A common pathway for detergent-assisted oligomerization of Aß42.

Amyloid beta (Aß) aggregation is a slow process without seeding or assisted nucleation. Sodium dodecyl sulfate (SDS) micelles stabilize Aß42 small oligomers (in the dimer to tetramer range); subsequent SDS removal leads to a 150-kD Aß42 oligomer. Dodecylphosphorylcholine (DPC) micelles also stabilize an Aß42 tetramer. Here we investigate the detergent-assisted oligomerization pathway by solid-state NMR spectroscopy and molecular dynamics simulations. SDS- and DPC-induced oligomers have the same structure, implying a common oligomerization pathway. An antiparallel ß-sheet formed by the C-terminal region, the only stable structure in SDS and DPC micelles, is directly incorporated into the 150-kD oligomer. Three Gly residues (at positions 33, 37, and 38) create holes that are filled by the SDS and DPC hydrocarbon tails, thereby turning a potentially destabilizing feature into a stabilizing factor. These observations have implications for endogenous Aß aggregation at cellular interfaces.

Oligomer Formation by Amyloid-ß42 in a Membrane-Mimicking Environment in Alzheimer's Disease.

The brains of Alzheimer's disease (AD) patients contain numerous amyloid plaques that are diagnostic of the disease. The plaques are primarily composed of the amyloidogenic peptides proteins Aß40 and Aß42, which are derived by the processing of the amyloid pre-cursor protein (APP) by two proteases called ß-secretase and γ-secretase. Aß42 differs from Aß40 in having two additional hydrophobic amino acids, ILE and ALA, at the C-terminus. A small percentage of AD is autosomal dominant (ADAD) and linked either to the genes for the presenilins, which are part of γ-secretase, or APP. Because ADAD shares most pathogenic features with widespread late-onset AD, Aß peptides have become the focus of AD research. Fibrils formed by the aggregation of these peptides are the major component of plaques and were initially targeted in AD therapy. However, the fact that the abundance of plaques does not correlate well with cognitive decline in AD patients has led investigators to examine smaller Aß aggregates called oligomers. The low levels and heterogeneity of Aß oligomers have made the determination of their structures difficult, but recent structure determinations of oligomers either formed or initiated in detergents have been achieved. We report here on the structures of these oligomers and suggest how they may be involved in AD.

Solvent Deuterium Oxide Isotope Effects on the Reactions of Organophosphorylated Acetylcholinesterase.

Organophosphates (OPs) are esters of substituted phosphates, phosphonates or phosphoramidates that react with acetylcholinesterase (AChE) by initially transferring the organophosphityl group to a serine residue in the enzyme active site, concomitant with loss of an alcohol or halide leaving group. With substituted phosphates, this transfer is followed by relatively slow hydrolysis of the organophosphoryl AChE, or dephosphorylation, that is often accompanied by an aging reaction that renders the enzyme irreversibly inactivated. Aging is a dealkylation that converts the phosphate triester to a diester. OPs are very effective AChE inhibitors and have been developed as insecticides and chemical warfare agents. We examined three reactions of two organophosphoryl AChEs, dimethyl- and diethylphosphorylated AChE, by comparing rate constants and solvent deuterium oxide isotope effects for hydrolysis, aging and oxime reactivation with pralidoxime (2-PAM). Our study was motivated (1) by a published x-ray crystal structure of diethylphosphorylated AChE, which showed severe distortion of the active site that was restored by the binding of pralidoxime, and (2) by published isotope effects for decarbamoylation that decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE. We previously reconciled these results by proposing a shift in the rate-limiting step from proton transfer for the small carbamoyl group to a likely conformational change in the distorted active site of the large carbamoyl enzyme. This proposal was tested but was not supported in this report. The smaller dimethylphosphoryl AChE and the larger diethylphosphoryl AChE gave similar isotope effects for both oxime reactivation and hydrolysis, and the isotope effect values of about two indicated that proton transfer was rate limiting for both reactions.

Cathepsin D regulates cerebral Aß42/40 ratios via differential degradation of Aß42 and Aß40.

