Resveratrol protects rats from Aβ-induced neurotoxicity by the reduction of iNOS expression and lipid peroxidation

Alzheimer disease (AD) is an age-dependent neurodegenerative disease characterized by the formation of β-amyloid (Aβ)-containing senile plaque. The disease could be induced by the administration of Aβ peptide, which was also known to upregulate inducible nitric oxide synthase (iNOS) and stimulate neuronal apoptosis. The present study is aimed to elucidate the cellular effect of resveratrol, a natural phytoestrogen with neuroprotective activities, on Aβ-induced hippocampal neuron loss and memory impairment. On adult Sprague-Dawley rats, we found the injection of Aβ could result in a significant impairment in spatial memory, a marked increase in the cellular level of iNOS and lipid peroxidation, and an apparent decrease in the expression of heme oxygenase-1 (HO-1). By combining the treatment with Aβ, resveratrol was able to confer a significant improvement in spatial memory, and protect animals from Aβ-induced neurotoxicity. These neurological protection effects of resveratrol were associated with a reduction in the cellular levels of iNOS and lipid peroxidation and an increase in the production of HO-1. Moreover, the similar neurological and cellular response were also observed when Aβ treatment was combined with the administration of a NOS inhibitor, N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME). These findings strongly implicate that iNOS is involved in the Aβ-induced lipid peroxidation and HO-1 downregulation, and resveratrol protects animals from Aβ-induced neurotoxicity by suppressing iNOS production.     

Pulmonary surfactant protein A protects lung epithelium from cytotoxicity of human β-defensin 3

Defensins are important molecules in the innate immune system that eliminate infectious microbes. They also exhibit cytotoxicity against host cells in higher concentrations. The mechanisms by which hosts protect their own cells from cytotoxicity of defensins have been poorly understood. We found that the cytotoxicity of human β-defensin 3 (hBD3) against lung epithelial cells was dose-dependently attenuated by pulmonary surfactant protein A (SP-A), a collectin implicated in host defense and regulation of inflammatory responses in the lung. The direct interaction between SP-A and hBD3 may be an important factor in decreasing this cytotoxicity because preincubation of epithelial cells with SP-A did not affect the cytotoxicity. Consistent with in vitro analysis, intratracheal administration of hBD3 to SP-A(-/-) mice resulted in more severe tissue damage compared with that in WT mice. These data indicate that SP-A protects lung epithelium from tissue injury caused by hBD3. Furthermore, we found that the functional region of SP-A lies within Tyr(161)-Lys(201). Synthetic peptide corresponding to this region, tentatively called SP-A Y161-G200, also inhibited cytotoxicity of hBD3 in a dose-dependent manner. The SP-A Y161-G200 is a candidate as a therapeutic reagent that prevents tissue injury during inflammation.     

CXCL8 protects human neurons from amyloid-β-induced neurotoxicity: relevance to Alzheimer's disease

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by amyloid-β (Aβ) deposition in senile plaques colocalized with activated microglia and astrocytes. Recent studies suggest that CXCL8 is involved in the AD pathogenesis. The objective of this study was to determine the cellular sources of CXCL8 in the central nervous system during AD pathogenesis, and investigate the effects of CXCL8 on neuronal survival and/or functions. Our results showed significantly higher CXCL8 levels in AD brain tissue lysates as compared to those of age-matched controls. Upon Aβ and/or pro-inflammatory cytokine stimulation, microglia, astrocytes and neurons were all capable of CXCL8 production in vitro. Although CXCL8-alone did not alter neuronal survival, it did inhibit Aβ-induced neuronal apoptosis and increased neuronal brain-derived neurotrophic factor (BDNF) production. We conclude that microglia, astrocytes and neurons, all contribute to the enhanced CXCL8 levels in the CNS upon Aβ and/or pro-inflammatory cytokine stimulation. Further, CXCL8 protects neurons possibly by paracrine or autocrine loop and regulates neuronal functions, therefore, may play a protective role in the AD pathogenesis.     

