Role of GSK-3beta activation and alpha7 nAChRs in Abeta(1-42)-induced tau phosphorylation in PC12 cells

β-amyloid peptide 1–42 (Aβ_1–42) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer's disease. Emerging evidence indicates that Aβ_1–42 can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Aβ_1–42-induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Aβ_1–42 increased phosphorylation of tau at serine-202 as detected by AT8 antibody. This Aβ_1–42-induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase-3β (GSK-3β) at tyrosine-216 (GSK-3β-pY216), which was partially inhibited by the GSK-3β inhibitor, CHIR98023. Aβ_1–42-induced tau phosphorylation and increase in GSK-3β-pY216 phosphorylation were also partially attenuated by α7 nicotinic acetylcholine receptor (α7 nAChR) selective ligands including agonist A-582941 and antagonists methyllycaconitine and α-bungarotoxin. The α7 nAChR agonist and the GSK-3β inhibitor had no additive effect. These observations suggest that α7 nAChR modulation can influence Aβ_1–42-induced tau phosphorylation, possibly involving GSK-3β. This study provides evidence of nAChR mechanisms underlying Aβ_1–42 toxicity and tau phosphorylation, which, if translated in vivo , could provide additional basis for the utility of α7 nAChR ligands in the treatment of Alzheimer's disease.     

α-Secretase-Derived Product of β-Amyloid Precursor Protein Is Decreased by Presenilin 1 Mutations Linked to Familial Alzheimer's Disease

Abstract: Recent reports indicate that missense mutations on presenilin (PS) 1 are likely responsible for the main early-onset familial forms of Alzheimer's disease (FAD). Consensual data obtained through distinct histopathological, cell biology, and molecular biology approaches have led to the conclusion that these PS1 mutations clearly trigger an increased production of the 42-amino-acid-long species of β-amyloid peptide (Aβ). Here we show that overexpression of wild-type PS1 in HK293 cells increases Aβ40 secretion. By contrast, FAD-linked mutants of PS1 trigger increased secretion of both Aβ40 and Aβ42 but clearly favor the production of the latter species. We also demonstrate that overexpression of the wild-type PS1 augments the α-secretase-derived C-terminally truncated fragment of β-amyloid precursor protein (APPα) recovery, whereas transfectants expressing mutated PS1 secrete drastically lower amounts of APPα when compared with cells expressing wild-type PS1. This decrease was also observed when comparing double transfectants overexpressing wild-type β-amyloid precursor protein and either PS1 or its mutated congener M146V-PS1. Altogether, our data indicate that PS mutations linked to FAD not only trigger an increased ratio of Aβ42 over total Aβ secretion but concomitantly down-regulate the production of APPα.     

Matrix metalloproteinase inhibition reduces oxidative stress associated with cerebral amyloid angiopathy in vivo in transgenic mice

Cerebral amyloid angiopathy (CAA), characterized by extracellular β-amyloid peptide (Aβ) deposits in vessel walls, is present in the majority of cases of Alzheimer's disease and is a major cause of hemorrhagic stroke. Although the molecular pathways activated by vascular Aβ are poorly understood, extracellular matrix metalloproteinases (MMP) and Aβ-induced oxidative stress appear to play important roles. We adapted fluorogenic assays for MMP activity and reactive oxygen species generation for use in vivo . Using multiphoton microcopy in APPswe/PS1dE9 and Tg-2576 transgenic mice, we observed strong associations between MMP activation, oxidative stress, and CAA deposition in leptomeningeal vessels. Antioxidant treatment with α-phenyl- N -tert-butyl-nitrone reduced oxidative stress associated with CAA (∼50% reduction) without affecting MMP activation. Conversely, a selection of agents that inhibit MMP by different mechanisms of action, including minocycline, simvastatin, and GM6001, reduced not only CAA-associated MMP activation (∼30–40% reduction) but also oxidative stress (∼40% reduction). The inhibitors of MMP did not have direct antioxidant effects. Treatment of animals with α-phenyl- N -tert-butyl-nitrone or minocycline did not have a significant effect on CAA progression rates. These data suggest a close association between Aβ-related MMP activation and oxidative stress in vivo and raise the possibility that treatment with MMP inhibitors may have beneficial effects by indirectly reducing the oxidative stress associated with CAA.     

