Identification and structural characterisation of carboxy-terminal polypeptides and antibody epitopes of Alzheimer's amyloid precursor protein using high-resolution mass spectrometry

Alzheimer's disease (AD) is the most common cause for human age-related dementia, characterised by formation of diffuse plaques in brain that are directly involved in AD pathogenesis. The major component of AD plaques is beta-amyloid, a 40 to 42 amino acid polypeptide derived from the amyloid precursor protein (APP) by proteolytic degradation involving the specific proteases, beta-and gamma-secretase acting at the N- and C- terminal cleavage site, respectively. In this study we have prepared polypeptides comprising the carboxy-terminal and transmembrane sequences of APP, by bacterial expression and chemical synthesis, as substrates for studying the C-terminal processing of APP and its interaction with the gamma-secretase complex. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used as a major tool for structure analysis. Immunisation of transgenic mouse models of AD with Abeta42 has been recently shown to be effective to inhibit and disaggregate Abeta-fibrils, and to reduce AD-related neuropathology and memory impairments. However, the mechanism underlying these therapeutic effects has been as yet unclear. Using proteolytic epitope excision from immune complexes in combination with FT-ICR-MS, we identified the epitope recognised by the therapeutically active antibody as the N-terminal Abeta(4-10) sequence; this soluble, nontoxic epitope opens new lead structures for AD vaccine development. A monoclonal antibody (Jonas; JmAb) directed against the cytosolic APP domain was used in studies of APP biochemistry and metabolism. Here we report the identification of the epitope recognised by the JmAb, using the combination of epitope excision and peptide mapping by FT-ICR-MS. The epitope was determined to be located at the C-terminal APP(740-747) sequence; it was confirmed by ELISA binding assays and authentic synthetic peptides and will be an efficient tool in the development of new specific vaccines. These results demonstrate high-resolution FT-ICR-MS as a powerful method for characterising biochemical pathways and molecular recognition structures of APP.     

Aggregation state and neurotoxic properties of alzheimer β-amyloid peptide

Alzheimer's disease (AD) represents the most common form of senile dementia and represents a tremendous health problem as the world population is aging. AD is characterized by the accumulation of amyloid β-peptide (Aβ) in the brain and the loss of cholinergic neurons in the basal forebrain. Accumulation of soluble and insoluble assemblies of Aβ in the brain is a crucial event in AD pathogenesis and the presence of amyloid plaques in the brain is required for definitive identification of AD. Yet, there is no correlation between amyloid plaques and the degree of dementia. In the past two decades researchers have devoted their effort to study and explain the mechanisms involved in the pathology of this devastating disease. Studies from different areas of the natural and medical sciences have provided important information towards the elucidation of some of the pathological processes that take place in AD. An aspect of crucial importance is the aggregation state of Aβ peptide and its role in neuropathology. Here, we discuss recent studies aimed at the identification of Aβ protein aggregates, the characterization of their toxic potential and the development of therapeutic strategies that target Aβ aggregation.     

Discovery of Chemicals to Either Clear or Indicate Amyloid Aggregates by Targeting Memory-Impairing Anti-Parallel Aβ Dimers

Amyloid-β (Aβ) oligomers are implicated in Alzheimer disease (AD). However, their unstable nature and heterogeneous state disrupts elucidation of their explicit role in AD progression, impeding the development of tools targeting soluble Aβ oligomers. Herein parallel and anti-parallel variants of Aβ(1–40) dimers were designed and synthesized, and their pathogenic properties in AD models characterized. Anti-parallel dimers induced cognitive impairments with increased amyloidogenesis and cytotoxicity, and this dimer was then used in a screening platform. Through screening, two FDA-approved drugs, Oxytetracycline and Sunitinib, were identified to dissociate Aβ oligomers and plaques to monomers in 5XFAD transgenic mice. In addition, fluorescent Astrophloxine was shown to detect aggregated Aβ in brain tissue and cerebrospinal fluid samples of AD mice. This screening platform provides a stable and homogeneous environment for observing Aβ interactions with dimer-specific molecules.     

