Amyloid Oligomers Research Articles

Structural basis of epitope recognition by anti-alpha-synuclein antibodies MJFR14-6-4-2

Alpha-synuclein (α-syn) inclusions in the brain are hallmarks of so-called Lewy body diseases. Lewy bodies contain mainly aggregated α-syn together with some other proteins. Monomeric α-syn lacks a well-defined three-dimensional structure, but it can aggregate into oligomeric and fibrillar amyloid species, which can be detected using specific antibodies. Here we investigate the aggregate specificity of monoclonal MJFR14-6-4-2 antibodies. We conclude that partial masking of epitope in unstructured monomer in combination with a high local concentration of epitopes is the main reason for MJFR14-6-4-2 selectivity towards aggregates. Based on the structural insight, we produced mutant α-syn that when fibrillated is unable to bind MJFR14-6-4-2. Using these fibrils as a tool for seeding cellular α-syn aggregation, provides superior signal/noise ratio for detection of cellular α-syn aggregates by MJFR14-6-4-2. Our data provide a molecular level understanding of specific recognition of toxic amyloid oligomers, which is critical for the development of inhibitors against synucleinopathies.

Intrathecal Immunoselective Nanopheresis for Alzheimer’s Disease: What and How? Why and When?

Nanotechnology is transforming therapeutics for brain disorders, especially in developing drug delivery systems. Intrathecal immunoselective nanopheresis with soluble monoclonal antibodies represents an innovative approach in the realm of drug delivery systems for Central Nervous System conditions, especially for targeting soluble beta-amyloid in Alzheimer’s disease. This review delves into the concept of intrathecal immunoselective nanopheresis. It provides an overall description of devices to perform this technique while discussing the nanotechnology behind its mechanism of action, its potential advantages, and clinical implications. By exploring current research and advancements, we aim to provide a comprehensive understanding of this novel method, addressing the critical questions of what it is, how it works, why it is needed, and when it should be applied. Special attention is given to patient selection and the optimal timing for therapy initiation in Alzheimer’s, coinciding with the peak accumulation of amyloid oligomers in the early stages. Potential limitations and alternative targets beyond beta-amyloid and future perspectives for immunoselective nanopheresis are also described.

Inflammasome activation aggravates choroidal neovascularization.

Inflammasome activation is implicated in diseases of aberrant angiogenesis such as age-related macular degeneration (AMD), though its precise role in choroidal neovascularization (CNV), a characteristic pathology of advanced AMD, is ill-defined. Reports on inhibition of inflammasome constituents on CNV are variable and the precise role of inflammasome in mediating pathological angiogenesis is unclear. Historically, subretinal injection of inflammasome agonists alone has been used to investigate retinal pigmented epithelium (RPE) degeneration, while the laser photocoagulation model has been used to study pathological angiogenesis in a model of CNV. Here, we report that the simultaneous introduction of any of several disease-relevant inflammasome agonists (Alu or B2 RNA, Alu cDNA, or oligomerized amyloid β (1-40)) exacerbates laser-induced CNV. These activities were diminished or abrogated by genetic or pharmacological targeting of inflammasome signaling constituents including P2rx7, Nlrp3, caspase-1, caspase-11, and Myd88, as well as in myeloid-specific caspase-1 knockout mice. Alu RNA treatment induced inflammasome activation in macrophages within the CNV lesion, and increased accumulation of macrophages in an inflammasome-dependent manner. Finally, IL-1β neutralization prevented inflammasome agonist-induced chemotaxis, macrophage trafficking, and angiogenesis. Collectively, these observations support a model wherein inflammasome stimulation promotes and exacerbates CNV and may be a therapeutic target for diseases of angiogenesis such as neovascular AMD.

