Aβ Oligomers Research Articles

Cutting-edge approach for Alzheimer’s disease detection in the early stages: an overview

Highlights Alzheimer’s disease (AD) is the leading cause of dementia. Selective memory impairment is a hallmark of AD. A novel blood test may identify the underlying toxin in AD. Amyloid-beta plaques outside of cells in the brain could not be harmful on their own. Alzheimer’s disease (AD) is the leading cause of dementia and a neurodegenerative ailment whose aetiology and pathophysiology are poorly understood. Selective memory impairment is a hallmark of AD and one of the first detectable symptoms in certain patients. Treatments exist to help with symptoms, but there is presently no cure, and the condition will worsen for all people eventually1. A novel blood test may identify the underlying toxin in AD, years before the onset of memory loss and cognitive decline. AD molecular pathology begins early on with the creation of toxic amyloid-peptide-beta (Aβ) oligomers2. Potentially elevated levels of phosphorylated tau181 (p-tau181) in the plasma are also associated with AD3. Impaired neural signalling, neuroinflammation, tau phosphorylation, and neurodegeneration are only some of the downstream impacts of these oligomers, and it is believed that they begin 10–20 years before the onset of symptoms4. However, new investigations have indicated that (Aβ) plaques are present in the brains of only about a third of Alzheimer’s patients, and that they are occasionally present in the brains of persons who exhibit no cognitive problems5. The Aβ plaques outside of cells in the brain could not be harmful on their own, but might be the result of other, more elusive toxins. The intricacies are still being worked out, but the concept has inspired scientists at the University of Washington to develop a very precise soluble oligomer binding assay (SOBA)6. Initially, 310 people’s blood plasma was used to test SOBA. The cognitive health of the subjects ranged from moderate impairment to full-blown AD. Blood plasma measurements of toxic Aβ oligomers were used by SOBA to identify all 53 people with Alzheimer’s, a diagnosis confirmed by autopsy. Also, 11 people in the control group had oligomers in their plasma tested by SOBA7. In retrospect, we know that 10 of the individuals had moderate cognitive impairment or Alzheimer’s. SOBA has the potential to be modified in the future to detect early signs of Parkinson’s disease, type II diabetes, and Lewy body dementia, all of which have been linked to protein misfolding. There have been several attempts to detect AD biomarkers, with mixed results. A blood screening that includes identifies A precursors indicated Alzheimer’s up to 30 years before the cognitive abnormalities ever started to manifest. Other researchers looked at the feasibility of using a novel blood testing tool called Simoa to assess tau concentrations and forecast the onset of AD8. More than 400 people’s blood was sampled by the team. They determined the quantity of p-tau181, a variant of tau associated with AD, in plasma (the blood’s liquid component). P-tau181 in plasma was shown to be different between healthy subjects and those with Alzheimer’s pathology verified in autopsy. Additionally, the test may be able to distinguish between Alzheimer’s pathology and that of a group of uncommon neurodegenerative disorders known as frontotemporal lobar degeneration. Results from the spinal fluid p-tau181 test and the positron emission tomography brain scan for Aβ protein were similar to those from the plasma p-tau181 test, both of which are considered to be reliable biomarkers for AD8,9. Now commonly used in clinical practice are blood tests screening for genes associated with AD. However, these tests are not very good at predicting who will get Alzheimer’s. With the use of a blood test, we could quickly screen a broader and more diversified pool of potential study participants. Improvements in early diagnosis of AD are a consequence of promising research such as SOBA and Simoa. Ethical approval Not applicable. Sources of funding No funding was received. Author contribution R.A.: conceptualization, data curation, writing – original draft preparation, and writing – reviewing and editing. B.K.D. and F.A.: data curation, writing – original draft preparation, and writing – reviewing and editing. T.B.E.: writing – reviewing and editing, visualization, and supervision. Conflicts of interest disclosure The authors declare that they have no conflicts of interest. Research registration unique identifying number (UIN) None. Guarantor T.B. Emran, PhD, Associate Professor, Department of Pharmacy, BGC Trust University Bangladesh, Chittagong 4381, Bangladesh. Tel: +880 303 356 193, fax: +880 312 550 224. https://orcid.org/0000-0003-3188-2272. Data availability The data in this correspondence article is not sensitive in nature and is accessible in the public domain. The data is, therefore, available and not of a confidential nature. Provenance and peer review Not commissioned, internally peer-reviewed.

