Oral Presentations OP01: Neurodegenerative Diseases. BCL‐W Plays a key neuroprotective role in Alzheimer's disease

The mechanisms underlying neuronal degeneration in Alzheimer's disease (AD) are very controversial and none more so than whether apoptosis plays a role. Although neurons in AD face a wide assortment of apoptogenic stimuli, the temporal dichotomy between the acuteness of apoptosis vs. the chronicity of AD suggests that apoptosis should be extremely rare in AD. In this regard, survival factor(s) must be involved. In this study, we investigated Bcl‐w, a pro‐survival member of the Bcl‐2 family. Although expressed at low levels in brains of control cases, Bcl‐w is significantly up‐regulated in AD as shown by both immunocytochemistry and immunoblot analysis. Astonishingly, increased Bcl‐w was found to be associated with neurofibrillary pathologies in AD, which was further demonstrated by an EM study. Since neuronal death in AD is thought to be triggered by increased production of amyloid‐β (Aβ), it was interesting to find that exposure of human M17 neuroblastoma cells to Aβ1–42 (1 nm ?10 μm ) dramatically up‐regulates Bcl‐w protein levels. Such increases may be a protective response that attenuates apoptotic processes. Consistent with this, transfected M17 cells overexpressing Bcl‐w were protected from both STS‐induced and Aβ‐induced apoptosis compared to vector‐transfected controls. Notably, both tau phosphorylation and p38 is inhibited in Bcl‐w transfected cells which may contribute to the neuroprotective role of Bcl‐w. Taken together, these set of in vitro and in vivo results suggest that Bcl‐w plays an important protective role in neurons in the AD brain.     

Neuroprotective properties of Bcl-w in Alzheimer disease

While there is a host of pro-apoptotic stimuli that target neurons in Alzheimer disease (AD), given the chronicity of the disease and the survival of many neurons, those neurons must either avoid or, at minimum, delay apoptotic death signaling. In this study, we investigated Bcl-w, a novel member of the Bcl-2 family that promotes cell survival. In AD, we found increased levels of Bcl-w associated with neurofibrillary pathology and punctate intracytoplasmic structures whereas, in marked contrast, there are only low diffuse levels of Bcl-w in the neuronal cytoplasm of age-matched control cases. Immunoblot analysis confirmed that Bcl-w levels were significantly increased in AD. By electron microscopy, we determined that the increased Bcl-w expression in AD was ultrastructurally localized to mitochondria and neurofibrillary pathology. To investigate the cause and consequence of Bcl-w up-regulation in neurons, we found that fibrillized amyloid-beta led to increased Bcl-w protein levels in M17 human neuroblastoma cells, and that overexpression of Bcl-w significantly protected neurons against staurosporine- and amyloid-beta-induced apoptosis. Taken together, these series of results suggest that Bcl-w may play an important protective role in neurons in the diseased brain and that this aspect could be therapeutically harnessed to afford neuroprotection.     

Neurodegeneration in an Aβ-induced model of Alzheimer's disease: the role of Cdk5

Cdk5 dysregulation is a major event in the neurodegenerative process of Alzheimer's disease (AD). In vitro studies using differentiated neurons exposed to Aβ exhibit Cdk5-mediated tau hyperphosphorylation, cell cycle re-entry and neuronal loss. In this study we aimed to determine the role of Cdk5 in neuronal injury occurring in an AD mouse model obtained through the intracerebroventricular (icv) injection of the Aβ_1–40 synthetic peptide. In mice icv-injected with Aβ, Cdk5 activator p35 is cleaved by calpains, leading to p25 formation and Cdk5 overactivation. Subsequently, there was an increase in tau hyperphosphorylation, as well as decreased levels of synaptic markers. Cell cycle reactivation and a significant neuronal loss were also observed. These neurotoxic events in Aβ-injected mice were prevented by blocking calpain activation with MDL28170 , which was administered intraperitoneally (ip). As MDL prevents p35 cleavage and subsequent Cdk5 overactivation, it is likely that this kinase is involved in tau hyperphosphorylation, cell cycle re-entry, synaptic loss and neuronal death triggered by Aβ. Altogether, these data demonstrate that Cdk5 plays a pivotal role in tau phosphorylation, cell cycle induction, synaptotoxicity, and apoptotic death in postmitotic neurons exposed to Aβ peptides in vivo , acting as a link between diverse neurotoxic pathways of AD.     