Sodium tanshinone IIA sulfonate protects against Aβ-induced cell toxicity through regulating Aβ process

Sodium tanshinone IIA sulfonate (STS) has been reported to prevent Alzheimer's disease (AD). However, the mechanism is still unknown. In this study, two in vitro models, Aβ-treated SH-SY5Y cells and SH-SY5Y human neuroblastoma cells transfected with APPsw (SH-SY5Y-APPsw cells), were employed to investigate the neuroprotective of STS. The results revealed that pretreatment with STS (1, 10 and 100 µmol/L) for 24 hours could protect against Aβ (10 µmol/L)-induced cell toxicity in a dose-dependent manner in the SH-SY5Y cells. Sodium tanshinone IIA sulfonate decreased the concentrations of reactive oxygen species, malondialdehyde, NO and iNOS, while increased the activities of superoxide dismutase and glutathione peroxidase in the SH-SY5Y cells. Sodium tanshinone IIA sulfonate decreased the levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in the SH-SY5Y cells. In addition, Western blot results revealed that the expressions of neprilysin and insulin-degrading enzyme were up-regulated in the SH-SY5Y cells after STS treatment. Furthermore, ELISA and Western blot results showed that STS could decrease the levels of Aβ. ELISA and qPCR results indicated that STS could increase α-secretase (ADAM10) activity and decrease β-secretase (BACE1) activity. In conclusion, STS could protect against Aβ-induced cell damage by modulating Aβ degration and generation. Sodium tanshinone IIA sulfonate could be a promising candidate for AD treatment.     

Rapamycin protects against Aβ-induced synaptotoxicity by increasing presynaptic activity in hippocampal neurons

The mammalian target of rapamycin (mTOR) is involved in the regulation of learning and memory. Recently, rapamycin has been shown to be neuroprotective in models for Alzheimer's disease in an autophagy-dependent manner. Here we show that rapamycin exerts neuroprotection via a novel mechanism that involves presynaptic activation. Rapamycin increases the frequency of miniature excitatory postsynaptic currents and calcium transients of rat hippocampal primary neurons by a mechanism that involves the up regulation of SV2, a presynaptic vesicular protein linked to neurotransmitter release. Under these conditions, rapamycin-treated hippocampal neurons are resistant to the synaptotoxic effect induced by Aβ oligomers, suggesting that enhancers of presynaptic activity can be therapeutic agents for Alzheimer's disease.     

Methyl B12 protects PC12 cells against cytotoxicity induced by Aβ25−35

Alzheimer's disease (AD) is the most common aging-associated dementia. The population of AD patients is increasing as the world age grows. Currently, there is no cure for AD. Given that methyl vitamin B12 (methylcobalamin) deficiency is related to AD and Aβ-induced oxidative damage and that methylcobalamin can scavenge reactive oxygen species (ROS) by direct or indirect ways, we studied the effect of methylcobalamin on the cytotoxicity of Aβ. PC12 cells were chronically exposed (24 hours) to Aβ_25-35 (25 μM) to establish an AD cell model. The cells were pretreated with or without methylcobalamin (1-100 μM) to investigate the role of methylcobalamin. Cell viability and apoptosis were tested, followed by testing of mitochondrial damage, oxidative stress, and mitochondrial calcium concentration. We observed that methylcobalamin improved the cell viability by decreasing the ratio of apoptosis cells in this AD cell model. Further experiments suggested that methylcobalamin functioned as an antioxidant to scavenge ROS, reducing the endoplasmic reticulum-mitochondria calcium flux through IP3R, preventing mitochondria dysfunction, ultimately protecting cells against apoptosis and cell death. Taken together, our results presented, for the first time, that methyl vitamin B12 can protect cells from Aβ-induced cytotoxicity and the mechanism was mainly relevant to the antioxidative function of methyl B12.     

ACS84, a novel hydrogen sulfide-releasing compound, protects against amyloid β-induced cell cytotoxicity

