The choroid plexus removes beta-amyloid from brain cerebrospinal fluid

beta-Amyloid (Abeta) concentration in the cerebrospinal fluid (CSF) of the brain may be regulated by the choroid plexus, which forms a barrier between blood and brain CSF. Abeta uptake from CSF was determined as its volume of distribution (V(D)) into isolated rat choroid plexus tissue. The V(D) of [125I]Abeta1-40 was corrected by subtraction of the V(D) of [14C]sucrose, a marker for extracellular space and diffusion. Abeta uptake into choroid plexus was time and temperature dependent. Uptake of [125I]Abeta was saturable. Abeta uptake was not affected by addition of transthyretin or apolipoprotein E3. In studies with primary culture monolayers of choroidal epithelial cells in Transwells, Abeta permeability across cells, corrected by [(14)C]sucrose, was greater from the CSF-facing membrane than from the blood-facing membrane. Similarly, cellular accumulation of [125I]Abeta was concentrative from both directions and was greater from the CSF-facing membrane, suggesting a bias for efflux. Overall, these results suggest the choroid plexus selectively cleanses Abeta from the CSF by an undetermined mechanism(s), potentially reducing Abeta from normal brains and the brains of Alzheimer's disease patients.     

Spontaneous in vitro formation of supramolecular beta-amyloid structures, "betaamy balls", by beta-amyloid 1-40 peptide.

The concentration of beta-amyloid peptide (Abeta), x-42 or x-40 amino acids long, increases in brain with the progression Alzheimer's disease (AD). These peptides are deposited extracellularly as highly insoluble fibrils that form densities of amyloid plaques. Abeta fibrillization is a complex polymerization process preceded by the formation of oligomeric and prefibrillar Abeta intermediates. In some of our in vitro studies, in which the kinetics of intermediate steps of fibril formation were examined, we used concentrations of synthetic Abeta that exceed what is normally employed in fibrillization studies, 300-600 microM. At these concentrations, in a cell free system and under physiological conditions, Abeta 1-40 peptide (Abeta40) forms fibrils that spontaneously assemble into clearly defined spheres, "betaamy balls", with diameters of approximately 20-200 microm. These supramolecular structures show weak birefringence with Congo red staining and high stability with prolonged incubation times (at least 2 weeks) at 30 degrees C, freezing, and dilution in H(2)O. At 600 microM, they are detected after incubation for approximately 20 h. Abeta peptide 1-42 (Abeta42) lacks the ability to form betaamy balls but accelerates Abeta40 betaamy ball formation at low stoichiometric levels (1:20 Abeta42:Abeta40 ratio). Abeta42 levels above this (=10-50% w/w) impede Abeta40 betaamy ball formation. Using light (LM) and electron microscopy (EM), this study examines the gross morphology and ultrastructure of Abeta40 betaamy balls and their time course of formation, in the absence and presence of Abeta42, along with some stability measures. As spheres of a misfolded protein, betaamy balls resemble both AD Abeta senile plaques and neuronal inclusion bodies associated with other neurodegenerative diseases.     

Differential physiologic responses of alpha7 nicotinic acetylcholine receptors to beta-amyloid1-40 and beta-amyloid1-42

The beta-amyloid peptides (Abeta), Abeta(1-40) and Abeta(1-42), have been implicated in Alzheimer's disease (AD) pathology. Although Abeta(1-42) is generally considered to be the pathological peptide in AD, both Abeta(1-40) and Abeta(1-42) have been used in a variety of experimental models without discrimination. Here we show that monomeric or oligomeric forms of the two Abeta peptides, when interact with the neuronal cation channel, alpha7 nicotinic acetylcholine receptors (alpha7nAChR), would result in distinct physiologic responses as measured by acetylcholine release and calcium influx experiments. While Abeta(1-42) effectively attenuated these alpha7nAChR-dependent physiology to an extent that was apparently irreversible, Abeta(1-40) showed a lower inhibitory activity that could be restored upon washings with physiologic buffers or treatment with alpha7nAChR antagonists. Our data suggest a clear pharmacological distinction between Abeta(1-40) and Abeta(1-42).     