Plasminogen decreases Aβ42 and Tau deposition, and shows multi-beneficial effects on Alzheimer's disease in mice and humans

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the world. The aggregation of both amyloid beta (Aβ) peptides extracellularly and Tau proteins intracellularly plays key roles in the pathological consequences of AD, which lead to cholinergic neurodegeneration and eventually death. Currently, there are no effective methods to stop the progression of AD. Using ex vivo, in vivo and clinical approaches, we investigated the functional effects of plasminogen on the widely used FAD, Aβ42 oligomer or Tau intracranial injection-induced AD mouse model and explored its therapeutic effects on patients with AD. The results show that intravenously injected plasminogen rapidly crosses the blood‒brain barrier (BBB); increases plasmin activity in the brain; colocalizes with and effectively promotes the clearance of Aβ42 peptide and Tau protein deposits ex vivo and in vivo; increases the choline acetyltransferase (ChAT) level and decreases the acetylcholinesterase (AChE) activity; and improves the memory functions. Clinically, when GMP-level plasminogen was administered to 6 AD patients for 1–2 weeks, their average scores on the Minimum Mental State Examination (MMSE), which is a standard scoring system used to measure the memory loss and cognitive deficits, were extremely significantly improved by 4.2 ± 2.23 points, e.g., an average increase from 15.5 ± 8.22 before treatment to 19.7 ± 7.09 after treatment. The preclinical study and pilot clinical study suggest that plasminogen is effective in treating AD and may be a promising drug candidate.

A peptide rich in glycine-serine-alanine repeats ameliorates Alzheimer-type neurodegeneration.

Repeated amino acid sequences in proteins are widely found, and the glycine-serine-alanine repeat is an element with a general propensity to form β-sheet aggregates as found in key pathological factors, in several neurodegenerative diseases. Such properties of this repeat may guide development of disease-modifying therapies for neurodegenerative disease. However, details of its role and underlying mechanism(s) remain largely unknown. Actions of specific glycine-serine-alanine repeat peptides (SNPs), especially SNP-9, on Alzheimer's disease (AD)-like abnormalities were evaluated in transgenic mice and Caenorhabditis elegans, andin rat and cell models. Entry of SNPs into the brain, SNP activity in neuronal cells and peptide entry into cells were analysed in vivo and in vitro. Cell-free systems and the yeast two-hybrid system were also used to explore possible targets of SNP-9, and interactions of potential targets with SNP-9 were confirmed in cell-based systems. We first identified SNP-9 as a potent neuroprotective peptide with the activity to decrease oligomeric amyloid β (Aβ) via co-assembling with the toxic Aβ oligomer to form hetero-oligomers. Also, calcyclin-binding protein was found to act as a SNP-9-binding protein, by screening of a human brain cDNA library. Such binding showed that SNP-9 could regulate the abnormal hyperphosphorylation of tau via calcyclin-bindingprotein. Our study provides a foundation for development of SNPs, especially SNP-9, as potential therapeutic interventions for AD. We propose SNP-9 as a potential therapeutic agent for the treatment of AD.

Residue-specific binding of Ni(II) ions influences the structure and aggregation of amyloid beta (Aβ) peptides

Alzheimer’s disease (AD) is the most common cause of dementia worldwide. AD brains display deposits of insoluble amyloid plaques consisting mainly of aggregated amyloid-β (Aβ) peptides, and Aβ oligomers are likely a toxic species in AD pathology. AD patients display altered metal homeostasis, and AD plaques show elevated concentrations of metals such as Cu, Fe, and Zn. Yet, the metal chemistry in AD pathology remains unclear. Ni(II) ions are known to interact with Aβ peptides, but the nature and effects of such interactions are unknown. Here, we use numerous biophysical methods—mainly spectroscopy and imaging techniques—to characterize Aβ/Ni(II) interactions in vitro, for different Aβ variants: Aβ(1–40), Aβ(1–40)(H6A, H13A, H14A), Aβ(4–40), and Aβ(1–42). We show for the first time that Ni(II) ions display specific binding to the N-terminal segment of full-length Aβ monomers. Equimolar amounts of Ni(II) ions retard Aβ aggregation and direct it towards non-structured aggregates. The His6, His13, and His14 residues are implicated as binding ligands, and the Ni(II)·Aβ binding affinity is in the low µM range. The redox-active Ni(II) ions induce formation of dityrosine cross-links via redox chemistry, thereby creating covalent Aβ dimers. In aqueous buffer Ni(II) ions promote formation of beta sheet structure in Aβ monomers, while in a membrane-mimicking environment (SDS micelles) coil–coil helix interactions appear to be induced. For SDS-stabilized Aβ oligomers, Ni(II) ions direct the oligomers towards larger sizes and more diverse (heterogeneous) populations. All of these structural rearrangements may be relevant for the Aβ aggregation processes that are involved in AD brain pathology.

