Aβ Variants Research Articles

Advances in Understanding and Managing Alzheimer's Disease: From Pathophysiology to Innovative Therapeutic Strategies.

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder characterized by the presence of amyloid-β (Aβ) plaques and tau-containing neurofibrillary tangles, leading to cognitive and physical decline. Representing the majority of dementia cases, AD poses a significant burden on healthcare systems globally, with onset typically occurring after the age of 65. While most cases are sporadic, about 10% exhibit autosomal forms associated with specific gene mutations. Neurofibrillary tangles and Aβ plaques formed by misfolded tau proteins and Aβ peptides contribute to neuronal damage and cognitive impairment. Currently, approved drugs, such as acetylcholinesterase inhibitors and N-methyl D-aspartate receptor agonists, offer only partial symptomatic relief without altering disease progression. A promising development is using lecanemab, a humanized IgG1 monoclonal antibody, as an immune therapeutic approach. Lecanemab demonstrates selectivity for polymorphic Aβ variants and binds to large soluble Aβ aggregates, providing a potential avenue for targeted treatment. This shift in understanding the role of the adaptive immune response in AD pathogenesis opens new possibilities for therapeutic interventions aiming to address the disease's intricate mechanisms. This review aims to summarize recent advancements in understanding Alzheimer's disease pathophysiology and innovative therapeutic approaches, providing valuable insights for both researchers and clinicians.

Simple improvements in vector design afford substantial gains in AAV delivery of aggregation-slowing Aβ variants

Adeno-associated virus (AAV) gene therapy for neurological disease has gained traction due to stunning advances in capsid evolution for CNS targeting. With AAV brain delivery now in focus, conventional improvements in viral expression vectors offer a complementary route for optimizing gene delivery. We previously introduced a novel AAV gene therapy to slow amyloid aggregation in the brain based on neuronal release of an Aβ sequence variant that inhibited fibrilization of wild-type Aβ. Here we explore three coding elements of the virally delivered DNA plasmid in an effort to maximize the production of therapeutic peptide in the brain. We demonstrate thatsimply replacing the Gaussia luciferase signal peptide with the mouse immunoglobulin heavy chain signal peptide increased release of variant Aβ by ∼5-fold. Sequence modifications within the expressed minigene further increased peptide release by promoting γ-secretase cleavage. Addition of a cytosolic fusion tag compatible with γ-secretase interaction allowed viral transduction to be tracked by immunostaining, independent from the variant Aβ peptide. Collectively these construct modifications increased neuronal production of therapeutic peptide by 10-fold upon intracranial AAV injection of neonatal mice. These findings demonstrate that modest changes in expression vector design can yield substantial gains in AAV efficiency for therapeutic applications.

Acute sleep deprivation in mice generates protein pathology consistent with neurodegenerative diseases.

Insufficient or disturbed sleep is strongly associated with adverse health conditions, including various neurodegenerative disorders. While the relationship between sleep and neurodegenerative disease is likely bidirectional, sleep disturbances often predate the onset of other hallmark clinical symptoms. Neuronal waste clearance is significantly more efficient during sleep; thus, disturbed sleep may lead to the accumulation of neuronal proteins that underlie neurodegenerative diseases. Key pathological features of neurodegenerative diseases include an accumulation of misfolded or misprocessed variants of amyloid beta (Aβ), tau, alpha synuclein (α-syn), and TarDNA binding protein 43 (TDP-43). While the presence of fibrillar protein aggregates of these neuronal proteins are characteristic of neurodegenerative diseases, the presence of small soluble toxic oligomeric variants of these different proteins likely precedes the formation of the hallmark aggregates. We hypothesized that sleep deprivation would lead to accumulation of toxic oligomeric variants of Aβ, tau, α-syn, and TDP-43 in brain tissue of wild-type mice. Adult mice were subjected to 6 h of sleep deprivation (zeitgeber 0-6) for 5 consecutive days or were left undisturbed as controls. Following sleep deprivation, brains were collected, and protein pathology was assessed in multiple brain regions using an immunostain panel of reagents selectively targeting neurodegenerative disease-related variants of Aβ, tau, α-syn, and TDP-43. Overall, sleep deprivation elevated levels of all protein variants in at least one of the brain regions of interest. The reagent PDTDP, targeting a TDP-43 variant present in Parkinson's disease, was elevated throughout the brain. The cortex, caudoputamen, and corpus callosum brain regions showed the highest accumulation of pathology following sleep deprivation. These data provide a direct mechanistic link between sleep deprivation, and the hallmark protein pathologies of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases.