BACKGROUND: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid ß-protein (Aß) and the microtubule-associated protein, tau, and has been genetically linked to late-onset Alzheimer disease (AD). Here, we sought to examine the consequences of genetic deletion of CatD on Aß proteostasis in vivo and to more completely characterize the degradation of Aß42 and Aß40 by CatD. METHODS: We quantified Aß degradation rates and levels of endogenous Aß42 and Aß40 in the brains of CatD-null (CatD-KO), heterozygous null (CatD-HET), and wild-type (WT) control mice. CatD-KO mice die by ~ 4 weeks of age, so tissues from younger mice, as well as embryonic neuronal cultures, were investigated. Enzymological assays and surface plasmon resonance were employed to quantify the kinetic parameters (KM, kcat) of CatD-mediated degradation of monomeric human Aß42 vs. Aß40, and the degradation of aggregated Aß42 species was also characterized. Competitive inhibition assays were used to interrogate the relative inhibition of full-length human and mouse Aß42 and Aß40, as well as corresponding p3 fragments. RESULTS: Genetic deletion of CatD resulted in 3- to 4-fold increases in insoluble, endogenous cerebral Aß42 and Aß40, exceeding the increases produced by deletion of an insulin-degrading enzyme, neprilysin or both, together with readily detectable intralysosomal deposits of endogenous Aß42-all by 3 weeks of age. Quite significantly, CatD-KO mice exhibited ~ 30% increases in Aß42/40 ratios, comparable to those induced by presenilin mutations. Mechanistically, the perturbed Aß42/40 ratios were attributable to pronounced differences in the kinetics of degradation of Aß42 vis-à-vis Aß40. Specifically, Aß42 shows a low-nanomolar affinity for CatD, along with an exceptionally slow turnover rate that, together, renders Aß42 a highly potent competitive inhibitor of CatD. Notably, the marked differences in the processing of Aß42 vs. Aß40 also extend to p3 fragments ending at positions 42 vs. 40. CONCLUSIONS: Our findings identify CatD as the principal intracellular Aß-degrading protease identified to date, one that regulates Aß42/40 ratios via differential degradation of Aß42 vs. Aß40. The finding that Aß42 is a potent competitive inhibitor of CatD suggests a possible mechanistic link between elevations in Aß42 and downstream pathological sequelae in AD.

Out-of-Register Parallel ß-Sheets and Antiparallel ß-Sheets Coexist in 150-kDa Oligomers Formed by Amyloid-ß(1-42).

We present solid-state NMR measurements of ß-strand secondary structure and inter-strand organization within a 150-kDa oligomeric aggregate of the 42-residue variant of the Alzheimer's amyloid-ß peptide (Aß(1-42)). We build upon our previous report of a ß-strand spanned by residues 30-42, which arranges into an antiparallel ß-sheet. New results presented here indicate that there is a second ß-strand formed by residues 11-24. Contrary to expectations, NMR data indicate that this second ß-strand is organized into a parallel ß-sheet despite the co-existence of an antiparallel ß-sheet in the same structure. In addition, the in-register parallel ß-sheet commonly observed for amyloid fibril structure does not apply to residues 11-24 in the 150-kDa oligomer. Rather, we present evidence for an inter-strand registry shift of three residues that likely alternate in direction between adjacent molecules along the ß-sheet. We corroborated this unexpected scheme for ß-strand organization using multiple two-dimensional NMR and 13C-13C dipolar recoupling experiments. Our findings indicate a previously unknown assembly pathway and inspire a suggestion as to why this aggregate does not grow to larger sizes.

A Second Look at the Crystal Structures of Drosophila melanogaster Acetylcholinesterase in Complex with Tacrine Derivatives Provides Insights Concerning Catalytic Intermediates and the Design of Specific Insecticides.