Soluble and insoluble protein aggregates, endoplasmic reticulum stress, and vascular dysfunction in Alzheimer's disease and cardiovascular diseases.

Dementia refers to a particular group of symptoms characterized by difficulties with memory, language, problem-solving, and other thinking skills that affect a person's ability to perform everyday activities. Alzheimer's disease (AD) is the most common form of dementia, affecting about 6.2 million Americans aged 65 years and older. Likewise, cardiovascular diseases (CVDs) are a major cause of disability and premature death, impacting 126.9 million adults in the USA, a number that increases with age. Consequently, CVDs and cardiovascular risk factors are associated with an increased risk of AD and cognitive impairment. They share important age-related cardiometabolic and lifestyle risk factors, that make them among the leading causes of death. Additionally, there are several premises and hypotheses about the mechanisms underlying the association between AD and CVD. Although AD and CVD may be considered deleterious to health, the study of their combination constitutes a clinical challenge, and investigations to understand the mechanistic pathways for the cause-effect and/or shared pathology between these two disease constellations remains an active area of research. AD pathology is propagated by the amyloid β(Aβ) peptides. These peptides give rise to small, toxic, and soluble Aβ oligomers (SPOs) that are nonfibrillar, and it is their levels that show a robust correlation with the extent of cognitive impairment. This review will elucidate the interplay between the effects of accumulating SPOs in AD and CVDs, the resulting ER stress response, and their role in vascular dysfunction. We will also address the potential underlying mechanisms, including the possibility that SPOs are among the causes of vascular injury in CVD associated with cognitive decline. By revealing common mechanistic underpinnings of AD and CVD, we hope that novel experimental therapeutics can be designed to reduce the burden of these devastating diseases. Graphical abstract Alzheimer's disease (AD) pathology leads to the release of Aβ peptides, and their accumulation in the peripheral organs has varying effects on various components of the cardiovascular system including endoplasmic reticulum (ER) stress and vascular damage. Image created with BioRender.com.

Molecular dynamics simulations reveal the importance of amyloid-beta oligomer β-sheet edge conformations in membrane permeabilization

Oligomeric aggregates of the amyloid-beta peptide(1-42) (Aβ42) are regarded as a primary cause of cytotoxicity related to membrane damage in Alzheimer's disease. However, a dynamical and structural characterization of pore-forming Aβ42 oligomers at atomic detail has not been feasible. Here, we used Aβ42 oligomer structures previously determined in a membrane-mimicking environment as putative model systems to study the pore formation process in phospholipid bilayers with all-atom molecular dynamics simulations. Multiple Aβ42 oligomer sizes, conformations, and N-terminally truncated isoforms were investigated on the multi-μs time scale. We found that pore formation and ion permeation occur via edge conductivity and exclusively for β-sandwich structures that feature exposed side-by-side β-strand pairs formed by residues 9-21 of Aβ42. The extent of pore formation and ion permeation depends on the insertion depth of hydrophilic residues 13-16 (HHQK domain) and thus on subtle differences in the overall stability, orientation, and conformation of the aggregates in the membrane. Additionally, we determined that backbone carbonyl and polar side-chain atoms from the edge strands directly contribute to the coordination sphere of the permeating ions. Furthermore, point mutations that alter the number of favorable side-chain contacts correlate with the ability of the Aβ42 oligomer models to facilitate ion permeation in the bilayer center. Our findings suggest that membrane-inserted, layered β-sheet edges are a key structural motif in pore-forming Aβ42 oligomers independent of their size and play a pivotal role in aggregate-induced membrane permeabilization.