Analysis of differential plaque depositions in the brains of Tg2576 and Tg-APPswe/PS1dE9 transgenic mouse models of Alzheimer disease

Adequate assessment of plaque deposition levels in the brain of mouse models of Alzheimer disease (AD) is required in many core issues of studies on AD, including studies on the mechanisms underlying plaque pathogenesis, identification of cellular factors modifying plaque pathology, and developments of anti-AD drugs. The present study was undertaken to quantitatively evaluate plaque deposition patterns in the brains of the two popular AD models, Tg2576 and Tg-APPswe/ PS1dE9 mice. Coronally-cut brain sections of Tg2576 and Tg-APPswe/PS1dE9 mice were prepared and plaque depositions were visualized by staining with anti- amyloid β peptides antibody. Microscopic images of plaque depositions in the prefrontal cortex, parietal cortex, piriform cortex and hippocampus were obtained and the number of plaques in each region was determined by a computer-aided image analysis method. A series of optical images representing a gradual increase of plaque deposition levels were selected in the four different brain regions and were assigned in each with a numerical grade of 1-6, where +1 was lowest and +6, highest, so that plaques per unit in mm(2) increased "sigmoidally" over the grading scales. Analyzing plaque depositions using the photographic plaque reference panels and a computer-aid image analysis method, it was demonstrated that the brains of Tg2576 mice started to accumulate predominantly small plaques, while the brains of Tg-APPswe/PS1dE9 mice deposited relatively large plaques.     

Discovery of Chemicals to Either Clear or Indicate Amyloid Aggregates by Targeting Memory‐Impairing Anti‐Parallel Aβ Dimers

Amyloid‐β (Aβ) oligomers are implicated in Alzheimer disease (AD). However, their unstable nature and heterogeneous state disrupts elucidation of their explicit role in AD progression, impeding the development of tools targeting soluble Aβ oligomers. Herein parallel and anti‐parallel variants of Aβ(1–40) dimers were designed and synthesized, and their pathogenic properties in AD models characterized. Anti‐parallel dimers induced cognitive impairments with increased amyloidogenesis and cytotoxicity, and this dimer was then used in a screening platform. Through screening, two FDA‐approved drugs, Oxytetracycline and Sunitinib, were identified to dissociate Aβ oligomers and plaques to monomers in 5XFAD transgenic mice. In addition, fluorescent Astrophloxine was shown to detect aggregated Aβ in brain tissue and cerebrospinal fluid samples of AD mice. This screening platform provides a stable and homogeneous environment for observing Aβ interactions with dimer‐specific molecules.     

Incubation with Cu(II) and Zn(II) salts enhances MALDI‐TOF mass spectra of amyloid‐beta and α‐synuclein toward in vivo analysis

Insoluble senile plaque aggregates are indicative of Alzheimer's disease pathology. A similar phenomenon occurs in Parkinson's disease with the build‐up of Lewy bodies. The analysis of senile plaques, and other brain samples, from Alzheimer's disease and Parkinson's disease patients by matrix‐assisted laser desorption/ionization mass spectrometry has advantages but also presents obstacles because of the nature of the processes utilized in isolation procedures and storage. Salts, buffers, and detergents necessary in the isolation of biological species may cause adducts and ion suppression that convolute the spectra obtained. We previously determined that amyloid‐beta from isolated senile plaque deposits fragment similarly to the synthetic 40 and 42 amino acid peptide when analyzed by matrix‐assisted laser desorption/ionization mass spectrometry. In addition, α‐synuclein also fragments predictably by in‐source decay. This provides information that may be applied to the identification and localization of amyloid‐beta and α‐synuclein in senile plaques and intact tissue sections. Ion suppression must still be accounted for when analyzing biological samples, which makes identifying fragments at lower abundance difficult. The addition of certain transition‐metal salts (Cu(II), Zn(II)) to the sample prior to analysis serves to “clean” the spectra and allow the peptide fragments produced to be observed with a much higher signal to noise and occasionally, improved resolution. We present a systematic study of incubation with different metal salts and their impact on the quality of the spectra, as well as the role of the binding of the metals to the model biological compounds, obtained for synthetic amyloid‐beta, synthetic α‐synuclein, and isolated senile plaques. The optimized sample preparation methods presented will provide for simpler and more thorough identification of these biologically relevant species in human‐derived samples.     