Oestradiol or genistein rescues neurons from amyloid beta-induced cell death by inhibiting activation of p38

Oestrogenic compounds have been postulated as neuroprotective agents. This prompted us to investigate their mechanism action in neurons in primary culture. Cells were pretreated with physiological concentrations of 17-β estradiol (0.2 n m ) or with nutritionally relevant concentrations of genistein (0.5 µ m ), and 48 h later treated with 5 µ m of amyloid beta (Aβ) for 24 h. We found that Aβ increased oxidative stress, measured as peroxide levels or oxidized glutathione/reduced glutathione ratio, which in turn, caused phosphorylation of p38 MAP kinase. Amyloid beta subsequently induced neuronal death. Inhibiting the MAP kinase pathway prevented cell death, confirming the role of p38 in the toxic effect of Aβ. All these effects were prevented when cells were pretreated for 48 h with oestradiol or genistein. Therefore, oestrogenic compounds rescue neurons from Aβ-induced cell death by preventing oxidative stress, which in turn inhibits the activation of p38, protecting neurons from cell death. Because hormone replacement therapy with oestradiol could cause serious setbacks, the potential therapeutic effect of phyto-oestrogens for the prevention of Aβ-associated neurodegenerative disorders should be more carefully studied in clinical research.     

Etazolate, a neuroprotective drug linking GABA(A) receptor pharmacology to amyloid precursor protein processing

Pharmacological modulation of the GABA_A receptor has gained increasing attention as a potential treatment for central processes affected in Alzheimer disease (AD), including neuronal survival and cognition. The proteolytic cleavage of the amyloid precursor protein (APP) through the α-secretase pathway decreases in AD, concurrent with cognitive impairment. This APP cleavage occurs within the β-amyloid peptide (Aβ) sequence, precluding formation of amyloidogenic peptides and leading to the release of the soluble N-terminal APP fragment (sAPPα) which is neurotrophic and procognitive. In this study, we show that at nanomolar-low micromolar concentrations, etazolate, a selective GABA_A receptor modulator, stimulates sAPPα production in rat cortical neurons and in guinea pig brains. Etazolate (20 nM–2 μM) dose-dependently protected rat cortical neurons against Aβ-induced toxicity. The neuroprotective effects of etazolate were fully blocked by GABA_A receptor antagonists indicating that this neuroprotection was due to GABA_A receptor signalling. Baclofen, a GABA_B receptor agonist failed to inhibit the Aβ-induced neuronal death. Furthermore, both pharmacological α-secretase pathway inhibition and sAPPα immunoneutralization approaches prevented etazolate neuroprotection against Aβ, indicating that etazolate exerts its neuroprotective effect via sAPPα induction. Our findings therefore indicate a relationship between GABA_A receptor signalling, the α-secretase pathway and neuroprotection, documenting a new therapeutic approach for AD treatment.     

β-Amyloid Peptides Increase the Binding and Internalization of Apolipoprotein E to Hippocampal Neurons

Abstract: The frequency of the ε4 allele of apolipoprotein E(apoE) is increased in late-onset and sporadic forms of Alzheimer's disease (AD). ApoE also binds to β-amyloid (Aβ) and both proteins are found in AD plaques. To further investigate the potential interaction of apoE and Aβ in the pathogenesis of AD, we have determined the binding, internalization, and degradation of human apoE isoforms in the presence and absence of Aβ peptides to rat primary hippocampal neurons. We demonstrate that the lipophilic Aβ peptides, in particular Aβ_1–42, Aβ_1–40, and Aβ_25–35, increase significantly apoE-liposome binding to hippocampal neurons. For each Aβ peptide, the increase was significantly greater for the apoE4 isoform than for the apoE3 isoform. The most effective of the Aβ peptides to increase apoE binding, Aβ_25–35, was further shown to increase significantly the internalization of both apoE3- and apoE4-liposomes, without affecting apoE degradation. Conversely, Aβ_1–40 uptake by hippocampal neurons was shown to be increased in the presence of apoE-liposomes, more so in the presence of the apoE4 than the apoE3 isoform. These results provide evidence that Aβ peptides interact directly with apoE lipoproteins, which may then be transported together into neuronal cells through apoE receptors.     