Microglia either promote or restrain TRAIL-mediated excitotoxicity caused by Aβ1−42 oligomers

BackgroundAlzheimer’s disease (AD) features progressive neurodegeneration and microglial activation that results in dementia and cognitive decline. The release of soluble amyloid (Aβ) oligomers into the extracellular space is an early feature of AD pathology. This can promote excitotoxicity and microglial activation. Microglia can adopt several activation states with various functional outcomes. Protective microglial activation states have been identified in response to Aβ plaque pathology in vivo. However, the role of microglia and immune mediators in neurotoxicity induced by soluble Aβ oligomers is unclear. Further, there remains a need to identify druggable molecular targets that promote protective microglial states to slow or prevent the progression of AD.MethodsHippocampal entorhinal brain slice culture (HEBSC) was employed to study mechanisms of Aβ1−42 oligomer-induced neurotoxicity as well as the role of microglia. The roles of glutamate hyperexcitation and immune signaling in Aβ-induced neurotoxicity were assessed using MK801 and neutralizing antibodies to the TNF-related apoptosis-inducing ligand (TRAIL) respectively. Microglial activation state was manipulated using Gi-hM4di designer receptor exclusively activated by designer drugs (DREADDs), microglial depletion with the colony-stimulating factor 1 receptor (CSF1R) antagonist PLX3397, and microglial repopulation (PLX3397 withdrawal). Proteomic changes were assessed by LC-MS/MS in microglia isolated from control, repopulated, or Aβ-treated HEBSCs.ResultsNeurotoxicity induced by soluble Aβ1−42 oligomers involves glutamatergic hyperexcitation caused by the proinflammatory mediator and death receptor ligand TRAIL. Microglia were found to have the ability to both promote and restrain Aβ-induced toxicity. Induction of microglial Gi-signaling with hM4di to prevent pro-inflammatory activation blunted Aβ neurotoxicity, while microglial depletion with CSF1R antagonism worsened neurotoxicity caused by Aβ as well as TRAIL. HEBSCs with repopulated microglia, however, showed a near complete resistance to Aβ-induced neurotoxicity. Comparison of microglial proteomes revealed that repopulated microglia have a baseline anti-inflammatory and trophic phenotype with a predicted pathway activation that is nearly opposite that of Aβ-exposed microglia. mTORC2 and IRF7 were identified as potential targets for intervention.ConclusionMicroglia are key mediators of both protection and neurodegeneration in response to Aβ. Polarizing microglia toward a protective state could be used as a preventative strategy against Aβ-induced neurotoxicity.

Poration of mitochondrial membranes by amyloidogenic peptides and other biological toxins.

Mitochondria are essential organelles known to serve broad functions, including in cellular metabolism, calcium buffering, signaling pathways and the regulation of apoptotic cell death. Maintaining the integrity of the outer (OMM) and inner mitochondrial membranes (IMM) is vital for mitochondrial health. Cardiolipin (CL), a unique dimeric glycerophospholipid, is the signature lipid of energy-converting membranes. It plays a significant role in maintaining mitochondrial architecture and function, stabilizing protein complexes and facilitating efficient oxidative phosphorylation (OXPHOS) whilst regulating cytochrome c release from mitochondria. CL is especially enriched in the IMM and at sites of contact between the OMM and IMM. Disorders of protein misfolding, such as Alzheimer's and Parkinson's diseases, involve amyloidogenic peptides like amyloid-β, tau and α-synuclein, which form metastable toxic oligomeric species that interact with biological membranes. Electrophysiological studies have shown that these oligomers form ion-conducting nanopores in membranes mimicking the IMM's phospholipid composition. Poration of mitochondrial membranes disrupts the ionic balance, causing osmotic swelling, loss of the voltage potential across the IMM, release of pro-apoptogenic factors, and leads to cell death. The interaction between CL and amyloid oligomers appears to favour their membrane insertion and pore formation, directly implicating CL in amyloid toxicity. Additionally, pore formation in mitochondrial membranes is not limited to amyloid proteins and peptides; other biological peptides, as diverse as the pro-apoptotic Bcl-2 family members, gasdermin proteins, cobra venom cardiotoxins and bacterial pathogenic toxins, have all been described to punch holes in mitochondria, contributing to cell death processes. Collectively, these findings underscore the vulnerability of mitochondria and the involvement of CL in various pathogenic mechanisms, emphasizing the need for further research on targeting CL-amyloid interactions to mitigate mitochondrial dysfunction.