Presenilin 1 Mutations Linked to Familial Alzheimer's Disease Increase the Intracellular Levels of Amyloid β-Protein 1–42 and Its N-Terminally Truncated Variant(s) Which Are Generated at Distinct Sites

Abstract: Mutations in the presenilin genes PS1 and PS2 cause the most common form of early-onset familial Alzheimer's disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid β-protein (Aβ) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuro-blastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1 (M146L or Δexon 10). The induction of mutated PS1 increased the intracellular levels of two distinct Aβ species ending at residue 42 that were likely to be Aβ1–42 and its N-terminally truncated variant(s) Aβx-42. The induction of mutated PS1 resulted in a higher level of intracellular Aβ1–42 than of intracellular Aβx-42, whereas extracellular levels of Aβ1–42 and Aβx-42 were increased proportionally. In addition, the intracellular generation of these Aβ42 species in wt and mutated PS1 -induced cells was completely blocked by brefeldin A, whereas it exhibited differential sensitivities to monensin: the increased accumulation of intracellular Aβx-42 versus inhibition of intracellular Aβ1–42 generation. These data strongly suggest that Aβx-42 is generated in a proximal Golgi, whereas Aβ1–42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific γ-secretase cleavage that occurs in the normal β-amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to β-secretase cleavage or (b) in the distinct sites where Aβx-42 and Aβ1–42 are generated.     

Generation and Regulation of β-Amyloid Peptide Variants by Neurons

Abstract: Studies of processing of the Alzheimer β-amyloid precursor protein (βAPP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways: the "β-secretase" pathway, which generates β-amyloid (Aβ_1–40/42; ∼4 kDa), and the "α-secretase" pathway, which generates a smaller fragment, the "p3" peptide (Aβ_17–40/42; ∼3 kDa). To determine whether similar processing events underlie βAPP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [³⁵S]methionine pulse-labeled primary neuronal cultures contained 4- and 3-kDa Aβ-related species. Radiosequencing analysis revealed that the 4-kDa band corresponded to conventional Aβ beginning at position Aβ(Asp¹), whereas both radio-sequencing and immunoprecipitation-mass spectrometry analyses indicated that the 3-kDa species in these conditioned media began with Aβ(Glu¹¹) at the N terminus, rather than Aβ(Leu¹⁷) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble βAPP_α release and decreased generation of both the 4-kDa Aβ and the 3-kDa N-truncated Aβ. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing Aβ secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant Aβ variant peptides and emphasize the role of protein phosphatases in modulating neuronal Aβ generation.     

Amyloid beta peptide and NMDA induce ROS from NADPH oxidase and AA release from cytosolic phospholipase A2 in cortical neurons

Increase in oxidative stress has been postulated to play an important role in the pathogenesis of a number of neurodegenerative diseases including Alzheimer's disease. There is evidence for involvement of amyloid-β peptide (Aβ) in mediating the oxidative damage to neurons. Despite yet unknown mechanism, Aβ appears to exert action on the ionotropic glutamate receptors, especially the N-methyl-D-aspartic acid (NMDA) receptor subtypes. In this study, we showed that NMDA and oligomeric Aβ_1–42 could induce reactive oxygen species (ROS) production from cortical neurons through activation of NADPH oxidase. ROS derived from NADPH oxidase led to activation of extracellular signal-regulated kinase 1/2, phosphorylation of cytosolic phospholipase A₂α (cPLA₂α), and arachidonic acid (AA) release. In addition, Aβ_1–42-induced AA release was inhibited by d (−)-2-amino-5-phosphonopentanoic acid and memantine, two different NMDA receptor antagonists, suggesting action of Aβ through the NMDA receptor. Besides serving as a precursor for eicosanoids, AA is also regarded as a retrograde messenger and plays a role in modulating synaptic plasticity. Other phospholipase A₂ products such as lysophospholipids can perturb membrane phospholipids. These results suggest an oxidative-degradative mechanism for oligomeric Aβ_1–42 to induce ROS production and stimulate AA release through the NMDA receptors. This novel mechanism may contribute to the oxidative stress hypothesis and synaptic failure that underline the pathogenesis of Alzheimer's disease.     