Towards Alzheimer's beta-amyloid vaccination.

Beta-amyloid pathology, the main hallmark of Alzheimer's disease (AD), has been linked to its conformational status and aggregation. We recently showed that site-directed monoclonal antibodies (mAbs) towards the N-terminal region of the human beta-amyloid peptide bind to preformed beta-amyloid fibrils (Abeta), leading to disaggregation and inhibition of their neurotoxic effect. Here we report the development of a novel immunization procedure to raise effective anti-aggregating amyloid beta-protein (AbetaP) antibodies, using as antigen filamentous phages displaying the only EFRH peptide found to be the epitope of these antibodies. Due to the high antigenicity of the phage no adjuvant is required to obtain high affinity anti-aggregating IgG antibodies in animals model, that exhibit identity to human AbetaP. Such antibodies are able to sequester peripheral AbetaP, thus avoiding passage through the blood brain barrier (BBB) and, as recently shown in a transgenic mouse model, to cross the BBB and dissolve already formed beta-amyloid plaques. To our knowledge, this is the first attempt to use as a vaccine a self-anti-aggregating epitope displayed on a phage, and this may pave the way to treat abnormal accumulation-peptide diseases, such as Alzheimer's disease or other amyloidogenic diseases.     

Radioiodinated clioquinol as a biomarker for beta-amyloid: Zn complexes in Alzheimer's disease

Neocortical beta-amyloid (Abeta) aggregates in Alzheimer's disease (AD) are enriched in transition metals that mediate assembly. Clioquinol (CQ) targets metal interaction with Abeta and inhibits amyloid pathology in transgenic mice. Here, we investigated the binding properties of radioiodinated CQ ([(125)I]CQ) to different in vitro and in vivo Alzheimer models. We observed saturable binding of [(125)I]CQ to synthetic Abeta precipitated by Zn(2+) (K(d)=0.45 and 1.40 nm for Abeta(1-42) and Abeta(1-40), respectively), which was fully displaced by free Zn(2+), Cu(2+), the chelator DTPA (diethylene triamine pentaacetic acid) and partially by Congo red. Sucrose density gradient of post-mortem AD brain indicated that [(125)I]CQ concentrated in a fraction enriched for both Abeta and Zn, which was modulated by exogenous addition of Zn(2+) or DTPA. APP transgenic (Tg2576) mice injected with [(125)I]CQ exhibited higher brain retention of tracer compared to non-Tg mice. Autoradiography of brain sections of these animals confirmed selective [(125)I]CQ enrichment in the neocortex. Histologically, both thioflavine-S (ThS)-positive and negative structures were labeled by [(125)I]CQ. A pilot SPECT study of [(123)I]CQ showed limited uptake of the tracer into the brain, which did however, appear to be more rapid in AD patients compared to age-matched controls. These data support metallated Abeta species as the neuropharmacological target of CQ and indicate that this drug class may have potential as in vivo imaging agents for Alzheimer neuropathology.     

Diurnal variation of leptin entry from blood to brain involving partial saturation of the transport system

