring future study.     

Glycation exacerbates the neuronal toxicity of β-amyloid

Accumulation evidence shows that β-amyloid (Aβ) is a neurotoxic and accumulation of Aβ is responsible for the pathology of Alzheimer''s disease (AD). However, it is currently not fully understood what makes Aβ toxic and accumulated. Previous studies demonstrate that Aβ is a suitable substrate for glycation, producing one form of the advanced glycation endproducts (AGEs). We speculated that Aβ-AGE formation may exacerbate the neurotoxicity. To explore whether the Aβ-AGE is more toxic than the authentic Aβ and to understand the molecular mechanisms, we synthesized glycated Aβ by incubating Aβ with methylglyoxal (MG) in vitro and identified the formation of glycated Aβ by fluorescence spectrophotometer. Then, we treated the primary hippocampal neurons cultured 8 days in vitro with Aβ-AGE or Aβ for 24 h. We observed that glycation exacerbated neurotoxicity of Aβ with upregulation of receptor for AGE (RAGE) and activation of glycogen synthase kinase-3 (GSK-3), whereas simultaneous application of RAGE antibody or GSK-3 inhibitor reversed the neuronal damages aggravated by glycated Aβ. Thereafter, we found that Aβ is also glycated with an age-dependent elevation of AGEs in Tg2576 mice, whereas inhibition of Aβ-AGE formation by subcutaneously infusion of aminoguanidine for 3 months significantly rescued the early cognitive deficit in mice. Our data reveal for the first time that the glycated Aβ is more toxic. We propose that the glycated Aβ with the altered secondary structure may be a more suitable ligand than Aβ for RAGE and subsequent activation of GSK-3 that can lead to cascade pathologies of AD, therefore glycated Aβ may be a new therapeutic target for AD.     

Spin trapping nitric oxide from neuronal nitric oxide synthase: A look at several iron–dithiocarbamate complexes

The free radical, nitric oxide (√NO), is responsible for a myriad of physiological functions. The ability to verify and study √NO in vivo is required to provide insight into the events taking place upon its generation and in particular the flux of √NO at relevant cellular sites. With this in mind, several iron-chelates (Fe²⁺(L)₂) have been developed, which have provided a useful tool for the study and identification of √NO through spin-trapping and electron paramagnetic resonance (EPR) spectroscopy. However, the effectiveness of √NO detection is dependent on the Fe²⁺(L)₂ complex. The development of more efficient and stable Fe²⁺(L)₂ chelates may help to better understand the role of √NO in vivo. In this paper, we present data comparing several proline derived iron–dithiocarbamate complexes with the more commonly used spin traps for √NO, Fe²⁺-di(N-methyl-D-glutamine-dithiocarbamate) (Fe²⁺(MGD)₂) and Fe²⁺-di(N-(dithiocarboxy)sarcosine) (Fe²⁺(DTCS)₂). We evaluate the apparent rate constant (k_app) for the reaction of √NO with these Fe²⁺(L)₂ complexes and the stability of the corresponding Fe²⁺(NO)(L)₂ in presence of NOS I.     

Control of nitric oxide synthase expression by transforming growth factor-β: Implications for homeostasis

Production of nitric oxide (NO) can be stimulated by inflammatory cytokines and bacterial lipopolysaccharide (LPS) in mammalian cells via an inducible nitric oxide synthase (iNOS). Conversely, the transforming growth factor-βs (TGF-βs) suppress NO production by reducing iNOS expression. Production of NO leads to disparate consequences, some beneficial and some damaging to the host, depending on the cell and context in which iNOS is induced. The TGF-βs counter these NO-mediated processes in macrophages, cardiac myocytes, smooth muscle cells, bone marrow cells, and retinal pigment epithelial cells. Autocrine or paracrine production of TGF-β may thus serve as a physiological counterbalance for iNOS expression, a mechanism which may be subverted by pathogens and tumors for their own survival. A greater understanding of the mechanisms and consequences of NO and TGF-β production may lead to effective therapeutic strategies in various diseases.     

Involvement of the nitric oxide signaling in modulation of naringin against intranasal manganese and intracerbroventricular β-amyloid induced neurotoxicity in rats

Manganese -induced aggregation of the amyloid-β peptide (Aβ) is a hallmark molecular feature of Alzheimer's disease (AD). The current study was designed to investigate the effects of chronic administration of naringin against β-A_1–42 and manganese induced experimental model. Wistar rats received intracerebroventricular (ICV) β-A_1–42 once, intranasal manganese, naringin and nitric oxide modulators for 21 days and behavioral alterations were assessed. Mitochondrial enzymes, oxidative parameters, TNF-α, β-A_1–42 acetylcholinesterase (AChE) levels and manganese concentration were measured. ICV β-A_1–42 and intranasal manganese treated rats showed a memory deficit and significantly increased in β-A_1–42 level and manganese concentration, mitochondrial oxidative damage, AChE level and inflammatory mediator in the hippocampus and cortex. Chronic administration of naringin (40 and 80 mg/kg) significantly improved memory performance and attenuated the oxidative damage and mitochondrial dysfunction in Aβ with Mn treated rats. In addition, naringin also attenuates the pro-inflammatory cytokines like TNF-α, AChE, Amyloid deposition and Mn concentration. Further, pretreatment of N(G)-Nitro-L-arginine methyl ester (L-NAME) with (5 mg/kg) with lower dose of naringin significantly potentiated its protective effect. These results demonstrate that naringin offers protection against ICV β-A_1–42 and intranasal manganese induced memory dysfunction possibly due to its antioxidant, anti-inflammatory, anti-amyloidogenesis therefore, could have a therapeutic potential in Alzheimer's disease.     