Cytochalasins Protect Hippocampal Neurons Against Amyloid β-Peptide Toxicity: Evidence that Actin Depolymerization Suppresses Ca2+ Influx

Abstract: Increasing data suggest that the amyloid β-peptide (Aβ), which accumulates in the brains of Alzheimer's victims, plays a role in promoting neuronal degeneration. Cell culture studies have shown that Aβ can be neurotoxic and recent findings suggest that the mechanism involves destabilization of cellular calcium homeostasis. We now report that cytochalasin D, a compound that depolymerizes actin microfilaments selectively, protects cultured rat hippocampal neurons against Aβ neurotoxicity. Cytochalasin D was effective at concentrations that depolymerized actin (10–100 n M ). The elevation of [Ca²⁺]_i induced by Aβ, and the enhancement of [Ca²⁺]_i responses to glutamate in neurons exposed to Aβ, were markedly attenuated in neurons pretreated with cytochalasin D. The protective effect of cytochalasin D appeared to result from a specific effect on actin filaments and reduction in calcium influx, because cytochalasin E, another actin filament-disrupting agent, also protected neurons against Aβ toxicity; the microtubule-disrupting agent colchicine was ineffective; cytochalasin D did not protect neurons against the toxicity of hydrogen peroxide. These findings suggest that actin filaments play a role in modulating [Ca²⁺]_i responses to neurotoxic insults and that depolymerization of actin can protect neurons against insults relevant to the pathogenesis of Alzheimer's disease.     

Opposing Actions of Thrombin and Protease Nexin-1 on Amyloid β-Peptide Toxicity and on Accumulation of Peroxides and Calcium in Hippocampal Neurons

Abstract: Amyloid β-peptide (Aβ) is the principal component of neuritic plaques in the brain in Alzheimer's disease (AD). Recent studies revealed that Aβ can be neurotoxic by a mechanism involving free radical production and loss of cellular ion homeostasis, thus implicating Aβ as a key factor in the pathogenesis of AD. However, other proteins are present in plaques in AD, including the protease thrombin and protease nexin-1 (PN1), a thrombin inhibitor. We therefore tested the hypothesis that thrombin and PN1 modify neuronal vulnerability to Aβ toxicity. In dissociated rat hippocampal cell cultures the toxicity of Aβ was significantly enhanced by coincubation with thrombin, whereas PN1 protected neurons against Aβ toxicity. Aβ induced an increase in levels of intracellular peroxides and calcium. Thrombin enhanced, and PN1 attenuated, the accumulation of peroxides and calcium induced by Aβ. Taken together, these data demonstrate that thrombin and PN1 have opposing effects on neuronal vulnerability to Aβ and suggest that thrombin and PN1 play roles in the pathogenesis of neuronal injury in AD.     

Protection of Flupirtine on β-Amyloid-Induced Apoptosis in Neuronal Cells In Vitro: Prevention of Amyloid-Induced Glutathione Depletion

Abstract: Effective drugs are not available to protect against β-amyloid peptide (Aβ)-induced neurotoxicity. Cortical neurons from rat embryos were treated with the toxic fragment Aβ25-35 at 1 µ M in the presence or absence of flupirtine, a triaminopyridine, successfully applied clinically as a nonopiate analgesic drug. Five days later 1 µ M Aβ25-35 caused reduction of cell viability to 31.1%. Preincubation of cells with flupirtine (1 or 5 µg/ml) resulted in a significant increase of the percentage of viable cells (74.6 and 65.4%, respectively). During incubation with Aβ25-35 the neurons undergo apoptosis as determined by appearance of the characteristic stepladder-like DNA fragmentation pattern and by the TUNEL technique. Aβ25-35-induced DNA fragmentation could be abolished by preincubation of the cells with 1 µg/ml flupirtine. Incubation with Aβ25-35 reduces the intraneuronal level of GSH from 21.4 to 7.4 nmol/10⁶ cells. This depletion could be partially prevented by preincubation of the cells with flupirtine. Thus, flupirtine may be adequate for the treatment of the neuronal loss in Alzheimer's disease (where Aβ accumulates in senile plaques) and probably other neurological diseases such as amyotrophic lateral sclerosis.     

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.     

A Role for 4-Hydroxynonenal, an Aldehydic Product of Lipid Peroxidation, in Disruption of Ion Homeostasis and Neuronal Death Induced by Amyloid β-Peptide