Hydrogen sulfide (H(2)S) is a novel neurotransmitter. We studied here the effect of ACS 84, a new H(2)S releasing compound, on the cytotoxicity induced by amyloid beta (Aβ) in microglia. Treatment with Aβ(1-40) (25μmol/L) for 24h significantly inhibited MTT reduction and increased lactate dehydrogenase release in BV-2 microglia cells. Pretreatment with ACS 84 (10μM) for 30min attenuated the above cytotoxicity caused by Aβ(1-40), suggesting that ACS 84 may protect microglia against Aβ(1-40)-induced cell injury. ACS 84 also significantly attenuated nitric oxide release and TNF-α production in BV-2 cells treated with Aβ peptides (Aβ(1-40) or Aβ(1-42)), but had no significant effect on the up-regulated protein expression of cyclooxygenase 2. These data suggest that ACS 84 may produce anti-inflammatory effect via inhibition of the release of inflammatory cytokines but not via suppression of the prostanoids production. Furthermore, pretreatment with ACS 84 also attenuated mitochondrial membrane potential loss (Δψ(m)) caused by Aβ(1-40) in both microglia and neurons. To examine the underlying signaling mechanism, we detected the phosphorylation of p38-, JNK- and ERK-MAPKs. It was found that Aβ(1-40) stimulated phosphorylation of all above three types of MAKPs. However, ACS 84 only attenuated the activation of p38 and JNK, but had no significant effect on that of ERK. Taken together, our data suggest that ACS 84 may protect Aβ-induced cell injury via anti-inflammation and preservation of mitochondrial function in a p38 and JNK dependent mechanism. Our work suggests that ACS 84 may have potential for the treatment of neurodegenerative diseases.     

Diammonium glycyrrhizinate upregulates PGC-1α and protects against Aβ1-42-induced neurotoxicity

Mitochondrial dysfunction is a hallmark of beta-amyloid (Aβ)-induced neurotoxicity in Alzheimer's disease (AD), and is considered an early event in AD pathology. Diammonium glycyrrhizinate (DG), the salt form of Glycyrrhizin, is known for its anti-inflammatory effects, resistance to biologic oxidation and membranous protection. In the present study, the neuroprotective effects of DG on Aβ(1-42)-induced toxicity and its potential mechanisms in primary cortical neurons were investigated. Exposure of neurons to 2 μM Aβ(1-42) resulted in significant viability loss and cell apoptosis. Accumulation of reactive oxygen species (ROS), decreased mitochondrial membrane potential, and activation of caspase-9 and caspase-3 were also observed after Aβ(1-42) exposure. All these effects induced by Aβ(1-42) were markedly reversed by DG treatment. In addition, DG could alleviate lipid peroxidation and partially restore the mitochondrial function in Aβ(1-42)-induced AD mice. DG also significantly increased the PGC-1α expression in vivo and in vitro, while knocking down PGC-1α partially blocked the protective effects, which indicated that PGC-1α contributed to the neuroprotective effects of DG. Furthermore, DG significantly decreased the escape latency and search distance and increased the target crossing times of Aβ(1-42)-induced AD mice in the Morris water maze test. Therefore, these results demonstrated that DG could attenuate Aβ(1-42)-induced neuronal injury by preventing mitochondrial dysfunction and oxidative stress and improved cognitive impairment in Aβ(1-42)-induced AD mice, indicating that DG exerted potential beneficial effects on AD.     

Elevated 17β-estradiol protects females from influenza A virus pathogenesis by suppressing inflammatory responses

Studies of the 1918 H1N1 influenza pandemic, the H5N1 avian influenza outbreak, and the 2009 H1N1 pandemic illustrate that sex and pregnancy contribute to severe outcome from infection, suggesting a role for sex steroids. To test the hypothesis that the sexes respond differently to influenza, the pathogenesis of influenza A virus infection was investigated in adult male and female C57BL/6 mice. Influenza infection reduced reproductive function in females and resulted in greater body mass loss, hypothermia, and mortality in females than males. Whereas lung virus titers were similar between the sexes, females had higher induction of proinflammatory cytokines and chemokines, including TNF-α, IFN-γ, IL-6, and CCL2, in their lungs than males. Removal of the gonads in both sexes eliminated the sex difference in influenza pathogenesis. Manipulation of testosterone or dihydrotestosterone concentrations in males did not significantly impact virus pathogenesis. Conversely, females administered high doses of estradiol had a ≥10-fold lower induction of TNF-α and CCL2 in the lungs and increased rates of survival as compared with females that had either low or no estradiol. The protective effects of estradiol on proinflammatory cytokines and chemokines, morbidity, and mortality were primarily mediated by signaling through estrogen receptor α (ERα). In summary, females suffer a worse outcome from influenza A virus infection than males, which can be reversed by administration of high doses of estradiol to females and reflects differences in the induction of proinflammatory responses and not in virus load.     