Simulating the anti-aggregative effect of fasudil in early dimerisation process of α-synuclein

The aggregation of the protein α-synuclein into amyloid deposits is associated with multiple neurological disorders, including Parkinson's disease. Soluble amyloid oligomers are reported to exhibit higher toxicity than insoluble amyloid fibrils, with dimers being the smallest toxic oligomer. Small molecule drugs, such as fasudil, have shown potential in targeting α-synuclein aggregation and reducing its toxicity. In this study, we use atomistic molecular dynamics simulations to demonstrate how fasudil affects the earliest stage of aggregation, namely dimerization. Our results show that the presence of fasudil reduces the propensity for intermolecular contact formation between protein chains. Consistent with previous reports, our analysis confirms that fasudil predominantly interacts with the negatively charged C-terminal region of α-synuclein. However, we also observe transient interactions with residues in the charged N-terminal and hydrophobic NAC regions. Our simulations indicate that while fasudil prominently interacts with the C-terminal region, it is the transient interactions with residues in the N-terminal and NAC regions that effectively block the formation of intermolecular contacts between protein chains and prevent early dimerization of this disordered protein.

Quinoline yellow acts as a novel amyloid fibrillation probe by using surface-enhanced Raman spectroscopy

Protein amyloid fibrillation is linked to a wide range of neurodegenerative diseases. Protein oligomer is an intermediate substance in the process of fibrillation, which is neurotoxic and formed by the aggregation of protein molecules under physiological stress. Early detection of protein oligomers could make timely intervention of protein fibrillation related diseases. Therefore, it is crucial to develop efficient inhibitors and probes for monitoring amyloid fibril formation. In this study, we developed a novel amyloid inhibitor quinoline yellow (QY), which was proved to be effective in inhibiting insulin protein fibrillation as demonstrated by fluorescence, morphology characterization and circular dichroism. When QY binds to insulin, it exerts inhibitory effects on the nucleation process and effectively impedes the formation of fibrillar fibrils. In addition, we present the application of surface-enhanced Raman spectroscopy (SERS) as an extremely sensitive technique for identifying amyloid oligomers. The investigation employed the probe QY, which demonstrated a linear reaction for identifying oligomers in the concentration range of 1.0–58.0 μM. Impressively, it showcased an exceptionally sensitive detection threshold of 0.2 μM. And also illustrating the binding sites and interaction mechanisms between small molecules of QY and insulin by SERS. The aforementioned methodology was also employed for the identification of insulin oligomers in human serum samples. Thereby, the proposed approach presenting a promising avenue with extensive implications in the realms of pharmaceutical exploration and disease diagnosis.

Search for potential Alzheimer’s disease therapeutics: Identification of inhibitors of amyloid oligomerization with high affinity for the zinc-binding site

The progression of Aβ peptide aggregation in the brain has been suggested to play a significant role in the pathogenesis and development of Alzheimer’s disease. This study is intended to provide insight into the interactions between the zinc-binding site of beta-amyloids and the zinc ion itself. The absence of zinc bonded to the beta-amyloid has been shown to potentially slow down the progression of Alzheimer's disease, so the goal is to provide an analysis of available drugs that can be repurposed and could profoundly impact Alzheimer's disease treatment. We address how and with what strength the existing compounds bind with beta-amyloid, potentially replacing or blocking zinc and preventing it from attaching to the amyloid. The analysis was performed using molecular operating environment software, which, starting from a filtered database, identified the drugs most likely to bind to the zinc-binding site on beta-amyloid.

Disulfide-Driven Charge and Hydrophobicity Rearrangement of Remodeled Membrane Proteins toward Amyloid-Type Aggregation.