The blood-brain barrier (BBB) regulates the amount of peripherally produced leptin reaching the brain. Knowing that the blood concentration of leptin has a circadian rhythm, we investigated whether the influx of leptin at the BBB followed the same pattern in three main sets of experiments. (a): The entry of 125I-leptin from blood to brain was measured in mice every 4 h, as indicated by the influx rate of 125I-leptin 1-10 min after an iv bolus injection. The blood concentration of endogenous leptin was measured at the same times. Blood leptin concentrations were higher at night and early morning (peak at 0800 h) and lower during the day (nadir at 1600 h). By contrast, the influx of 125I-leptin was fastest at 2000 h and slowest at 0400 h. Addition of unlabeled leptin (1 microg/mouse) significantly decreased the influx rate of 125I-leptin at all time points, indicating saturability of the transport system. The unlabeled leptin also abolished the diurnal variation of the influx of 125I-leptin. (b): The entry of 125I-leptin into spinal cord was faster than that into brain and showed a different diurnal pattern. The greatest influx occurred at 2400 h and the slowest at 0800 h. In spinal cord, unlike brain, unlabeled leptin (1 microg/mouse) neither inhibited the influx of 125I-leptin nor abolished the diurnal rhythm. (c): Higher concentrations of unlabeled leptin (5 microg/mouse) inhibited the uptake of 125I-leptin in spinal cord as well as in brain, but not in muscle. This experiment measured uptake 10 min after iv injection at 0600 h (beginning of the light cycle) and 1800 h (beginning of the dark cycle). Thus, influx of 125I-leptin into the CNS shows diurnal variation, indicating a circadian rhythm in the transport system at the BBB, saturation of the leptin transport system shows differences between the brain and spinal cord, and blood concentrations of leptin suggest that partial saturation of the transport system occurs at physiological concentrations of circulating leptin, contributing to the differing diurnal patterns in brain and spinal cord. Together, the results show that the BBB is actively involved in the neuroendocrine regulation of feeding behavior.     

Different mechanisms influencing permeation of PDGF-AA and PDGF-BB across the blood-brain barrier

Platelet-derived growth factor (PDGF) exerts neurotrophic and neuromodulatory effects on the CNS. To determine the permeability of the blood-brain barrier (BBB) to PDGF, we examined the blood-to-brain influx of radioactively labeled PDGF isoforms (PDGF-AA and PDGF-BB) by multiple-time regression analysis after intravenous (i.v.) injection and by in-situ perfusion, and also determined the physicochemical characteristics which affect their permeation across the BBB, including lipophilicity (measured by octanol:buffer partition coefficient), hydrogen bonding (measured by differences in octanol : buffer and isooctane : buffer partition coefficients), serum protein binding (measured by capillary electrophoresis), and stability of PDGF in blood 10 min after i.v. injection (measured by HPLC). After i.v. bolus injection, neither 125I-PDGF-AA nor 125I-PDGF-BB crossed the BBB, their influx rates being similar to that of the vascular marker 99mTc-albumin. 125I-PDGF-AA degraded significantly faster in blood than 125I-PDGF-BB. PDGF-BB, however, was completely bound to a large protein in serum whereas PDGF-AA showed no binding. Thus, degradation might explain the poor blood-to-brain influx of PDGF-AA, whereas protein binding could explain the poor influx of circulating PDGF-BB. Despite their lack of permeation in the intact mouse, both 125I-PDGF-AA and 125I-PDGF-BB entered the brain by perfusion in blood-free buffer, and the significantly faster rate of 125I-PDGF-AA than 125I-PDGF-BB may be explained by the lower hydrogen bonding potential of 125I-PDGF-AA. Thus, the lack of significant distribution of PDGF from blood to brain is not because of the intrinsic barrier function of the BBB but probably because of degradation and protein binding. Information from these studies could be useful in the design of analogues for delivery of PDGF as a therapeutic agent.     

