Zinc-induced aggregation of Abeta (10-21) potentiates its action on voltage-gated potassium channel

Zinc may play an important role in the pathogenesis of Alzheimer's disease (AD) through influencing the conformation and neurotoxicity of amyloid beta-proteins (Abeta). Zn(2+) induces rapid aggregation of synthetic or endogenous Abeta in a pH-dependent fashion. Here we show for the first time that Zn(2+)-induced aggregation of Abeta (10-21) potentiates its action on outward potassium currents in hippocampal CA1 pyramidal neurons. Using the whole-cell voltage-clamp technique, we showed that Abeta (10-21) blocked the fast-inactivating outward potassium current (I(A)) in a concentration- and aggregation-dependent manner, but with no effect on the delayed rectifier potassium current (I(K)). Both the unaggregated and aggregated forms of Abeta (10-21) significantly shifted the activation curve and the inactivation curve of I(A) to more negative potentials. But the aggregated form has more effects than the unaggregated form. These data indicated that aggregation of amyloid fragments by zinc ions is required in order to obtain full modulatory effects on potassium channel currents.     

A new evidence for DNA nicking property of amyloid beta-peptide (1-42): relevance to Alzheimer's disease

Alzheimer's disease (AD) is a complex neurodegenerative disorder with a progressive mental deterioration manifested by memory loss. No definite etiology has been established for AD to date. Amyloid beta (Abeta) protein plays a central role in the pathology of AD through multiple pathways like oxidative stress, apoptosis etc. Recently, our laboratory first time has evidenced localization of Abeta immunoreactivity in apoptotic nuclei of degenerating AD brain hippocampal neurons and also showed that Abeta (1-42) binds and alters the helicity of DNA. The present study provided fundamental data on DNA nicking induced by Abeta. The results showed that Abeta (1-42) has DNA nicking activity similar to nucleases. Further, magnesium ion (1mM) enhanced DNA nicking activity of Abeta. The data on Abeta solution stability on DNA nicking revealed that the oligomers of Abeta (1-42) peptides showed more DNA nicking activity compared to monomers and fibrillar forms. The nuclease specific inhibitor aurintricarboxylic acid prevented the DNA nicking property of Abeta. Transmission electron microscopy (TEM) studies revealed that Abeta causes open circular and linear forms in supercoiled DNA and also clearly evidenced the physical association of protein-DNA complex. The above data indicated that Abeta mimics endonuclease behavior. Our finding of DNA nicking activity of Abeta peptides has biological significance in terms of causing direct DNA damage.     

Small heat shock proteins differentially affect Abeta aggregation and toxicity

beta-Amyloid (Abeta) is the primary protein component of senile plaques in Alzheimer's disease (AD) and has been implicated in neurotoxicity associated with the disease. Abeta aggregates readily in vitro and in vivo, and its toxicity has been linked to its aggregation state. Prevention of Abeta aggregation has been investigated as a means to prevent Abeta toxicity associated with AD. Recently we found that Hsp20 from Babesia bovis prevented both Abeta aggregation and toxicity [S. Lee, K. Carson, A. Rice-Ficht, T. Good, Hsp20, a novel alpha-crystallin, prevents Abeta fibril formation and toxicity, Protein Sci. 14 (2005) 593-601.]. In this work, we examined the mechanism of Hsp20 interaction with Abeta1-40 and compared its activity to that of other small heat shock proteins, carrot Hsp17.7 and human Hsp27. While all three small heat shock proteins were able to prevent Abeta aggregation, only Hsp20 was able to attenuate Abeta toxicity in cultured SH-SY5Y cells. Understanding the mechanism of the Hsp20-Abeta interaction may provide insights into the design of the next generation of Abeta aggregation and toxicity inhibitors.     

