A hallmark of Alzheimer's disease (AD) brain is the amyloid β (Aβ) plaque, which is comprised of Aβ peptides. Multiple lines of evidence suggest that Aβ oligomers are more toxic than other peptide forms. We sought to develop a robust assay to quantify oligomers from CSF. Antibody 19.3 was compared in one-site and competitive ELISAs for oligomer binding specificity. A two-site ELISA for oligomers was developed using 19.3 coupled to a sensitive, bead-based fluorescent platform able to detect single photons of emitted light. The two-site ELISA was >2500× selective for Aβ oligomers over Aβ monomers with a limit of detection ∼0.09 pg/ml in human CSF. The lower limit of reliable quantification of the assay was 0.18 pg/ml and the antibody pairs recognized Aβ multimers comprised of either synthetic standards, or endogenous oligomers isolated from confirmed human AD and healthy control brain. Using the assay, a significant 3- to 5-fold increase in Aβ oligomers in human AD CSF compared with comparably aged controls was demonstrated. The increase was seen in three separate human cohorts, totaling 63 AD and 54 controls. CSF oligomers ranged between 0.1 and 10 pg/ml. Aβ oligomer levels did not strongly associate with age or gender, but had an inverse correlation with MMSE score. The C statistic for the Aβ oligomer ROC curve was 0.86, with 80% sensitivity and 88% specificity to detect AD, suggesting reasonable discriminatory power for the AD state and the potential for utility as a diagnostic marker.

Many proteins and peptides can adhere to plastic surfaces, including polypropylene. In the case of CSF with an overall low protein concentration, such an effect may interfere with the accuracy of analysis e.g. of protein biomarkers. A substance that reduces protein adherence to plastic surfaces, like Tween-20, may enable the demonstration of more clinically-relevant levels of biomarkers. Similarly aged healthy elderly volunteers (n = 27) or patients diagnosed with early Alzheimer's disease (AD, n = 14) were lumbar punctured. CSF was collected either directly into small cryovials in ice-water and then frozen, or 5 mL was collected into a tube containing 25 m L 10% Tween-20 in PBS (final Tween concentration 0.05%), and then aliquoted and frozen. Polypropylene tubes were used throughout and all samples were stored at -80°C until analysis. Amyloid-b 42, Ab 40, total tau protein and phospho-181 tau were analysed using commercial kits. In these limited groups, 0.05% Tween-20 had no significant effect on the levels of total tau or phospho-181 tau protein measured in human CSF, even though both forms of tau were elevated in early AD compared to controls. Aβ42, but not Aβ40, was significantly reduced in CSF from patients with AD compared to controls, and addition of Tween resulted in increased measurements for both peptides in both groups in the range 20–40%. However, although the ratio of total tau or phospho-181 tau to Aβ42 or Aβ40 decreased in the presence of Tween-20, the relationship between the control and AD group medians remained approximately the same, with or without Tween-20. Addition of Tween-20 to CSF may provide a more accurate measurement of Aβ40 and Aβ42 levels in human CSF, but as the relationships between the groups do not alter, there does not seem to be any clinical advantage or disadvantage for discriminating the diagnostic groups by the addition of Tween-20 to our samples.

Current therapeutic strategies for Alzheimer's disease (AD) target brain amyloid beta (Aβ) reduction. Oligomeric Aβ is thought to be the most toxic Aβ species however analytically validated assays which are quantitative, selective and sensitive enough to measure cerebrospinal fluid (CSF) oligomers have been difficult to develop. An ELISA was developed with these properties, using an Aβ oligomer-specific antibody. Antibody pairs were tested for Aβ oligomer and monomer selectivity using standard, sandwich ELISA methodology on an Envision® chemilluminesence platform. The pair with the best selectivity (1000-fold) and sensitivity (sub-ng/mL) for oligomers, humanized monoclonal antibody 19.3 and mouse monoclonal antibody 82E1, was not sensitive enough to detect endogenous oligomers in human CSF. The 19.3/82E1 assay was migrated to the Erenna® platform, which coupled one antibody to a solution-phase magnetic bead and the other to a cleavable, fluorescent dye. Individual dye molecules were counted as a measure of detected oligomers. CSF from clinically-confirmed AD and age-matched controls was tested in blinded fashion using this assay. Monomeric Aβ40 and Aβ42 levels were also measured by ELISA. Additional studies confirming 19.3 oligomer selectivity included size-exclusion chromatography and one-site competitive ELISA; employing either synthetic oligomers or extracts from APPswe brain at ages of oligomer formation prior to plaque. The oligomer assay had a limit of detection (2x background) for Aβ oligomers at 0.04 pg/mL and a limit of reliable quantification (with CV < 20% over background) of 0.4 pg/mL. In addition, the assay demonstrated 5000-fold selectivity for oligomeric versus monomeric Aβ. The new platform enabled differentiation of CSF between 40 confirmed AD and 32 age-matched controls, with respective mean oligomer signals of 2 pg/mL and 0.56 pg/mL. In addition to cognitive score, the respective CSF Aβ monomer levels confirmed diagnosis as the AD patients had significantly reduced Aβ42, but unchanged Aβ40 versus controls. Aβ oligomers were elevated approximately 4-fold in AD CSF compared to control, consistent with previous reports using relative measures. Future work using larger sample sets will correlate oligomers with other fluid and cognitive AD biomarkers. In addition, the pharmacodynamic response of CSF Aβ oligomers following therapeutic intervention will be examined.