Glutamine Synthetase-Induced Enhancement of β-Amyloid Peptide Aβ(1–40) Neurotoxicity Accompanied by Abrogation of Fibril Formation and Aβ Fragmentation

Abstract: β-Amyloid peptide (Aβ) is the main constituent in both senile plaques and diffuse deposits in Alzheimer's diseased brains. It was previously shown that synthetic Aβs were able to form free radical species in aqueous solution and cause both oxidative damage to cell proteins and inactivation of key metabolic enzymes. We also previously demonstrated that an interaction of Aβ(1–40) with the oxidatively sensitive enzyme glutamine synthetase (GS) resulted in both inactivation of GS and an increase of Aβ toxicity to hippocampal cell cultures. In the present study the enhancement of Aβ toxicity during interaction with GS was found to be accompanied by abrogation of fibril formation and partial fragmentation of Aβ(1–40). HPLC elution profiles demonstrated the production of several peptide fragments. Analysis of the amino acid sequence of the major fragments identified them as the first 15 and the last six amino acids of Aβ(1–40). The fragmentation of Aβ was inhibited by immunoprecipitation of GS.     

Proinflammatory cytokine interferon-γ increases induction of indoleamine 2,3-dioxygenase in monocytic cells primed with amyloid β peptide 1–42: implications for the pathogenesis of Alzheimer's disease

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme of the kynurenine pathway of tryptophan metabolism, ultimately leading to production of the excitotoxin quinolinic acid (QUIN) by monocytic cells. In the Tg2576 mouse model of Alzheimer's disease, systemic inflammation induced by lipopolysaccharide leads to an increase in IDO expression and QUIN production in microglia surrounding amyloid plaques. We examined whether the IDO over-expression in microglia could be mediated by brain proinflammatory cytokines induced during the peripheral inflammation using THP-1 cells and peripheral blood mononuclear cells (PBMC) as models for microglia. THP-1 cells pre-treated with 5–25 μM amyloid β peptide (Aβ) (1–42) but not with Aβ (1–40) or Aβ (25–35) became an activated state as indicated by their morphological changes and enhanced adhesiveness. IDO expression was only slightly increased in the reactive cells but strongly enhanced following treatment with proinflammatory cytokine interferon-γ (IFN-γ) but not with interleukin-1β, tumor necrosis factor-α, or interleukin-6 at 100 U/mL. The concomitant addition of Aβ (1–42) with IFN-γ was totally ineffective, indicating that Aβ pre-treatment is prerequisite for a high IDO expression. The priming effect of Aβ (1–42) for the IDO induction was also observed for PBMC. These findings suggest that IFN-γ induces IDO over-expression in the primed microglia surrounding amyloid plaques.     

Rescue of Aβ1–42-induced memory impairment in day-old chick by facilitation of astrocytic oxidative metabolism: implications for Alzheimer's disease

Administration of small oligomeric β-amyloid (Aβ)_1–42 45 min before one-trial bead discrimination learning in day-old chicks abolishes consolidation of learning 30 min post-training (Gibbs et al. Neurobiol. Aging , in press). Administration of the β₃-adrenergic agonist CL316243, which specifically stimulates astrocytic but not neuronal glucose uptake, rescues Aβ impaired memory. Weakly reinforced training can be consolidated by various metabolic substrates and we have demonstrated neuronal dependence on oxidative metabolism of glucose soon after training versus astrocytic glucose dependence 20 min later. Based on these findings we examined whether different metabolic substrates were able to counteract memory inhibition by Aβ_1–42. Although lactate, the medium-chain fatty acid octanoate, and the ketone body β-hydroxybutyrate consolidated weakly reinforced training when injected close to learning, none of them were able to salvage Aβ-impaired memory; at this early time. All three metabolites and the astrocytic-specific acetate consolidated weak learning and rescued Aβ-impaired memory when injected 10–20 min post-training. However, neither glucose nor insulin rescued memory when injected at 20 min. Rescue of memory by providing astrocytes with alternative substrates for oxidative metabolism suggests that Aβ_1–42 exerts its amnestic effects specifically by impairing astrocytic glycolysis.     