Humoral immune response to fibrillar beta-amyloid peptide

The beta-amyloid peptide (Abeta) is a normal product of the proteolytic processing of its precursor (beta-APP). Normally, it elicits a very low humoral immune response; however, the aggregation of monomeric Abeta to form fibrillar Abeta amyloid creates a neo-epitope, to which antibodies are generated. Rabbits were injected with fibrillar human Abeta(1-42), and the resultant antibodies were purified and their binding properties characterized. The antibodies bound to an epitope in the first eight residues of Abeta and required a free amino terminus. Additional residues did not affect the affinity of the epitope as long as the peptide was unaggregated; the antibody bound Abeta residues 1-8, 1-11, 1-16, 1-28, 1-40, and 1-42 with similar affinities. In contrast, the antibodies bound approximately 1000-fold more tightly to fibrillar Abeta(1-42). Their enhanced affinity did not result from their bivalent nature: monovalent Fab fragments exhibited a similar affinity for the fibrils. Nor did it result from the particulate nature of the epitope: monomeric Abeta(1-16) immobilized on agarose and soluble Abeta(1-16) exhibited similar affinities for the antifibrillar antibodies. In addition, antibodies raised to four nonfibrillar peptides corresponding to internal Abeta sequences did not exhibit enhanced affinity for fibrillar Abeta(1-42). Antibodies directed to the C-terminus of Abeta bound poorly to fibrillar Abeta(1-42), which is consistent with models where the carboxyl terminus is buried in the interior of the fibril and the amino terminus is on the surface. When used as an immunohistochemical probe, the antifibrillar Abeta(1-42) IgG exhibited enhanced affinity for amyloid deposits in the cerebrovasculature. We hypothesize either that the antibodies recognize a specific conformation of the eight amino-terminal residues of Abeta, which is at least 1000-fold more favored in the fibril than in monomeric peptides, or that affinity maturation of the antibodies produces an additional binding site for the amino-terminal residues of an adjacent Abeta monomer. In vivo this specificity would direct the antibody primarily to fibrillar vascular amyloid deposits even in the presence of a large excess of monomeric Abeta or its precursor. This observation may explain the vascular meningeal inflammation that developed in Alzheimer's disease patients immunized with fibrillar Abeta. Passive immunization with an antibody directed to an epitope hidden in fibrillar Abeta and in the transmembrane region of APP might be a better choice in the search for an intervention to remove Abeta monomers without provoking an inflammatory response.     

Monoclonal antibodies that target pathological assemblies of Abeta

Amyloid beta (Abeta) immunotherapy for Alzheimer's disease has shown initial success in mouse models of Alzheimer's disease and in human patients. However, because of meningoencephalitis in clinical trials of active vaccination, approaches using therapeutic antibodies may be preferred. As a novel antigen to generate monoclonal antibodies, the current study has used Abeta oligomers (amyloid beta-derived diffusible ligands, ADDLs), pathological assemblies known to accumulate in Alzheimer's disease brain. Clones were selected for the ability to discriminate Alzheimer's disease from control brains in extracts and tissue sections. These antibodies recognized Abeta oligomers and fibrils but not the physiologically prevalent Abeta monomer. Discrimination derived from an epitope found in assemblies of Abeta1-28 and ADDLs but not in other sequences, including Abeta1-40. Immunoneutralization experiments showed that toxicity and attachment of ADDLs to synapses in culture could be prevented. ADDL-induced reactive oxygen species (ROS) generation was also inhibited, establishing this response to be oligomer-dependent. Inhibition occurred whether ADDLs were prepared in vitro or obtained from Alzheimer's disease brain. As conformationally sensitive monoclonal antibodies that selectively immunoneutralize binding and function of pathological Abeta assemblies, these antibodies provide tools by which pathological Abeta assemblies from Alzheimer's disease brain might be isolated and evaluated, as well as offering a valuable prototype for new antibodies useful for Alzheimer's disease therapeutics.     

Abeta(1-40) peptide radiopharmaceuticals for brain amyloid imaging: (111)In chelation, conjugation to poly(ethylene glycol)-biotin linkers, and autoradiography with Alzheimer's disease brain sections