The pathophysiological process leading to Alzheimer's disease (AD) is poorly understood, though changes accompanying the deposition of the pathological hallmarks, plaque and neurofibrillary tangles, are better defined. Emphasis in research is increasingly attempting to shift away from the disease process when substantial neurodegeneration has taken place, towards the preclinical phase. Patients diagnosed with early AD (n=14) were lumbar punctured at 6 month intervals for 2 years. Results were compared with those from similarly-aged healthy elderly volunteers (n=27), lumbar punctured only once. CSF was collected directly into small polypropylene cryovials in ice-water and then frozen. All samples were stored at -80°C until analysis. Beta-amyloid42, Aβ 40, total tau protein and phospho-181 tau were analysed using commercial kits. A battery of cognitive tests was also performed. In these limited groups, there were significant differences between patients and controls at baseline for all biomarkers except Aβ 40, but minimal change in progression in any of these levels over the next 2 years. There were also significant differences at baseline between patients and controls for all cognitive tests. Over the subsequent two years, performance of patients on some tests (like the mini-mental state examination) was highly significantly reduced compared to baseline. By comparison, others (like the clock test) indicated less change and were only weakly significant, while some were not significantly changed, probably because of poor performance already at baseline. Changes in CSF biomarkers compared to controls must have taken place prior to diagnosis of AD. This was also true of the ability to perform certain neuropsychological tests, though some clearly employ brain abilities that must have been more intact at baseline, but which subsequently deteriorated. The battery of data overall helps to show changes in various aspects of the pathological process during early AD.

Accumulation of small soluble assemblies of amyloid-β (Aβ)(42) in the brain is thought to play a key role in the pathogenesis of Alzheimer's disease. As a result, there has been much interest in finding small molecules that inhibit the formation of synaptotoxic Aβ(42) oligomers that necessitates sensitive methods for detecting the initial steps in the oligomerization of Aβ(42). Modeling suggests that oligomerized Aβ(42) adopts a conformation in which the C-terminus is embedded in the center, whereas the N-terminus is exposed at the periphery of the oligomer. Here we report that an inverse change in Aβ(42) C-terminal and N-terminal epitope accessibility provides the basis of a sensitive method for assessing early steps in Aβ(42) oligomerization. Using ELISA and AlphaLISA, we found that Aβ(42) C-terminal immunoreactivity decreased in a time- and concentration-dependent manner under conditions favoring oligomerization. This reduction was accompanied by an increase in the N-terminal immunoreactivity, suggesting that assemblies with multiple exposed N-terminal epitopes were detected. Importantly the assay generates a robust window between monomers and oligomers at as low as 1 nM Aβ(42). Using this assay, known oligomerization inhibitors produced a dose-dependent unmasking of the Aβ(42) C-terminal epitope. After automation, the assay proved to be highly reproducible and effective for high throughput screening of small molecules that inhibit Aβ(42) oligomerization.

We have developed a novel series of pyrrolidine derived BACE-1 inhibitors. The potency of the weak initial lead structure was enhanced using library-based SAR methods. The series was then further advanced by rational design while maintaining a minimal ligand binding efficiency threshold. Ultimately, the co-crystal structure was obtained revealing that these inhibitors interacted with the enzyme in a unique fashion. In all, the potency of the series was enhanced by 4 orders of magnitude from the HTS lead with concomitant increases in physical properties needed for series advancement. The progression of these developments in a systematic fashion is described.