As a common pathological hallmark, protein aggregation into amyloids is a highly complicated phenomenon, attracting extensive research interest for elucidating its structural details and formation mechanisms. Membrane deposition and disulfide-driven protein misfolding play critical roles in amyloid-type aggregation, yet the underlying molecular process remains unclear. Here, we employed sum frequency generation (SFG) vibrational spectroscopy to comprehensively investigate the remodeling process of lysozyme, as the model protein, into amyloid-type aggregates at the cell membrane interface. It was discovered that disulfide reduction concurrently induced the transition of membrane-bound lysozyme from predominantly α-helical to antiparallel β-sheet structures, under a mode switch of membrane interaction from electrostatic to hydrophobic, and subsequent oligomeric aggregation. These findings shed light on the systematic understanding of dynamic molecular mechanisms underlying membrane-interactive amyloid oligomer formation.

Effects of Oral ALZ-801/Valiltramiprosate on Plasma Biomarkers, Brain Hippocampal Volume, and Cognition: Results of 2-Year Single-Arm, Open-Label, Phase 2 Trial in APOE4 Carriers with Early Alzheimer's Disease.

ALZ-801/valiltramiprosate is a small-molecule oral inhibitor of beta amyloid (Aβ) aggregation and oligomer formation being studied in a phase 2 trial in APOE4 carriers with early Alzheimer's disease (AD) to evaluate treatment effects on fluid and imaging biomarkers and cognitive assessments. The single-arm, open-label phase 2 trial was designed to evaluate the effects of the ALZ-801 265 mg tablet taken twice daily (after 2weeks once daily) on plasma fluid AD biomarkers, hippocampal volume (HV), and cognition over 104 weeks in APOE4 carriers. The study enrolled subjects aged 50-80 years, with early AD [Mini-Mental State Examination (MMSE) ≥ 22, Clinical Dementia Rating-Global (CDR-G) 0.5 or 1], apolipoprotein E4 (APOE4) genotypes including APOE4/4 and APOE3/4 genotypes, and positive cerebrospinal fluid (CSF) AD biomarkers or prior amyloid scans. The primary outcome was plasma p-tau181, HV evaluated by magnetic resonance imaging (MRI) was the key secondary outcome, and plasma Aβ42 and Aβ40 were the secondary biomarker outcomes. The cognitive outcomes were the Rey Auditory Verbal Learning Test and the Digit Symbol Substitution Test. Safety and tolerability evaluations included treatment-emergent adverse events and amyloid-related imaging abnormalities (ARIA). The study was designed and powered to detect 15% reduction from baseline in plasma p-tau181 at the 104-week endpoint. A sample size of 80 subjects provided adequate power to detect this difference at a significance level of 0.05 using a two-sided paired t-test. The enrolled population of 84 subjects (31 homozygotes and 53 heterozygotes) was 52% females, mean age 69 years, MMSE 25.7 [70% mild cognitive impairment (MCI), 30% mild AD] with 55% on cholinesterase inhibitors. Plasma p-tau181 reduction from baseline was significant (31%, p = 0.045) at 104 weeks and all prior visits; HV atrophy was significantly reduced (p = 0.0014) compared with matched external controls from an observational Early AD study. Memory scores showed minimal decline from baseline over 104 weeks and correlated significantly with decreased HV atrophy (Spearman's 0.44, p = 0.002). Common adverse events were COVID infection and mild nausea, and no drug-related serious adverse events were reported. Of 14 early terminations, 6 were due to nonserious treatment-emergent adverse events and 1 death due to COVID. There was no vasogenic brain edema observed on MRI over 104 weeks. The effect of ALZ-801 on reducing plasma p-tau181 over 2 years demonstrates target engagement and supports its anti-Aβ oligomer action that leads to a robust decrease in amyloid-induced brain neurodegeneration. The significant correlation between reduced HV atrophy and cognitive stability over 2 years suggests a disease-modifying effect of ALZ-801 treatment in patients with early AD. Together with the favorable safety profile with no events of vasogenic brain edema, these results support further evaluation of ALZ-801 in a broader population of APOE4 carriers, who represent two-thirds of patients with AD. https://clinicaltrials.gov/study/NCT04693520 .

