Abeta ion channels. Prospects for treating Alzheimer's disease with Abeta channel blockers

The main pathological features in the Alzheimer's brain are progressive depositions of amyloid protein plaques among nerve cells, and neurofibrillary tangles within the nerve cells. The major components of plaques are Abeta peptides. Numerous reports have provided evidence that Abeta peptides are cytotoxic and may play a role in the pathogenesis of AD. An increasing number of research reports support the concept that the Abeta-membrane interaction event may be followed by the insertion of Abeta into the membrane in a structural configuration which forms an ion channel. This review summarizes experimental procedures which have been designed to test the hypothesis that the interaction of Abeta with a variety of membranes, both artificial and natural, results in the subsequent formation of Abeta ion channels We describe experiments, by ourselves and others, that support the view that Abeta is cytotoxic largely due to the action of Abeta channels in the cell membrane. The interaction of Abeta with the surface of the cell membrane may results in the activation of a chain of processes that, when large enough, become cytotoxic and induce cell death by apoptosis. Remarkably, the blockage of Abeta ion channels at the surface of the cell absolutely prevents the activation of these processes at different intracellular levels, thereby preserving the life of the cells. As a prospect for therapy for Alzheimer's disease, our findings at cellular level may be testable on AD animal models to elucidate the potential role and the magnitude of the contribution of the Abeta channels for induction of the disease.     

Early and late cytotoxic effects of external application of the Alzheimer's Abeta result from the initial formation and function of Abeta ion channels

Extracellular application of the Alzheimer's beta-amyloid (Abeta) peptide evokes a series of cellular responses that leads to the death of cells by apoptosis. Some responses to freshly prepared Abeta occur immediately, including changes in intracellular calcium concentration and changes in membrane permeability and phosphatidylserine asymmetry. We show here that the cytotoxic action of externally applied Abeta, such as caspase activation and apoptotic loss of cell viability, occurs and persists even several days after Abeta is removed from the medium. We find that the mechanism for this persistent cytotoxic action of extracellular Abeta is based on the sustained activity of active Abeta ion channels that remain incorporated in the cell membrane. To confirm this assessment, we blocked the late cytotoxic action of Abeta with the classically known Abeta channel blockers zinc and tromethamine. To further validate this conclusion, we developed a specific peptide segment from the sequence forming the mouth of the Abeta channel to block Abeta Ca2+ channels acutely and to block late Abeta effects on caspase activation and apoptosis. This is the first report of a specific Abeta channel blocker compound, NA4, which efficaciously and potently blocks the most known cellular responses to Abeta.     

The Alzheimer's peptides Abeta40 and 42 adopt distinct conformations in water: a combined MD / NMR study

The role of peptides Abeta40 and Abeta42 in the early pathogenesis of Alzheimer's disease (AD) is frequently emphasized in the literature. It is known that Abeta42 is more prone to aggregation than Abeta40, even though they differ in only two (IA) amino acid residues at the C-terminal end. A direct comparison of the ensembles of conformations adopted by the monomers in solution has been limited by the inherent flexibility of the unfolded peptides. Here, we characterize the conformations of Abeta40 and Abeta42 in water by using a combination of molecular dynamics (MD) and measured scalar (3)J(HNHalpha) data from NMR experiments. We perform replica exchange MD (REMD) simulations and find that classical forcefields reproduce the NMR data quantitatively when the sampling is extended to the microseconds time-scale. Using the quantitative agreement of the NMR data as a validation of the model, we proceed to compare the conformational ensembles of the Abeta40 and Abeta42 peptide monomers. Our analysis confirms the existence of structured regions within the otherwise flexible Abeta peptides. We find that the C terminus of Abeta42 is more structured than that of Abeta40. The formation of a beta-hairpin in the sequence (31)IIGLMVGGVVIA involving short strands at residues 31-34 and 38-41 (in bold) reduces the C-terminal flexibility of the Abeta42 peptide and may be responsible for the higher propensity of this peptide to form amyloids.     