Retinal pathological features and proteome signatures of Alzheimer’s disease

Alzheimer’s disease (AD) pathologies were discovered in the accessible neurosensory retina. However, their exact nature and topographical distribution, particularly in the early stages of functional impairment, and how they relate to disease progression in the brain remain largely unknown. To better understand the pathological features of AD in the retina, we conducted an extensive histopathological and biochemical investigation of postmortem retina and brain tissues from 86 human donors. Quantitative examination of superior and inferior temporal retinas from mild cognitive impairment (MCI) and AD patients compared to those with normal cognition (NC) revealed significant increases in amyloid β-protein (Aβ42) forms and novel intraneuronal Aβ oligomers (AβOi), which were closely associated with exacerbated retinal macrogliosis, microgliosis, and tissue atrophy. These pathologies were unevenly distributed across retinal layers and geometrical areas, with the inner layers and peripheral subregions exhibiting most pronounced accumulations in the MCI and AD versus NC retinas. While microgliosis was increased in the retina of these patients, the proportion of microglial cells engaging in Aβ uptake was reduced. Female AD patients exhibited higher levels of retinal microgliosis than males. Notably, retinal Aβ42, S100 calcium-binding protein B+ macrogliosis, and atrophy correlated with severity of brain Aβ pathology, tauopathy, and atrophy, and most retinal pathologies reflected Braak staging. All retinal biomarkers correlated with the cognitive scores, with retinal Aβ42, far-peripheral AβOi and microgliosis displaying the strongest correlations. Proteomic analysis of AD retinas revealed activation of specific inflammatory and neurodegenerative processes and inhibition of oxidative phosphorylation/mitochondrial, and photoreceptor-related pathways. This study identifies and maps retinopathy in MCI and AD patients, demonstrating the quantitative relationship with brain pathology and cognition, and may lead to reliable retinal biomarkers for noninvasive retinal screening and monitoring of AD.

Aza-Residue Modulation of Cyclic d,l-α-Peptide Nanotube Assembly with Enhanced Anti-Amyloidogenic Activity.

Transient soluble oligomers of amyloid-β (Aβ) are considered among the most toxic species in Alzheimer's disease (AD). Soluble Aβ oligomers accumulate early prior to insoluble plaque formation and cognitive impairment. The cyclic d,l-α-peptide CP-2 (1) self-assembles into nanotubes and demonstrates promising anti-amyloidogenic activity likely by a mechanism involving engagement of soluble oligomers. Systematic replacement of the residues in peptide 1 with aza-amino acid counterparts was performed to explore the effects of hydrogen bonding on propensity to mitigate Aβ aggregation and toxicity. Certain azapeptides exhibited improved ability to engage, alter the secondary structure, and inhibit aggregation of Aβ. Moreover, certain azapeptides disassembled preformed Aβ fibrils and protected cells from Aβ-mediated toxicity. Substitution of the l-norleucine3 and d-serine6 residues in peptide 1 with aza-norleucine and aza-homoserine provided, respectively, nontoxic [azaNle3]-1 (4) and [azaHse6]-1 (7), that significantly abated symptoms in a transgenic Caenorhabditis elegans AD model by decreasing Aβ oligomer levels.

Lipids uniquely alter the secondary structure and toxicity of amyloid beta 1-42 aggregates.

Abrupt aggregation of amyloid β1-42 (Aβ) peptide is a hallmark of Alzheimer's disease (AD), a severe pathology that affects more than 44million people worldwide. A growing body of evidence suggests that lipids can uniquely alter rates of Aβ1-42 aggregation. However, it remains unclear whether lipids only alter rates of protein aggregation or also uniquely modify the secondary structure and toxicity of Aβ1-42 oligomers and fibrils. In this study, we investigated the effect of phosphatidylcholine (PC), cardiolipin (CL), and cholesterol (Chol) on Aβ1-42 aggregation. We found that PC, CL and Chol strongly accelerated the rate of fibril formation compared to the rate of Aβ1-42 aggregation in the lipid-free environment. Furthermore, anionic CL enabled the strongest acceleration of Aβ1-42 aggregation compared to zwitterionic PC and uncharged Chol. We also found that PC, CL and Chol uniquely altered the secondary structure of early-, middle- and late-stage Aβ1-42 aggregates. Specifically, CL and Chol drastically increased the amount of parallel β-sheet in Aβ1-42 oligomers and fibrils grown in the presence of these lipids. This caused a significant increase in the toxicity of Aβ:CL and Aβ:Chol compared to the toxicity of Aβ:PC and Aβ1-42 aggregates formed in the lipid-free environment. These results demonstrate that toxicity of Aβ aggregates correlates with the amount of their β-sheet content, which, in turn, is determined by the chemical structure of lipids present at the stage of Aβ1-42 aggregation.