Exploring Tau Fibril-Disaggregating and Antioxidating Molecules Binding to Membrane-Bound Amyloid Oligomers Using Machine Learning-Enhanced Docking and Molecular Dynamics.

Intracellular tau fibrils are sources of neurotoxicity and oxidative stress in Alzheimer's. Current drug discovery efforts have focused on molecules with tau fibril disaggregation and antioxidation functions. However, recent studies suggest that membrane-bound tau-containing oligomers (mTCOs), smaller and less ordered than tau fibrils, are neurotoxic in the early stage of Alzheimer's. Whether tau fibril-targeting molecules are effective against mTCOs is unknown. The binding of epigallocatechin-3-gallate (EGCG), CNS-11, and BHT-CNS-11 to in silico mTCOs and experimental tau fibrils was investigated using machine learning-enhanced docking and molecular dynamics simulations. EGCG and CNS-11 have tau fibril disaggregation functions, while the proposed BHT-CNS-11 has potential tau fibril disaggregation and antioxidation functions like EGCG. Our results suggest that the three molecules studied may also bind to mTCOs. The predicted binding probability of EGCG to mTCOs increases with the protein aggregate size. In contrast, the predicted probability of CNS-11 and BHT-CNS-11 binding to the dimeric mTCOs is higher than binding to the tetrameric mTCOs for the homo tau but not for the hetero tau-amylin oligomers. Our results also support the idea that anionic lipids may promote the binding of molecules to mTCOs. We conclude that tau fibril-disaggregating and antioxidating molecules may bind to mTCOs, and that mTCOs may also be useful targets for Alzheimer's drug design.

An NMR Toolkit to Probe Amyloid Oligomer Inhibition in Neurodegenerative Diseases: From Ligand Screening to Dissecting Binding Topology and Mechanisms of Action.

The aggregation of amyloid peptides and proteins into toxic oligomers is a hallmark of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Machado-Joseph's disease, and transmissible spongiform encephalopathies. Inhibition of amyloid oligomers formation and interactions with biological counterparts, as well as the triggering of non-toxic amorphous aggregates, are strategies towards preventive interventions against these pathologies. NMR spectroscopy addresses the need for structural characterization of amyloid proteins and their aggregates, their binding to inhibitors, and rapid screening of compound libraries for ligand identification. Here we briefly discuss the solution experiments constituting the NMR spectroscopist's toolkit and provide examples of their application.

Oligomeric amyloid-β targeted contrast agent for MRI evaluation of Alzheimer's disease mouse models.

Oligomeric amyloid beta (oAβ) is a toxic factor that acts in the early stage of Alzheimer's disease (AD) and may initiate the pathologic cascade. Therefore, detecting oAβ has a crucial role in the early diagnosis, monitoring, and treatment of AD. The purpose of this study was to evaluate MRI signal changes in different mouse models and the time-dependent signal changes using our novel gadolinium (Gd)-dodecane tetraacetic acid (DOTA)- ob5 aptamer contrast agent. We developed an MRI contrast agent by conjugating Gd-DOTA-DNA aptamer called ob5 to evaluate its ability to detect oAβ deposits in the brain using MRI. A total of 10 control mice, 9 3xTg AD mice, and 11 APP/PS/Tau AD mice were included in this study, with the age of each model being 16 or 36 weeks. A T1-weighted image was acquired at the time points before (0min) and after injection of the contrast agent at 5, 10, 15, 20, and 25min. The analyses were performed to compare MRI signal differences among the three groups and the time-dependent signal differences in different mouse models. Both 3xTg AD and APP/PS/Tau AD mouse models had higher signal enhancement than control mice at all scan-time points after injection of our contrast media, especially in bilateral hippocampal areas. In particular, all Tg AD mouse models aged 16 weeks showed a higher contrast enhancement than those aged 36 weeks. For 3xTg AD and APP/PS/Tau AD groups, the signal enhancement was significantly different among the five time points (0min, 5min, 10min, 15min, 20min, and 25min) in multiple ROI areas, typically in the bilateral hippocampus, left thalamus, and left amygdala. The findings of this study suggest that the expression of the contrast agent in different AD models demonstrates its translational flexibility across different species. The signal enhancement peaked around 15-20min after injection of the contrast agent. Therefore, our novel contrast agent targeting oAβ has the potential ability to diagnose early AD and monitor the progression of AD.