Inhibitors of amyloid toxicity based on beta-sheet packing of Abeta40 and Abeta42

Amyloid fibrils associated with Alzheimer's disease and a wide range of other neurodegenerative diseases have a cross beta-sheet structure, where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. The surface of the beta-sheet has pronounced ridges and grooves when the individual beta-strands have a parallel orientation and the amino acids are in-register with one another. Here we show that in Abeta amyloid fibrils, Met35 packs against Gly33 in the C-terminus of Abeta40 and against Gly37 in the C-terminus of Abeta42. These packing interactions suggest that the protofilament subunits are displaced relative to one another in the Abeta40 and Abeta42 fibril structures. We take advantage of this corrugated structure to design a new class of inhibitors that prevent fibril formation by placing alternating glycine and aromatic residues on one face of a beta-strand. We show that peptide inhibitors based on a GxFxGxF framework disrupt sheet-to-sheet packing and inhibit the formation of mature Abeta fibrils as assayed by thioflavin T fluorescence, electron microscopy, and solid-state NMR spectroscopy. The alternating large and small amino acids in the GxFxGxF sequence are complementary to the corresponding amino acids in the IxGxMxG motif found in the C-terminal sequence of Abeta40 and Abeta42. Importantly, the designed peptide inhibitors significantly reduce the toxicity induced by Abeta42 on cultured rat cortical neurons.     

Abeta42 overproduction associated with structural changes in the catalytic pore of gamma-secretase: common effects of Pen-2 N-terminal elongation and fenofibrate

gamma-Secretase is an atypical aspartyl protease that cleaves amyloid beta-precursor protein to generate Abeta peptides that are causative for Alzheimer disease. gamma-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1, and Pen-2. Pen-2 directly binds to transmembrane domain 4 of PS and confers proteolytic activity on gamma-secretase, although the mechanism of activation and its role in catalysis remain unknown. Here we show that an addition of amino acid residues to the N terminus of Pen-2 specifically increases the generation of Abeta42, the longer and more aggregable species of Abeta. The effect of the N-terminal elongation of Pen-2 on Abeta42 generation was independent of the amino acid sequences, the expression system and the presenilin species. In vitro gamma-secretase assay revealed that Pen-2 directly affects the Abeta42-generating activity of gamma-secretase. The elongation of Pen-2 N terminus caused a reduction in the water accessibility of the luminal side of the catalytic pore of PS1 in a similar manner to that caused by an Abeta42-raising gamma-secretase modulator, fenofibrate, as determined by substituted cysteine accessibility method. These data suggest a unique mechanism of Abeta42 overproduction associated with structural changes in the catalytic pore of presenilins caused commonly by the N-terminal elongation of Pen-2 and fenofibrate.     

CLAC binds to aggregated Abeta and Abeta fragments, and attenuates fibril elongation

Deposition of amyloid beta-peptide (Abeta) into amyloid plaques is one of the invariant neuropathological features of Alzheimer's disease. Proteins that codeposit with Abeta are potentially important for the pathogenesis, and a recently discovered plaque-associated protein is the collagenous Alzheimer amyloid plaque component (CLAC). In this study, we investigated the molecular interactions between Abeta aggregates and CLAC using surface plasmon resonance spectroscopy and a solid-phase binding immunoassay. We found that CLAC binds to Abeta with high affinity, that the central region of Abeta is necessary and sufficient for CLAC interaction, and that the aggregation state of Abeta as well as the presence of negatively charged residues is important. We also show that this binding results in a reduced rate of fibril elongation. Taken together, we suggest that CLAC becomes involved at an intermediate stage in the pathogenesis by binding to Abeta fibrils, including fibrils formed from peptides with truncated N- or C-termini, and thereby slows their growth.     