The amyloid plaques of Alzheimer's disease (AD) are formed by the neuropeptide Abeta(1)(-)(42/43), and carboxyl terminal truncated forms of this neuropeptide, designated Abeta(1)(-)(40), bind to amyloid plaques of AD autopsy tissue sections. Therefore, Abeta(1)(-)(40) is a potential peptide radiopharmaceutical that could be used for imaging brain amyloid in living subjects with AD, should this neuropeptide be made transportable through the blood-brain barrier (BBB). To accomplish this, the neuropeptide must be modified to enable (i) attachment to a BBB drug targeting system and (ii) labeling with a radionuclide, e.g., 111-indium, suitable for brain imaging by external detection modalities such as single photon emission computed tomography (SPECT). The present studies describe the synthesis of an Abeta(1)(-)(40) analogue that contains a biotin at the amino terminus and a diethylenetriaminepentaacetic acid (DTPA) moiety conjugated to one of the internal lysine residues. The DTPA-[N-biotin]-Abeta(1)(-)(40) was purified by gel filtration fast-protein liquid chromatography (FPLC) using two Superose 12HR columns in series, and the structure of the purified peptide was confirmed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The binding of the [(111)In]DTPA-[N-biotin]-Abeta(1)(-)(40) to amyloid plaques of AD autopsy tissue sections was demonstrated by film and emulsion autoradiography. A poly(ethylene glycol) (PEG) linker of 3400 Da molecular mass, designated PEG(3400), was inserted between the Abeta(1)(-)(40) and the biotin moiety, but this modification diminishes binding of Abeta(1)(-)(40) to the AD amyloid plaques. In summary, these studies describe a novel formulation of biotinylated Abeta(1)(-)(40) that allows radiolabeling with 111-indium. The peptide radiopharmaceutical may be conjugated to an avidin-based BBB drug targeting system to enable transport through the BBB and imaging of brain amyloid in vivo.     

Cysteine protease inhibitors effectively reduce in vivo levels of brain beta-amyloid related to Alzheimer's disease

Abnormal accumulation of neurotoxic beta-amyloid peptides (Abeta) in brain represents a key factor in the progression of Alzheimer's disease (AD). Identification of small molecules that effectively reduce brain levels of Abeta is important for development of Abeta-lowering agents for AD. In this study, we demonstrate that in vivo Abeta levels in brain are significantly reduced by the cysteine protease inhibitor E64d and the related CA074Me inhibitor, which inhibits cathepsin B. Direct infusion of these inhibitors into brains of guinea pigs resulted in reduced levels of Abeta by 50-70% after 30 days of treatment. Substantial decreases in Abeta also occurred after only 7 days of inhibitor infusion, with a reduction in both Abeta40 and Abeta42 peptide forms. A prominent decrease in Abeta peptides was observed in brain synaptosomal nerve terminal preparations after CA074Me treatment. Analyses of APP-derived proteolytic fragments showed that CA074Me reduced brain levels of the CTFbeta fragment, and increased amounts of the sAPPalpha fragment. These results suggest that CA074Me inhibits Abeta production by modulating APP processing. Animals appeared healthy after treatment with these inhibitors. These results, showing highly effective in vivo decreases in brain Abeta levels by these cysteine protease inhibitors, indicate the feasibility of using related compounds for lowering Abeta in AD.     

Anti-amyloidogenic activity of tannic acid and its activity to destabilize Alzheimer's beta-amyloid fibrils in vitro

Inhibition of the accumulation of amyloid beta-peptide (Abeta) and the formation of beta-amyloid fibrils (fAbeta) from Abeta, as well as the destabilization of preformed fAbeta in the CNS would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). We previously reported that nordihydroguaiaretic acid (NDGA) and wine-related polyphenols inhibit fAbeta formation from Abeta(1-40) and Abeta(1-42) as well as destabilizing preformed fAbeta(1-40) and fAbeta(1-42) dose-dependently in vitro. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of polymeric polyphenol, tannic acid (TA) on the formation, extension, and destabilization of fAbeta(1-40) and fAbeta(1-42) at pH 7.5 at 37 degrees C in vitro. We next compared the anti-amyloidogenic activities of TA with myricetin, rifampicin, tetracycline, and NDGA. TA dose-dependently inhibited fAbeta formation from Abeta(1-40) and Abeta(1-42), as well as their extension. Moreover, it dose-dependently destabilized preformed fAbetas. The effective concentrations (EC50) of TA for the formation, extension and destabilization of fAbetas were in the order of 0-0.1 microM. Although the mechanism by which TA inhibits fAbeta formation from Abeta as well as destabilizes preformed fAbeta in vitro is still unclear, it could be a key molecule for the development of therapeutics for AD.     