Zinc-amyloid beta interactions on a millisecond time-scale stabilize non-fibrillar Alzheimer-related species

The role of zinc, an essential element for normal brain function, in the pathology of Alzheimer's disease (AD) is poorly understood. On one hand, physiological and genetic evidence from transgenic mouse models supports its pathogenic role in promoting the deposition of the amyloid beta-protein (Abeta) in senile plaques. On the other hand, levels of extracellular ("free") zinc in the brain, as inferred by the levels of zinc in cerebrospinal fluid, were found to be too low for inducing Abeta aggregation. Remarkably, the release of transient high local concentrations of zinc during rapid synaptic events was reported. The role of such free zinc pulses in promoting Abeta aggregation has never been established. Using a range of time-resolved structural and spectroscopic techniques, we found that zinc, when introduced in millisecond pulses of micromolar concentrations, immediately interacts with Abeta 1-40 and promotes its aggregation. These interactions specifically stabilize non-fibrillar pathogenic related aggregate forms and prevent the formation of Abeta fibrils (more benign species) presumably by interfering with the self-assembly process of Abeta. These in vitro results strongly suggest a significant role for zinc pulses in Abeta pathology. We further propose that by interfering with Abeta self-assembly, which leads to insoluble, non-pathological fibrillar forms, zinc stabilizes transient, harmful amyloid forms. This report argues that zinc represents a class of molecular pathogens that effectively perturb the self-assembly of benign Abeta fibrils, and stabilize harmful non-fibrillar forms.     

Increased expression of the receptor for advanced glycation end products in neurons and astrocytes in a triple transgenic mouse model of Alzheimer's disease

The receptor for advanced glycation end products (RAGE) has been reported to have a pivotal role in the pathogenesis of Alzheimer''s disease (AD). This study investigated RAGE levels in the hippocampus and cortex of a triple transgenic mouse model of AD (3xTg-AD) using western blotting and immunohistochemical double-labeling to assess cellular localization. Analysis of western blots showed that there were no differences in the hippocampal and cortical RAGE levels in 10-month-old adult 3xTg-AD mice, but significant increases in RAGE expression were found in the 22- to 24-month-old aged 3xTg-AD mice compared with those of age-matched controls. RAGE-positive immunoreactivity was observed primarily in neurons of aged 3xTg-AD mice with very little labeling in non-neuronal cells, with the notable exception of RAGE presence in astrocytes in the hippocampal area CA1. In addition, RAGE signals were co-localized with the intracellular amyloid precursor protein (APP)/amyloid beta (Aβ) but not with the extracellular APP/Aβ. In aged 3xTg-AD mice, expression of human tau was observed in the hippocampal area CA1 and co-localized with RAGE signals. The increased presence of RAGE in the 3xTg-AD animal model showing critical aspects of AD neuropathology indicates that RAGE may contribute to cellular dysfunction in the AD brain.     

The amyloid-beta (Abeta) peptide pattern in cerebrospinal fluid in Alzheimer's disease: evidence of a novel carboxyterminally elongated Abeta peptide

The patterns of amyloid beta (Abeta) peptides in human cerebrospinal fluid (CSF) and brain homogenates were studied by surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry, and the results were compared with those obtained by Abeta-SDS-PAGE/immunoblot. Apart from the peptides known in the literature to occur in the CSF, we postulate the existence of a novel, previously not described peptide, either Abeta1-45 or Abeta2-46. This peptide was observed exclusively in a pool of samples originating from patients with AD, i.e. CSF and postmortem brain homogenates, but not in either the pooled CSF samples nor the pooled brain homogenates of the non-demented controls. Similarly to our previous results, Abeta1-42 was decreased in the CSF in AD. Expectedly, brain homogenates of the control subjects did not show the presence of Abeta peptides. Compared with Abeta-SDS-PAGE/immunoblot, SELDI-TOF enabled more precise analysis of Abeta peptides in the human material. We conclude that SELDI-TOF offers a promising tool for dementia expression pattern profiling using a minute amount of a biological sample.     