Amino-terminally elongated Aβ peptides are generated by the secreted metalloprotease ADAMTS4 and deposit in a subset of Alzheimer's disease brains.

The aggregation and deposition of amyloid-β (Aβ) peptides in the brain is thought to be the initial driver in the pathogenesis of Alzheimer's disease (AD). Aside from full-length Aβ peptides starting with an aspartate residue in position 1, both N-terminally truncated and elongated Aβ peptides are produced by various proteases from the amyloid precursor protein (APP) and have been detected in brain tissues and body fluids. Recently, we demonstrated that the particularly abundant N-terminally truncated Aβ4-x peptides are generated by ADAMTS4, a secreted metalloprotease that is exclusively expressed in the oligodendrocyte cell population. In this study, we investigated whether ADAMTS4 might also be involved in the generation of N-terminally elongated Aβ peptides. We used cell-free and cell-based assays in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) and electrochemiluminescence sandwich immunoassays to identify and quantify N-terminally elongated Aβ peptide variants. Antibodies against these Aβ variants were characterised by peptide microarrays and employed for the immunohistochemical analyses of human brain samples. In this study, we discovered additional ADAMTS4 cleavage sites in APP. These were located N-terminal to Asp-(1) in the Aβ peptide sequence between residues Glu-(-7) and Ile-(-6) as well as Glu-(-4) and Val-(-3), resulting in the release of N-terminally elongated Aβ-6-x and Aβ-3-x peptides, of which the latter serve as a component in a promising Aβ-based plasma biomarker. Aβ-6/-3-40 peptides were detected in supernatants of various cell lines and in the cerebrospinal fluid (CSF), and ADAMTS4 enzyme activity promoted the release of Aβ-6/-3-x peptides. Furthermore, by immunohistochemistry, a subset of AD cases displayed evidence of extracellular and vascular localization of N-terminally elongated Aβ-6/-3-x peptides. The current findings implicate ADAMTS4 in both the pathological process of Aβ peptide aggregation and in the early detection of amyloid pathology in AD.

An N-terminal acidic β-sheet domain is responsible for the metal-accumulation properties of amyloid-β protofibrils: a molecular dynamics study

The influence of metal ions on the structure of amyloid-β\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\beta $$\\end{document} (Aβ) protofibril models was studied through molecular dynamics to explore the molecular mechanisms underlying metal-induced Aβ aggregation relevant in Alzheimer’s disease (AD). The models included 36-, 48-, and 188-mers of the Aβ42 sequence and two disease-modifying variants. Primary structural effects were observed at the N-terminal domain, as it became susceptible to the presence of cations. Specially when β-sheets predominate, this motif orients N-terminal acidic residues toward one single face of the β-sheet, resulting in the formation of an acidic region that attracts cations from the media and promotes the folding of the N-terminal region, with implications in amyloid aggregation. The molecular phenotype of the protofibril models based on Aβ variants shows that the AD-causative D7N mutation promotes the formation of N-terminal β-sheets and accumulates more Zn2+, in contrast to the non-amyloidogenic rodent sequence that hinders the β-sheets and is more selective for Na+ over Zn2+ cations. It is proposed that forming an acidic β-sheet domain and accumulating cations is a plausible molecular mechanism connecting the elevated affinity and concentration of metals in Aβ fibrils to their high content of β-sheet structure at the N-terminal sequence.Graphic abstract

The Compartmentalization of Amyloid-β in Idiopathic Normal Pressure Hydrocephalus Brain Biopsies.