β-amyloid decreases detectable endothelial nitric oxide synthase in human erythrocytes: a role for membrane acetylcholinesterase

Until few years ago, many studies of Alzheimer's disease investigated the effects of this syndrome in the central nervous system. Only recently, the detection of amyloid beta peptide (Aβ) in the blood has evidenced the necessity to extend studies on extraneuronal cells, particularly on erythrocytes. Aβ is also present in brain capillaries, where it interacts with the erythrocytes, inducing several metabolic and functional alterations. Recently, functionally active endothelial type nitric oxide synthase (eNOS) was discovered in human erythrocytes. The goal of the present study was to evidence the effect of Aβ on erythrocyte eNOS. We found that Aβ following to 24-h exposure causes a decrease in the immune staining of erythrocyte eNOS. Concurrently, Aβ alters erythrocyte cell morphology, decreases nitrites and nitrates levels, and affects membrane acetylcholinesterase activity. Propidium, an acetylcholinesterase inhibitor, was able to reverse the effects elicited by Aβ. These events could contribute to the vascular alterations associated with Alzheimer's disease disease. Copyright ? 2012 John Wiley & Sons, Ltd.     

A peptide that binds specifically to the β-amyloid of Alzheimer's disease: selection and assessment of anti-β-amyloid neurotoxic effects

The accumulation of the amyloid-β peptide (Aβ) into amyloid plaques, an essential event in Alzheimer''s disease (AD) pathogenesis, has caused researchers to seek compounds that physiologically bind Aβ and modulate its aggregation and neurotoxicity. In order to develop new Aβ-specific peptides for AD, a randomized 12-mer peptide library with Aβ_1-10 as the target was used to identify peptides in the present study. After three rounds of selection, specific phages were screened, and their binding affinities to Aβ_1-10 were found to be highly specific. Finally, a special peptide was synthesized according to the sequences of the selected phages. In addition, the effects of the special peptide on Aβ aggregation and Aβ-mediated neurotoxicity in vitro and in vivo were assessed. The results show that the special peptide not only inhibited the aggregation of Aβ into plaques, but it also alleviated Aβ-induced PC12 cell viability and apoptosis at appropriate concentrations as assessed by the cell counting kit-8 assay and propidium iodide staining. Moreover, the special peptide exhibited a protective effect against Aβ-induced learning and memory deficits in rats, as determined by the Morris water maze task. In conclusion, we selected a peptide that specifically binds Aβ_1-10 and can modulate Aβ aggregation and Aβ-induced neuronal damage. This opens up possibilities for the development of a novel therapeutic approach for the treatment of AD.     

β Common receptor integrates the erythropoietin signaling in activation of endothelial nitric oxide synthase

Erythropoietin (EPO), the key hormone for erythropoiesis, also increases nitric oxide (NO) bioavailability in endothelial cells (ECs), yet the definitive mechanisms are not fully understood. Increasing evidence has demonstrated that β common receptor (βCR) plays a crucial role in EPO-mediated non-hematopoietic effects. We investigated the role of βCR in EPO-induced endothelial NO synthase (eNOS) activation in bovine aortic ECs (BAECs) and the molecular mechanisms involved. Results of confocal microscopy and immunoprecipitation analyses revealed that βCR was colocalized and interacted with EPO receptor (EPOR) in ECs. Inhibition of βCR or EPOR by neutralizing antibodies or small interfering RNA abolished the EPO-induced NO production. Additionally, blockage of βCR abrogated the EPO-induced increase in the phosphorylation of eNOS, Akt, Src, or Janus kinase 2 (JAK2). Immunoprecipitation analysis revealed that treatment with EPO increased the interaction between βCR and eNOS, which was suppressed by inhibition of Src, JAK2, or Akt signaling with specific pharmacological inhibitors. Furthermore, EPO-induced EC proliferation, migration, and tube formation were blocked by pretreatment with βCR antibody and Src, JAK2, or PI3K/Akt inhibitors. Moreover, in vivo experiments showed that EPO increased the level of phosphorylated eNOS, Src, JAK2, and Akt, as well as βCR-eNOS association in aortas and promoted the angiogenesis in Matrigel plug, which was diminished by βCR or EPOR neutralizing antibodies. Our findings suggest that βCR may play an integrative role in the EPO signaling-mediated activation of eNOS in ECs.     