Amyloid beta peptide (Abeta42) activates PLC-delta1 promoter through the NF-kappaB binding site

The abnormal deposition of amyloid beta peptide (Abeta) is a hallmark of Alzheimer's disease (AD). Phospholipase C-delta1 (PLC-delta1) is also known to abnormally accumulate in the brains of AD patients, but no report has addressed the relationship between these two events. This study investigated the effect of Abeta42 on the PLC-delta1 expression in human neuroblastoma cell lines. The PLC-delta1 mRNA level was increased by treatment with Abeta42 in a RT-PCR analysis. In the reporter assay, Abeta42 was found to activate the PLC-delta1 promoter activity in a dose-dependent manner. A novel NF-kappaB binding site in the PLC-delta1 promoter appeared to be responsible for the Abeta42 activity. First, the dominant negative forms of the NF-kappaB activating molecules, dominant negative TGF-beta activated kinase 1 (dnTAK1) and dnNIK (dominant negative NF-kappaB-inducing kinase), abolished the Abeta42 activity in the reporter assay. Second, the Abeta42 augmented a factor binding on the NF-kappaB site in the electrophoretic mobility shift assay (EMSA), which was abolished by a molar excess of the unlabeled consensus NF-kappaB oligonucleotide. These results suggest that the PLC-delta1 promoter is under the control of NF-kappaB, which mediates the expression of PLC-delta due to the Abeta42 treatment.     

Characterizations of distinct amyloidogenic conformations of the Abeta (1-40) and (1-42) peptides

Major constituents of the amyloid plaques found in the brain of Alzheimer's patients are the 39-43 residue beta-amyloid (Abeta) peptides. Extensive in vitro as well as in vivo biochemical studies have shown that the 40- and 42-residue Abeta peptides play major roles in the neurodegenerative pathology of Alzheimer's disease. Although the two Abeta peptides share common aggregation properties, the 42-residue peptide is more amyloidogenic and more strongly associated with amyloid pathology. Thus, characterizations of the two Abeta peptides are of critical importance in understanding the molecular mechanism of Abeta amyloid formation. In this report, we present combined CD and NMR studies of the monomeric states of the two peptides under both non-amyloidogenic (<5 degrees C) and amyloid-forming conditions (>5 degrees C) at physiological pH. Our CD studies of the Abeta peptides showed that initially unfolded Abeta peptides at low temperature (<5 degrees C) gradually underwent conformational changes to more beta-sheet-like monomeric intermediate states at stronger amyloidogenic conditions (higher temperatures). Detailed residue-specific information on the structural transition was obtained by using NMR spectroscopy. Residues in the N-terminal (3-12) and 20-22 regions underwent conformational changes to more extended structures at the stronger amyloidogenic conditions. Almost identical structural transitions of those residues were observed in the two Abeta peptides, suggesting a similar amyloidogenic intermediate for the two peptides. The 42-residue Abeta (1-42) peptide was, however, more significantly structured at the C-terminal region (39-42), which may lead to the different aggregation propensity of the two peptides.     

FRAT1 peptide decreases Abeta production in swAPP(751) cells

Recently, LiCl has been shown to inhibit amyloid beta peptide secretion in association with diminished glycogen synthase kinase beta (GSK3beta) activity. However, it remains unclear if direct inhibition of GSK3beta activity will result in decreased Abeta production. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) protein is a negative regulator of GSK3alpha/beta kinase activity. To examine whether direct inhibition of GSK3alpha/beta kinase activity can lower Abeta production, a FRAT1 peptide was expressed in swAPP(751) cells that produce high levels of Abeta. Our data demonstrate that cellular expression of FRAT1 peptide in swAPP(751) cells increases both GSK3alpha and beta phosphorylation on Ser21 and Ser9, respectively, while inhibiting kinase activity of both isoforms. Moreover, as a result of FRAT1 expression, the production of both total Abeta and Abeta(1-42) was significantly decreased. Thus, we provide evidence that direct regulation of GSK3alpha/beta by FRAT1 peptide significantly decreases Abeta production in swAPP(751) cells.     