Screening Carbon Nano Materials for Preventing Amyloid Protein Aggregation by Adopting a Facile Method.

The soluble-to-toxic transformation of intrinsically disordered amyloidogenic proteins such as amyloid beta (Aβ), α-synuclein, mutant Huntingtin Protein (mHTT) and islet amyloid polypeptide (IAPP) among others are associated with disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and Type 2 Diabetes (T2D), respectively. The dissolution of mature fibrils and toxic amyloidogenic intermediates, including oligomers, continues to be the pinnacle in the treatment of neurodegenerative disorders. Yet, methods to effectively and quantitatively report on the interconversion between amyloid monomers, oligomers and mature fibrils fall short. Here we describe a simplified method that implements the use of gel electrophoresis to address the transformation between soluble monomeric amyloid proteins and mature amyloid fibrils. The technique implements an optimized but well-known, simple, inexpensive, and quantitative assessment previously used to assess the oligomerization of amyloid monomers and subsequent amyloid fibrils. This method facilitates the screening of small molecules that disintegrate oligomers and fibrils into monomers, dimers, and trimers and/or retain amyloid proteins in their monomeric forms. Most importantly, our optimized method diminishes existing barriers associated with existing (alternative) techniques to evaluate fibril formation and intervention.

Oligomeric amyloid beta prevents myelination in a clusterin-dependent manner.

White matter loss is a well-documented phenomenon in Alzheimer's disease (AD) patients that has been recognized for decades. However, the underlying reasons for the failure of oligodendrocyte progenitor cells (OPCs) to repair myelin deficits in these patients remain elusive. A single nucleotide polymorphism (SNP) in Clusterin has been identified as a risk factor for late-onset Alzheimer's disease and linked to a decrease in white matter integrity in healthy adults, but its specific role in oligodendrocyte function and myelin maintenance in Alzheimer's disease pathology remains unclear. To investigate the impact of Clusterin on OPCs in the context of Alzheimer's disease, we employed a combination of immunofluorescence and transmission electron microscopy techniques, primary culture of OPCs, and an animal model of Alzheimer's disease. Our findings demonstrate that Clusterin, a risk factor for late-onset AD, is produced by OPCs and inhibits their differentiation into oligodendrocytes. Specifically, we observed upregulation of Clusterin in OPCs in the 5xFAD mouse model of AD. We also found that the phagocytosis of debris, including amyloid beta (Aβ), myelin, and apoptotic cells leads to the upregulation of Clusterin in OPCs. In vivo experiments confirmed that Aβ oligomers stimulate Clusterin upregulation and that OPCs are capable of phagocytosing Aβ. Furthermore, we discovered that Clusterin significantly inhibits OPC differentiation and hinders the production of myelin proteins. Finally, we demonstrate that Clusterin inhibits OPC differentiation by reducing the production of IL-9 by OPCs. Our data suggest that Clusterin may play a key role in the impaired myelin repair observed in AD and could serve as a promising therapeutic target for addressing AD-associated cognitive decline.

Primary Nucleation of Polymorphic α-Synuclein Dimers Depends on Copper Concentrations and Definite Copper-Binding Site.