Over recent decades, crystallographic software for data processing and structure refinement has improved dramatically, resulting in more accurate and detailed crystal structures. It is, therefore, sometimes valuable to have a second look at "old" diffraction data, especially when earlier interpretation of the electron density maps was rather difficult. Here, we present updated crystal structures of Drosophila melanogaster acetylcholinesterase (DmAChE) originally published in [Harel et al., Prot Sci (2000) 9:1063-1072], which reveal features previously unnoticed. Thus, previously unmodeled density in the native active site can be interpreted as stable acetylation of the catalytic serine. Similarly, a strong density in the DmAChE/ZA complex originally attributed to a sulfate ion is better interpreted as a small molecule that is covalently bound. This small molecule can be modeled as either a propionate or a glycinate. The complex is reminiscent of the carboxylate butyrylcholinesterase complexes observed in crystal structures of human butyrylcholinesterases from various sources, and demonstrates the remarkable ability of cholinesterases to stabilize covalent complexes with carboxylates. A very strong peak of density (10 σ) at covalent distance from the Cß of the catalytic serine is present in the DmAChE/ZAI complex. This can be undoubtedly attributed to an iodine atom, suggesting an unanticipated iodo/hydroxyl exchange between Ser238 and the inhibitor, possibly driven by the intense X-ray irradiation. Finally, the binding of tacrine-derived inhibitors, such as ZA (1DX4) or the iodinated analog, ZAI (1QON) results in the appearance of an open channel that connects the base of the active-site gorge to the solvent. This channel, which arises due to the absence of the conserved tyrosine present in vertebrate cholinesterases, could be exploited to design inhibitors specific to insect cholinesterases. The present study demonstrates that updated processing of older diffraction images, and the re-refinement of older diffraction data, can produce valuable information that could not be detected in the original analysis, and strongly supports the preservation of the diffraction images in public data banks.

Rate-limiting step in the decarbamoylation of acetylcholinesterases with large carbamoyl groups.

Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) by initially transferring the carbamoyl group to a serine residue in the enzyme active site accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme. This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4 min to more than 30 days. Therefore, carbamates are effective AChE inhibitors that have been developed as insecticides and as therapeutic agents. In this report, we review recent data showing that decarbamoylation rate constants are independent of the ester leaving group for a series of carbamic acid esters with the same carbamoyl group and that decarbamoylation rate constants decreased by 800-fold when the alkyl substituents on the carbamoyl group increased in size from N-monomethyl- to N,N-diethyl-. We also review data showing that solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change in the distorted active site in N,N-diethylcarbamoyl AChE. The nature of such a conformational change is suggested from X-ray crystal structures of AChE phosphorylated by paraoxon.

VEGF receptor-1 modulates amyloid ß 1-42 oligomer-induced senescence in brain endothelial cells.

Aggregated amyloid ß (Aß) peptides in the Alzheimer's disease (AD) brain are hypothesized to trigger several downstream pathologies, including cerebrovascular dysfunction. Previous studies have shown that Aß peptides can have antiangiogenic properties, which may contribute to vascular dysfunction in the early stages of the disease process. We have generated data showing that brain endothelial cells (ECs) exposed to toxic Aß1-42 oligomers can readily enter a senescence phenotype. To determine the effect of Aß oligomers on brain ECs, we treated early passaged human brain microvascular ECs and HUVECs with high MW Aß1-42 oligomers (5 µM, for 72 h). For controls, we used no peptide treatment, 5 µM Aß1-42 monomers, and 5 µM Aß1-42 fibrils, respectively. Brain ECs treated with Aß1-42 oligomers showed increased senescence-associated ß-galactosidase staining and increased senescence-associated p21/p53 expression. Treatment with either Aß1-42 monomer or Aß1-42 fibrils did not induce senescence in this assay. We then measured vascular endothelial growth factor receptor (VEGFR) expression in the Aß1-42 oligomer-treated ECs, and these cells showed significantly increased VEGFR-1 expression and decreased VEGFR-2 levels. Overexpression of VEGFR-1 in brain ECs readily induced senescence, suggesting a direct role of VEGFR-1 signaling events in this paradigm. More importantly, small interfering RNA-mediated knockdown of VEGFR-1 expression in brain ECs was able to prevent up-regulation of p21 protein expression and significantly reduced induction of senescence following Aß1-42 oligomer treatment. Our studies show that exposure to Aß1-42 oligomers may impair vascular functions by altering VEGFR-1 expression and causing ECs to enter a senescent phenotype. Altered VEGFR expression has been documented in brains of AD patients and suggests that this pathway may play a role in AD disease pathogenesis. These studies suggest that modulating VEGFR-1 expression and signaling events could potentially prevent senescence and rejuvenate EC functions, and provides us with a novel target to pursue for prevention and treatment of cerebrovascular dysfunction in AD.-Angom, R. S., Wang, Y., Wang, E., Pal, K., Bhattacharya, S., Watzlawik, J. O., Rosenberry, T. L., Das, P., Mukhopadhyay, D. VEGF receptor-1 modulates amyloid ß 1-42 oligomer-induced senescence in brain endothelial cells.

Decarbamoylation of acetylcholinesterases is markedly slowed as carbamoyl groups increase in size.

Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) by initially transferring the carbamoyl group to a serine residue in the enzyme active site accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme. This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4 min to more than 30 days. Therefore, carbamates are effective AChE inhibitors that have been developed as insecticides and as therapeutic agents. We show here, in contrast to a previous report, that decarbamoylation rate constants are independent of the leaving group for a series of carbamates with the same carbamoyl group. When the alkyl substituents on the carbamoyl group increased in size from N-monomethyl- to N,N-dimethyl-, N-ethyl-N-methyl-, or N,N-diethyl-, the decarbamoylation rate constants decreased by 4-, 70-, and 800-fold, respectively. We suggest that this relationship arises as a result of active site distortion, particularly in the acyl pocket of the active site. Furthermore, solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change in the distorted active site.

Distinct spatiotemporal accumulation of N-truncated and full-length amyloid-ß42 in Alzheimer's disease.

Accumulation of amyloid-ß peptides is a dominant feature in the pathogenesis of Alzheimer's disease; however, it is not clear how individual amyloid-ß species accumulate and affect other neuropathological and clinical features in the disease. Thus, we compared the accumulation of N-terminally truncated amyloid-ß and full-length amyloid-ß, depending on disease stage as well as brain area, and determined how these amyloid-ß species respectively correlate with clinicopathological features of Alzheimer's disease. To this end, the amounts of amyloid-ß species and other proteins related to amyloid-ß metabolism or Alzheimer's disease were quantified by enzyme-linked immunosorbent assays (ELISA) or theoretically calculated in 12 brain regions, including neocortical, limbic and subcortical areas from Alzheimer's disease cases (n = 19), neurologically normal elderly without amyloid-ß accumulation (normal ageing, n = 13), and neurologically normal elderly with cortical amyloid-ß accumulation (pathological ageing, n = 15). We observed that N-terminally truncated amyloid-ß42 and full-length amyloid-ß42 accumulations distributed differently across disease stages and brain areas, while N-terminally truncated amyloid-ß40 and full-length amyloid-ß40 accumulation showed an almost identical distribution pattern. Cortical N-terminally truncated amyloid-ß42 accumulation was increased in Alzheimer's disease compared to pathological ageing, whereas cortical full-length amyloid-ß42 accumulation was comparable between Alzheimer's disease and pathological ageing. Moreover, N-terminally truncated amyloid-ß42 were more likely to accumulate more in specific brain areas, especially some limbic areas, while full-length amyloid-ß42 tended to accumulate more in several neocortical areas, including frontal cortices. Immunoprecipitation followed by mass spectrometry analysis showed that several N-terminally truncated amyloid-ß42 species, represented by pyroglutamylated amyloid-ß11-42, were enriched in these areas, consistent with ELISA results. N-terminally truncated amyloid-ß42 accumulation showed significant regional association with BACE1 and neprilysin, but not PSD95 that regionally associated with full-length amyloid-ß42 accumulation. Interestingly, accumulations of tau and to a greater extent apolipoprotein E (apoE, encoded by APOE) were more strongly correlated with N-terminally truncated amyloid-ß42 accumulation than those of other amyloid-ß species across brain areas and disease stages. Consistently, immunohistochemical staining and in vitro binding assays showed that apoE co-localized and bound more strongly with pyroglutamylated amyloid-ß11-x fibrils than full-length amyloid-ß fibrils. Retrospective review of clinical records showed that accumulation of N-terminally truncated amyloid-ß42 in cortical areas was associated with disease onset, duration and cognitive scores. Collectively, N-terminally truncated amyloid-ß42 species have spatiotemporal accumulation patterns distinct from full-length amyloid-ß42, likely due to different mechanisms governing their accumulations in the brain. These truncated amyloid-ß species could play critical roles in the disease by linking other clinicopathological features of Alzheimer's disease.