Blood-brain barrier transport of a novel micro 1-specific opioid peptide,H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA)

The purpose of this study was to clarify the mechanism of the blood-brain barrier (BBB) transport of H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA), which is a novel dermorphin analog with high affinity for the micro 1-opioid receptor. The in vivo BBB permeation influx rate of [125I]TAPA after an i.v. bolus injection (7.3 pmol/g body weight) into mice was estimated to be 0.265 +/- 0.025 microL/(min.g of brain). The influx rate of [125I]TAPA was reduced 70% by the coadministration of unlabeled TAPA (33 nmol/g of brain), suggesting the existence of a specific transport system for TAPA at the BBB. In order to elucidate the BBB transport mechanism of TAPA, a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4) was used as an in vitro model of the BBB. The acid-resistant binding of [125I]TAPA, which represents the internalization of the peptide into cells, was temperature- and concentration-dependent with a half-saturation constant of 10.0 +/- 1.7 microm. The acid-resistant binding of TAPA was significantly inhibited by 2,4-dinitrophenol, dansylcadaverine (an endocytosis inhibitor) and poly-l-lysine and protamine (polycations). These results suggest that TAPA is transported through the BBB by adsorptive-mediated endocytosis, which is triggered by binding of the peptide to negatively charged sites on the surface of brain capillary endothelial cells. Blood-brain barrier transport via adsorptive-mediated endocytosis plays a key role in the expression of the potent opioid activity of TAPA in the CNS.     

Degradation of beta-amyloid by proteolytic antibody light chains

Deposition of beta-amyloid (Abeta) is considered an important early event in the pathogenesis of Alzheimer's disease (AD). Clearance of Abeta thus represents a potential therapeutic approach. Antibody-mediated clearance of Abeta by vaccination inhibited and cleared Abeta deposition in animal models; however, inflammatory side effects were observed in humans. An alternative potentially noninflammatory approach to facilitate clearance is to proteolytically cleave Abeta. We screened 12 proteolytic recombinant antibody fragments for potential alpha-secretase activity, a naturally occurring enzyme that cleaves between the Lys16 and Leu17 residues of the amyloid precursor protein (APP). We utilized the synthetic alpha-secretase substrate, benzyloxycarbonyl-l-lysine o-nitrophenyl ester (Z-lys-o-Np) as a preliminary screen for alpha-secretase activity. Two antibody light chain fragments that hydrolyzed Z-lys-o-Np were identified. Abeta hydrolysis was studied using mass spectrometry to identify the cleavage patterns of the antibodies. The recombinant antibody light chain antibody fragment, c23.5, showed alpha-secretase-like activity, producing the 1-16 and 17-40 amino acid fragments of Abeta. The second light chain antibody fragment, hk14, demonstrated carboxypeptidase-like activity, cleaving sequentially from the carboxyl terminal of Abeta. These antibody light chains provide a novel route toward engineering efficient therapeutic antibodies capable of cleaving Abeta in vivo.     

IL-4-induced selective clearance of oligomeric beta-amyloid peptide(1-42) by rat primary type 2 microglia

A hallmark of immunopathology associated with Alzheimer's disease is the presence of activated microglia (MG) surrounding senile plaque deposition of beta-amyloid (Abeta) peptides. Abeta peptides are believed to be potent activators of MG, which leads to Alzheimer's disease pathology, but the role of MG subtypes in Abeta clearance still remains unclear. In this study, we found that IL-4 treatment of rat primary-type 2 MG enhanced uptake and degradation of oligomeric Abeta(1-42) (o-Abeta(1-42)). IL-4 treatment induced significant expression of the scavenger receptor CD36 and the Abeta-degrading enzymes neprilysin (NEP) and insulin-degrading enzyme (IDE) but reduced expression of certain other scavenger receptors. Of cytokines and stimulants tested, the anti-inflammatory cytokines IL-4 and IL-13 effectively enhanced CD36, NEP, and IDE. We demonstrated the CD36 contribution to IL-4-induced Abeta clearance: Chinese hamster ovary cells overexpressing CD36 exhibited marked, dose-dependent degradation of (125)I-labeled o-Abeta(1-42) compared with controls, the degradation being blocked by anti-CD36 Ab. Also, we found IL-4-induced clearance of o-Abeta(1-42) in type 2 MG from CD36-expressing WKY/NCrj rats but not in cells from SHR/NCrj rats with dysfunctional CD36 expression. NEP and IDE also contributed to IL-4-induced degradation of Abeta(1-42), because their inhibitors, thiorphan and insulin, respectively, significantly suppressed this activity. IL-4-stimulated uptake and degradation of o-Abeta(1-42) were selectively enhanced in type 2, but not type 1 MG that express CD40, which suggests that the two MG types may play different neuroimmunomodulating roles in the Abeta-overproducing brain. Thus, selective o-Abeta(1-42) clearance, which is induced by IL-4, may provide an additional focus for developing strategies to prevent and treat Alzheimer's disease.     