Amyloid-β (Aβ) is one of the hallmark lesions of Alzheimer's disease (AD). During the disease process, Aβ undergoes biochemical changes, producing toxic Aβ variants, proposed to be detected within the neurons. Idiopathic normal pressure hydrocephalus (iNPH) causes cognitive impairment, gait, and urinary symptoms in elderly, that can be reversed by a ventriculo-peritoneal shunt. Majority of iNPH subjects display different Aβ variants in their brain biopsies, obtained during shunting. To study the cellular compartmentalization of different Aβ variants in brain biopsies from iNPH subjects. We studied the cellular localization of different proteoforms of Aβ using antibodies towards different amino acid sequences or post-translational modifications of Aβ, including clones 4G8, 6F/3D, unmodified- (7H3D6), pyroglutamylated- (N3pE), phosphorylated-(1E4E11) Aβ and Aβ protein precursor (AβPP), in brain biopsies from 3 iNPH subjects, using immunohistochemistry and light microscopy (LM), light microscopy on semi-thin sections (LMst), and electron microscopy (EM). In LM all Aβ variants were detected. In LMst and EM, the Aβ 4G8, 6F/3D, and the pyroglutamylated Aβ were detected. The AβPP was visualized by all methods. The Aβ labelling was located extracellularly with no specific signal within the intracellular compartment, whereas the AβPP was seen both intra- and extracellularly. The Aβ markers displayed extracellular localization when visualized by three assessment techniques, reflecting the pathological extracellular accumulation of Aβ in the human brain. No intracellular Aβ pathology was seen. AβPP was visualized in intra- and extracellularly, which corresponds to the localization of the protein in the membranes of cells and organelles.

Traumatic Brain Injury in Mice Generates Early-Stage Alzheimer’s Disease Related Protein Pathology that Correlates with Neurobehavioral Deficits

Traumatic brain injury (TBI) increases the long-term risk of neurodegenerative diseases, including Alzheimer’s disease (AD). Here, we demonstrate that protein variant pathology generated in brain tissue of an experimental TBI mouse model is similar to protein variant pathology observed during early stages of AD, and that subacute accumulation of AD associated variants of amyloid beta (Aβ) and tau in the TBI mouse model correlated with behavioral deficits. Male C57BL/6 mice were subjected to midline fluid percussion injury or to sham injury, after which sensorimotor function (rotarod, neurological severity score), cognitive deficit (novel object recognition), and affective deficits (elevated plus maze, forced swim task) were assessed post-injury (DPI). Protein pathology at 7, 14, and 28 DPI was measured in multiple brain regions using an immunostain panel of reagents selectively targeting different neurodegenerative disease-related variants of Aβ, tau, TDP-43, and alpha-synuclein. Overall, TBI resulted in sensorimotor deficits and accumulation of AD-related protein variant pathology near the impact site, both of which returned to sham levels by 14 DPI. Individual mice, however, showed persistent behavioral deficits and/or accumulation of toxic protein variants at 28 DPI. Behavioral outcomes of each mouse were correlated with levels of seven different protein variants in ten brain regions at specific DPI. Out of 21 significant correlations between protein variant levels and behavioral deficits, 18 were with variants of Aβ or tau. Correlations at 28 DPI were all between a single Aβ or tau variant, both of which are strongly associated with human AD cases. These data provide a direct mechanistic link between protein pathology resulting from TBI and the hallmarks of AD.

Conformation of Pyroglutamated Amyloid β (3-40) and (11-40) Fibrils - Extended or Hairpin?

Amyloid β (Aβ) is a hallmark protein of Alzheimer's disease. One physiologically important Aβ variant is formed by initial N-terminal truncation at a glutamic acid position (either E3 or E11), which is subsequently cyclized to a pyroglutamate (either pE3 or pE11). Both forms have been found in high concentrations in the core of amyloid plaques and are likely of high importance in the pathology of Alzheimer's disease. However, the molecular structure of the fibrils of these variants is not entirely clear. Solid-state NMR spectroscopy studies have reported a molecular contact between Gly25 and Ile31, which would disagree with the conventional hairpin model of wildtype (WT-)Aβ1-40 fibrils, most often described in the literature. We investigated the conformation of the monomeric unit of pE3-Aβ3-40 and pE11-Aβ11-40 (and for comparison also wildtype (WT)-Aβ1-40) fibrils to find out whether the hairpin or a newly suggested extended structure dominates the structure of the Aβ monomers in these fibrils. To this end, solid-state NMR spectroscopy was applied probing the inter-residual contacts between Phe19/Leu34, Ala21/Leu34, and especially Gly25/Ile31 using suitable isotopic labeling schemes. In the second part, the flexible turn of the Aβ40 peptides was replaced by a (3-(3-aminomethyl)phenylazo)phenylacetic acid (AMPP)-based photoswitch, which can predefine the peptide conformation to either an extended (trans) or hairpin (cis) conformation. This enables simultaneous spectroscopic assessment of the conformation of the AMPP-photoswitch, allowing in situ structural investigations during fibrillation in contrast to structural techniques such as NMR spectroscopy or cryo-EM, which can only be applied to stable conformers. Both methods confirm an extended structure for the peptidic monomers in fibrils of all investigated Aβ variants. Especially the Gly25/Ile31 contact is a decisive indicator for the extended structure along with the characteristic absorption spectra of trans-AMPP-Aβ.