Increased Activity-Regulating and Neuroprotective Efficacy of α-Secretase-Derived Secreted Amyloid Precursor Protein Conferred by a C-Terminal Heparin-Binding Domain

Abstract: Proteolytic cleavage of β-amyloid precursor protein (βAPP) by α-secretase results in release of one secreted form (sAPP) of APP (sAPPα), whereas cleavage by β-secretase releases a C-terminally truncated sAPP (sAPPβ) plus amyloid β-peptide (Aβ). βAPP mutations linked to some inherited forms of Alzheimer's disease may alter its processing such that levels of sAPPα are reduced and levels of sAPPβ increased. sAPPαs may play important roles in neuronal plasticity and survival, whereas Aβ can be neurotoxic. sAPPα was ∼100-fold more potent than sAPPβ in protecting hippocampal neurons against excitotoxicity, Aβ toxicity, and glucose deprivation. Whole-cell patch clamp and calcium imaging analyses showed that sAPPβ was less effective than sAPPα in suppressing synaptic activity, activating K⁺ channels, and attenuating calcium responses to glutamate. Using various truncated sAPPα and sAPPβ APP695 products generated by eukaryotic and prokaryotic expression systems, and synthetic sAPP peptides, the activity of sAPPα was localized to amino acids 591–612 at the C-terminus. Heparinases greatly reduced the actions of sAPPαs, indicating a role for a heparin-binding domain at the C-terminus of sAPPα in receptor activation. These findings indicate that alternative processing of βAPP has profound effects on the bioactivity of the resultant sAPP products and suggest that reduced levels of sAPPα could contribute to neuronal degeneration in Alzhiemer's disease.     

Neuroprotective effects of donepezil through inhibition of GSK-3 activity in amyloid-β-induced neuronal cell death

Acetylcholinesterase inhibitors (AChE-inhibitors) are used for the treatment of Alzheimer's disease. Recently, the AChE-inhibitor donepezil was found to have neuroprotective effects. However, the protective mechanisms of donepezil have not yet been clearly identified. We investigated the neuroprotective effects of donepezil and other AChE-inhibitors against amyloid-β1–42 (Aβ42)-induced neurotoxicity in rat cortical neurons. To evaluate the neuroprotective effects of AChE-inhibitors, primary cultured cortical neurons were pre-treated with several concentrations of AChE-inhibitors for 24 h and then treated with 20 μM Aβ42 for 6 h. In addition to donepezil, other AChE-inhibitors (galantamine and huperizine A) also showed increased neuronal cell viability against Aβ42 toxicity in a concentration-dependent manner. However, we demonstrated that donepezil has a more potent effect in inhibiting glycogen synthase kinase-3 (GSK-3) activity compared with other AChE-inhibitors. The neuroprotective effects of donepezil were blocked by LY294002 (10 μM), a phosphoinositide 3 kinase inhibitor, but only partially by mecamylamine (10 μM), a blocker of nicotinic acetylcholine receptors. Additionally, donepezil's neuroprotective mechanism was related to the enhanced phosphorylation of Akt and GSK-3β and reduced phosphorylation of tau and glycogen synthase. These results suggest that donepezil prevents Aβ42-induced neurotoxicity through the activation of phosphoinositide 3 kinase/Akt and inhibition of GSK-3, as well as through the activation of nicotinic acetylcholine receptors.     