Glutaminyl cyclase inhibition attenuates pyroglutamate Abeta and Alzheimer's disease-like pathology

Because of their abundance, resistance to proteolysis, rapid aggregation and neurotoxicity, N-terminally truncated and, in particular, pyroglutamate (pE)-modified Abeta peptides have been suggested as being important in the initiation of pathological cascades resulting in the development of Alzheimer's disease. We found that the N-terminal pE-formation is catalyzed by glutaminyl cyclase in vivo. Glutaminyl cyclase expression was upregulated in the cortices of individuals with Alzheimer's disease and correlated with the appearance of pE-modified Abeta. Oral application of a glutaminyl cyclase inhibitor resulted in reduced Abeta(3(pE)-42) burden in two different transgenic mouse models of Alzheimer's disease and in a new Drosophila model. Treatment of mice was accompanied by reductions in Abeta(x-40/42), diminished plaque formation and gliosis and improved performance in context memory and spatial learning tests. These observations are consistent with the hypothesis that Abeta(3(pE)-42) acts as a seed for Abeta aggregation by self-aggregation and co-aggregation with Abeta(1-40/42). Therefore, Abeta(3(pE)-40/42) peptides seem to represent Abeta forms with exceptional potency for disturbing neuronal function. The reduction of brain pE-Abeta by inhibition of glutaminyl cyclase offers a new therapeutic option for the treatment of Alzheimer's disease and provides implications for other amyloidoses, such as familial Danish dementia.     

FAD mutants unable to increase neurotoxic Abeta 42 suggest that mutation effects on neurodegeneration may be independent of effects on Abeta

Strong support for a primary causative role of the Abeta peptides in the development of Alzheimer's disease (AD) neurodegeneration derives from reports that presenilin familial AD (FAD) mutants alter amyloid precursor protein processing, thus increasing production of neurotoxic Abeta 1-42 (Abeta 42). This effect of FAD mutants is also reflected in an increased ratio of peptides Abeta 42 over Abeta 1-40 (Abeta 40). In the present study, we show that several presenilin 1 FAD mutants failed to increase production of Abeta 42 or the Abeta 42/40 ratio. Our data suggest that the mechanism by which FAD mutations promote neurodegeneration and AD may be independent of their effects on Abeta production.     

Efficiency of histidine-associating compounds for blocking the alzheimer's Abeta channel activity and cytotoxicity

The opening of the Alzheimer's Aβ channel permits the flux of calcium into the cell, thus critically disturbing intracellular ion homeostasis. Peptide segments that include the characteristic histidine (His) diad, His¹³ and His¹⁴, efficiently block the Aβ channel activity, blocking Aβ cytotoxicity. We hypothesize that the vicinal His-His peptides coordinate with the rings of His in the mouth of the pore, thus blocking the flow of calcium ions through the channel, with consequent blocking of Aβ cytotoxicity. To test this hypothesis, we studied Aβ ion channel activity and cytotoxicity after the addition of compounds that are known to have His association capacity, such as Ni²⁺, imidazole, His, and a series of His-related compounds. All compounds were effective at blocking both Aβ channel and preventing Aβ cytotoxicity. The efficiency of protection of His-related compounds was correlated with the number of imidazole side chains in the blocker compounds. These data reinforce the premise that His residues within the Aβ channel sequence are in the pathway of ion flow. Additionally, the data confirm the contribution of the Aβ channel to the cytotoxicity of exogenous Aβ.     

Two-dimensional structure of beta-amyloid(10-35) fibrils

Beta-amyloid (Abeta) peptides are the main protein component of the pathognomonic plaques found in the brains of patients with Alzheimer's disease. These heterogeneous peptides adopt a highly organized fibril structure both in vivo and in vitro. Here we use solid-state NMR on stable, homogeneous fibrils of Abeta(10-35). Specific interpeptide distance constraints are determined with dipolar recoupling NMR on fibrils prepared from a series of singly labeled peptides containing (13)C-carbonyl-enriched amino acids, and skipping no more that three residues in the sequence. From these studies, we demonstrate that the peptide adopts the structure of an extended parallel beta-sheet in-register at pH 7.4. Analysis of DRAWS data indicates interstrand distances of 5.3 +/- 0.3 A (mean +/- standard deviation) throughout the entire length of the peptide, which is compatible only with a parallel beta-strand in-register. Intrastrand NMR constraints, obtained from peptides containing labels at two adjacent amino acids, confirm the secondary structural findings obtained using DRAWS. Using peptides with (13)C incorporated at the carbonyl position of adjacent amino acids, structural transitions from alpha-helix to beta-sheet were observed at residues 19 and 20, but using similar techniques, no evidence for a turn could be found in the putative turn region comprising residues 25-29. Implications of this extended parallel organization for Abeta(10-35) for overall fibril formation, stability, and morphology based upon specific amino acid contacts are discussed.     