The primary nucleation process of α-synuclein (AS) that forms toxic oligomeric species is the early stage of the pathological cause of Parkinson's disease. It is well-known that copper influences this primary nucleation process. While significant efforts have been made to solve the structures of polymorphic AS fibrils, the structures of AS oligomers and the copper-bound AS oligomers at the molecular level and the effect of copper concentrations on the primary nucleation are elusive. Here, we propose and demonstrate new molecular mechanism pathways of primary nucleation of AS that are tuned by distinct copper concentrations and by a specific copper-binding site. We present the polymorphic AS dimers bound to different copper-binding sites at the atomic resolution in high- and low-copper concentrations, using extensive molecular dynamics simulations. Our results show the complexity of the primary nucleation pathways that rely on the copper concentrations and the copper binding site. From a broader perspective, our study proposes a new strategy to control the primary nucleation of other toxic amyloid oligomers in other neurodegenerative diseases.

The protective effects of Chromofungin in oligomeric amyloid β42 (Aβ42)-induced toxicity in neurons in Alzheimer's disease.

Oligomeric Aβ42 is considered to play a harmful role in the pathophysiology of Alzheimer's disease (AD). Prolonged exposure to oligomeric Aβ42 could induce neuronal damage including cellular senescence. Amelioration of Aβ42-induced cellular senescence has been considered as a promising strategy for the treatment of AD. Chromofungin, a chromogranin A-derived peptide, has displayed various biological functions in different types of cells and tissues. However, the effects of Chromofungin on oligomeric Aβ42-induced cellular senescence have not been previously reported. In the current study, we report a novel function of Chromofungin by showing that treatment with Chromofungin could ameliorate Aβ42-induced neurotoxicity in M17 neuronal cells. The Cell Counting Kit-8 (CCK-8) assay and the lactate dehydrogenase (LDH) release experiments revealed that 0.5 and 1 mM are the optimal concentrations of Chromofungin for cell culture in M17 cells. Challenging with oligomeric Aβ42 (5 μM) for 7 and 14 days led to a significant decrease in telomerase activity, which was rescued by Chromofungin dose-dependently. Additionally, the senescence-associated β-galactosidase (SA-β-gal) staining assay demonstrated that Chromofungin mitigated oligomeric Aβ42-induced cellular senescence. Correspondingly, treatment with Chromofungin reversed the gene expression of human telomerase reverse transcriptase (hTERT), telomeric repeat-binding factor 2 (TERF2), and p21 against oligomeric Aβ42 in M17 neurons. Interestingly, Chromofungin attenuated oligomeric Aβ42-induced oxidative stress (OS) in M17 cells by reducing the production of intracellular reactive oxygen species (ROS) but increasing the levels of intracellular superoxide dismutase (SOD). Importantly, the presence of Chromofungin reduced the expression of cyclooxygenase2 (COX-2) as well as the generation of prostaglandin E2 (PGE2). Transduction with Ad-COX-2 impaired the effects of Chromofungin on telomerase activity and the profile of cellular senescence. Our findings suggest that Chromofungin might act as a potential agent for the treatment of AD.

Crystal Violet Selectively Detects Aβ Oligomers but Not Fibrils In Vitro and in Alzheimer's Disease Brain Tissue.

Deposition of extracellular Amyloid Beta (Aβ) and intracellular tau fibrils in post-mortem brains remains the only way to conclusively confirm cases of Alzheimer's Disease (AD). Substantial evidence, though, implicates small globular oligomers instead of fibrils as relevant biomarkers of, and critical contributors to, the clinical symptoms of AD. Efforts to verify and utilize amyloid oligomers as AD biomarkers in vivo have been limited by the near-exclusive dependence on conformation-selective antibodies for oligomer detection. While antibodies have yielded critical evidence for the role of both Aβ and tau oligomers in AD, they are not suitable for imaging amyloid oligomers in vivo. Therefore, it would be desirable to identify a set of oligomer-selective small molecules for subsequent development into Positron Emission Tomography (PET) probes. Using a kinetics-based screening assay, we confirm that the triarylmethane dye Crystal Violet (CV) is oligomer-selective for Aβ42 oligomers (AβOs) grown under near-physiological solution conditions in vitro. In postmortem brains of an AD mouse model and human AD patients, we demonstrate that A11 antibody-positive oligomers but not Thioflavin S (ThioS)-positive fibrils colocalize with CV staining, confirming in vitro results. Therefore, our kinetic screen represents a robust approach for identifying new classes of small molecules as candidates for oligomer-selective dyes (OSDs). Such OSDs, in turn, provide promising starting points for the development of PET probes for pre-mortem imaging of oligomer deposits in humans.