Substrate-bound beta-amyloid peptides inhibit cell adhesion and neurite outgrowth in primary neuronal cultures

Accumulation of the beta-amyloid protein (Abeta) in the brain is an important step in the pathogenesis of Alzheimer's disease. However, the mechanism of Abeta toxicity remains unclear. Abeta can bind to the extracellular matrix, a structure that regulates adhesive events such as neurite outgrowth and synaptogenesis. The binding of Abeta to the extracellular matrix suggests that Abeta may disrupt cell-substrate interactions. Therefore, the effect of substrate-bound Abeta on the growth of isolated chick sympathetic and mouse cortical neurons was examined. Abeta1-40 and Abeta1-42 had dose-dependent effects on cell morphology. When tissue culture plates were coated with 0.1-10 ng/well Abeta, neurite outgrowth increased. Higher amounts of Abeta peptides (> or =3 microg/well) inhibited outgrowth. The inhibitory effect was related to aggregation of the peptide, as preincubation of Abeta1-40 for 24 h at 37 degrees C (a process known to increase amyloid fibril formation) was necessary for inhibition of neurite outgrowth. Abeta29-42, but not Abeta1-28, also inhibited neurite outgrowth at high concentrations, demonstrating that the inhibitory domain is located within the hydrophobic C-terminal region. Abeta1-40, Abeta1-42, and Abeta29-42 also inhibited cell-substrate adhesion, indicating that the effect on neurite outgrowth may have been due to inhibition of cell adhesion. The results suggest that accumulation of Abeta may disrupt cell-adhesion mechanisms in vivo.     

Synthesis and characterization of styrylchromone derivatives as beta-amyloid imaging agents

Several promising agents have been synthesized and evaluated for in vivo imaging probes of beta-amyloid plaques in Alzheimer's disease (AD) brain. Recently, we have developed flavone derivatives, which possess the basic structure of the 2-phenylchromone, as useful candidates for amyloid imaging agents. In an attempt to further develop novel tracers, we synthesized and evaluated a series of 2-styrylchromone derivatives, which replace the 2-phenyl substituent of flavone backbone with the 2-styryl. A series of radioiodinated styrylchromone derivatives were designed and synthesized. The binding affinities for amyloid plaques were assessed by in vitro binding assay using pre-formed synthetic Abeta(1-40) aggregates. The new series of styrylchromone derivatives showed high binding affinity to Abeta aggregates at the K(d) values of 32.0, 17.5 and 8.7nM for [(125)I]6, [(125)I]9, and [(125)I]12, respectively. In biodistribution studies using normal mice, [(125)I]6 and [(125)I]9 examined in normal mice displayed high brain uptakes with 4.9 and 2.8%ID/g at 2min post injection. The radioactivity washed out from the brain rapidly (1.6 and 1.0%ID/g at 60min post injection for [(125)I]6 and [(125)I]9, respectively). But [(125)I]12 did not show marked brain uptake, and the washout rate from the brain was relatively slow throughout the time course (1.1 and 1.4%ID/g at 2 and 30min post injection, respectively). Although additional modifications are necessary to improve the brain uptake and rapid clearance of non-specifically bound radiotracer, the styrylchromone backbone may be useful as a backbone structure to develop novel beta-amyloid imaging agents.     