Abstract: Peroxidation of membrane lipids results in release of the aldehyde 4-hydroxynonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. Because accumulating data indicate that free radicals mediate injury and death of neurons in Alzheimer's disease (AD) and because amyloid β-peptide (Aβ) can promote free radical production, we tested the hypothesis that HNE mediates Aβ25-35-induced disruption of neuronal ion homeostasis and cell death. Aβ induced large increases in levels of free and protein-bound HNE in cultured hippocampal cells. HNE was neurotoxic in a time- and concentration-dependent manner, and this toxicity was specific in that other aldehydic lipid peroxidation products were not neurotoxic. HNE impaired Na⁺,K⁺-ATPase activity and induced an increase of neuronal intracellular free Ca²⁺ concentration. HNE increased neuronal vulnerability to glutamate toxicity, and HNE toxicity was partially attenuated by NMDA receptor antagonists, suggesting an excitotoxic component to HNE neurotoxicity. Glutathione, which was previously shown to play a key role in HNE metabolism in nonneuronal cells, attenuated the neurotoxicities of both Aβ and HNE. The antioxidant propyl gallate protected neurons against Aβ toxicity but was less effective in protecting against HNE toxicity. Collectively, the data suggest that HNE mediates Aβ-induced oxidative damage to neuronal membrane proteins, which, in turn, leads to disruption of ion homeostasis and cell degeneration.     

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.     

In Vitro Induction of Apoptosis or Differentiation by Dopamine in an Immortalized Olfactory Neuronal Cell Line

Abstract: Amyloid β-peptide (Aβ) is deposited as insoluble fibrils in the brain parenchyma and cerebral blood vessels in Alzheimer's disease (AD). In addition to neuronal degeneration, cerebral vascular alterations indicative of damage to vascular endothelial cells and disruption of the blood-brain barrier occur in AD. Here we report that Aβ25-35 can impair regulatory functions of endothelial cells (ECs) from porcine pulmonary artery and induce their death. Subtoxic exposures to Aβ25-35 induced albumin transfer across EC monolayers and impaired glucose transport into ECs. Cell death induced by Aβ25-35 was of an apoptotic form, characterized by DNA condensation and fragmentation, and prevented by inhibitors of macromolecular synthesis and endonucleases. The effects of Aβ25-35 were specific because Aβ1-40 also induced apoptosis in ECs with the apoptotic cells localized to the microenvironment of Aβ1-40 aggregates and because astrocytes did not undergo similar changes after exposure to Aβ25-35. Damage and death of ECs induced by Aβ25-35 were attenuated by antioxidants, a calcium channel blocker, and a chelator of intracellular calcium, indicating the involvement of free radicals and dysregulation of calcium homeostasis. The data show that Aβ induces increased permeability of EC monolayers to macromolecules, impairs glucose transport, and induces apoptosis. If similar mechanisms are operative in vivo, then Aβ and other amyloidogenic peptides may be directly involved in vascular EC damage documented in AD and other disorders that involve vascular amyloid accumulation.     

Amyloid β Peptide (25–35) Inhibits Na+-Dependent Glutamate Uptake in Rat Hippocampal Astrocyte Cultures

Abstract: Large numbers of neuritic plaques surrounded by reactive astrocytes are characteristic of Alzheimer's disease (AD). There is a large body of research supporting a causal role for the amyloid β peptide (Aβ), a main constituent of these plaques, in the neuropathology of AD. Several hypotheses have been proposed to explain the toxicity of Aβ including free radical injury and excitotoxicity. It has been reported that treatment of neuronal/astrocytic cultures with Aβ increases the vulnerability of neurons to glutamate-induced cell death. One mechanism that may explain this finding is inhibition of the astrocyte glutamate transporter by Aβ. The aim of the current study was to determine if Aβs inhibit astrocyte glutamate uptake and if this inhibition involves free radical damage to the transporter/astrocytes. We have previously reported that Aβ can generate free radicals, and this radical production was correlated with the oxidation of neurons in culture and inhibition of astrocyte glutamate uptake. In the present study, Aβ (25–35) significantly inhibited l -glutamate uptake in rat hippocampal astrocyte cultures and this inhibition was prevented by the antioxidant Trolox. Decreases in astrocyte function, in particular l -glutamate uptake, may contribute to neuronal degeneration such as that seen in AD. These results lead to a revised excitotoxicity/free radical hypothesis of Aβ toxicity involving astrocytes.     