Accumulating evidence suggests that diffusible oligomers of Aβ (ADDLs) may be responsible for the dementia associated with Alzheimer's disease (AD). ADDLs bind to hippocampal and cortical neurons, impair neuronal function and induce deficits in cognition. One therapeutic approach for the treatment of AD is the development of antibodies that reduce Aβ in brain and thereby modulate ADDL levels. The monoclonal antibody m266 is known to bind monomers and dimers of Aβ in the periphery. Since some antibody may penetrate the blood-brain-barrier and modulate central Aβ levels, we evaluated m266 levels in the CSF and brain of hAPP over-expressing mice, its association with existing plaques and ability to attenuate the deposition of ADDLs into plaques. To assess antibody levels in brain, 12-month-old transgenic mice were administered 125I-labeled m266 (8mpk), the CSF and brain collected and processed for analysis. In addition, m266 was evaluated in a transgenic model of ADDL deposition. Recent studies have shown that ADDLs, when infused into the transgenic mouse brain, incorporate into growing plaques and seed new plaques. To investigate the ability of m266 to abate ADDL formation and deposition in brain, biotin-labeled ADDLs were infused into the hippocampus of mice to label existing plaques. The animals were then treated weekly, for 4 weeks with m266 or vehicle (8mpk; IV injection) and the deposition of endogenous ADDLs into plaques assessed with immunofluorescence. Two hours after IV infusion of 125I-labeled m266, ∼0.1% of the antibody was detected in the CSF and ∼0.18% in brain. Although m266 was present in the transgenic mouse brain, immunohistochemical studies revealed that the antibody was not associated with plaques; evidence that m266 was not directly acting to remove existing Aβ deposits. Evaluation of m266 in a model that determines the rate that ADDLs are added to existing plaques further demonstrated that the antibody did not attenuate the deposition of new material in the transgenic brain. These data suggest that m266 was not capable of abating the formation and deposition of ADDLs in the cortex and hippocampus of transgenic mice. Data from human clinical trials will assess the impact of m266 on cognition in patients with AD.

A novel series of heteroaromatic BACE-1 inhibitors is described. These inhibitors interact with the enzyme in a unique fashion that allows for potent binding in a non-traditional paradigm. In addition to the elucidation of their binding profile, a pH dependent effect on the binding affinity as a result of the intrinsic pKa of these inhibitors and the pH of the BACE-1 enzyme binding assay is discussed.

Synaptic degeneration, including impairment of synaptic plasticity and loss of synapses, is an important feature of Alzheimer disease pathogenesis. Increasing evidence suggests that these degenerative synaptic changes are associated with an accumulation of soluble oligomeric assemblies of amyloid beta (A beta) known as ADDLs. In primary hippocampal cultures ADDLs bind to a sub-population of neurons. However the molecular basis of this cell type-selective interaction is not understood. Here, using siRNA screening technology, we identified alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits and calcineurin as candidate genes potentially involved in ADDL-neuron interactions. Immunocolocalization experiments confirmed that ADDL binding occurs in dendritic spines that express surface AMPA receptors, particularly the calcium-impermeable type II AMPA receptor subunit (GluR2). Pharmacological removal of the surface AMPA receptors or inhibition of AMPA receptors with antagonists reduces ADDL binding. Furthermore, using co-immunoprecipitation and photoreactive amino acid cross-linking, we found that ADDLs interact preferentially with GluR2-containing complexes. We demonstrate that calcineurin mediates an endocytotic process that is responsible for the rapid internalization of bound ADDLs along with surface AMPA receptor subunits, which then both colocalize with cpg2, a molecule localized specifically at the postsynaptic endocytic zone of excitatory synapses that plays an important role in activity-dependent glutamate receptor endocytosis. Both AMPA receptor and calcineurin inhibitors prevent oligomer-induced surface AMPAR and spine loss. These results support a model of disease pathogenesis in which A beta oligomers interact selectively with neurotransmission pathways at excitatory synapses, resulting in synaptic loss via facilitated endocytosis. Validation of this model in human disease would identify therapeutic targets for Alzheimer disease.