Phosphorylation-state dependent intraneuronal sorting of Aβ differentially impairs autophagy and the endo-lysosomal system

ABSTRACT Progressive accumulation of amyloid-β (Aβ) aggregates in extracellular plaques is a characteristic hallmark of Alzheimer disease (AD). Aβ is also found in intraneuronal deposits and associated with alterations of the endo-lysosomal system and impairment of macroautophagy/autophagy. Here, we assessed the effect of Aβ phosphorylation on neuronal autophagy and the endo-lysosomal pathway. Analysis of APP-PSEN1dE9 transgenic mice revealed a phosphorylation-state dependent intraneuronal accumulation of Aβ species in endo-lysosomal and autophagy-related compartments. Cell biological studies further demonstrate a differential uptake and sorting of phosphorylated Aβ variants in cultured neurons, and phosphorylation-state specific effects of Aβ variants on neuronal autophagy and lysosomal function. While Aβ phosphorylated at serine residue 8 accumulated in autophagosomes, Aβ phosphorylated at serine residue 26 showed efficient transport to lysosomes. The selective sorting of phosphorylated Aβ species caused differential impairment of vesicular transport and lysosomal function associated with neurotoxicity. Thus, the relative occurrence of phosphorylated Aβ species and their intraneuronal accumulation could contribute to AD pathogenesis, and to the commonly observed aberrations of the vesicular transport system already at the early stages of the disease. Abbreviations AD: Alzheimer disease; APP: amyloid beta precursor protein; ATG: autophagy related; Aβ: amyloid-β; CTSD: cathepsin D; DAPI: 4’,6-diamidino-2-phenylindole; EEA1: early endosome antigen 1; FA: formic acid; GFP: green fluorescent protein; LAMP2: lysosomal-associated membrane protein 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP2: microtubule-associated protein 2; nmAβ: non-modified amyloid-β; npAβ: non-phosphorylated amyloid-β; pAβ: phosphorylated amyloid-β; p-Ser26Aβ: amyloid-β phosphorylated at serine residue 26; p-Ser8Aβ: amyloid-β phosphorylated at serine residue 8; RAB: RAB, member RAS oncogene family; RFP: red fluorescent protein; SQSTM1/p62: sequestome 1; YFP: yellow fluorescent protein.

Atomistic molecular simulations of Aβ-Zn conformational ensembles.

The amyloid-forming Aβ peptide is able to interact with metal cations to form very stable complexes that influence fibril formation and contribute to the onset of Alzheimer's disease. Multiple structures of peptides derived from Aβ in complex with different metals have been resolved experimentally to provide an atomic-level description of the metal-protein interactions. However, Aβ is intrinsically disordered, and hence more amenable to an ensemble description. Molecular dynamics simulations can now reach the timescales needed to generate ensembles for these type of complexes. However, this requires accurate force fields both for the protein and the protein-metal interactions. Here we use state-of-the-art methods to generate force field parameters for the Zn(II) cations in a set of complexes formed by different Aβ variants and combine them with the Amber99SB*-ILDN optimized force field. Upon comparison of NMR experiments with the simulation results, further optimized with a Bayesian/Maximum entropy approach, we provide an accurate description of the molecular ensembles for most Aβ-metal complexes. We find that the resulting conformational ensembles are more heterogeneous than the NMR models deposited in the Protein Data Bank.