Methyl dynamics of the amyloid-beta peptides Abeta40 and Abeta42

To probe the role of side chain dynamics in Abeta aggregation, we studied the methyl dynamics of native Abeta40 and Abeta42 by measuring cross relaxation rates with interleaved data collection. The methyl groups in the C-terminus are in general more rigid in Abeta42 than in Abeta40, consistent with previous results from backbone (15)N dynamics. This lends support to the hypothesis that a rigid C-terminus in Abeta42 may serve as an internal aggregation seed. Interestingly, two methyl groups of V18 located in the central hydrophobic cluster are more mobile in Abeta42 than in Abeta40, most likely due to the paucity of V18 intra-molecular interactions in Abeta42. V18 may then be more available for inter-molecular interactions to form Abeta42 aggregates. Thus, the side chain mobility of the central hydrophobic cluster may play an important role in Abeta aggregation and may contribute to the difference in aggregation propensity between Abeta40 and Abeta42.     

Abeta(25-35) and Abeta(1-40) act on different calcium channels in CA1 hippocampal neurons

The acute effects of beta-amyloid (25-35) and (1-40) on high voltage activated calcium channels were compared in CA1 pyramidal cells of adult mouse hippocampal slices using the whole-cell patch-clamp recording. Bath application of oligomeric beta-amyloid (25-35) reversibly increased the barium current (I(Ba)) to 1.61 (normalized amplitude), while oligomeric beta-amyloid (1-40) reversibly enhanced the I(Ba) to 1.74. Reverse-sequence beta-amyloid [(35-25) and (40-1)] had no effect. The effect of beta-amyloid (25-35) was blocked by nifedipine, a selective antagonist of L-type calcium channels. In contrast, the effect of beta-amyloid (1-40) was not blocked by nifedipine and I(Ba) was enhanced to 4.96. It is concluded that these oligomeric peptides may act through different types of calcium channels and/or receptors. The toxicity of Abeta(25-35) implicates a potentiation of L-type calcium channels while the one of Abeta(1-40) is related to an increase of non-L-type calcium channels, which may involve an increase in transmitter release.     

Acetylcholinesterase triggers the aggregation of PrP 106-126

Acetylcholinesterase (AChE), a senile plaque component, promotes amyloid-beta-protein (Abeta) fibril formation in vitro. The presence of prion protein (PrP) in Alzheimer's disease (AD) senile plaques prompted us to assess if AChE could trigger the PrP peptides aggregation as well. Consequently, the efficacy of AChE on the PrP peptide spanning-residues 106-126 aggregation containing a coumarin fluorescence probe (coumarin-PrP 106-126) was studied. Kinetics of coumarin-PrP 106-126 aggregation showed a significant increase of maximum size of aggregates (MSA), which was dependent on AChE concentration. AChE-PrP 106-126 aggregates showed the tinctorial and optical amyloid properties as determined by polarized light and electronic microscopy analysis. A remarkable inhibition of MSA was obtained with propidium iodide, suggesting that AChE triggers PrP 106-126 and Abeta aggregation through a similar mechanism. Huprines (AChE inhibitors) also significantly decreased MSA induced by AChE as well, unveiling the potential interest for some AChE inhibitors as a novel class of potential anti-prion drugs.     

Enhanced G-protein activation by a mixture of Abeta(25-35), Abeta(1-40/42) and zinc

Beta-amyloid peptides (Abetas) bind to several G-protein coupled receptor proteins and stimulate GTPase activity in neurons. In this study we determined the effects of Abeta(1-42), Abeta(1-40), Abeta(25-35) and their mixtures on [(35)S]GTP binding in rat brain cortical membranes in the absence and presence of zinc. We found that the Abetas alone induced a concentration-dependent activation of G-proteins (IC50 approximately 10(-6) m), while aggregated Abeta fibrils only affected GTP binding at concentrations above 10(-5) m. Mixing Abeta(25-35) with Abeta(1-42) or Abeta(1-40) induced a several-fold increase in GTP-binding. This potentiation followed a bell shaped curve with a maximum at 50 : 50 ratios. No potentiating effect could be seen by mixing Abeta(1-40) and Abeta(1-42) or highly aggregated Abetas. Zinc had no effect on Abeta(1-40/42) but strongly potentiated the Abeta(25-35) or the mixed peptides-induced GTP-binding. Changes in secondary structure accompanied the mixed peptides or the peptide/zinc complexes induced potentiation, revealing that structural alterations are behind the increased biological action. These concentration dependent potentiating effects of zinc and the peptide mixtures could be physiologically important at brain regions where peptide fragments and/or zinc are present at elevated concentrations.     