Mechanism of docosahexaenoic acid-induced inhibition of in vitro Aβ1–42 fibrillation and Aβ1–42-induced toxicity in SH-S5Y5 cells

The mechanism of the effect of docosahexaenoic acid (DHA; C22:6, n -3), one of the essential brain nutrients, on in vitro fibrillation of amyloid β (Aβ_1–42), Aβ_1–42-oligomers and its toxicity imparted to SH-S5Y5 cells was studied with the use of thioflavin T fluorospectroscopy, laser confocal microfluorescence, and transmission electron microscopy. The results clearly indicated that DHA inhibited Aβ_1–42-fibrill formation with a concomitant reduction in the levels of soluble Aβ_1–42 oligomers. The polymerization (into fibrils) of preformed oligomers treated with DHA was inhibited, indicating that DHA not only obstructs their formation but also inhibits their transformation into fibrils. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12.5%), Tris–Tricine gradient(4–20%) gel electrophoresis and western blot analyses revealed that DHA inhibited at least 2 species of Aβ_1–42 oligomers of 15–20 kDa, indicating that it hinders these on-pathway tri/tetrameric intermediates during fibrillation. DHA also reduced the levels of dityrosine and tyrosine intrinsic fluorescence intensity, indicating DHA interrupts the microenvironment of tyrosine in the Aβ_1–42 backbone. Furthermore, DHA protected the tyrosine from acrylamide collisional quenching, as indicated by decreases in Stern–Volmer constants. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction efficiency and immunohistochemical examination suggested that DHA inhibits Aβ_1–42-induced toxicity in SH-S5Y5 cells. Taken together, these data suggest that by restraining Aβ_1–42 toxic tri/tetrameric oligomers, DHA may limit amyloidogenic neurodegenerative diseases, Alzheimer's disease.     

Alzheimer's β-Amyloid Peptide 1–42 Induces a Phagocytic Response in Murine Microglia

Abstract: β-Amyloid (Aβ) peptides are a key component of the senile plaques that characterize Alzheimer's disease. Cytokine-producing microglia have been shown to be intimately associated with amyloid deposits and have also been implicated as scavengers responsible for clearing Aβ deposits. However, little is known about the initial activation of these microglia or the effect of Aβ on phagocytosis. Murine BV-2 microglia were used to assess the effect of synthetic Aβ 1–42 on phagocytosis by quantifying uptake of fluorescent microspheres, acetylated low-density lipoproteins, and zymosan particles by flow cytometry. Aβ 1–42 stimulated microglial phagocytosis in a time- and dose-dependent manner. Aβ fibrils produced the greatest potentiation, and once activated, phagocytosis remained elevated after removal of Aβ from the cultures. Aβ-stimulated phagocytosis could be blocked if proteoglycans were first complexed to Aβ fibrils. These data suggest that Aβ fibrils act as an immune signal to stimulate microglial phagocytosis and that extracellular matrix molecules may modify Aβ function.     

Retinoid X receptor-α mediates (R )-flurbiprofen's effect on the levels of Alzheimer's β-amyloid

Alzheimer's disease (AD) is characterized by the formation of extracellular senile plaques in the brain, whose major component is a small peptide called β-amyloid (Aβ). Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) has been found beneficial for AD and several reports suggest that NSAIDs reduce the generation of Aβ, especially the more amyloidogenic form Aβ42. However, the exact mechanism underlying NSAIDs' effect on AD risk remains largely inconclusive and all clinical trials using NSAIDs for AD treatment show negative results so far. Recent studies have shown that some NSAIDs can bind to certain nuclear receptors, suggesting that nuclear receptors may be involved in NSAID's effect on AD risk. Here we find that ( R )-flurbiprofen, the R -enantiomer of the racemate NSAID flurbiprofen, can significantly reduce Aβ secretion, but at the same time, increases the level of intracellular Aβ. In addition, we find that a nuclear receptor, retinoid X receptor α (RXRα), can regulate Aβ generation and that down-regulation of RXRα significantly increases Aβ secretion. We also show that ( R )-flurbiprofen can interfere with the interaction between RXRα and 9- cis -retinoid acid, and that 9- cis -retinoid acid decreases ( R )-flurbiprofen's reduction of Aβ secretion. Moreover, the modulation effect of ( R )-flurbiprofen on Aβ is abolished upon RXRα down-regulation. Together, these results suggest that RXRα can regulate Aβ generation and is also required for ( R )-flurbiprofen-mediated Aβ generation.     