Neuroprotective amyloid β N-terminal peptides differentially alter human α7- and α7β2-nicotinic acetylcholine (nACh) receptor single-channel properties.

Oligomeric amyloid β 1-42 (oAβ1-42) exhibits agonist-like action at human α7- and α7β2-containing nicotinic receptors. The N-terminal amyloid β1-15 fragment (N-Aβ fragment) modulates presynaptic calcium and enhances hippocampal-based synaptic plasticity via α7-containing nicotinic receptors. Further, the N-Aβ fragment and its core sequence, the N-amyloid-beta core hexapeptide (N-Aβcore), protect against oAβ1-42-associated synapto- and neurotoxicity. Here, we investigated how oAβ1-42, the N-Aβ fragment, and the N-Aβcore regulate the single-channel properties of α7- and α7β2-nicotinic receptors. Single-channel recordings measured the impact of acetylcholine, oAβ1-42, the N-Aβ fragment, and the N-Aβcore on the unitary properties of human α7- and α7β2-containing nicotinic receptors expressed in nicotinic-null SH-EP1 cells. Molecular dynamics simulations identified potential sites of interaction between the N-Aβ fragment and orthosteric α7+/α7- and α7+/β2- nicotinic receptor binding interfaces. The N-Aβ fragment and N-Aβcore induced α7- and α7β2-nicotinic receptor single-channel openings. Relative to acetylcholine, oAβ1-42 preferentially enhanced α7β2-nicotinic receptor single-channel open probability and open-dwell times. Co-application with the N-Aβcore neutralized these effects. Further, administration of the N-Aβ fragment alone, or in combination with acetylcholine or oAβ1-42, selectively enhanced α7-nicotinic receptor open probability and open-dwell times (compared to acetylcholine or oAβ1-42). Amyloid-beta peptides demonstrate functional diversity in regulating α7- and α7β2-nicotinic receptor function, with implications for a wide range of nicotinic receptor-mediated functions in Alzheimer's disease. The effects of these peptides on α7- and/or α7β2-nicotinic receptors revealed complex interactions with these subtypes, providing novel insights into the neuroprotective actions of amyloid β-derived fragments against the toxic effects of oAβ1-42.

Localized surface plasmon resonance and atomic force microscopy study of model lipid membranes and their interactions with amyloid and melatonin

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid plaques in the brain. The toxicity of amyloid to neuronal cell surfaces arises from interactions between small intermediate aggregates, namely amyloid oligomers, and the cell membrane. The nature of these interactions changes with age and disease progression. In our previous work, we demonstrated that both membrane composition and nanoscale structure play crucial roles in amyloid toxicity, and that membrane models mimicking healthy neuron were less affected by amyloid than model membranes mimicking AD neuronal membranes. This understanding introduces the possibility of modifying membrane properties with membrane-active molecules, such as melatonin, to protect them from amyloid-induced damage. In this study, we employed atomic force microscopy and localized surface plasmon resonance to investigate the protective effects of melatonin. We utilized synthetic lipid membranes that mimic the neuronal cellular membrane at various stages of AD and explored their interactions with amyloid-β (1–42) in the presence of melatonin. Our findings reveal that the early diseased membrane model is particularly vulnerable to amyloid binding and subsequent damage. However, melatonin exerts its most potent protective effect on this early-stage membrane. These results suggest that melatonin could act at the membrane level to alleviate amyloid toxicity, offering the most protection during the initial stages of AD.