From brain to food: analysis of phosphatidylcholins, lyso-phosphatidylcholins and phosphatidylcholin-plasmalogens derivates in Alzheimer's disease human post mortem brains and mice model via mass spectrometry

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by extracellular senile plaques mainly consisting of Aβ, a 40-42 amino acid long peptide, and intracellular neurofibrillary tangles, accompanied by an excessive loss of synapses. Recently evidence accumulated that nutrition, especially polyunsaturated fatty acids, influences AD pathogenesis. Especially mid-life food habits with the consumption of specific fatty acids (FA) appear to influence the disease risk. The timely separation between food intake and disease makes a direct correlation with detailed analysis of eating habits combined with accurate food analysis nearly unattainable. A possible solution to circumvent these difficulties is to investigate the FA composition in human post mortem brain. In this study we focused on the main phospholipids phosphatidylcholin (PC), phosphatidylcholin-plasmalogen (PC-PL) and lyso-phosphatidylcholin (lyso-PC) in AD brains compared to control brains. Frontal cortices, temporal cortices and cerebellum of 30 AD (mean 78 years) and 14 control aged matched brains (mean 77.4 years) as well as APP transgenic mice compared to control mice were analyzed using an AB Sciex 4000 Qtrap mass spectrometer utilizing a FIA MS/MS method. PC, PC-PL and lyso-PC metabolites were analyzed in respect to saturation level and FA composition. As expected, the majority of the lipid species showed no significant differences, but interestingly a few species revealed a highly significant reduction in AD brains. These FAs are potential candidates for further food analysis in respect to AD pathology. Additionally, we show that the method applied with multiple reaction monitoring (MRM) used for this study is suitable for semi quantitative analysis of small amounts (10 μl) of brain tissue.     

Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer's disease amyloid-β protein

Background: Amyloid plaques composed of the fibrillar form of the amyloid-β protein (Aβ) are the defining neuropathological feature of Alzheimer's disease (AD). A detailed understanding of the time course of amyloid formation could define steps in disease progression and provide targets for therapeutic intervention. Amyloid fibrils, indistinguishable from those derived from an AD brain, can be produced in vitro using a seeded polymerization mechanism. In its simplest form, this mechanism involves a cooperative transition from monomeric Aβ to the amyloid fibril without the buildup of intermediates. Recently, however, a transient species, the Aβ amyloid protofibril, has been identified. Here, we report studies of Aβ amyloid protofibril and its seeded transition into amyloid fibrils using atomic force microscopy.Results: Seeding of the protofibril-to-fibril transition was observed. Preformed fibrils, but not protofibrils, effectively seeded this transition. The assembly state of Aβ influenced the rate of seeded growth, indicating that protofibrils are fibril assembly precursors. The handedness of the helical surface morphology of fibrils depended on the chirality of Aβ. Finally, branched and partially wound fibrils were observed.Conclusions: The temporal evolution of morphologies suggests that the protofibril-to-fibril transition is nucleation-dependent and that protofibril winding is involved in that transition. Fibril unwinding and branching may be essential for the post-nucleation growth process. The protofibrillar assembly intermediate is a potential target for AD therapeutics aimed at inhibiting amyloid formation and AD diagnostics aimed at detecting presymptomatic disease.     

Amyloidosis in Alzheimer’s Disease: Pathogeny,Etiology, and Related Therapeutic Directions

The amyloid hypothesis of Alzheimer’s disease has long been the predominant theory, suggesting that Alzheimer’s disease is caused by the accumulation of amyloid beta protein (Aβ) in the brain, leading to neuronal toxicity in the central nervous system (CNS). Because of breakthroughs in molecular medicine, the amyloid pathway is thought to be central to the pathophysiology of Alzheimer’s disease (AD). Currently, it is believed that altered biochemistry of the Aβ cycle remains a central biological feature of AD and is a promising target for treatment. This review provides an overview of the process of amyloid formation, explaining the transition from amyloid precursor protein to amyloid beta protein. Moreover, we also reveal the relationship between autophagy, cerebral blood flow, ACHE, expression of LRP1, and amyloidosis. In addition, we discuss the detailed pathogenesis of amyloidosis, including oxidative damage, tau protein, NFTs, and neuronal damage. Finally, we list some ways to treat AD in terms of decreasing the accumulation of Aβ in the brain.     