Traumatic Brain Injury Increases β-Amyloid Peptide 1-42 in Cerebrospinal Fluid

Abstract: The β-amyloid peptides, Aβ1-42 and Aβ1-40, were quantified in ventricular CSF taken daily for up to 3 weeks from six individuals with severe traumatic brain injury (TBI). There was considerable interindividual variability in the levels of Aβ peptides, but in general Aβ1-42 levels equalled or exceeded those of Aβ1-40. Averaging the daily totals of our trauma cohort revealed that the levels of Aβ1-42 and Aβ1-40 rose after injury, peaking in the first week and then declining toward control levels over the next 2 weeks. Aβ1-42 levels were on average two to three times higher in the trauma cohort than in CSF from nontrauma samples. Compared with nontrauma samples, the Aβ1-40/Aβ1-42 ratio decreased about fivefold in the trauma patients, further indicative of increased Aβ1-42 levels. The ratio remained low at all time points studied. No change was measured in the levels of β-amyloid precursor protein during the same interval. These results suggest that Aβ1-42 becomes elevated in the CSF after severe brain trauma.     

Apoptotic Cell Death Induced by β-Amyloid1–42 Peptide Is Cell Type Dependent

Abstract: β-Amyloid peptide (Aβ), a proteolytic fragment of the β-amyloid precursor protein, is a major component of senile plaques in the brain of Alzheimer's disease patients. This neuropathological feature is accompanied by increased neuronal cell loss in the brain and there is evidence that Aβ is directly neurotoxic. In the present study reduced cell viability in four different neuroblastoma cell types was observed after treatment with human Aβ_1–42 for 1 day. Of the cell types tested rat PC12 and human IMR32 cells were most susceptible to Aβ toxicity. Chromosomal condensation and fragmentation of nuclei were seen in PC12, NB₂a, and B104 cells but not in IMR32 cells irrespective of their high sensitivity to Aβ. Electrophoretic analysis of cellular DNA confirmed internucleosomal DNA fragmentation typical for apoptosis in all cell types except IMR32. These findings suggest that the form of Aβ-induced cell death (necrosis or apoptosis) may depend on the cell type.     

SIRT1 activation by curcumin pretreatment attenuates mitochondrial oxidative damage induced by myocardial ischemia reperfusion injury

Ischemia reperfusion (IR) injury (IRI) is harmful to the cardiovascular system and causes mitochondrial oxidative stress. Silent information regulator 1 (SIRT1), a type of histone deacetylase, contributes to IRI. Curcumin (Cur) is a strong natural antioxidant and is the active component in Curcuma longa; Cur has protective effects against IRI and may regulate the activity of SIRT1. This study was designed to investigate the protective effect of Cur pretreatment on myocardial IRI and to elucidate this potential mechanism. Isolated and in vivo rat hearts and cultured neonatal rat cardiomyocytes were subjected to IR. Prior to this procedure, the hearts or cardiomyocytes were exposed to Cur in the absence or presence of the SIRT1 inhibitor sirtinol or SIRT1 siRNA. Cur conferred a cardioprotective effect, as shown by improved postischemic cardiac function, decreased myocardial infarct size, decreased myocardial apoptotic index, and several biochemical parameters, including the up-regulation of the antiapoptotic protein Bcl2 and the down-regulation of the proapoptotic protein Bax. Sirtinol and SIRT1 siRNA each blocked the Cur-mediated cardioprotection by inhibiting SIRT1 signaling. Cur also resulted in a well-preserved mitochondrial redox potential, significantly elevated mitochondrial superoxide dismutase activity, and decreased formation of mitochondrial hydrogen peroxide and malondialdehyde. These observations indicated that the IR-induced mitochondrial oxidative damage was remarkably attenuated. However, this Cur-elevated mitochondrial function was reversed by sirtinol or SIRT1 siRNA treatment. In summary, our results demonstrate that Cur pretreatment attenuates IRI by reducing IR-induced mitochondrial oxidative damage through the activation of SIRT1 signaling.     