Ferrocene tripeptide Gly-Pro-Arg conjugates: Synthesis and inhibitory effects on Alzheimer’s Aβ1–42 fibrillogenesis and Aβ-induced cytotoxicity in vitro

Alzheimer’s disease (AD) is the most common cause of dementia, and currently there is no clinical treatment to cure it or to halt its progression. Aggregation and fibril formation of β-amyloid peptides (Aβ) are central events in the pathogenesis of AD. Many efforts have been spent on the development of effective inhibitors to prevent Aβ fibrillogenesis and cause disaggregation of preformed Aβ fibrils. In this study, the conjugates of ferrocene and Gly-Pro-Arg (GPR) tripeptide, Boc-Gly-Pro-Arg(NO₂)-Fca-OMe (4, GPR–Fca) and Fc-Gly-Pro-Arg-OMe (7, Fc–GPR) (Fc: ferrocene; Fca: ferrocene amino acid) were synthesized by HOBT/HBTU protocol in solution. These ferrocene GPR conjugates were employed to inhibit Aβ_1–42 fibrillogenesis and to disaggregate preformed Aβ fibrils. The inhibitory properties of ferrocene GPR conjugates on Aβ_1–42 fibrillogenesis were evaluated by thioflavin T (ThT) fluorescence assay, and confirmed by atomic force microscopy (AFM) analysis. The interaction between the ferrocene GPR conjugates and Aβ_1–42 was monitored by electrochemical means. Our results showed that both GPR and GPR–Fca can significantly inhibit the fibril formation of Aβ_1–42, and cause disaggregation of the preformed fibrils. As expected, GPR–Fca shows stronger inhibitory effect on Aβ_1–42 fibrillogenesis than that of its parent peptide GPR. In contrast, Fc–GPR shows no inhibitory effect on fibrillogenesis of Aβ_1–42. Furthermore, GPR–Fca demonstrates significantly protection against Aβ-induced cytotoxicity and exhibits high resistance to proteolysis and good lipophilicity.     

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

Transforming growth factor-β₁ (TGF-β₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β₁ promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β₁-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),¹ diabetic cardiomyopathy² and hypertension.³ Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β₁.⁴ TGF-β₁ signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.^{5, 6} Elevated TGF-β₁ is observed in post-MI rat heart⁷ and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.⁸ The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.^{9, 10}Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.^{11, 12, 13, 14} Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.^{15, 16} Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.^{17, 18, 19, 20} The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.^{11, 12, 13, 14, 15, 16, 17, 18, 21, 22}Recent studies have demonstrated that TGF-β₁ may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells^{15, 23, 24} but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.²⁵ Moreover, it has recently been reported that autophagy can negatively¹⁵ and positively^{25, 26, 27} regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β₁-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart.     

Parkin protects dopaminergic neurons from excessive Wnt/β-catenin signaling

Parkinson’s disease (PD) is caused by degeneration of the dopaminergic (DA) neurons of the substantia nigra but the molecular mechanisms underlying the degenerative process remain elusive. Several reports suggest that cell cycle deregulation in post-mitotic neurons could lead to neuronal cell death. We now show that Parkin, an E3 ubiquitin ligase linked to familial PD, regulates β-catenin protein levels in vivo. Stabilization of β-catenin in differentiated primary ventral midbrain neurons results in increased levels of cyclin E and proliferation, followed by increased levels of cleaved PARP and loss of DA neurons. Wnt3a signaling also causes death of post-mitotic DA neurons in parkin null animals, suggesting that both increased stabilization and decreased degradation of β-catenin results in DA cell death. These findings demonstrate a novel regulation of Wnt signaling by Parkin and suggest that Parkin protects DA neurons against excessive Wnt signaling and β-catenin-induced cell death.     

Ellagic acid promotes Aβ42 fibrillization and inhibits Aβ42-induced neurotoxicity

Smaller, soluble oligomers of β-amyloid (Aβ) play a critical role in the pathogenesis of Alzheimer’s disease (AD). Selective inhibition of Aβ oligomer formation provides an optimum target for AD therapy. Some polyphenols have potent anti-amyloidogenic activities and protect against Aβ neurotoxicity. Here, we tested the effects of ellagic acid (EA), a polyphenolic compound, on Aβ42 aggregation and neurotoxicity in vitro. EA promoted Aβ fibril formation and significant oligomer loss, contrary to previous results that polyphenols inhibited Aβ aggregation. The results of transmission electron microscopy (TEM) and Western blot displayed more fibrils in Aβ42 samples co-incubated with EA in earlier phases of aggregation. Consistent with the hypothesis that plaque formation may represent a protective mechanism in which the body sequesters toxic Aβ aggregates to render them harmless, our MTT results showed that EA could significantly reduce Aβ42-induced neurotoxicity toward SH-SY5Y cells. Taken together, our results suggest that EA, an active ingredient in many fruits and nuts, may have therapeutic potential in AD.     

