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.     

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(?)?=?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) [Leu(17) - Val(18) - Phe(19) - Phe(20) - Ala(21)] and glycine zipper motifs within the peptide fragment Aβ(17-35) were critical in binding and in fibrillogenesis confirming that nNOS was amyloidogenic catalyst.     

Effects of treadmill exercise on brain insulin signaling and β-amyloid in intracerebroventricular streptozotocin induced-memory impairment in rats

[Purpose]

The purpose of the study is to explore effect of 6 weeks treadmill exercise on brain insulin signaling and β-amyloid(Aβ).

[Methods]

The rat model of Alzheimer’s disease(AD) used in the present study was induced by the intracerebroventricular(ICV) streptozotocin(STZ). To produce the model of animal with AD, STZ(1.5mg/kg) was injected to a cerebral ventricle of both cerebrums of Sprague-Dawley rat(20 weeks). The experimental animals were divided into ICV-Sham(n=7), ICV-STZ CON(n=7), ICV-STZ EXE(n=7). Treadmill exercise was done for 30 min a day, 5 days a week for 6 weeks. Passive avoidance task was carried out before and after treadmill exercise.

[Results]

The results of this study show that treadmill exercise activated Protein kinase B(AKT)/ Glycogen synthase kinase 3α (GSK3α), possibly via activation of insulin receptor(IR) and insulin receptor substrate(IRS) and reduced Aβ in the brain of ICV-STZ rats. More interestingly, treadmill exercise improved cognitive function of ICV-STZ rats. Finally, physical exercise or physical activity gave positive influences on brain insulin signaling pathway.

[Conclusion]

Therefore, treadmill exercise can be applied to improve AD as preventive and therapeutic method.     

Exogenous seeding of cerebral β-amyloid deposition in βAPP-transgenic rats

Deposition of the amyloid-β (Aβ) peptide in senile plaques and cerebral Aβ angiopathy (CAA) can be stimulated in Aβ-precursor protein (APP)-transgenic mice by the intracerebral injection of dilute brain extracts containing aggregated Aβ seeds. Growing evidence implicates a prion-like mechanism of corruptive protein templating in this phenomenon, in which aggregated Aβ itself is the seed. Unlike prion disease, which can be induced de novo in animals that are unlikely to spontaneously develop the disease, previous experiments with Aβ seeding have employed animal models that, as they age, eventually will generate Aβ lesions in the absence of seeding. In the present study, we first established that a transgenic rat model expressing human APP (APP21 line) does not manifest endogenous deposits of Aβ within the course of its median lifespan (30?months). Next, we injected 3-month-old APP21 rats intrahippocampally with dilute Alzheimer brain extracts containing aggregated Aβ. After a 9-month incubation period, these rats had developed senile plaques and CAA in the injected hippocampus, whereas control rats remained free of such lesions. These findings underscore the co-dependence of agent and host in governing seeded protein aggregation, and show that cerebral Aβ-amyloidosis can be induced even in animals that are relatively refractory to the spontaneous origination of parenchymal and vascular deposits of Aβ.     

Antistress and angioprotective influence of nitric oxide in epilepsy-prone rats of Krushinskiĭ-Molodkina strain

In rats of Krushinsky-Molodkina strain (KMR), the audiogenic stress induced epileptiform seizure and development of acute disturbances of cerebral circulation of hemorrhagic nature. The inhibitor of NO-synthase (L-NNA) increased the severity of clinical symptoms, mortality, and the intensity of intracranial hemorrhages. In contrast, L-arginine elevated the resistance of KMRs to acoustic stress. The intensity of nitrergic innervation was analyzed in preparations of the middle cerebral artery with the use of histochemical NADPH-diaphorase staining. In preparations of control KMRs, a net of NADPH-positive perivascular nerve fibers was found, while in experimental KMRs, in an hour after sound stimulation, such fibers practically were not revealed. Preliminary exposure of KMRs in hypoxic conditions (1 hour in hypobaric chamber at simulated altitude of 5000 m above the sea level) decreased the development of stress lesions. The protective effect of hypoxic training disappeared after the administration of NO-synthase inhibitor (L-NNA). The study demonstrated participation of nitric oxide (NO) in adaptive reactions of cerebral hemodynamics linked with the significant increase of cerebral blood flow.     