The Curcumin Derivative GT863 Protects Cell Membranes in Cytotoxicity by Aβ Oligomers

In Alzheimer's disease (AD), accumulation of amyloid β-protein (Aβ) is one of the major mechanisms causing neuronal cell damage. Disruption of cell membranes by Aβ has been hypothesized to be the important event associated with neurotoxicity in AD. Curcumin has been shown to reduce Aβ-induced toxicity; however, due to its low bioavailability, clinical trials showed no remarkable effect on cognitive function. As a result, GT863, a derivative of curcumin with higher bioavailability, was synthesized. The purpose of this study is to clarify the mechanism of the protective action of GT863 against the neurotoxicity of highly toxic Aβ oligomers (Aβo), which include high-molecular-weight (HMW) Aβo, mainly composed of protofibrils in human neuroblastoma SH-SY5Y cells, focusing on the cell membrane. The effect of GT863 (1 μM) on Aβo-induced membrane damage was assessed by phospholipid peroxidation of the membrane, membrane fluidity, membrane phase state, membrane potential, membrane resistance, and changes in intracellular Ca2+ ([Ca2+]i). GT863 inhibited the Aβo-induced increase in plasma-membrane phospholipid peroxidation, decreased membrane fluidity and resistance, and decreased excessive [Ca2+]i influx, showing cytoprotective effects. The effects of GT863 on cell membranes may contribute in part to its neuroprotective effects against Aβo-induced toxicity. GT863 may be developed as a prophylactic agent for AD by targeting inhibition of membrane disruption caused by Aβo exposure.

Construction of a DNA Nanoassembly Based on Spatially Ordered Recognition Elements for Inhibiting β-Amyloid Aggregation.

A β-amyloid (Aβ) aggregation process is a spontaneous process where the original random coil or helical structure changes into a regularly arranged β-sheet structure. The development of inhibitors with the features of low cost, high efficiency, and biosafety by targeting Aβ self-aggregation is significant for Alzheimer's disease treatment. However, the issues of low inhibition efficiency under low concentrations of inhibitors and biological toxicity are currently to be addressed. To resolve the above problems, a DNA nanoassembly (HCR-Apt) based on spatially ordered recognition elements was constructed by targeted disruption of Aβ ordered arrangement. It was discovered that HCR-Apt could inhibit effectively the fibrillation of Aβ40 monomers and oligomers at substoichiometric ratios. This may be due to orderly arrangement of aptamers in rigid nanoskeletons for enhancing the recognition interaction between aptamers and Aβ40. The strong interaction between HCR-Apt and Aβ40 limited the flexible conformational conversion of Aβ40 molecules, thereby inhibiting their self-assembly. Computational simulations and experimental analysis revealed the interactions of Apt42 with Aβ40, which explained different inhibition effects on the fibrillation of Aβ40 monomers and oligomers. Furthermore, the analysis of tyrosine intrinsic fluorescence spectra and surface plasmon resonance imaging showed that the interaction of HCR-Apt and Aβ40 was stronger than that of Apt42 and Aβ40. These findings contributed to establishing a promising method of boosting the recognition interaction by orderly arrangement of recognition elements. Taken together, this work is expected to provide a simple and efficient strategy for inhibiting Aβ aggregation, expanding aptamer's application potential in neurodegenerative diseases.

Brain region-specific myelinogenesis is not directly linked to amyloid-β in APP/PS1 transgenic mice

Alzheimer's disease (AD) is characterized by aggregating amyloid beta-protein (Aβ). Recent evidence has shown that insufficient myelinogenesis contributes to AD-related functional deficits. However, it remains unclear whether Aβ, in either plaque or soluble form, could alter myelinogenesis in AD brains. By cell-lineage tracing and labeling, we found both myelinogenesis and Aβ deposits displayed a region-specific pattern in the 13-month-old APP/PS1 transgenic mouse brains. Aβ plaques cause focal demyelination, but only about 15% Aβ plaques are closely associated with newly formed myelin in the APP/PS1 brains. Further, the Aβ plaque total area and the amount of new myelin are not linearly correlated across different cortical regions, suggesting that Aβ plaques induce demyelination but may not exclusively trigger remyelination. To understand the role of soluble Aβ in regulating myelinogenesis, we chose to observe the visual system, wherein soluble Aβ is detectable but without the presence of Aβ plaques in the APP/PS1 retina, optic nerve, and optic tract. Interestingly, newly-formed myelin density was not significantly altered in the APP/PS1 optic nerves and optic tracts as compared to the wildtype controls, suggesting soluble Aβ probably does not change myelinogenesis. Further, treatment of purified oligodendrocyte precursor cells (OPCs) with soluble Aβ (oligomers) for 48 h did not change the cell densities of MBP positive cells and PDGFRα positive OPCs in vitro. Consistently, injection of soluble Aβ into the lateral ventricles did not alter myelinogenesis in the corpus callosum of NG2-CreErt; Tau-mGFP mice significantly. Together, these findings indicate that the region-dependent myelinogenesis in AD brains is not directly linked to Aβ, but rather probably a synergic result in adapting to AD pathology.