Spontaneous aggregation and cytotoxicity of the beta-amyloid Abeta1-40: a kinetic model

The time dependency of the spontaneous aggregation of the fibrillogenic -Amyloid peptide, A^1–40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-A antibody 6E10, raised against residues 1–17, at concentrations of 200–300 nM delayed significantly the aggregation of 50 M amyloid peptide. The anti–A antibody 4G8, raised against residues 17–24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39–43 of the full-length A was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the A^1–40–induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of A^1–40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of A^1–40 in vitro and possibly in vivo.     

Abeta42 is more rigid than Abeta40 at the C terminus: implications for Abeta aggregation and toxicity

Abeta40 and Abeta42 are the major forms of amyloid beta peptides (Abeta) in the brain. Although Abeta42 differs from Abeta40 by only two residues, Abeta42 is much more prone to aggregation and more toxic to neurons than Abeta40. To probe whether dynamics contribute to such dramatic difference in function, backbone ps-ns dynamics of native Abeta monomers were characterized by 15N spin relaxation at 273.3 K and 800 MHz. Abeta42 aggregates much faster than Abeta40 in the NMR tube. The effect of Abeta aggregation was removed from the relaxation measurement by interleaved data collection. R1, R2 and nuclear Overhauser enhancement (NOE) values are similar in Abeta40 and Abeta42, except at the C terminus, indicating Abeta42 and Abeta40 monomers have identical global motions. Comparisons of the spectral density function J(0.87omegaH) and order parameters (S2) indicate that the Abeta42 C terminus is more rigid than the Abeta40 C terminus. At 280.4 K and 287.6 K, the Abeta42 C terminus remains more rigid than the Abeta40 C terminus, suggesting such a dynamical difference is likely present at the physiological temperature. The Abeta42 monomer likely has less configurational entropy due to restricted motion in the C terminus and may pay a smaller entropic price to form fibrils than the Abeta40 monomer. We hypothesize that the entropic difference between Abeta40 and Abeta42 monomers might partly account for the fact that Abeta42 is the major Abeta species in parenchymal senile plaques in most Alzheimer's diseased brains in spite of the predominance of Abeta40 in plasma. The increased rigidity of the Abeta42 C terminus is likely due to its pre-ordering for beta-conformation present in soluble oligomers and fibrils. The Abeta42 C terminus may therefore serve as an internal seed for aggregation.     

Zinc binding agonist effect on the recognition of the beta-amyloid (4-10) epitope by anti-beta-amyloid antibodies

Amyloid plaques associated to Alzheimer's disease present a high content of zinc ions. We previously showed that the N-terminal region of the amyloid peptide Abeta constitutes an autonomous zinc-binding domain. This region encompasses the previously identified epitope Abeta(4-10) targeted by antibodies capable to reduce amyloid deposition, but the influence of Abeta/Zn binding on the epitope recognition remains unknown. We demonstrate here the effect of Zn2+ ions on the recognition of peptides sharing the sequence of the Abeta N-terminal domain, by two monoclonal antibodies recognizing the beta-amyloid(4-10) epitope. The presence of Zn2+, but not of other cations, increased the recognition of the (1-16) peptide, while it was without effect on the recognition of the (1-10) peptide. These findings show a zinc-induced conformational change of the (1-16)-N-terminal region of AP3, which results in a better accessibility of the Abeta(4-10) epitope to the anti-Abeta antibodies, and suggest a role of zinc in epitope-based vaccination approaches.     