Synaptic degeneration, including impairment of synaptic plasticity and loss of synapses, is thought to be involved in the early processes of Alzheimer's disease. Increasing evidence indicates that these degenerative changes are likely caused by the binding of soluble Aβ oligomers to excitatory synapses. To date, the exact mechanism(s) by which Aβ oligomers bind to synapses and cause degenerative synaptotoxicity remains elusive. A recent siRNA screen with murine N2A cells suggested that Aβ-derived diffusible ligands (ADDLs) bound to the AMPA receptor, a major class of glutamate receptor that mediates fast excitatory synaptic transmission, on the surface of neurons. Subsequent ex vivo studies with primary rat hippocampal neurons showed that ADDLs bound to dendritic spines containing AMPA receptor subunits. The ADDL binding was shown to be dependent on the presence of surface AMPA receptor, as treatments inducing internalization of AMPA receptors markedly reduced ADDL binding. Using an optimized affinity pull-down method, we also showed that AMPA receptors were present in the receptor complex bound to ADDLs. These results suggested that the binding of ADDLs to the receptor complex required the normal conformation of the AMPA receptor. Interestingly, we also found that while acute ADDL treatment induced a significant loss of surface AMPA receptor subunits from hippocampal neurons, repeated and chronic applications caused a striking aggregation of the AMPA receptor sbunit2 (gluR2) on the surface of dendrites as well as severe synapse loss. These results show that AMPA receptors are among the key components on the synaptic surface that are adversely affected by Aβ oligomers and suggest that oligomer-induced changes to synaptic morphology and the AMPA receptor signaling pathway may underlay some of the cognitive deficits seen in patients with Alzheimer's disease.

A high-throughput screen at 100 microM inhibitor concentration for the BACE-1 enzyme revealed a novel spiropiperidine iminohydantoin aspartyl protease inhibitor template. An X-ray cocrystal structure with BACE-1 revealed a novel mode of binding whereby the inhibitor interacts with the catalytic aspartates via bridging water molecules. Using the crystal structure as a guide, potent compounds with good brain penetration were designed.

Abnormal production and accumulation of amyloid-beta peptide (A beta) plays a major role in the pathogenesis of Alzheimer's disease (AD). beta-secretase (BACE1) is responsible for the cleavage at the beta-site in amyloid beta protein precursor (A beta PP/APP) to generate the N-terminus of A beta. Here we report the stepwise identification and characterization of a novel APP-beta-site mutant, "NFEW (APP-NFEV) in vitro and in cells. In vitro, the APP_NFEV exhibits 100-fold enhanced cleavage rate relative to the "wild-type" substrate (APPwt) and 10-fold increase relative to the Swedish-type mutation variant (APPsw). In cells, it was preferably cleaved among 24 APP beta-site mutations tested. More importantly, the APP_NFEV mutant failed to generate any detectable A beta peptides in BACEI-KO mouse fibroblast cells. The production of A beta peptides was restored by co-transfecting human BACE1, demonstrating that BACE1 is the only enzyme responsible for the processing of APP-NFEV in these cells. Analysis of APP_NFEV cleavage products secreted in the media revealed that in cells BACE I cleaves APP-NFEV at the position between NF and EV, identical to that observed in vitro. A BACE inhibitor blocked the processing of the APP_NFEV beta-site in vitro and in cells. Our data indicates that the "NFEW mutant is not only an enhanced substrate for BACE1 in vitro, but also a specific substrate for BACE1 in cells.

Abnormal production and accumulation of amyloid-beta peptide (Abeta) plays a major role in the pathogenesis of Alzheimer's disease (AD). beta-secretase (BACE1) is responsible for the cleavage at thebeta-site in amyloid beta protein precursor (AbetaPP/APP) to generate the N-terminus of Abeta. Here we report the stepwise identification and characterization of a novel APP-beta-site mutant, "NFEV" (APP_NFEV) in vitro and in cells. In vitro, the APP_NFEV exhibits 100-fold enhanced cleavage rate relative to the "wild-type" substrate (APPwt) and 10-fold increase relative to the Swedish-type mutation variant (APPsw). In cells, it was preferably cleaved among 24 APP beta-site mutations tested. More importantly, the APP_NFEV mutant failed to generate any detectable Abeta peptides in BACE1-KO mouse fibroblast cells. The production of Abeta peptides was restored by co-transfecting human BACE1, demonstrating that BACE1 is the only enzyme responsible for the processing of APP_NFEV in these cells. Analysis of APP_NFEV cleavage products secreted in the media revealed that in cells BACE1 cleaves APP_NFEV at the position between NF and EV, identical to that observed in vitro. A BACE inhibitor blocked the processing of the APP_NFEV beta-site in vitro and in cells. Our data indicates that the "NFEV" mutant is not only an enhanced substrate for BACE1 in vitro, but also a specific substrate for BACE1 in cells.