Comparison of the Binding of Reversible Inhibitors to Human Butyrylcholinesterase and Acetylcholinesterase: A Crystallographic, Kinetic and Calorimetric Study.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) hydrolyze the neurotransmitter acetylcholine and, thereby, function as coregulators of cholinergic neurotransmission. Although closely related, these enzymes display very different substrate specificities that only partially overlap. This disparity is largely due to differences in the number of aromatic residues lining the active site gorge, which leads to large differences in the shape of the gorge and potentially to distinct interactions with an individual ligand. Considerable structural information is available for the binding of a wide diversity of ligands to AChE. In contrast, structural data on the binding of reversible ligands to BChE are lacking. In a recent effort, an inhibitor competition approach was used to probe the overlap of ligand binding sites in BChE. Here, we extend this study by solving the crystal structures of human BChE in complex with five reversible ligands, namely, decamethonium, thioflavin T, propidium, huprine, and ethopropazine. We compare these structures to equivalent AChE complexes when available in the protein data bank and supplement this comparison with kinetic data and observations from isothermal titration calorimetry. This new information now allows us to define the binding mode of various ligand families and will be of importance in designing specific reversible ligands of BChE that behave as inhibitors or reactivators.

Blood-Based Oligomeric and Other Protein Variant Biomarkers to Facilitate Pre-Symptomatic Diagnosis and Staging of Alzheimer's Disease.

Oligomeric forms of amyloid-ß (Aß), tau, and TDP-43 play important roles in Alzheimer's disease (AD), and therefore are promising biomarkers. We previously generated single chain antibody fragments (scFvs) that selectively bind disease-related variants of these proteins including A4, C6T, and E1, which bind different oligomeric Aß variants; D11C, which binds oligomeric tau; and AD-TDP1 and AD-TDP2, which bind disease related TDP-43 variants. To determine the utility of these disease-related variants as early biomarkers, we first analyzed 11 human sera samples obtained ∼2 years prior to an initial mild cognitive impairment (MCI) diagnosis. While the subsequent diagnoses for the cases covered several different conditions, all samples had elevated protein variant levels relative to the plasma controls although with different individual biomarker profiles. We then analyzed a set of longitudinal human plasma samples from four AD (encompassing time points prior to MCI diagnosis and continuing until after conversion to AD) and two control cases. Pre-MCI samples were characterized by high TDP-43 variant levels, MCI samples by high Aß variant levels, and AD samples by high Aß and tau variant levels. Sample time points ranged from ∼7 years pre-MCI to ∼9 years after AD conversion. Bivariate correlations showed a negative correlation with TDP-43 levels and positive correlations with cumulative Aß and oligomeric tau levels indicating an increase in neurodegenerative processes with time in AD. Detection of disease related protein variants not only readily selects AD cases from controls, but also stages progression of AD and holds promise for a pre-symptomatic blood-based biomarker profile for AD.

Development of acetophenone ligands as potential neuroimaging agents for cholinesterases.

Association of cholinesterase with ß-amyloid plaques and tau neurofibrillary tangles in Alzheimer's disease offers an opportunity to detect disease pathology during life. Achieving this requires development of radiolabelled cholinesterase ligands with high enzyme affinity. Various fluorinated acetophenone derivatives bind to acetylcholinesterase with high affinity, including 2,2,2-trifluoro-1-(3-dimethylaminophenyl)ethanone (1) and 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (2). Such compounds also offer potential for incorporation of radioactive fluorine (18F) for Positron Emission Tomography (PET) imaging of cholinesterases in association with Alzheimer's disease pathology in the living brain. Here we describe the synthesis of two meta-substituted chlorodifluoroacetophenones using a Weinreb amide strategy and their rapid conversion to the corresponding trifluoro derivatives through nucleophilic substitution by fluoride ion, in a reaction amenable to incorporating 18F for PET imaging. In vitro kinetic analysis indicates tight binding of the trifluoro derivatives to cholinesterases. Compound 1 has a Ki value of 7nM for acetylcholinesterase and 1300nM for butyrylcholinesterase while for compound 2 these values are 0.4nM and 26nM, respectively. Tight binding of these compounds to cholinesterase encourages their development for PET imaging detection of cholinesterase associated with Alzheimer's disease pathology.

Hopeahainol A binds reversibly at the acetylcholinesterase (AChE) peripheral site and inhibits enzyme activity with a novel higher order concentration dependence.