α2-Macroglobulin Attenuates β-Amyloid Peptide 1–40 Fibril Formation and Associated Neurotoxicity of Cultured Fetal Rat Cortical Neurons

Abstract: β-Amyloid peptides (Aβ) are deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of Aβ in cultured neurons is dependent on its aggregation state, but the factors contributing to aggregation and fibril formation are poorly understood. In the present study, we investigated whether α₂-macroglobulin (α₂M), a protein present in neuritic plaques and elevated in Alzheimer's disease brain, is a potential regulatory factor for Aβ fibril formation. Previous studies in our laboratory have shown that α₂M is an Aβ binding protein. We now report that, in contrast to another plaque-associated protein, α₁-antichymotrypsin, α₂M coincubated with Aβ significantly reduces aggregation and fibril formation in vitro. Additionally, cultured fetal rat cortical neurons are less vulnerable to the toxic actions of aged Aβ following pretreatment with α₂M. We postulate that α₂M is able to maintain Aβ in a soluble state, preventing fibril formation and associated neurotoxicity.     

Protein Kinase C Activation Potentiates the Rapid Secretion of the Amyloid Precursor Protein from Rat Cortical Synaptosomes

Abstract : In this study we have used the presynaptic-rich rat cerebrocortical synaptosomal preparation to investigate the proteolytic cleavage of the amyloid precursor protein (AβPP) by the α-secretase pathway within the βA4 domain to generate a soluble secreted N-terminal fragment (AβPP_s). AβPP was detected in crude cortical synaptosomal membranes, although at a lower density than that observed in whole-tissue homogenates. Protein kinase C (PKC) activation induced a translocation of the conventional PKC isoform β1 and novel PKCε from cytosol to membrane fractions, but there was no alteration in the proportion of AβPP associated with the Tritonsoluble and -insoluble fractions. AβPP_s was constitutively secreted from cortical synaptosomes, with this secretion being enhanced significantly by the direct activation of PKC with phorbol ester. The PKC-induced secretion of AβPP_s was only partially blocked by the PKC inhibitor GF109203X (2.5 μ M ), whereas the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein was significantly inhibited by GF109203X. The differential sensitivities of the MARCKS phosphorylation and AβPP_s secretion to GF109203X may imply that different PKC isoforms are involved in these two events in the synaptosomal system. These findings strongly suggest that the α-secretase activity leading to the secretion of AβPP_s can occur at the level of the presynaptic terminal.     

Inhibition of Alzheimer's amyloid toxicity with a tricyclic pyrone molecule in vitro and in vivo

Small β-amyloid (Aβ) 1–42 aggregates are toxic to neurons and may be the primary toxic species in Alzheimer's disease (AD). Methods to reduce the level of Aβ, prevent Aβ aggregation, and eliminate existing Aβ aggregates have been proposed for treatment of AD. A tricyclic pyrone named CP2 is found to prevent cell death associated with Aβ oligomers. We studied the possible mechanisms of neuroprotection by CP2. Surface plasmon resonance spectroscopy shows a direct binding of CP2 with Aβ42 oligomer. Circular dichroism spectroscopy reveals monomeric Aβ42 peptide remains as a random coil/α-helix structure in the presence of CP2 over 48 h. Atomic force microscopy studies show CP2 exhibits similar ability to inhibit Aβ42 aggregation as that of Congo red and curcumin. Atomic force microscopy closed-fluid cell study demonstrates that CP2 disaggregates Aβ42 oligomers and protofibrils. CP2 also blocks Aβ fibrillations using a protein quantification method. Treatment of 5× familial Alzheimer's disease mice, a robust Aβ42-producing animal model of AD, with a 2-week course of CP2 resulted in 40% and 50% decreases in non-fibrillar and fibrillar Aβ species, respectively. Our results suggest that CP2 might be beneficial to AD patients by preventing Aβ aggregation and disaggregating existing Aβ oligomers and protofibrils.