Zinc transporter-3 (ZnT3) protein is responsible for loading zinc into presynaptic vesicles and consequently controls the availability of zinc at the glutamatergic synapse. ZnT3 has been shown to decline with age and in Alzheimer's disease (AD) and is crucially involved in learning and memory. In this study, we utilised whole animal behavioural analyses in the ZnT3 KO mouse line, together with electrophysiological analysis of long-term potentiation in brain slices from ZnT3 KO mice, to show that metal chaperones (clioquinol, 30 mg/kg/day for 6 weeks) can prevent the age-dependent cognitive phenotype that characterises these animals. This likely occurs as a result of a homeostatic restoration of synaptic protein expression, as clioquinol significantly restored levels of various pre- and postsynaptic proteins that are critical for normal cognition, including PSD-95; AMPAR and NMDAR2b. We hypothesised that this clioquinol-mediated restoration of synaptic health resulted from a selective increase in synaptic zinc content within the hippocampus. While we demonstrated a small regional increase in hippocampal zinc content using synchrotron x-ray fluorescence microscopy, further sub-region analyses are required to determine whether this effect is seen in other regions of the hippocampal formation that are more closely linked to the synaptic plasticity effects observed in this study. These data support our recent report on the use of a different metal chaperone (PBT2) to prevent normal age-related cognitive decline and demonstrate that metal chaperones are efficacious in preventing the zinc-mediated cognitive decline that characterises ageing and disease.

Human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase (RT) coordinates DNA polymerization and ribonuclease H (RNase H) activities using two discrete active sites embedded within a single heterodimeric polyprotein. We have identified a novel thiophene diketo acid, 4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid, that selectively inhibits polymerase-independent RNase H cleavage (IC50 = 3.2 μm) but has no effect on DNA polymerization (IC50 > 50 μm). The activity profile of the diketo acid is shown to be distinct from previously described compounds, including the polymerase inhibitor foscarnet and the putative RNase H inhibitor 4-chlorophenylhydrazone. Both foscarnet and the hydrazone inhibit RNase H cleavage and DNA polymerization activities of RT, yet neither inhibits the RNase H activity of RT containing a mutation in the polymerase active site (D185N) or an isolated HIV-1 RNase H domain chimera containing the α-C helix from Escherichia coli RNase HI, suggesting these compounds affect RNase H indirectly. In contrast, the diketo acid inhibits the RNase H activity of the isolated RNase H domain as well as full-length RT, and inhibition is not affected by the polymerase active site mutation. In isothermal titration calorimetry studies using the isolated RNase H domain, binding of the diketo acid is independent of nucleic acid but strictly requires Mn2+implying a direct interaction between the inhibitor and the RNase H active site. These studies demonstrate that inhibition of HIV-1 RNase H may occur by either direct or indirect mechanisms, and they provide a framework for identifying novel agents such as 4-[5-(benzoylamino)thien- 2-yl]-2,4-dioxobutanoic acid that specifically targets RNase H.

The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3' end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metal-dependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg(2+). The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.

Diketo acids such as L-731,988 are potent inhibitors of HIV-1 integrase that inhibit integration and viral replication in cells. These compounds exhibit the unique ability to inhibit the strand transfer activity of integrase in the absence of an effect on 3' end processing. To understand the reasons for this distinct inhibitory profile, we developed a scintillation proximity assay that permits analysis of radiolabeled inhibitor binding and integrase function. High-affinity binding of L-731,988 is shown to require the assembly of a specific complex on the HIV-1 long terminal repeat. The interaction of L-731,988 with the complex and the efficacy of L-731, 988 in strand transfer can be abrogated by the interaction with target substrates, suggesting competition between the inhibitor and the target DNA. The L-731,988 binding site and that of the target substrate are thus distinct from that of the donor substrate and are defined by a conformation of integrase that is only adopted after assembly with the viral end. These results elucidate the basis for diketo acid inhibition of strand transfer and have implications for integrase-directed HIV-1 drug discovery efforts.