Modulation of aggregation and structural polymorphisms of β-amyloid fibrils in cellular environments by pyroglutamate-3 variant cross-seeding

Amyloidogenic deposition of β-amyloid (Aβ) peptides in human brain involves not only the wild-type Aβ (wt-Aβ) sequences, but also posttranslationally modified Aβ (PTM-Aβ) variants. Recent studies hypothesizes that the PTM-Aβ variants may trigger the deposition of wt-Aβ, which underlies the pathology of Sporadic Alzheimer's disease. Among PTM-Aβ variants, the pyroglutamate-3-Aβ (pyroE3-Aβ) has attracted much attention because of their significant abundances and broad distributions in senile plaques and dispersible and soluble oligomers. pyroE3-specific antibodies are being tested as potential anti-Aβ drugs in clinical trials. However, evidence that support the triggering effect of pyroE3-Aβ on wt-Aβ in cells remain lacking, which diminishes its pathological relevance. We show here that cross-seeding with pyroE3-Aβ40 leads to accelerated extracellular and intracellular aggregation of wt-Aβ40 in different neuronal cells. Cytotoxicity levels are elevated through the cross-seeded aggregation, comparing with the self-seeded aggregation of wt-Aβ40 or the static presence of pyroE3-Aβ40 seeds. For the extracellular deposition in mouse neuroblastoma Neuro2a (N2a) cells, the cytotoxicity elevation correlates positively with the seeding efficiency. Besides aggregation rates, cross-seeding with pyroE3-Aβ40 also modulates the molecular level structural polymorphisms of the resultant wt-Aβ40 fibrils. Using solid-state nuclear magnetic resonance (ssNMR) spectroscopy, we identified key structural differences between the parent pyroE3/ΔE3 and wt-Aβ40 fibrils within their fibrillar cores. Structural propagation from seeds to daughter fibrils is demonstrated to be more pronounced in the extracellular seeding in N2a cells by comparing the ssNMR spectra from different seeded wt-Aβ40 fibrils, but less significant in the intracellular seeding process in human neuroblastoma SH-SY5Y cells.

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.

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.

Matrix Development for the Detection of Phosphorylated Amyloid-β Peptides by MALDI-TOF-MS.

Amyloid-β (Aβ) peptides, including post-translationally modified variants thereof, are believed to play a key role in the onset and progression of Alzheimer's disease. Suggested modified Aβ species with potential disease relevance include Aβ peptides phosphorylated at serine in position eight (pSer8-Aβ) or 26 (pSer26-Aβ). However, the published studies on those Aβ peptides essentially relied on antibody-based approaches. Thus, complementary analyses by mass spectrometry, as shown for other modified Aβ variants, will be necessary not only to unambiguously verify the existence of phosphorylated Aβ species in brain samples but also to reveal their exact identity as to phosphorylation sites and potential terminal truncations. With the aim of providing a novel tool for addressing this still-unresolved issue, we developed a customized matrix formulation, referred to as TOPAC, that allows for improved detection of synthetic phosphorylated Aβ species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. When TOPAC was compared with standard matrices, we observed higher signal intensities but minimal methionine oxidation and phosphate loss for intact pSer8-Aβ(1-40) and pSer26-Aβ(1-40). Similarly, TOPAC also improved the mass spectrometric detection and sequencing of the proteolytic cleavage products pSer8-Aβ(1-16) and pSer26-Aβ(17-28). We expect that TOPAC will facilitate future efforts to detect and characterize endogenous phosphorylated Aβ species in biological samples and that it may also find its use in phospho-proteomic approaches apart from applications in the Aβ field.

Exploring Combination Therapies in AD: Additive Effects of the QPCT Inhibitor Varoglutamstat and Aducanumab on Aβ Pathology and Biomarkers In Vivo