Natural product inhibitors of AChE are of interest both because they offer promise as inexpensive drugs for symptomatic relief in Alzheimer's disease and because they may provide insights into the structural features of the AChE catalytic site. Hopeahainol A is an uncharged polyphenol AChE inhibitor from the stem bark of Hopea hainanensis with a constrained, partially dearomatized bicyclic core. Molecular modeling indicates that hopeahainol A binds at the entrance of the long but narrow AChE active site gorge because it is too bulky to be accommodated within the gorge without severe distortion of the gorge as depicted in AChE crystal structures. We conducted inhibitor competition experiments in which AChE inhibition was measured with hopeahainol A together with either edrophonium (which binds at the base of the gorge) or thioflavin T (which binds to the peripheral or P-site near the gorge mouth). The results agreed with the molecular modeling and indicated that hopeahainol A at lower concentrations (<200 µM) bound only to the P-site, as hopeahainol A and thioflavin T were unable to form a ternary complex with AChE while hopeahainol A and edrophonium did form a ternary complex with essentially no competition between them. Inhibition increased to a striking extent at higher concentrations of hopeahainol A, with plots analogous to classic Dixon plots showing a dependence on hopeahainol A concentrations to the third- or fourth order. The inhibition at higher hopeahainol A concentrations was completely reversed on dilution and blocked by bound edrophonium. We hypothesize that bound hopeahainol A induces conformational changes in the AChE active site that allow binding of additional hopeahainol A molecules, a phenomenon that would be unprecedented for a reversible inhibitor that apparently forms no covalent bonds with AChE.

Opposing roles of the triggering receptor expressed on myeloid cells 2 and triggering receptor expressed on myeloid cells-like transcript 2 in microglia activation.

Mutations in triggering receptor expressed on myeloid cells 2 (TREM2), which has been proposed to regulate the inflammatory responses and the clearance of apoptotic neurons and/or amyloid-ß, are genetically linked to increased risk for late-onset Alzheimer's disease (AD). Interestingly, a missense variant in TREM-like transcript 2 (TREML2), a structurally similar protein encoded by the same gene cluster with TREM2 on chromosome 6, has been shown to protect against AD. However, the molecular mechanisms by which TREM2 and TREML2 regulate the pathogenesis of AD, and their functional relationship, if any, remain unclear. Here, we show that lipopolysaccharide (LPS) stimulation significantly suppressed TREM2 but increased TREML2 expression in mouse brain. Consistent with this in vivo result, LPS or oligomeric amyloid-ß treatment down regulated TREM2 but up-regulated TREML2 expression in primary microglia. Most important, modulation of TREM2 or TREML2 levels had opposing effects on inflammatory responses with enhancement or suppression of LPS-induced proinflammatory cytokine gene expression observed on TREM2 or TREML2 down regulation, respectively. In addition, the proliferation of primary microglia was significantly decreased when TREM2 was down regulated, whereas it was increased on TREML2 knockdown. Together, our results suggest that several microglial functions are strictly regulated by TREM2 and TREML2, whose dysfunctions likely contribute to AD pathogenesis by impairing brain innate immunity. Our findings provide novel mechanistic insights into the functions of TREM2 and TREML2 in microglia and have implications on designing new therapeutic strategies to treat AD.

Antiparallel ß-Sheet Structure within the C-Terminal Region of 42-Residue Alzheimer's Amyloid-ß Peptides When They Form 150-kDa Oligomers.

Understanding the molecular structures of amyloid-ß (Aß) oligomers and underlying assembly pathways will advance our understanding of Alzheimer's disease (AD) at the molecular level. This understanding could contribute to disease prevention, diagnosis, and treatment strategies, as oligomers play a central role in AD pathology. We have recently presented a procedure for production of 150-kDa oligomeric samples of Aß(1-42) (the 42-residue variant of the Aß peptide) that are compatible with solid-state nuclear magnetic resonance (NMR) analysis, and we have shown that these oligomers and amyloid fibrils differ in intermolecular arrangement of ß-strands. Here we report new solid-state NMR constraints that indicate antiparallel intermolecular alignment of ß-strands within the oligomers. Specifically, 150-kDa Aß(1-42) oligomers with uniform (13)C and (15)N isotopic labels at I32, M35, G37, and V40 exhibit ß-strand secondary chemical shifts in 2-dimensional (2D) finite-pulse radiofrequency-driven recoupling NMR spectra, spatial proximities between I32 and V40 as well as between M35 and G37 in 2D dipolar-assisted rotational resonance spectra, and close proximity between M35 H(α) and G37 H(α) in 2D CHHC spectra. Furthermore, 2D dipolar-assisted rotational resonance analysis of an oligomer sample prepared with 30% labeled peptide indicates that the I32-V40 and M35-G37 contacts are between residues on different molecules. We employ molecular modeling to compare the newly derived experimental constraints with previously proposed geometries for arrangement of Aß molecules into oligomers.