Plantainoside B in Bacopa monniera Binds to Aβ Aggregates Attenuating Neuronal Damage and Memory Deficits Induced by Aβ.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by dementia. The most characteristic pathological changes in AD brain include extracellular amyloid-β (Aβ) accumulation and neuronal loss. Particularly, cholinergic neurons in the nucleus basalis of Meynert are some of the first neuronal groups to degenerate; accumulating evidence suggests that Aβ oligomers are the primary form of neurotoxicity. Bacopa monniera is a traditional Indian memory enhancer whose extract has shown neuroprotective and Aβ-reducing effects. In this study, we explored the low molecular weight compounds from B. monniera extracts with an affinity to Aβ aggregates, including its oligomers, using Aβ oligomer-conjugated beads and identified plantainoside B. Plantainoside B exhibited evident neuroprotective effects by preventing Aβ attachment on the cell surface of human induced pluripotent stem cell (hiPSC)-derived cholinergic neurons. Moreover, it attenuated memory impairment in mice that received intrahippocampal Aβ injections. Furthermore, radioisotope experiments revealed that plantainoside B has affinity to Aβ aggregates including its oligomers and brain tissue from a mouse model of Aβ pathology. In addition, plantainoside B could delay the Aβ aggregation rate. Accordingly, plantainoside B may exert neuroprotective effects by binding to Aβ oligomers, thus interrupting the binding of Aβ oligomers to the cell surface. This suggests its potential application as a theranostics in AD, simultaneously diagnostic and therapeutic drugs.

Detecting physiological concentrations of Alzheimer's associated amyloid-β oligomers with a cell-based response.

Alzheimer's disease (AD) is characterized by blood-brain barrier (BBB) breakdown and neuronal cell death, associated with aggregated amyloid-β (Aβ) plaques deposited around brain vasculature and within the brain. Changes in the brain occur up to 20 years before symptoms arise; however, there is currently no pre-symptomatic detection method available for AD. An intact BBB requires tight junctions (TJ) that render relatively high transendothelial electrical resistance (TEER). AD-associated breakdown of the BBB correlates with vascular Aβ deposition, suggesting that aggregated, pathological Aβ can modulate TEER. We explore leveraging this cellular response for early disease diagnosis via detection of early Aβ aggregates. Primary human brain microvascular endothelial cells (HBMVECs) were cultured on a suspended membrane and supplemented to mimic the BBB. Once TEER values reflected TJ formation, HBMVEC monolayers were treated with oligomeric Ab at picomolar to nanomolar concentrations, and TEER was monitored. Oligomeric Aβ at picomolar and nanomolar concentrations, but not monomer, induced a reduction of TEER and disruption of TJs. These results demonstrate that pathogenic Aβ aggregates are uniquely responsible for inducing endothelial monolayer permeability and reducing monolayer electrical resistance, and this response can be detected at physiological concentrations. This work also validates that introduction of physiologically active Aβ oligomers can increase permeability of an endothelial monolayer, while non-pathogenetic monomer is inert. These results provide evidence that oligomers, the pathogenic form of Aβ, selectively break down the BBB. Future work will include establishing a threshold of Aβ oligomer concentrations that reduce TEER to establish cellular sensitivity, and sensitizing cells to obtain a more pronounced response. Translation of this experimental system to a cell-based biosensor platform will lay the groundwork for the development of cost-effective, early AD detection.

Comparing the structure and stability of diverse amyloid beta oligomers using fluorescence correlation spectroscopy.

Soluble oligomers of amyloid beta (Aβ) play a central role in Alzheimer's Disease pathology, however the detailed mechanisms of Aβ oligomer toxicity remain under investigation. One challenge in measuring oligomer size or cytotoxicity is the variability between published protocols that describe distinct oligomers, and the likely scenario that there are multiple pathological oligomers rather than a single disease-causing complex. Given the heavy use of dilution and incubation in these protocols, a systematic review covering how different oligomeric concentrations and preparation protocols affect oligomeric size and aggregation propensity is essential for standardizing assays of amyloid toxicity. To begin addressing this gap, we have systematically characterized and compared two distinct amyloid beta oligomers derived from either a homogeneous detergent or from cell media using fluorescence correlation spectroscopy (FCS). We observe temporal changes in size and stability of these oligomers over a wide range of concentrations and attempt to codify their similarities and differences. Significant changes between oligomer populations are observed over hours, with more diluted complexes falling apart. These observations also demonstrate that protein oligomer behavior is highly specific to their environments, which must be accounted for in biological assays, especially in the presence of cell media. Accounting for such discrepancies will also enable high-throughput screening of compounds that inhibit Aβ oligomer cytotoxicity with high reproducibility, which is a crucial component of drug discovery in the context of neurodegeneration and aging.