Estrogens Attenuate and Corticosterone Exacerbates Excitotoxicity, Oxidative Injury, and Amyloid β-Peptide Toxicity in Hippocampal Neurons

Abstract: Steroid hormones, particularly estrogens and glucocorticoids, may play roles in the pathogenesis of neurodegenerative disorders, but their mechanisms of action are not known. We report that estrogens protect cultured hippocampal neurons against glutamate toxicity, glucose deprivation, FeSO₄ toxicity, and amyloid β-peptide (Aβ) toxicity. The toxicity of each insult was significantly attenuated in cultures pretreated for 2 h with 100 n M -10 µ M 17β-estradiol, estriol, or progesterone. In contrast, corticosterone exacerbated neuronal injury induced by glutamate, FeSO₄, and Aβ. Several other steroids, including testosterone, aldosterone, and vitamin D, had no effect on neuronal vulnerability to the different insults. The protective actions of estrogens and progesterone were not blocked by actinomycin D or cycloheximide. Lipid peroxidation induced by FeSO₄ and Aβ was significantly attenuated in neurons and isolated membranes pretreated with estrogens and progesterone, suggesting that these steroids possess antioxidant activities. Estrogens and progesterone also attenuated Aβ- and glutamate-induced elevation of intracellular free Ca²⁺ concentrations. We conclude that estrogens, progesterone, and corticosterone can directly affect neuronal vulnerability to excitotoxic, metabolic, and oxidative insults, suggesting roles for these steroids in several different neurodegenerative disorders.     

Aβ aggregation and possible implications in Alzheimer's disease pathogenesis

Amyloid β protein (Aβ) has been associated with Alzheimer's disease (AD) because it is a major component of the extracellular plaque found in AD brains. Increased Aβ levels correlate with the cognitive decline observed in AD. Sporadic AD cases are thought to be chiefly associated with lack of Aβ clearance from the brain, unlike familial AD which shows increased Aβ production. Aβ aggregation leading to deposition is an essential event in AD. However, the factors involved in Aβ aggregation and accumulation in sporadic AD have not been completely characterized. This review summarizes studies that have examined the factors that affect Aβ aggregation and toxicity. By necessity these are studies that are performed with recombinant-derived or chemically synthesized Aβ. The studies therefore are not done in animals but in cell culture, which includes neuronal cells, other mammalian cells and, in some cases, non-mammalian cells that also appear susceptible to Aβ toxicity. An understanding of Aβ oligomerization may lead to better strategies to prevent AD.     

Mitochondrial Free Radical Signal in Ceramide-Dependent Apoptosis: A Putative Mechanism for Neuronal Death in Parkinson''s Disease

Abstract: We investigated the potential role of different proteases in the death of cultured rat hippocampal pyramidal neurons induced by β-amyloid(Aβ) (25–35). Both Aβ(25–35)- and staurosporine-induced death of these neurons appeared to involve apoptosis, as indicated using Hoechst 33342 and terminal dUDP nick end labeling staining, whereas NMDA-induced death appeared more complex. Two irreversible inhibitors of the interleukin-1β converting enzyme (ICE) and related proteases, Z-Val-Ala-Asp-CH₂F and acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone, blocked neuronal death produced by Aβ(25–35), staurosporine, and NMDA to differing extents. Furthermore, MDL 28,170, a selective inhibitor of the calcium-regulated protease calpain, also inhibited death induced by all agents. Aβ(25–35) and staurosporine stimulated the breakdown of the protein spectrin, a calpain substrate. Spectrin breakdown was inhibited by MDL 28,170 but not by ICE inhibitors. Leupeptin was only effective in preventing NMDA-induced death. These results support the role of apoptosis in neuronal death due to Aβ(25–35) treatment and also suggest a role for calcium-regulated proteases in this process.     

Processing of Amyloid Precursor Protein in Human Primary Neuron and Astrocyte Cultures

Abstract: Increased production of amyloid β peptide (Aβ) is highly suspected to play a major role in Alzheimer's disease (AD) pathogenesis. Because Aβ deposits in AD senile plaques appear uniquely in the brain and are fairly restricted to humans, we assessed amyloid precursor protein (APP) metabolism in primary cultures of the cell types associated with AD senile plaques: neurons, astrocytes, and microglia. We find that neurons secrete 40% of newly synthesized APP, whereas glia secrete only 10%. Neuronal and astrocytic APP processing generates five C-terminal fragments similar to those observed in human adult brain, of which the most amyloidogenic higher-molecular-weight fragments are more abundant. The level of amyloidogenic 4-kDa Aβ exceeds that of nonamyloidogenic 3-kDa Aβ in both neurons and astrocytes. In contrast, microglia make more of the smallest C-terminal fragment and no detectable Aβ. We conclude that human neurons and astrocytes generate higher levels of amyloidogenic fragments than microglia and favor amyloidogenic processing compared with previously studied culture systems. Therefore, we propose that the higher amyloidogenic processing of APP in neurons and astrocytes, combined with the extended lifespan of individuals, likely promotes AD pathology in aging humans.     

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.