Change of cholinergic transmission and memory deficiency induced by injection of β-amyloid protein into NBM of rats

The change of cholinergic transmission of p-amyloid protein (P-AP) treated rats was studied by intracerebral microdialysis sampling combined with HPLC analysis. β-AP1-40 was injected into nucleus basalis magnocellularis (NBM). Passive avoidance response test (step-down test) and delayed alternation task were used for memory testing. The impairment of memory after injection of β-AP1-40 into NBM exhibited mainly the deficiency of short-term working memory. One week after injection of β-AP1-40 the release of acetylcholine (ACh) from frontal cortex of freely-moving rats decreased significantly, and the response of cholinergic nerve ending to the action of high [K+] solution was rather weak. In control animals the percentage of increase of ACh-release during behavioral performance was 57%, while in β-AP1-40-treated rats it was 34%. The temporary increase of the ACh-release of the rat put into a new place was also significantly diminished in β-AP1-40 -treated rats. The results show that the injection of      

Are cyclooxygenase-2 and nitric oxide involved in the dyskinesia of Parkinson's disease induced by l-DOPA?

Inflammatory mechanisms are proposed to play a role in l-DOPA-induced dyskinesia. Cyclooxygenase-2 (COX2) contributes to inflammation pathways in the periphery and is constitutively expressed in the central nervous system. Considering that inhibition of nitric oxide (NO) formation attenuates l-DOPA-induced dyskinesia, this study aimed at investigating if a NO synthase (NOS) inhibitor would change COX2 brain expression in animals with l-DOPA-induced dyskinesia. To this aim, male Wistar rats received unilateral 6-hydroxydopamine microinjection into the medial forebrain bundle were treated daily with l-DOPA (21 days) combined with 7-nitroindazole or vehicle. All hemi-Parkinsonian rats receiving l-DOPA showed dyskinesia. They also presented increased neuronal COX2 immunoreactivity in the dopamine-depleted dorsal striatum that was directly correlated with dyskinesia severity. Striatal COX2 co-localized with choline-acetyltransferase, calbindin and DARPP-32 (dopamine-cAMP-regulated phosphoprotein-32), neuronal markers of GABAergic neurons. NOS inhibition prevented l-DOPA-induced dyskinesia and COX2 increased expression in the dorsal striatum. These results suggest that increased COX2 expression after l-DOPA long-term treatment in Parkinsonian-like rats could contribute to the development of dyskinesia.     

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.     

Change of cholinergic transmission and memory deficiency induced by injection of β-amyloid protein into NBM of rats

The change of cholinergic transmission of ?-amyloid protein (β-AP) treated rats was studied by intracerebral microdialysis sampling combined with HPLC analysis. β-AP_1—40 was injected into nucleus basalis magnocellularis (NBM). Passive avoidance response test (step-down test) and delayed alternation task were used for memory testing. The impairment of memory after injection of β-AP_1—40 into NBM exhibited mainly the deficiency of short-term working memory. One week after injection of β-AP_1—40 the release of acetylcholine (ACh) from frontal cortex of freely-moving rats decreased significantly, and the response of cholinergic nerve ending to the action of high [K⁺] solution was rather weak. In control animals the percentage of increase of ACh-release during behavioral performance was 57%, while in β-AP_1—40-treated rats it was 34%. The temporary increase of the ACh-release of the rat put into a new place was also significantly diminished in β-AP_1—40-treated rats. The results show that the injection of β-AP_1—40 into NBM impairs the cholinergic transmission in frontal cortex, and the impairment of cholinergic transmission may be the main cause of the deficit of working memory.     

The effect of the use of copper carbonate and copper nanoparticles in the diet of rats on the level of β-amyloid and acetylcholinesterase in selected organs

BackgroundCopper has an important role in nervous system function, as a cofactor of many enzymes and in the synthesis of neurotransmitters. Both the dose and the chemical form of copper can determine the impact of this element on metabolism, the neurological system and the immune system.AimsThe aim of the study was to determine whether and in what form the addition of copper changes the level of amyloid beta and acetylcholinesterase level in selected rat tissues.MethodsThirty, healthy, male, albino Wistar rats aged 7 weeks were randomly divided into 3 groups. Three experimental treatments were used to evaluate the effects of different levels and sources of Cu (6.5 mg kg of diet) in the diet: Cu₀ – rats fed a diet without Cu supplementation; Cu_salt – rats fed a diet with CuCO₃ (6.5 mg kg of diet) during two months of feeding; Cu_NPs - rats fed a diet with Cu nanoparticles (6.5 mg kg of diet) during two months of feeding. In blood serum and tissue homogenates there rated the indicators proving the potential neurodegenerative effect and epigenetic DNA damage induced by chemical form of copper or lack of additional copper supplementation in diet were determined. There were analysed: level of acetylcholinesterase, β-amyloid, low-density lipoprotein receptor-related protein 1, apyrimidinic endonuclease, thymidine glycosidase, alkylpurine-DNA-N-glycosylase and glycosylated acetylcholinesterase.ResultsIrrespective of the form of copper added, it was found to increase acetylcholinesterase level in the brain, spleen and liver, as well as in the blood plasma of the rats. Copper in the form of CuCO₃ was found to increase acetylcholinesterase level in the kidneys. The addition of both forms of copper caused a marked increase in the plasma concentration of β-amyloid in comparison with the diet with no added Cu. The addition of both forms of copper caused a marked increase in the plasma concentration of β-amyloid in comparison with the diet with no added Cu.ConclusionsA lack of added Cu in the diet of rats reduces the concentration of amyloid-β in the blood, whereas administration of copper, in the form of either CuNPs or CuCO₃, increases the level of this peptide in the blood. The use of copper in the form of CuNPs in the diet of rats does not increase the level of β-amyloid more than the use of the carbonate form of this element. The use of CuNPs or CuCO₃ in the diet of rats increases acetylcholinesterase level in the brain, spleen, liver, and blood. CuNPs in the diet of rats were not found to increase acetylcholinesterase level to a greater extent than Cu⁺² carbonate.     