AbstractBackgroundPyroglutamate‐3Aβ (N3pE), a toxic Aβ variant formed by the activity of glutaminyl gyclase (QPCT) has been shown to play a pivotal role in the development and progression of Alzheimer’s disease (AD). Recently, we demonstrated that a combination of the QPCT inhibitor varoglutamstat (PQ912) with the N3pE‐specific antibody m6 has an additive effect on lowering of N3pE in vivo. Here, we analyzed whether a similar additive effect could be achieved when combining varoglutamstat with the Aβ‐aggregate‐specific antibody aducanumab.MethodNine‐months old APPxhQC mice were treated with either varoglutamstat (1.6 g/kg chow, ad libitum), chimeric aducanumab (chAdu, ≈10 mg/kg i.p. weekly) or a combination of both for 16 weeks. Brain Aβ accumulation (ELISA and immunohistochemistry) and AD biomarkers in the water‐soluble brain fractions (neurogranin, BACE‐1, YKL‐40; ELISA) were analyzed and compared to a vehicle‐treated group.ResultTreatment with either varoglutamstat or chAdu significantly reduced the accumulation of both total Aβ and N3pE in the brain. The effect of chAdu was more pronounced for total Aβ (‐35% vs. ‐21% for varoglutamstat), while varoglutamstat treatment resulted in a stronger decrease of N3pE (‐28% vs. ‐19% for chAdu). The combination treatment generally led to a stronger decrease of Aβ vs. single agent treatment (Bliss combination index (CI) = 0.88 for soluble Aβ42 and 1.23 for insoluble Aβ42). For insoluble N3pE, the observed effect was nearly additive (CI = 1.09). Treatment with chAdu, varoglutamstat or both had different effects on the analyzed AD biomarkers. chAdu treatment significantly decreased BACE‐1 protein levels (‐30%), while varoglutamstat treatment significantly reduced levels of the brain inflammation marker YKL‐40 (‐27%), which is in line with earlier clinical Phase 2a study results.ConclusionOur data indicate that a combination of agents with different modes of action such as aducanumab, an Aβ aggregate‐specific antibody and varoglutamstat, a small molecule designed to block formation of toxic, aggregation‐prone N3pE, can act additively to decrease total Aβ and N3pE levels in the brain. Our results also provide a rationale for investigating different potential combination regimens, including an initial antibody‐mediated Aβ clearance followed by long‐term suppression of N3pE formation and inflammation by varoglutamstat (“treat and maintain”).

VIVIAD, A Phase 2b Study Investigating Varoglutamstat in Patients with MCI and Mild AD: Dose Selection and Interim Safety Results

AbstractBackgroundVaroglutamstat (PQ912), a small molecule glutaminyl cyclase (QPCT) inhibitor, reduces the brain levels of pyroglutamate‐3‐Aβ (N3pE‐Aβ), a toxic Aβ variant shown to play a pivotal role in the development and progression of Alzheimer’s disease (AD). Encouraging results reported in a prior Phase 2a study (NCT02389413) led to the initiation of a state‐of‐the‐art Phase 2b trial investigating multiple cognitive, safety and biomarker endpoints.MethodsVIVIAD (NCT04498650) is a multicentre randomized, placebo‐controlled, double‐blind, parallel group dose finding Phase 2b study in patients with cognitive impairment (MCI) and mild AD. Objectives are to evaluate the longterm efficacy (primary endpoint Cogstate NTB), safety and tolerability of oral varoglutamstat. The study will recruit 250 patients. The first 90 patients are randomised 1:1:1 (600 mg twice daily, (BID) or 300 mg BID varoglutamstat or placebo BID). An independent DSMB will unblind safety results after 90 patients have completed 24 weeks of treatment in June 2022 and decide on the varoglutamstat dose to be carried forward. The data remain blinded outside the DSMB. After the DSMB decision, all patients will be randomised 1:1 between the final dose of varoglutamstat and placebo and continue treatment up to 48‐96 weeks dependent on study entry date.ResultsThe study is currently enrolling patients in 22 AD study centers in 5 European countries (160 patients are randomised as of April 25, 2022). The safety data providing the base for the DSMB dose decision, the DSMB decision itself and the baseline characteristics of the patients enrolled into the trial so far will be reported at AAIC.ConclusionThe state‐of‐the art Phase 2b study VIVIAD is designed to yield important results in early AD for varoglutamstat, the first small molecule and only project in clinical development selectively targeting the de novo production of neurotoxic N3pE‐Aβ. The DSMB decision represents an important milestone within the Phase 2b study as it will recommend the dose of varoglutamstat considered to be safe for being carried forward to the study completion.

A Review of Application of Aβ42/40 Ratio in Diagnosis and Prognosis of Alzheimer's Disease.

The number of patients with Alzheimer's disease (AD) and non-Alzheimer's disease (non-AD) has drastically increased over recent decades. The amyloid cascade hypothesis attributes a vital role to amyloid-β protein (Aβ) in the pathogenesis of AD. As the main pathological hallmark of AD, amyloid plaques consist of merely the 42 and 40 amino acid variants of Aβ (Aβ42 and Aβ40). The cerebrospinal fluid (CSF) biomarker Aβ42/40 has been extensively investigated and eventually integrated into important diagnostic tools to support the clinical diagnosis of AD. With the development of highly sensitive assays and technologies, blood-based Aβ42/40, which was obtained using a minimally invasive and cost-effective method, has been proven to be abnormal in synchrony with CSF biomarker values. This paper presents the recent progress of the CSF Aβ42/40 ratio and plasma Aβ42/40 for AD as well as their potential clinical application as diagnostic markers or screening tools for dementia.