Independent generation of Abeta42 and Abeta38 peptide species by gamma-secretase

Proteolytic processing of the amyloid precursor protein by beta- and gamma-secretase generates the amyloid-beta (Abeta) peptides, which are principal drug targets in Alzheimer disease therapeutics. gamma-Secretase has imprecise cleavage specificity and generates the most abundant Abeta40 and Abeta42 species together with longer and shorter peptides such as Abeta38. Several mechanisms could explain the production of multiple Abeta peptides by gamma-secretase, including sequential processing of longer into shorter Abeta peptides. A novel class of gamma-secretase modulators (GSMs) that includes some non-steroidal anti-inflammatory drugs has been shown to selectively lower Abeta42 levels without a change in Abeta40 levels. A signature of GSMs is the concomitant increase in shorter Abeta peptides, such as Abeta38, leading to the suggestion that generation of Abeta42 and Abeta38 peptide species by gamma-secretase is coordinately regulated. However, no evidence for or against such a precursor-product relationship has been provided. We have previously shown that stable overexpression of aggressive presenilin-1 (PS1) mutations associated with early-onset familial Alzheimer disease attenuated the cellular response to GSMs, resulting in greatly diminished Abeta42 reductions as compared with wild type PS1. We have now used this model system to investigate whether Abeta38 production would be similarly affected indicating coupled generation of Abeta42 and Abeta38 peptides. Surprisingly, treatment with the GSM sulindac sulfide increased Abeta38 production to similar levels in four different PS1 mutant cell lines as compared with wild type PS1 cells. This was confirmed with the structurally divergent GSMs ibuprofen and indomethacin. Mass spectrometry analysis and high resolution urea gel electrophoresis further demonstrated that sulindac sulfide did not induce detectable compensatory changes in levels of other Abeta peptide species. These data provide evidence that Abeta42 and Abeta38 species can be independently generated by gamma-secretase and argue against a precursor-product relationship between these peptides.     

Mutations that reduce aggregation of the Alzheimer's Abeta42 peptide: an unbiased search for the sequence determinants of Abeta amyloidogenesis

The primary component of amyloid plaque in the brains of Alzheimer's patients is the 42 residue amyloid-beta-peptide (Abeta42). Although the amino acid residue sequence of Abeta42 is known, the molecular determinants of Abeta amyloidogenesis have not been elucidated. To facilitate an unbiased search for the sequence determinants of Abeta aggregation, we developed a genetic screen that couples a readily observable phenotype in E. coli to the ability of a mutation in Abeta42 to reduce aggregation. The screen is based on our finding that fusions of the wild-type Abeta42 sequence to green fluorescent protein (GFP) form insoluble aggregates in which GFP is inactive. Cells expressing such fusions do not fluoresce. To isolate variants of Abeta42 with reduced tendencies to aggregate, we constructed and screened libraries of Abeta42-GFP fusions in which the sequence of Abeta42 was mutated randomly. Cells expressing GFP fusions to soluble (non-aggregating) variants of Abeta42 exhibit green fluorescence. Implementation of this screen enabled the isolation of 36 variants of Abeta42 with reduced tendencies to aggregate. The sequences of most of these variants are consistent with previous models implicating hydrophobic regions as determinants of Abeta42 aggregation. Some of the variants, however, contain amino acid substitutions not implicated in pre-existing models of Abeta amyloidogenesis.     