Fusion antibody for Alzheimer's disease with bidirectional transport across the blood-brain barrier and abeta fibril disaggregation

Delivery of monoclonal antibody therapeutics across the blood-brain barrier is an obstacle to the diagnosis or therapy of CNS disease with antibody drugs. The immune therapy of Alzheimer's disease attempts to disaggregate the amyloid plaque of Alzheimer's disease with an anti-Abeta monoclonal antibody. The present work is based on a three-step model of immune therapy of Alzheimer's disease: (1) influx of the anti-Abeta monoclonal antibody across the blood-brain barrier in the blood to brain direction, (2) binding and disaggregation of Abeta fibrils in brain, and (3) efflux of the anti-Abeta monoclonal antibody across the blood-brain barrier in the brain to blood direction. This is accomplished with the genetic engineering of a trifunctional fusion antibody that binds (1) the human insulin receptor, which mediates the influx from blood to brain across the blood-brain barrier, (2) the Abeta fibril to disaggregate amyloid plaque, and (3) the Fc receptor, which mediates the efflux from brain to blood across the blood-brain barrier. This fusion protein is a new antibody-based therapeutic for Alzheimer's disease that is specifically engineered to cross the human blood-brain barrier in both directions.     

Polymorphic fibril formation by residues 10-40 of the Alzheimer's beta-amyloid peptide

We report investigations of the morphology and molecular structure of amyloid fibrils comprised of residues 10-40 of the Alzheimer's beta-amyloid peptide (Abeta(10-40)), prepared under various solution conditions and degrees of agitation. Omission of residues 1-9 from the full-length Alzheimer's beta-amyloid peptide (Abeta(1-40)) did not prevent the peptide from forming amyloid fibrils or eliminate fibril polymorphism. These results are consistent with residues 1-9 being disordered in Abeta(1-40) fibrils, and show that fibril polymorphism is not a consequence of disorder in residues 1-9. Fibril morphology was analyzed by atomic force and electron microscopy, and secondary structure and inter-side-chain proximity were probed using solid-state NMR. Abeta(1-40) fibrils were found to be structurally compatible with Abeta(10-40): Abeta(1-40) fibril fragments were used to seed the growth of Abeta(10-40) fibrils, with propagation of fibril morphology and molecular structure. In addition, comparison of lyophilized and hydrated fibril samples revealed no effect of hydration on molecular structure, indicating that Abeta(10-40) fibrils are unlikely to contain bulk water.     

Alternative pathways for production of beta-amyloid peptides of Alzheimer's disease

This highlight article describes three Alzheimer's disease (AD) studies presented at the 5th General Meeting of the International Proteolysis Society that address enzymatic mechanisms for producing neurotoxic beta-amyloid (Abeta) peptides. One group described the poor kinetics of BACE 1 for cleaving the wild-type (WT) beta-secretase site of APP found in most AD patients. They showed that cathepsin D displays BACE 1-like specificity and cathepsin D is 280-fold more abundant in human brain than BACE 1. Nevertheless, as BACE 1 and cathepsin D show poor activity towards the WT beta-secretase site, they suggested continuing the search for additional beta-secretase(s). The second group reported cathepsin B as an alternative beta-secretase possessing excellent kinetic efficiency and specificity for the WT beta-secretase site. Significantly, inhibitors of cathepsin B improved memory, with reduced amyloid plaques and decreased Abeta(40/42) in brains of AD animal models expressing amyloid precursor protein containing the WT beta-secretase site. The third group addressed isoaspartate and pyroglutamate (pGlu) posttranslational modifications of Abeta. Results showed that cathepsin B, but not BACE 1, efficiently cleaves the WT beta-secretase isoaspartate site. Furthermore, cyclization of N-terminal Glu by glutaminyl cyclase generates highly amyloidogenic pGluAbeta(3-40/42). These presentations suggest cathepsin B and glutaminyl cyclase as potential new AD therapeutic targets.