The Hidden Role of Non-Canonical Amyloid β Isoforms in Alzheimer's Disease.

Recent advances have placed the pro-inflammatory activity of amyloid β (Aβ) on microglia cells as the focus of research on Alzheimer's Disease (AD). Researchers are confronted with an astonishing spectrum of over 100 different Aβ variants with variable length and chemical modifications. With the exception of Aβ1-42 and Aβ1-40, the biological significance of most peptides for AD is as yet insufficiently understood. We therefore aim to provide a comprehensive overview of the contributions of these neglected Aβ variants to microglia activation. First, the impact of Aβ receptors, signaling cascades, scavenger mechanisms, and genetic variations on the physiological responses towards various Aβ species is described. Furthermore, we discuss the importance of different types of amyloid precursor protein processing for the generation of these Aβ variants in microglia, astrocytes, oligodendrocytes, and neurons, and highlight how alterations in secondary structures and oligomerization affect Aβ neurotoxicity. In sum, the data indicate that gene polymorphisms in Aβ-driven signaling pathways in combination with the production and activity of different Aβ variants might be crucial factors for the initiation and progression of different forms of AD. A deeper assessment of their interplay with glial cells may pave the way towards novel therapeutic strategies for individualized medicine.

Amyloid beta and its naturally occurring N-terminal variants are potent activators of human and mouse formyl peptide receptor 1

Formyl peptide receptors (FPRs) may contribute to inflammation in Alzheimer's disease through interactions with neuropathological Amyloid beta (Aβ) peptides. Previous studies reported activation of FPR2 by Aβ1-42, but further investigation of other FPRs and Aβ variants is needed. This study provides a comprehensive overview of the interactions of mouse and human FPRs with different physiologically relevant Aβ-peptides using transiently transfected cells in combination with calcium imaging. We observed that, in addition to hFPR2, all other hFPRs also responded to Aβ1-42, Aβ1-40, and the naturally occurring variants Aβ11-40 and Aβ17-40. Notably, Aβ11-40 and Aβ17-40 are very potent activators of mouse and human FPR1, acting at nanomolar concentrations. Buffer composition and aggregation state are extremely crucial factors that critically affect the interaction of Aβ with different FPR subtypes. To investigate the physiological relevance of these findings, we examined the effects of Aβ11-40 and Aβ17-40 on the human glial cell line U87. Both peptides induced a strong calcium flux at concentrations that are very similar to those obtained in experiments for hFPR1 in HEK cells. Further immunocytochemistry, qPCR, and pharmacological experiments verified that these responses were primarily mediated through hFPR1. Chemotaxis experiments revealed that Aβ11-40 but not Aβ17-40 evoked cell migration, which argues for a functional selectivity of different Aβ peptides. Together, these findings provide the first evidence that not only hFPR2 but also hFPR1 and hFPR3 may contribute to neuroinflammation in Alzheimer's disease through an interaction with different Aβ variants.

Deep‐Learning‐Assisted Stratification of Amyloid Beta Mutants Using Drying Droplet Patterns

The development of simple and accurate methods to predict mutations in proteins remains an unsolved challenge in modern biochemistry. It is discovered that critical information about primary and secondary peptide structures can be inferred from the stains left behind by their drying droplets. To analyze the complex stain patterns, deep-learning neuronal networks are challenged with polarized light microscopy images derived from the drying droplet deposits of a range of amyloid beta (1-42) (Aβ42 ) peptides. These peptides differ in a single amino acid residue and represent hereditary mutants of Alzheimer's disease. Stain patterns are not only reproducible but also result in comprehensive stratification of eight amyloid beta (Aβ) variants with predictive accuracies above 99%. Similarly, peptide stains of a range of distinct Aβ42 peptide conformations are identified with accuracies above 99%. The results suggest that a method as simple as drying a droplet of a peptide solution onto a solid surface may serve as an indicator of minute, yet structurally meaningful differences in peptides' primary and secondary structures. Scalable and accurate detection schemes for stratification of conformational and structural protein alterations are critically needed to unravel pathological signatures in many human diseases such as Alzheimer's and Parkinson's disease.