Alzheimer's Abeta fused to green fluorescent protein induces growth stress and a heat shock response

The 42 amino acid Alzheimer's Abeta peptide is involved in the progression of Alzheimer's disease. Here we describe the effects of intracellular Abeta, produced through its attachment to either end of a green fluorescent protein, in yeast. Cells producing Abeta exhibited a lower growth yield and a heat shock response, showing that Abeta fusions promote stress in cells and supporting the notion that intracellular Abeta is a toxic molecule. These studies have relevance in understanding the role of Abeta in the death of neuronal cells, and indicate that yeast may be a new tractable model system for the screening for inhibitors of the stress caused by Abeta.     

Abeta peptide interactions with isoflurane, propofol, thiopental and combined thiopental with halothane: a NMR study

Abeta peptide is the major component of senile plaques (SP) which accumulates in AD (Alzheimer's disease) brain. Reports from different laboratories indicate that anesthetics interact with Abeta peptide and induce Abeta oligomerization. The molecular mechanism of Abeta peptide interactions with these anesthetics was not determined. We report molecular details for the interactions of uniformly (15)N labeled Abeta40 with different anesthetics using 2D nuclear magnetic resonance (NMR) experiments. At high concentrations both isoflurane and propofol perturb critical amino acid residues (G29, A30 and I31) of Abeta peptide located in the hinge region leading to Abeta oligomerization. In contrast, these three specific residues do not interact with thiopental and subsequently no Abeta oligomerization was observed. However, studies with combined anesthetics (thiopental and halothane), showed perturbation of these residues (G29, A30 and I31) and subsequently Abeta oligomerization was found. Perturbation of these specific Abeta residues (G29, A30 and I31) by different anesthetics could play an important role to induce Abeta oligomerization.     

ApoE promotes the proteolytic degradation of Abeta

Apolipoprotein E is associated with age-related risk for Alzheimer's disease and plays critical roles in Abeta homeostasis. We report that ApoE plays a role in facilitating the proteolytic clearance of soluble Abeta from the brain. The endolytic degradation of Abeta peptides within microglia by neprilysin and related enzymes is dramatically enhanced by ApoE. Similarly, Abeta degradation extracellularly by insulin-degrading enzyme is facilitated by ApoE. The capacity of ApoE to promote Abeta degradation is dependent upon the ApoE isoform and its lipidation status. The enhanced expression of lipidated ApoE, through the activation of liver X receptors, stimulates Abeta degradation. Indeed, aged Tg2576 mice treated with the LXR agonist GW3965 exhibited a dramatic reduction in brain Abeta load. GW3965 treatment also reversed contextual memory deficits. These data demonstrate a mechanism through which ApoE facilitates the clearance of Abeta from the brain and suggest that LXR agonists may represent a novel therapy for AD.     

Analysis of the secondary structure of beta-amyloid (Abeta42) fibrils by systematic proline replacement

Amyloid fibrils in Alzheimer's disease mainly consist of 40- and 42-mer beta-amyloid peptides (Abeta40 and Abeta42) that exhibit aggregative ability and neurotoxicity. Although the aggregates of Abeta peptides are rich in intermolecular beta-sheet, the precise secondary structure of Abeta in the aggregates remains unclear. To identify the amino acid residues involved in the beta-sheet formation, 34 proline-substituted mutants of Abeta42 were synthesized and their aggregative ability and neurotoxicity on PC12 cells were examined. Prolines are rarely present in beta-sheet, whereas they are easily accommodated in beta-turn as a Pro-X corner. Among the mutants at positions 15-32, only E22P-Abeta42 extensively aggregated with stronger neurotoxicity than wild-type Abeta42, suggesting that the residues at positions 15-21 and 24-32 are involved in the beta-sheet and that the turn at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of Abeta42. The C-terminal proline mutants (A42P-, I41P-, and V40P-Abeta42) hardly aggregated with extremely weak cytotoxicity, whereas the C-terminal threonine mutants (A42T- and I41T-Abeta42) aggregated potently with significant cytotoxicity. These results indicate that the hydrophobicity of the C-terminal two residues of Abeta42 is not related to its aggregative ability and neurotoxicity, rather the C-terminal three residues adopt the beta-sheet. These results demonstrate well the large difference in aggregative ability and neurotoxicity between Abeta42 and Abeta40. In contrast, the proline mutants at the N-terminal 13 residues showed potent aggregative ability and neurotoxicity similar to those of wild-type Abeta42. The identification of the beta-sheet region of Abeta42 is a basis for designing new aggregation inhibitors of Abeta peptides.     