Ex situ atomic force microscopy analysis of beta-amyloid self-assembly and deposition on a synthetic template

The beta-amyloid (Abeta) deposition, which is the conversion of soluble Abeta peptides to insoluble plaques on a surface, is an essential pathological process in Alzheimer's disease (AD). The identification and characterization of possible environmental factors that may influence amyloid deposition in vivo are important to unveil the underlying etiology of AD. According to the amyloid cascade hypothesis, diffuse plaques are initial and visual deposits in the early event of AD, leading to amyloid plaques. To study amyloid deposition and growth in vitro, we prepared a synthetic template by immobilizing Abeta seeds on an N-hydroxysuccinimide ester-activated solid surface. According to our analysis with an ex situ atomic force microscope, the formation of amyloid plaque-like aggregates was mediated by the interaction between Abeta in a solution and on a synthetic template, suggesting that Abeta oligomers function well as seeds for amyloid deposition. It was observed that insoluble amyloid aggregates formed on the template surface serve as a sink of soluble Abeta in a solution as well as mediate the formation of intermediates in the pathway of amyloid fibrillization in a solution. Relative seeding efficiencies of fresh monomers, oligomers, and fully grown fibrils were analyzed by measuring the deposited plaque volume and its height distribution through atomic force microscopy. The result revealed that oligomeric forms of Abeta act more efficiently as seeds than monomers or fibrils do. Fluorescence spectroscopy with thioflavin T confirmed that amyloid aggregate formation proceeds in a concentration-dependent manner. Analysis with Fourier transform infrared spectroscopy indicated a progressive transition of soluble Abeta42 monomer to amyloid fibrils having antiparallel beta-sheet structure on the template. Furthermore, studies on the interaction between Abeta40 and 42, two major variants of Abeta derived from the amyloid precursor protein, showed that amyloid aggregate formation on the surface was accelerated further by the homogeneous association of soluble Abeta42 onto Abeta42 seeds than by other combinations. A slightly acidic condition was found to be unfavorable for amyloid formation. This study gives insight into understanding the effects of environmental factors on amyloid formation via the use of a synthetic template system.     

Differentiating Aβ40 and Aβ42 in amyloid plaques with a small molecule fluorescence probe

Differentiating amyloid beta (Aβ) subspecies Aβ40 and Aβ42 has long been considered an impossible mission with small-molecule probes. In this report, based on recently published structures of Aβ fibrils, we designed iminocoumarin–thiazole (ICT) fluorescence probes to differentiate Aβ40 and Aβ42, among which Aβ42 has much higher neurotoxicity. We demonstrated that ICTAD-1 robustly responds to Aβ fibrils, evidenced by turn-on fluorescence intensity and red-shifting of emission peaks. Remarkably, ICTAD-1 showed different spectra towards Aβ40 and Aβ42 fibrils. In vitro results demonstrated that ICTAD-1 could be used to differentiate Aβ40/42 in solutions. Moreover, our data revealed that ICTAD-1 could be used to separate Aβ40/42 components in plaques of AD mouse brain slides. In addition, two-photon imaging suggested that ICTAD-1 was able to cross the BBB and label plaques in vivo. Interestingly, we observed that ICTAD-1 was specific toward plaques, but not cerebral amyloid angiopathy (CAA) on brain blood vessels. Given Aβ40 and Aβ42 species have significant differences of neurotoxicity, we believe that ICTAD-1 can be used as an important tool for basic studies and has the potential to provide a better diagnosis in the future.