Structural basis of C-terminal β-amyloid peptide binding by the antibody ponezumab for the treatment of Alzheimer's disease

Alzheimer's disease, the most common cause of dementia in the elderly and characterized by the deposition and accumulation of plaques, is composed in part of β-amyloid (Aβ) peptides, loss of neurons, and the accumulation of neurofibrillary tangles. Here, we describe ponezumab, a humanized monoclonal antibody, and show how it binds specifically to the carboxyl (C)-terminus of Aβ40. Ponezumab can label Aβ that is deposited in brain parenchyma found in sections from Alzheimer's disease casualties and in transgenic mouse models that overexpress Aβ. Importantly, ponezumab does not label full-length, non-cleaved amyloid precursor protein on the cell surface. The C-terminal epitope of the soluble Aβ present in the circulation appears to be available for ponezumab binding because systemic administration of ponezumab greatly elevates plasma Aβ40 levels in a dose-dependent fashion after administration to a mouse model that overexpress human Aβ. Administration of ponezumab to transgenic mice also led to a dose-dependent reduction in hippocampal amyloid load. To further explore the nature of ponezumab binding to Aβ40, we determined the X-ray crystal structure of ponezumab in complex with Aβ40 and found that the Aβ40 carboxyl moiety makes extensive contacts with ponezumab. Furthermore, the structure-function analysis supported this critical requirement for carboxy group of AβV40 in the Aβ-ponezumab interaction. These findings provide novel structural insights into the in vivo conformation of the C-terminus of Aβ40 and the brain Aβ-lowering efficacy that we observed following administration of ponezumab in transgenic mouse models.     

Reduction of histone cytotoxicity by the Alzheimer β-amyloid peptide precursor

In a search for Alzheimer β-amyloid peptide precursor ligands, Potempska et al. (Arch. Biochem. Biophys. (1993) 304, 448) found that histones bind with high affinity and specificity to the secreted precursor. Because exogenous histones can be cytotoxic, we compared the effects of histones on the viability of cells which produce little β-amyloid peptide precursor (U-937) to those on cells that produce twenty times as much precursor (COS-7). Addition of purified histones caused necrosis of U-937 cells (histone H4, LD₅₀=1.5 μM). Extracellular Aβ precursor in the submicromolar range prevented histone-induced U-937 cell necrosis. Cell-surface precursor also reduced histone toxicity: COS-7 cells were less sensitive to the toxic effects of histone H4 (LD₅₀=5.4 μM). COS-7 cells in which the expression of an APP mRNA-directed ribozyme reduced the synthesis of the protein by up to 80% were more sensitive to histone H4 (LD₅₀=3.2 μM) than cells that expressed the vector alone. Histone H4 binds to cell-associated Aβ precursor. Cells expressing the Aβ precursor-directed ribozyme bound less ¹²⁵I-labeled histone H4 than those expressing the vector alone. In the limited extracellular space of tissues in vivo, both secreted and cell-surface Aβ precursor protein may play significant roles in trapping chromatin or histones and removing them from the extracellular milieu.     

Alzheimer's disease: Effects of β-amyloid on mitochondria

The impairment of the respiratory chain or defects in the detoxification system can decrease electron transfer efficiency, reduce ATP production, and increase reactive oxygen species (ROS) production by mitochondria. Accumulation of ROS results in oxidative stress, a hallmark of neurodegenerative diseases such as Alzheimer's disease (AD). β-amyloid has been implicated in the pathogenesis of AD, and its accumulation may lead to degeneration of neuronal or non-neuronal cells. There is evidence that β-amyloid interacts with mitochondria but little is known concerning the significance of this interaction in the physiopathology of AD. This review explores possible mechanisms of β-amyloid-induced mitochondrial toxicity.     

Importance of nitric oxide synthase in the anchoring structures of Neobenedenia girellae – an important fish parasite

Specimens of Neobenedenia girellae, recognized as one of the most important pathogen of fish, were collected in order to find evidence of the nitric oxide synthase (NOS) and its possible role in haptor control. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western Blotting technique were used on a homogenate of parasites; immunohistochemistry technique was applied on whole mount parasites, which were thereby investigated with confocal laser scanning microscope. Images obtained by immunohistochemistry show a strong evidence of the enzyme presence in the posterior body of N. girellae specimens, colocalizing with α-tubulin; focusing on the distribution one could speculate that the constitutive form of NOS is expressed. Samples investigated with Western Blotting show a moderate evidence of NOS presence too. The investigation of the role of NOS in regulating the most important attaching structures of N. girellae is important not only from the standpoint of biochemical knowledge but also in terms of development of new pharmacological strategies for the eradication of these parasites from aquacultures.     

Can modulation of endothelial nitric oxide synthase explain the vasculoprotective actions of statins?

Statin-based drugs are the mainstay of therapy for coronary artery disease. Recent insights into the cellular mechanisms of this class of drugs suggest that modulation of nitric oxide synthesis contributes to their beneficial actions. These effects are independent of their lipid-lowering effects and result in enhanced nitric oxide release.