Secretion and differential localization of the proteolytic cleavage products Abeta40 and Abeta42 of the Alzheimer amyloid precursor protein in human fetal myogenic cells

Abeta peptides are major components of the amyloid plaques that characterize Alzheimer's disease. These peptides are proteolytic cleavage products of the amyloid precursor protein (APP) and are generated by beta- and gamma-secretases. Here we show by multiparameter immunofluorescence imaging in muscle cells that localization of the Abeta40 and Abeta42 cleavage products reveals different myocyte types in a three-dimensional culture system. These myocyte types are heterogeneous by selective intracellular concentration of either Abeta40 or Abeta42 in vesicular structures, whilst only the Abeta40 peptide is secreted as indicated by Western blot analysis. This cellular pattern of APP proteolysis and Abeta peptide secretion correlates with lack of L-APP mRNA splice isoforms. Differential secretion and intracellular accumulation of Abeta peptides is characteristic for the early myocyte development and might be related to cell fusion.     

Identification and characterization of key kinetic intermediates in amyloid beta-protein fibrillogenesis

Amyloid beta-protein (Abeta) assembly into toxic oligomeric and fibrillar structures is a seminal event in Alzheimer's disease, therefore blocking this process could have significant therapeutic benefit. A rigorous mechanistic understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Prior studies have shown that Abeta fibrillogenesis involves conformational changes leading to the formation of extended beta-sheets and that an alpha-helix-containing intermediate may be involved. However, the significance of this intermediate has been a matter of debate. We report here that the formation of an oligomeric, alpha-helix-containing assembly is a key step in Abeta fibrillogenesis. The generality of this phenomenon was supported by conformational studies of 18 different Abeta peptides, including wild-type Abeta(1-40) and Abeta(1-42), biologically relevant truncated and chemically modified Abeta peptides, and Abeta peptides causing familial forms of cerebral amyloid angiopathy. Without exception, fibrillogenesis of these peptides involved an oligomeric alpha-helix-containing intermediate and the kinetics of formation of the intermediate and of fibrils was temporally correlated. The kinetics varied depending on amino acid sequence and the extent of peptide N- and C-terminal truncation. The pH dependence of helix formation suggested that Asp and His exerted significant control over this process and over fibrillogenesis in general. Consistent with this idea, Abeta peptides containing Asp-->Asn or His-->Gln substitutions showed altered fibrillogenesis kinetics. These data emphasize the importance of the dynamic interplay between Abeta monomer conformation and oligomerization state in controlling fibrillogenesis kinetics.     

Ion channel formation by Alzheimer's disease amyloid beta-peptide (Abeta40) in unilamellar liposomes is determined by anionic phospholipids

Incorporation of Alzheimer's disease amyloid beta-proteins (AbetaPs) across natural and artificial bilayer membranes leads to the formation of cation-selective channels. To study the peptide-membrane interactions involved in channel formation, we used cation reporter dyes to measure AbetaP-induced influx of Na+, Ca2+, and K+ into liposomes formed from phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylcholine (PC). We found that Abeta40, but not Abeta40-1 or Abeta28, caused a dose-dependent increase in the concentration of each cation in the lumen of liposomes formed from the acidic phospholipids PS and PI. The Abeta40-induced changes in cation concentration, which we attribute to ion entry through Abeta40 channels, were not observed when using liposomes formed from the neutral phospholipid PC. Using mixtures of phospholipids, the magnitude of the AbetaP40-induced ion entry increased with the acidic phospholipid content of the liposomes, with entry being observed with as little as 5% PS or PI. Thus, while negatively charged phospholipids are required for formation of cation-permeable channels by Abeta40, a small amount is sufficient to support the process. These results have implications for the mechanisms of AbetaP cytotoxicity, suggesting that even a small amount of externalized negative charge could render cells susceptible to the deleterious effects of unregulated ion influx through AbetaP channels.