A small molecule fluorescence probe ICTAD-1 was rationally designed for differentiating Aβ40 and Aβ42 in solutions and in Aβ plaques.     

[124I]IBETA: A New Aβ Plaque Positron Emission Tomography Imaging Agent for Alzheimer’s Disease

Several fluorine-18-labeled PET β-amyloid (Aβ) plaque radiotracers for Alzheimer’s disease (AD) are in clinical use. However, no radioiodinated imaging agent for Aβ plaques has been successfully moved forward for either single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. Radioiodinated pyridyl benzofuran derivatives for the SPECT imaging of Aβ plaques using iodine-123 and iodine-125 are being pursued. In this study, we assess the iodine-124 radioiodinated pyridyl benzofuran derivative 5-(5-[¹²⁴I]iodobenzofuran-2-yl)-N,N-dimethylpyridin-2-amine ([¹²⁴I]IBETA) (Ki = 2.36 nM) for utilization in PET imaging for Aβ plaques. We report our findings on the radioiododestannylation reaction used to prepare [^124/125I]IBETA and evaluate its binding to Aβ plaques in a 5 × FAD mouse model and postmortem human AD brain. Both [¹²⁵I]IBETA and [¹²⁴I]IBETA are produced in >25% radiochemical yield and >85% radiochemical purity. The in vitro binding of [¹²⁵I]IBETA and [¹²⁴I]IBETA in transgenic 5 × FAD mouse model for Aβ plaques was high in the frontal cortex, anterior cingulate, thalamus, and hippocampus, which are regions of high Aβ accumulation, with very little binding in the cerebellum (ratio of brain regions to cerebellum was >5). The in vitro binding of [¹²⁵I]IBETA and [¹²⁴I]IBETA in postmortem human AD brains was higher in gray matter containing Aβ plaques compared to white matter (ratio of gray to white matter was >5). Anti-Aβ immunostaining strongly correlated with [¹²⁴/¹²⁵I]IBETA regional binding in both the 5 × FAD mouse and postmortem AD human brains. The binding of [¹²⁴/¹²⁵I]IBETA in 5 × FAD mouse and postmortem human AD brains was displaced by the known Aβ plaque imaging agent, Flotaza. Preliminary PET/CT studies of [¹²⁴I]IBETA in the 5 × FAD mouse model suggested [¹²⁴I]IBETA was relatively stable in vivo with a greater localization of [¹²⁴I]IBETA in the brain regions with a high concentration of Aβ plaques. Some deiodination was observed at later time points. Therefore, [¹²⁴I]IBETA may potentially be a useful PET radioligand for Aβ plaques in brain studies.     

Natural Compounds against Alzheimer’s Disease: Molecular Recognition of Aβ1–42 Peptide by Salvia sclareoides Extract and its Major Component,Rosmarinic Acid,as Investigated by NMR

Amyloid peptides, Aβ1–40 and Aβ1–42, represent major molecular targets to develop potential drugs and diagnostic tools for Alzheimer’s Disease (AD). In fact, oligomeric and fibrillar aggregates generated by these peptides are amongst the principal components of amyloid plaques found post mortem in patients suffering from AD. Rosmarinic acid has been demonstrated to be effective in preventing the aggregation of amyloid peptides in vitro and to delay the progression of the disease in animal models. Nevertheless, no information is available about its molecular mechanism of action. Herein, we report the NMR characterization of the interaction of Salvia sclareoides extract and that of its major component, rosmarinic acid, with Aβ1–42 peptide, whose oligomers have been described as the most toxic Aβ species in vivo. Our data shed light on the structural determinants of rosmarinic acid–Aβ1–42 oligomers interaction, thus allowing the elucidation of its mechanism of action. They also provide important information for the rational design of new compounds with higher affinity for Aβ peptides to generate new anti‐amyloidogenic molecules and/or molecular tools for the specific targeting of amyloid aggregates in vivo. In addition, we identified methyl caffeate, another natural compound present in different plants and human diet, as a good ligand of Aβ1–42 oligomers, which also shows anti‐amyloidogenic activity. Finally, we demonstrated the possibility to exploit STD‐NMR and trNOESY experiments to screen extracts from natural sources for the presence of Aβ peptide ligands.     