Acetylcholinesterase complexes with the natural product inhibitors dihydrotanshinone I and territrem B: binding site assignment from inhibitor competition and validation through crystal structure determination.

Acetylcholinesterase (AChE) is a critical enzyme that regulates neurotransmission by degrading the neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for both therapeutic drugs that treat Alzheimer's disease and organophosphate (OP) chemical warfare agents that cripple the nervous system and cause death through paralysis. We are exploring a strategy to design compounds that bind tightly at or near a peripheral or P-site near the mouth of the AChE active site gorge and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. Here, we review our recent reports on two uncharged, natural product inhibitors of AChE, dihydrotanshinone I and territrem B, that have relatively high affinities for the enzyme. We describe an inhibitor competition assay and comment on the structures of these inhibitors in complex with recombinant human acetylcholinesterase as determined by X-ray crystallography. Our results reveal that dihydrotanshinone I binding is specific to only the P-site, while territrem B binding spans the P-site and extends into the acylation or A-site at the base of the gorge.

The natural product dihydrotanshinone I provides a prototype for uncharged inhibitors that bind specifically to the acetylcholinesterase peripheral site with nanomolar affinity.

Cholinergic synaptic transmission often requires extremely rapid hydrolysis of acetylcholine by acetylcholinesterase (AChE). AChE is inactivated by organophosphates (OPs) in chemical warfare nerve agents. The resulting accumulation of acetylcholine disrupts cholinergic synaptic transmission and can lead to death. A potential long-term strategy for preventing AChE inactivation by OPs is based on evidence that OPs must pass through a peripheral site or P-site near the mouth of the AChE active site gorge before reacting with a catalytic serine in an acylation site or A-site at the base of the gorge. An ultimate goal of this strategy is to design compounds that bind tightly at or near the P-site and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site with ligands and potential drugs that selectively restrict access, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. We apply here an inhibitor competition assay that can correctly determine whether an AChE inhibitor binds to the P-site, the A-site, or both sites. We have used this assay to examine three uncharged, natural product inhibitors of AChE, including aflatoxin B1, dihydrotanshinone I, and territrem B. The first two of these inhibitors are predicted by the competition assay to bind selectively to the P-site, while territrem B is predicted to span both the P- and A-sites. These predictions have recently been confirmed by X-ray crystallography. Dihydrotanshinone I, with an observed binding constant (KI) of 750 nM, provides a good lead compound for the development of high-affinity, uncharged inhibitors with specificity for the P-site.

The Alzheimer's amyloid-ß(1-42) peptide forms off-pathway oligomers and fibrils that are distinguished structurally by intermolecular organization.

Increasing evidence suggests that soluble aggregates of amyloid-ß (Aß) initiate the neurotoxicity that eventually leads to dementia in Alzheimer's disease. Knowledge on soluble aggregate structures will enhance our understanding of the relationship between structures and toxicities. Our group has reported a stable and homogeneous preparation of Aß(1-42) oligomers that has been characterized by various biophysical techniques. Here, we have further analyzed this species by solid state nuclear magnetic resonance (NMR) spectroscopy and compared NMR results to similar observations on amyloid fibrils. NMR experiments on Aß(1-42) oligomers reveal chemical shifts of labeled residues that are indicative of ß-strand secondary structure. Results from two-dimensional dipolar-assisted rotational resonance experiments indicate proximities between I31 aliphatic and F19 aromatic carbons. An isotope dilution experiment further indicates that these contacts between F19 and I31 are intermolecular, contrary to models of Aß oligomers proposed previously by others. For Aß(1-42) fibrils, we observed similar NMR lineshapes and inter-side-chain contacts, indicating similar secondary and quaternary structures. The most prominent structural differences between Aß(1-42) oligomers and fibrils were observed through measurements of intermolecular (13)C-(13)C dipolar couplings observed in PITHIRDS-CT experiments. PITHIRDS-CT data indicate that, unlike fibrils, oligomers are not characterized by in-register parallel ß-sheets. Structural similarities and differences between Aß(1-42) oligomers and fibrils suggest that folded ß-strand peptide conformations form early in the course of self-assembly and that oligomers and fibrils differ primarily in schemes of intermolecular organization. Distinct intermolecular arrangements between Aß(1-42) oligomers and fibrils may explain why this oligomeric state appears off-pathway for monomer self-assembly to fibrils.