Autophagy and Self-Preservation: A Step Ahead from Cell Plasticity?

Silencing the SPINK-related gene Kazal1 in hydra gland cells induces an excessive autophagy of both gland and digestive cells, leading to animal death. Moreover, during regeneration, autophagosomes are immediately detected in regenerating tips, where Kazal1 expression is lowered. When Kazal1 is completely silenced, hydra no longer survive the amputation stress (Chera S, de Rosa R, Miljkovic-Licina M, Dobretz K, Ghila L, Kaloulis K, and Galliot B. Silencing of the hydra serine protease inhibitor Kazal1 gene mimics the human Spink1 pancreatic phenotype. J Cell Sci 2006; 119:846-57). These results highlight the essential digestive and cytoprotective functions played by Kazal1 in hydra. In mammals, autophagy of exocrine pancreatic cells is also induced upon SPINK1/Spink3 inactivation, whereas SPINK3 is activated in injured pancreatic cells. Hence SPINKs, by preventing an excessive autophagy, appear to act as key players of the stress-induced self-preservation program. In hydra, this program is a prerequisite to the early cellular transition, whereby digestive cells of the regenerating tips transform into a head-organizer center. Enhancing the self-preservation program in injured tissues might therefore be the condition for unmasking their potential cell and/or developmental plasticity.

Addendum to:

Silencing of the Hydra Serine Protease Inhibitor Kazal1 Gene Mimics the HumanSpink1 Pancreatic Phenotype

S. Chera, R. de Rosa, M. Miljkovic-Licina, K. Dobretz, L. Ghila, K. Kaloulis and B. Galliot

J Cell Sci 2006; 119:846-57     

Glutathione Cycle Impairment Mediates Aβ-induced Cell Toxicity

Membrane lipid peroxidation processes yield products that may react with proteins to cause oxidative modification. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP⁺-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. When exposed to lipid peroxidation products, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE) and lipid hydroperoxide, ICDH was susceptible to oxidative damage, which was indicated by the loss of activity and the formation of carbonyl groups. The structural alterations of modified enzymes were indicated by the change in thermal stability, intrinsic tryptophan fluorescence and binding of the hydrophobic probe 8-anilino 1-napthalene sulfonic acid. Upon exposure to 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH), which induces lipid peroxidation in membrane, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed in U937 cells. Using immunoprecipitation and immunoblotting, we were able to isolate and positively identify HNE adduct in mitochondrial ICDH from AAPH-treated U937 cells. The lipid peroxidation-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Inhibition of Bax protects neuronal cells from oligomeric A�� neurotoxicity

Although oligomeric β-amyloid (Aβ) has been suggested to have an important role in Alzheimer disease (AD), the mechanism(s) of how Aβ induces neuronal cell death has not been fully identified. The balance of pro- and anti-apoptotic Bcl-2 family proteins (e.g., Bcl-2 and Bcl-w versus Bad, Bim and Bax) has been known to have a role in neuronal cell death and, importantly, expression levels of these proteins are reportedly altered in the vulnerable neurons in AD. However, the roles of apoptotic proteins in oligomeric Aβ-induced cell death remain unclear in vivo or in more physiologically relevant models. In addition, no study to date has examined whether Bax is required for the toxicity of oligomeric Aβ. Here, we found that treatment with oligomeric Aβ increased Bim levels but decreased Bcl-2 levels, leading to the activation of Bax and neuronal cell death in hippocampal slice culture and in vivo. Furthermore, the inhibition of Bax activity either by Bax-inhibiting peptide or bax gene knockout significantly prevented oligomeric Aβ-induced neuronal cell death. These findings are first to demonstrate that Bax has an essential role in oligomeric Aβ-induced neuronal cell death, and that the targeting of Bax may be a therapeutic approach for AD.     