Oligomer Formation by Amyloid-β42 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 Aβ40 and Aβ42, which are derived by the processing of the amyloid pre-cursor protein (APP) by two proteases called β-secretase and γ-secretase. Aβ42 differs from Aβ40 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, Aβ 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 Aβ aggregates called oligomers. The low levels and heterogeneity of Aβ 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.     

In Silico Analysis of Metabolites from Peruvian Native Plants as Potential Therapeutics against Alzheimer’s Disease

Background: Despite research on the molecular bases of Alzheimer’s disease (AD), effective therapies against its progression are still needed. Recent studies have shown direct links between AD progression and neurovascular dysfunction, highlighting it as a potential target for new therapeutics development. In this work, we screened and evaluated the inhibitory effect of natural compounds from native Peruvian plants against tau protein, amyloid beta, and angiotensin II type 1 receptor (AT1R) pathologic AD markers. Methods: We applied in silico analysis, such as virtual screening, molecular docking, molecular dynamics simulation (MD), and MM/GBSA estimation, to identify metabolites from Peruvian plants with inhibitory properties, and compared them to nicotinamide, telmisartan, and grapeseed extract drugs in clinical trials. Results: Our results demonstrated the increased bioactivity of three plants’ metabolites against tau protein, amyloid beta, and AT1R. The MD simulations indicated the stability of the AT1R:floribundic acid, amyloid beta:rutin, and tau:brassicasterol systems. A polypharmaceutical potential was observed for rutin due to its high affinity to AT1R, amyloid beta, and tau. The metabolite floribundic acid showed bioactivity against the AT1R and tau, and the metabolite brassicasterol showed bioactivity against the amyloid beta and tau. Conclusions: This study has identified molecules from native Peruvian plants that have the potential to bind three pathologic markers of AD.     

Anionic Oligothiophenes Compete for Binding of X‐34 but not PIB to Recombinant Aβ Amyloid Fibrils and Alzheimer's Disease Brain‐Derived Aβ

Deposits comprised of amyloid‐β (Aβ) are one of the pathological hallmarks of Alzheimer's disease (AD) and small hydrophobic ligands targeting these aggregated species are used clinically for the diagnosis of AD. Herein, we observed that anionic oligothiophenes efficiently displaced X‐34, a Congo Red analogue, but not Pittsburgh compound B (PIB) from recombinant Aβ amyloid fibrils and Alzheimer's disease brain‐derived Aβ. Overall, we foresee that the oligothiophene scaffold offers the possibility to develop novel high‐affinity ligands for Aβ pathology only found in human AD brain, targeting a different site than PIB.     

Direct identification of proteins from T47D cells and murine brain tissue by matrix-assisted laser desorption/ionization post-source decay/collision-induced dissociation

The purpose of this study is to determine the feasibility of the direct matrix-assisted laser desorption/ionization (MALDI) identification of proteins in fixed T47D breast cancer cells and murine brain tissues. The ability to identify proteins from cells and tissue may lead to biomarkers that effectively predict the onset of defined disease states, and their dynamic behavior could be an important hint for drug target discoveries. Direct tissue application of trypsin allows protein identification in cells and tissues, while maintaining spatial integrity and intracellular organization. Using a chemical printer, matrix was co-registered on trypsinized human T47D breast cancer cells and cryo-preserved sections of murine brain tissue, followed by MALDI post-source decay (PSD) or MALDI collision-induced dissociation (CID), respectively. Mass-to-charge (m/z) data from the cells and brain tissues were processed using Mascot software interrogation of the National Center for Biotechnology Information (NCBI) database. Histone H2B was identified from cultured T47D human breast cancer cells. Tubulin beta2 was identified from mouse brain cortex following an induced stroke. These results suggest that MALDI PSD/CID, combined with bioinformatics, can be used for the direct identification of proteins from cells and tissues. Refinements in preparation techniques may improve this approach to provide a tool for quantitative proteomics and clinical analysis.