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.     

α-bisabolol β-D-fucopyranoside as a potential modulator of β-amyloid peptide induced neurotoxicity: An in vitro & in silico study

Alzheimer’s disease (AD) is a multifaceted neurodegenerative disorder affecting the elderly people. For the AD treatment, there is inefficiency in the existing medication, as these drugs reduce only the symptoms of the disease. Since multiple pathological proteins are involved in the development of AD, searching for a single molecule targeting multiple AD proteins will be a new strategy for the management of AD. In view of this, the present study was designed to synthesize and evaluate the multifunctional neuroprotective ability of the sesquiterpene glycoside α-bisabolol β-D-fucopyranoside (ABFP) against multiple targets like acetylcholinesterase, oxidative stress and β-amyloid peptide aggregation induced cytotoxicity. In silico computational docking and simulation studies of ABFP with acetylcholinesterase (AChE) showed that it can interact with Asp74 and Thr75 residues of the enzyme. The in vitro studies showed that the compound possess significant ability to inhibit the AChE enzyme apart from exhibiting antioxidant, anti-aggregation and disaggregation properties. In addition, molecular dynamics simulation studies proved that the interacting residue between Aβ peptide and ABFP was found to be involved in Leu34 and Ile31. Furthermore, the compound was able to protect the Neuro2 a cells against Aβ_25-35 peptide induced toxicity. Overall, the present study evidently proved ABFP as a neuroprotective agent, which might act as a multi-target compound for the treatment of Alzheimer’s disease.     

Lutein protects against β-amyloid peptide-induced oxidative stress in cerebrovascular endothelial cells through modulation of Nrf-2 and NF-κb

In the present study, we determined the protective role of lutein against Aβ _25–35 peptide-induced oxidative stress and apoptosis in bEND.3 cells. Cell viability was determined through MTT assay. Reactive oxygen species, lipid peroxides, and antioxidant enzyme activities were evaluated to analyze the oxidative stress status. NF-κB and Nrf-2 downstream target protein expressions were determined through western blot. Apoptosis was analyzed through caspase activities and subG1 accumulation. The results showed that Aβ _25–35 significantly increased (p < 0.001) oxidative stress biomarkers. Aβ _25–35 significantly up-regulated NF-κB nuclear expression and down-regulated Nrf-2 levels and HO-1 and, NQO-1 expressions. Aβ _25–35 induced apoptosis through decreasing mitochondrial membrane potential and increasing caspase 9 and 3 activities. Lutein pre-treatment significantly (p < 0.001) improved cell viability and decreased ROS levels (p < 0.001) and lipid peroxidation (p < 0.01). Lutein prevented Aβ _25–35-induced NF-κB nuclear expressions and up-regulated Nrf-2 expressions. Further, lutein also improved mitochondrial membrane potential and down-regulated caspase activities and subG1 accumulation. The present study shows the protective role of lutein against Aβ _25–35-induced toxicity by modulating Nrf-2 and NF-κB expressions in cerebrovascular endothelial cells.     

In vitro and in vivo comparisons of the effects of the fruiting body and mycelium of Antrodia camphorata against amyloid β-protein-induced neurotoxicity and memory impairment

Antrodia camphorata is a particular and precious medicinal mushroom, and its fruiting body was found to provide more efficient protection from oxidative stress and inflammation than its mycelium because of its higher content of triterpenoids, total phenols, and so on. In the previous in vitro studies, the mycelium of A. camphorata is proven to provide strong neuroprotection in neuron cells and suggested to have the potential of protection against neurotoxicity of amyloid β-protein (Aβ) known as the risk factor toward Alzheimer's disease (AD) development. However, the in vivo study and the comparison study with the fruiting body have not yet been investigated. This study compared the effect of the fruiting body and mycelium of A. camphorata on alleviating the Aβ40-induced neurocytotoxicity in the in vitro Aβ-damaged neuron cell model (PC-12 cell treated with Aβ40) and memory impairment in the in vivo AD animal model induced with a continuous brain infusion of Aβ40. In the results of in vitro and in vivo studies, the fruiting body possessed stronger anti-oxidative and anti-inflammatory abilities for inhibiting neurocytotoxicity in Aβ40-treated PC-12 cells and Aβ40 accumulation in Aβ40-infused brain than mycelium. Moreover, hyperphosphorylated tau (p-tau) protein expression, known as an important AD risk factor, was suppressed by the treatment of fruiting body rather than that of mycelium in the in vitro and in vivo studies. These comparisons supported the reasons why the fruiting body resulted in a more significant improvement effect on working memory ability than mycelium in the AD rats.     