A molecular imprint of intracellular amyloid-beta ApoE interaction correlates with its toxicity molecular imprint of intracellular amyloid beta ApoE interaction.

ApoE isoforms are major genetic risk factors related to Alzheimer's disease (AD), which is associated with the aggregation of the amyloid beta (Aβ) peptide. However, a molecular-level understanding of how ApoE is involved in AD pathology is still lacking. Here we employ a fluorescent lifetime-based assay that reports a specific ApoE-dependent modification of Aβ, both in vitro and in neuronal cells. We explore this phenomenon in several types of cells where the change of lifetime of Aβ oligomers correlates with the relative ApoE content, with the highest effect displayed by primary-cultured rat astrocytes. At the single-molecule level, ApoE-induced modification also produces distinct Aβ oligomers with increased affinity to artificial lipid bilayers. A known peptide inhibitor of Aβ-ApoE interaction, LVFFA, diminishes both the lifetime modification and toxicity of Aβ oligomers in a dose-dependent manner. The correlation between the short lifetime component and Aβ toxicity was corroborated by experiments using two nontoxic Aβ variants. Our data, therefore, establish a clear correlation between the lifetime modification of Aβ and its toxicity. Finally, neuronal stem cells derived from cells obtained from Alzheimer's patients also show the short-lifetime component, the changed oligomeric state, and the increased membrane affinity, which is likely to be relevant for AD. In summary, our assay can provide a direct readout of the toxic ApoE-Aβ interaction in vitro and in cells, which can provide a platform for AD drug discovery.

Using giant plasma membrane vesicles (GMPVs) to study age-dependent changes in amyloid-β oligomer toxicity.

Aging is a major risk factor associated with several human ailments, including some forms of cancer, autoimmunity, and neurodegeneration. Longitudinal changes in the multicellular organisms, including issues with genomic stability and protein oxidation, have been extensively reported. However, age-dependent changes in plasma membrane lipids and how they contribute to age-related phenotypes are less understood. Moreover, mechanistic understanding about how age-dependent lipidic changes can seed or exacerbate the toxicity of aggregating protein, such as amyloid-β (Aβ) in Alzheimer's disease, is of particular interest. Indeed, certain isolated detergents, fatty acids, and lipids have been shown to nucleate soluble Aβ oligomers (Aβos) with notable cytotoxicity, but little has been done to understand how changes in complex mixtures such as plasma membrane lipids scaffold toxic oligomers in a longitudinal manner. To deconvolute how cellular age can influence the cytotoxic properties of Aβos, giant-plasma membrane derived vesicles (GPMVs) will be collected from cultured cells at various passage numbers and at senescence. Those GMPVs will then be used as lipidic scaffolds for soluble Aβos and the Aβo-GPMV complexes will be treated to cultured cells to monitor their responses. Specifically, Aβo-GPMV toxicity will be monitored by observing changes in cellular respiration, senescence, and/or cell death. The driving hypothesis is that GPMVs derived from aged cells (at high-passage numbers) will seed enhanced toxicity of Aβ oligomers compared to the young (low/medium passage number) counterparts.

Exploring the structural conformations of amyloid-beta oligomers on self-assembled monolayers and in water under confinement, a molecular dynamics study.