WNT5A signaling contributes to Aβ-induced neuroinflammation and neurotoxicity

Neurodegenration is a pathological hallmark of Alzheimer's disease (AD), but the underlying molecular mechanism remains elusive. Here, we present evidence that reveals a crucial role of Wnt5a signaling in this process. We showed that Wnt5a and its receptor Frizzled-5 (Fz5) were up-regulated in the AD mouse brain, and that beta-amyloid peptide (Aβ), a major constituent of amyloid plaques, stimulated Wnt5a and Fz5 expression in primary cortical cultures; these observations indicate that Wnt5a signaling could be aberrantly activated during AD pathogenesis. In support of such a possibility, we observed that inhibition of Wnt5a signaling attenuated while activation of Wnt5a signaling enhanced Aβ-evoked neurotoxicity, suggesting a role of Wnt5a signaling in AD-related neurodegeneration. Furthermore, we also demonstrated that Aβ-induced neurotoxicity depends on inflammatory processes, and that activation of Wnt5a signaling elicited the expression of proinflammatory cytokines IL-1β and TNF-α whereas inhibition of Wnt5a signaling attenuated the Aβ-induced expression of the cytokines in cortical cultures. Our findings collectively suggest that aberrantly up-regulated Wnt5a signaling is a crucial pathological step that contributes to AD-related neurodegeneration by regulating neuroinflammation.     

Heme oxygenase-1 protects against Alzheimer's amyloid-β1-42-induced toxicity via carbon monoxide production

Heme oxygenase-1 (HO-1), an inducible enzyme up-regulated in Alzheimer''s disease, catabolises heme to biliverdin, Fe²⁺ and carbon monoxide (CO). CO can protect neurones from oxidative stress-induced apoptosis by inhibiting Kv2.1 channels, which mediates cellular K⁺ efflux as an early step in the apoptotic cascade. Since apoptosis contributes to the neuronal loss associated with amyloid β peptide (Aβ) toxicity in AD, we investigated the protective effects of HO-1 and CO against Aβ_1-42 toxicity in SH-SY5Y cells, employing cells stably transfected with empty vector or expressing the cellular prion protein, PrP^c, and rat primary hippocampal neurons. Aβ_1-42 (containing protofibrils) caused a concentration-dependent decrease in cell viability, attributable at least in part to induction of apoptosis, with the PrP^c-expressing cells showing greater susceptibility to Aβ_1-42 toxicity. Pharmacological induction or genetic over-expression of HO-1 significantly ameliorated the effects of Aβ_1-42. The CO-donor CORM-2 protected cells against Aβ_1-42 toxicity in a concentration-dependent manner. Electrophysiological studies revealed no differences in the outward current pre- and post-Aβ_1-42 treatment suggesting that K⁺ channel activity is unaffected in these cells. Instead, Aβ toxicity was reduced by the L-type Ca²⁺ channel blocker nifedipine, and by the CaMKKII inhibitor, STO-609. Aβ also activated the downstream kinase, AMP-dependent protein kinase (AMPK). CO prevented this activation of AMPK. Our findings indicate that HO-1 protects against Aβ toxicity via production of CO. Protection does not arise from inhibition of apoptosis-associated K⁺ efflux, but rather by inhibition of AMPK activation, which has been recently implicated in the toxic effects of Aβ. These data provide a novel, beneficial effect of CO which adds to its growing potential as a therapeutic agent.Amongst the earliest of events leading to neuronal loss in Alzheimer''s disease (AD) is the loss of functional synapses,^{1, 2, 3} apparent long before deposition of amyloid β peptide (Aβ)-containing plaques.⁴ Although other parts of the neurone (e.g. the axon or soma) appear intact, their health at this early stage of disease progression is not clear. However, neurones ultimately die in AD and there is clear evidence that numerous events indicative of apoptosis occur even at early stages of disease progression.^{5, 6, 7, 8} Thus, targeting of apoptotic mechanisms may be of therapeutic value in AD as well as in other neurodegenerative disorders. Furthermore, apoptosis is established as a mechanism of neuronal loss following other types of pathological stresses including ischemia associated with stroke,⁹ which can predispose individuals to the development of AD.^{10, 11, 12}Apoptosis is strongly influenced by intracellular K⁺ levels¹³ which regulate caspase activation, mitochondrial membrane potential and volume, osmolarity and cell volume.^{13, 14} K⁺ loss via K⁺ channels is a key early stage in apoptosis,^{15, 16, 17, 18, 19} and K⁺ channel inhibitors can protect against apoptosis triggered by numerous insults including oxidative stress.^{20, 21} Evidence suggests a particularly important role for the voltage-gated channel Kv2.1 in this process: expression of dominant negative Kv2.1 constructs (thus lacking functional Kv2.1 channels) protects against oxidant-induced apoptosis, and over-expression of Kv2.1 increases susceptibility to apoptosis.^{22, 23} Pro-apoptotic agents cause a rapid increase in the surface expression of Kv2.1 channels,²⁴ but whether or not this occurs in AD remains to be determined. Alternative pathways recently reported to promote cell death include activation of the AMP-dependent protein kinase (AMP kinase) which can act either as a Tau kinase²⁵ or to inhibit the mTOR pathway²⁶ and thus contribute to neurodegeneration.Heme oxygenases (HO) are enzymes widely distributed throughout the body. In the central nervous system, HO-2 is constitutively expressed in neurones and astrocytes, while HO-1 is inducible in both cell types.^{27, 28, 29, 30} Both HO-1 and HO-2 break down heme to liberate biliverdin, ferrous iron (Fe²⁺) and carbon monoxide (CO). This catalysis is of biological significance since it is crucial to iron and bile metabolism, and also generates a highly effective antioxidant in bilirubin (from biliverdin via bilirubin reductase). Numerous stimuli can induce HO-1 gene expression,³¹ including oxidative stress³² and Aβ peptides.³³ Importantly, HO-1 is strikingly up-regulated in AD patients, a finding considered indicative of oxidative stress.^{27, 34, 35} Induction of HO-1 is clearly a neuroprotective response (although in some cases can exert detrimental effects²⁷). However, there is growing evidence that CO can be neuroprotective, for example against the damage of focal ischemia.³⁶ Our recent studies have demonstrated that CO provides protection against oxidant-induced apoptosis by selectively inhibiting Kv2.1.^{23, 37} In the present study, we have investigated whether HO-1, or its product CO, can provide protection against Aβ-induced toxicity in the human neuroblastoma, SH-SY5Y, and in rat primary hippocampal neurones, and whether this involves regulation of K⁺ channels. We show that both HO-1 and CO protect cells against the toxicity of protofibrillar Aβ_1-42 but that protection does not arise from inhibition of apoptosis-associated K⁺ efflux, but rather by inhibition of AMPK activation.     