Activation of the neutrophil and loss of plasma glutathione during Mg-deficiency – modulation by nitric oxide synthase inhibition

Sprague-Dawley rats (200 g) were fed either a Mg-deficient or Mg-sufficient diet for 3 weeks. An enriched neutrophil fraction (>85%) was isolated from the blood by sodium metrizoate/dextran gradient centrifugation. Using the superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay, the basal activity of neutrophils isolated from the Mg-deficient rats were found elevated 5 fold after two weeks, and up to 7 fold after three weeks on the diet. Upon challenge by phorbol myristate acetate (PMA), unlike the Mg-sufficient cells, the Mg-deficient cells exhibited no significant activation. Treatment of the Mg-deficient rats with the nitric oxide (NO)-synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) in the drinking water, significantly attenuated the basal superoxide producing activity of the neutrophils and partially restored its response to PMA challenge. In association with the neutrophil activation. Mg-deficiency resulted in 70% decrease in plasma glutathione and 220% increase in Fe-promoted, thiobarbituric acid reactive substance (TBARS) levels; both changes were significantly attenuated by L-NAME treatment. The results suggest that neutrophils from Mg-deficient rats are activated endogenously to generate oxy-radicals which might directly mediate the in vivo peroxidative indices during Mg-deficiency. Furthermore, the neutrophil activity was lowered by NO-synthase inhibition suggesting that NO overproduction during Mg-deficiency participates in the neutrophil activation process.     

The protective effects of tanshinone IIA on neurotoxicity induced by β-amyloid protein through calpain and the p35/Cdk5 pathway in primary cortical neurons

The characteristic pathological change of Alzheimer's disease (AD) include deposits of β-amyloid protein (Aβ) in brain, neurofibrillary tangles (NFTs), as well as a few neuronal loss. Evidence shows that Aβ causes calcium influx and induces the cleavage of p35 into p25. Furthermore, the binding of p25 to cyclin-dependent kinase 5 (Cdk5) constitutively activates Cdk5. The p25/Cdk5 complex then hyperphosphorylates tau. Tanshinone IIA (tanIIA), a natural product extracted from Chinese herbal medicine Salvia miltiorrhiza BUNGE, has been reported to exert antioxidative activity. However, its neuroprotective activity remains unclear. The present study determined whether tanIIA protects neurons against Aβ(25-35)-induced cytotoxicity and detected the association of this protective effect with calpain and the p35/Cdk5 pathway. The results showed that tanIIA protected neurons against the neurotoxicity of Aβ(25-35), increased the viability of neurons, decreased expression of phosphorylated tau in neurons induced by Aβ(25-35), improved the impairment of the cell ultrastructure (such as nuclear condensation and fragmentation, and neurofibril collapse). Further more, we found that tanIIA maintained the normal expression of p35 on peripheral membranes, and decreased p25 expression in the cytoplasm. TanIIA also inhibited the translocation of Cdk5 from the nucleus into the cytoplasm of primary neurons induced by Aβ(25-35). These data suggested that tanIIA possessed neuroprotective action and the protection may involve in calpain and the p35/Cdk5 pathway.     

The prophylactic action of (−)-Epicatechin against alloxan induced diabetes in rats

(?)-Epicatechin (1) was isolated from the bark of an Indian medicinal plant $\text{Pterocarpus}$$\text{marsupium}$ Roxb. the water extract of which is used as an antidiabetic drug (2). (?)-Epicatechin administration to albino rats of either sex in doses of 30 mg/kg (i.p.) for two days prior to alloxan (150 mg/kg i.p.) administration, and continued for next 24 hours was able to protect the animals against the diabetogenic actions of alloxan. The protection by (?)-epicatechin may be due to scavenging of the deleterious and highly reactive hydroxyl radical which is generated by alloxan.     

Involvement of β-Carotene in the Antioxidant Defense of the Bacterial Cell

Effects of recombinant -carotene on the resistance of E. coli culture to menadione and paraquat were studied. The presence of -carotene in E. coli cells prevented, to a considerable extent, an increase in superoxide dismutase activity (induced by redox mediators) without affecting the culture growth. These findings suggest that -carotene is involved in the defense of cells against oxidative stress.