Parallel increase in p70 kinase activation and tau phosphorylation (S262) with Abeta overproduction

This study set out to search for a link between overproduction of Abeta and p70S6 kinase (p70S6K) phosphorylation/activation. Results showed that levels of p-p70S6K at T421/S424 and T389 are significantly increased in mouse N2a neuroblastoma cells carrying human APP with Swedish mutation (APPswe), and in transgenic APPswe/PS1 (A246E) mice as compared with respective controls, corresponding to the increase of tau phosphorylation at S262. This parallel increase in p70S6K activation and tau phosphorylation could be demonstrated by treating wild-type N2a cells with Abeta25-35. Our results suggest that the Abeta deposition in senile plaques in Alzheimer disease brains might be a primary event that activates p70S6K and phosphorylates tau at S262, resulting in microtubule disruption.     

Amyloid peptide Abeta(1-42) binds selectively and with picomolar affinity to alpha7 nicotinic acetylcholine receptors

We have recently reported evidence that a very high affinity interaction between the beta-amyloid peptide Abeta(1-42) and the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) may be a precipitating event in the formation of amyloid plaques in Alzheimer's disease. In the present study, the kinetics for the binding of Abeta(1-42) to alpha7nAChR and alpha4beta2nAChR were determined using the subtype-selective nicotinic receptor ligands [(3)H]methyllycaconitine and [(3)H]cytisine. Synaptic membranes prepared from rat and guinea pig cerebral cortex and hippocampus were used as the source of receptors. Abeta(1-42) bound to the alpha7nAChR with exceptionally high affinity, as indicated by K(i) values of 4.1 and 5.0 pM for rat and guinea pig receptors, respectively. When compared with the alpha7nAChR, the affinity of Abeta(1-42) for the alpha4beta2nAChR was approximately 5,000-fold lower, as indicated by corresponding K(i) values of 30 and 23nM. The results of this study support the concept that an exceptionally high affinity interaction between Abeta(1-42) and alpha7nAChR could serve as a precipitating factor in the formation of amyloid plaques and thereby contribute to the selective degeneration of cholinergic neurons that originate in the basal forebrain and project to the cortex and hippocampus.     

Alzheimer's amyloid beta-peptide (1-42) induces cell death in human neuroblastoma via bax/bcl-2 ratio increase: an intriguing role for methionine 35

Nucleolin associates with various DNA repair, recombination, and replication proteins, and possesses DNA helicase, strand annealing, and strand pairing activities. Examination of nuclear protein extracts from human somatic cells revealed that nucleolin and Rad51 co-immunoprecipitate. Furthermore, purified recombinant Rad51 associates with in vitro transcribed and translated nucleolin. Electroporation-mediated introduction of anti-nucleolin antibody resulted in a 10- to 20-fold reduction in intra-plasmid homologous recombination activity in human fibrosarcoma cells. Additionally, introduction of anti-nucleolin antibody sensitized cells to death induced by the topoisomerase II inhibitor, amsacrine. Introduction of anti-Rad51 antibody also reduced intra-plasmid homologous recombination activity and induced hypersensitivity to amsacrine-induced cell death. Co-introduction of anti-nucleolin and anti-Rad51 antibodies did not produce additive effects on homologous recombination or on cellular sensitivity to amsacrine. The association of the two proteins raises the intriguing possibility that nucleolin binding to Rad51 may function to regulate homologous recombinational repair of chromosomal DNA.     

Eliminating membrane depolarization caused by the Alzheimer peptide Abeta(1-42, aggr.)