Oligomeric Aβ-induced synaptic dysfunction in Alzheimer’s disease

Alzheimer’s disease (AD) is a devastating disease characterized by synaptic and neuronal loss in the elderly. Compelling evidence suggests that soluble amyloid-β peptide (Aβ) oligomers induce synaptic loss in AD. Aβ-induced synaptic dysfunction is dependent on overstimulation of N-methyl-D-aspartate receptors (NMDARs) resulting in aberrant activation of redox-mediated events as well as elevation of cytoplasmic Ca²⁺, which in turn triggers downstream pathways involving phospho-tau (p-tau), caspases, Cdk5/dynamin-related protein 1 (Drp1), calcineurin/PP2B, PP2A, Gsk-3β, Fyn, cofilin, and CaMKII and causes endocytosis of AMPA receptors (AMPARs) as well as NMDARs. Dysfunction in these pathways leads to mitochondrial dysfunction, bioenergetic compromise and consequent synaptic dysfunction and loss, impaired long-term potentiation (LTP), and cognitive decline. Evidence also suggests that Aβ may, at least in part, mediate these events by causing an aberrant rise in extrasynaptic glutamate levels by inhibiting glutamate uptake or triggering glutamate release from glial cells. Consequent extrasynaptic NMDAR (eNMDAR) overstimulation then results in synaptic dysfunction via the aforementioned pathways. Consistent with this model of Aβ-induced synaptic loss, Aβ synaptic toxicity can be partially ameliorated by the NMDAR antagonists (such as memantine and NitroMemantine). PSD-95, an important scaffolding protein that regulates synaptic distribution and activity of both NMDA and AMPA receptors, is also functionally disrupted by Aβ. PSD-95 dysregulation is likely an important intermediate step in the pathological cascade of events caused by Aβ. In summary, Aβ-induced synaptic dysfunction is a complicated process involving multiple pathways, components and biological events, and their underlying mechanisms, albeit as yet incompletely understood, may offer hope for new therapeutic avenues.