Alzheimer's disease (AD) is a devastating neurological disorder, affecting millions of people worldwide. The aggregation of amyloid-beta (Aβ) peptides and the intermediate protein tau into amyloid fibrils is believed to be a critical event in the pathogenesis of AD. Amyloid fibrils represent the endpoints of aggregation, and it is widely believed that their precursors—the soluble oligomers—are the more important neurotoxins. However, there is limited structural information available on Aβ oligomers. Although atomic force microscopy (AFM) is a widely-used method employed to study the conformation of Aβ oligomers, the resolution of AFM is limited and unable to discern atomistic structural features. Here, we construct a self-assembled monolayer (SAM), mimicking the surface properties of mica (used in AFM), with varying surface charge densities. Then, we apply the enhanced sampling algorithm REST2 (replica exchange with solute tempering) from the NAMD package, to efficiently sample the protein conformations on the SAM surface. Our simulations have resulted in cross β-sheet formation between Aβ monomers, similar to that observed experimentally. In particular, we find that residues 17-21 and 35-40 are involved in β-sheet formation, the former of which has been observed in the Aβ fibril structure. We also observe the prominence of the intramolecular Asp23-Lys28 salt bridge, which has been found to play a significant role in the formation of Aβ fibrils. Further, we notice that an asymmetric charge distribution on the SAM surface tends to result in greater β-sheet formation. Our simulations of Aβ monomers in TIP4P water, confined using a harmonic wall, similarly result in cross β-sheet formation among the monomers. Confinement has been found to increase the conformational stability of proteins, suggesting the possibility of Aβ formation in the crowded environment of the cell.

The purely thermodynamic anti-prionic mode of action for the treatment of neurodegenerative diseases - realized by target protein specific all-D-peptides.

Neurodegenerative protein-misfolding diseases, like Alzheimer's (AD) and Parkinson's disease (PD), are driven by prion-like self-replicating and propagating protein assemblies of Aβ, α-synuclein, and many more. Therefore, we have developed the so-called anti-prionic mode of action (MoA). It is realized by all-D-enantiomeric peptide ligands that bind the monomeric protein of interest with high affinity, thereby stabilizing the physiological intrinsically disordered monomer structure. We call this purely thermodynamic driven mode of action “anti-prionic”, because it is eliminating already existing prion-like aggregates by disassembling them into non-toxic and functional monomers. I will summarize the current, and yet unpublished, progress in realizing the anti-prionic MoA for the target proteins α-synuclein and Aβ. Briefly, the most promising all-D-enantiomeric ligand for α-synuclein, SVD-1a, disassembles preformed α-synuclein fibrils (PFF) as shown by AFM, DLS and SEC analysis. SPR and NMR demonstrate picomolar affinity of SVD-1a to α-synuclein monomers, while keeping them in their physiological intrinsically disordered conformation. The all-D-enantiomeric ligand for Aβ, RD2, disassembles Aβ oligomers obtained from brain tissue of former AD patients. A clinical phase Ib, double-bind, placebo-controlled study with 20 ATN positive patients treated once daily orally with RD2 or placebo for 4 weeks with an additional 4 weeks follow up period demonstrates good safety and tolerability, and yields interesting results on efficacy. This is valuable information for the scheduled phase II study. I will also acknowledge the many contributors of both developments that are too many to be included here in the abstract. Conclusion: The unique anti-prionic mode of action for the treatment of AD, PD and other protein misfolding diseases is promising.

Monitoring oligomeric forms of amyloid beta in the early stages of aggregation using hybrid X-ray footprinting mass spectrometry with inline fluorescence.

The aggregation-prone Amyloid Beta (Aβ) peptide has been the target of the majority of efforts to develop Alzheimer's Disease therapeutics, most of which have failed or have shown mixed results. It is no longer widely believed that buildup of amyloid fibrils is a causal factor in cognitive decline associated with the disease, and a growing body of evidence now suggests that low molecular weight oligomeric forms of Aβ may be the culprits in disease progression. However, while numerous Aβ fibril structures have been characterized, primarily by NMR and cryo-EM, obtaining structural information on oligomeric forms of Aβ that presumably precede and/or seed fibril formation has proved challenging. These transient forms are heterogeneous, and depend heavily on experimental conditions such as buffer, temperature, concentration, and degree of quiescence during measurement. Here, we present a new approach to delineating structural features of early-stage low molecular weight Aβ oligomers using a solvent accessibility assay in conjunction with inline fluorescence measurements. In this method, inline fluorescence is collected nearly simultaneously from samples during collection of X-ray footprinting mass spectrometry (XFMS) data. In the XFMS method, X-ray irradiation dissociates solvent water to produce hydroxyl radicals, which covalently modify solvent accessible side chains. Liquid chromatography-mass spectrometry is subsequently used to analyze the covalent modifications produced, and the data provide a “water map” at the single residue level, which is used to determine protein structure. This new hybrid approach circumvents issues in batch-to-batch variability of Aβ aggregation which has made this peptide so difficult to study, and provides a method to combine residue-specific structural information nearly simultaneously with global protein structure readouts from either intrinsic protein fluorescence or fluorophores such as Thioflavin T.