A high-throughput screen found compounds that eliminate the dramatic membrane depolarization caused by the aggregated Alzheimer Abeta1-42 peptide, which activates mGluR1 receptors. The library was composed of known biologically active compounds; the cell-based assay measured the changes of membrane potential with a slow-acting voltage-sensitive dye. We found 10 potentially useful compounds, some of which reduce the Abeta-induced membrane depolarization up to 96%. Interestingly, the active compounds include specific tyrosine kinase inhibitors and inhibitors of certain chloride channels. We deduce that mGluR1 receptors, activated by Abeta1-42 or otherwise, can control the membrane potential via downstream activation of certain tyrosine kinases and certain ion channels. Dopaminergic and serotonergic agonists that emerged from the screen presumably compensate for the Abeta-induced membrane depolarization by themselves causing a hyperpolarization. The hit compounds, whose pharmacokinetics are known, show promise for the restoration of cognitive function in the treatment of early and mid-stage Alzheimer's disease.     

Human but not rat amylin shares neurotoxic properties with Abeta42 in long-term hippocampal and cortical cultures

Type 2 diabetes mellitus (DM) and Alzheimer's disease (AD) share epidemiological and biochemical features. Both are characterized by insoluble protein aggregates with a fibrillar conformation--amylin in Type 2 DM pancreatic islets, and Abeta in AD brain. To determine whether amylin shares neurotoxic properties with Abeta, we incubated hippocampal and cortical neurons with Abeta42 and human amylin. Different from non-amyloidogenic rat amylin, both caused a dose-, time- and cell type-specific neurotoxicity supporting the notion of a similar toxic mechanism. Depending on the cell type, this finding is also supported by co-incubation of human amylin and Abeta.     

Formation of toxic Abeta(1-40) fibrils on GM1 ganglioside-containing membranes mimicking lipid rafts: polymorphisms in Abeta(1-40) fibrils

The abnormal aggregation and deposition of amyloid β protein (Aβ) on neuronal cells are critical to the onset of Alzheimer's disease. The entity (oligomers or fibrils) of toxic Aβ species responsible for the pathogenesis of the disease has been controversial. We have reported that the Aβ aggregates on ganglioside-rich domains of neuronal PC12 cells as well as in raft-like model membranes. Here, we identified toxic Aβ(1-40) aggregates formed with GM1-ganglioside-containing membranes. Aβ(1-40) was incubated with raft-like liposomes composed of GM1/cholesterol/sphingomyelin at 1:2:2 and 37 °C. After a lag period, toxic amyloid fibrils with a width of 12 nm were formed and subsequently laterally assembled with slight changes in their secondary structure as confirmed by viability assay, thioflavin-T fluorescence, circular dichroism, and transmission electron microscopy. In striking contrast, Aβ fibrils formed without membranes were thinner (6.7 nm) and much less toxic because of weaker binding to cell membranes and a smaller surface hydrophobicity. This study suggests that toxic Aβ(1-40) species formed on membranes are not soluble oligomers but amyloid fibrils and that Aβ(1-40) fibrils exhibit polymorphisms.     

Induction of anti-inflammatory immune response by an adenovirus vector encoding 11 tandem repeats of Abeta1-6: toward safer and effective vaccines against Alzheimer's disease

Induction of an immune response to amyloid beta-protein (Abeta) is effective in treating animal models of Alzheimer's disease. Human clinical trials of vaccination with synthetic Abeta (AN1792), however, were halted due to brain inflammation, presumably induced by T cell-mediated immune responses. We have developed an adenovirus vector as a "possibly safer" vaccine. Here, we show that an adenovirus vector encoding 11 tandem repeats of Abeta1-6 can induce an immune response against amyloid beta-protein. Much higher titers against amyloid beta-protein were observed when an adenovirus vector encoding GM-CSF was co-administered. Immunoglobulin isotyping revealed a predominant IgG1 response, indicating anti-inflammatory Th2 type. Immunohistochemical analysis revealed no inflammation-related pathology in the brain of mice immunized with the adenovirus vector. Induced antibodies strongly reacted with amyloid plaques in the brain, demonstrating functional activity of the antibodies. Thus, the adenovirus vector encoding 11 tandem repeats of Abeta1-6 may be a safer alterative to peptide-based vaccines.