Aβ Fibrils Research Articles

A smart adaptable metal sequestering peptide conjugate to modulate Aβ fibrillar aggregation.

The aggregation of amyloid β peptide (Aβ) in the presence of elevated levels of transition-metal ions, e.g., Fe3+, Cu2+, Zn2+, is accountable for enhanced cellular toxicity in Alzheimer's disease. Many strategies are reported to inhibit either Cu2+, Zn2+, or Fe3+-induced Aβ fibrillation, focused on one metal. Herein, a taurine-containing adaptable metal sequestering peptide (AMSP) has been developed as the modulator of any of the cited metal-induced Aβ-aggregation in vitro. We designed the peptide conjugate comprising VFFA as a recognition motif and a taurine moiety coupled with a pendant chain of glutamic acid such that the -SO3H groups of taurine lie nearby 13His and 14His of Aβ, and sequester such metal ions that construct the salt bridge preponderantly via13His-metal-14His composition as well as bridges with 6His of Aβ. We checked the modulation of fibrillar aggregates of Aβ in the presence of metal ions by monitoring the fibril accumulation using several biophysical methods. The results established that non-aggregating AMSP sequesters Zn2+ preferably, along with Fe3+ and Cu2+ ions from the metal-Aβ complex at the physiological condition, efficiently inhibiting Aβ aggregation. While such adaptable metal binders that can chelate various metals are new, AMSP inhibits aggregation through selective recognition and metal scavenging.

A mitochondria targeting, de novo designed, aggregation-induced emission probe for selective detection of neurotoxic amyloid-β aggregates.

A striking issue is the scarcity of imaging probes for the early diagnosis of Alzheimer's disease. For the development of Aβ biomarkers, a mitochondria targeting, de novo designed, aggregation-induced emission (AIE) probe Cou-AIE-TPP+ is constructed by engineering the aromatic coumarin framework into the bridge of electron donor-acceptor-donor tethered with a lipophilic cationic triphenylphosphonium (TPP+) group. The synthesized Cou-AIE-TPP+ probe exhibits biocompatibility, noncytotoxicity, and a huge Stokes shift (124 nm in PBS). Cou-AIE-TPP+ has respectable fluorescence augmentation inside the aggregated Aβ40 in comparison with monomeric Aβ40 with a high binding affinity (Kd = 83 nM) to Aβ40 aggregates, is capable of detecting the kinetics of amyloid aggregation, and is superior to the gold standard probe thioflavin T. Fluorescence lifetime and brightness are also augmented when the probe Cou-AIE-TPP+ binds with Aβ aggregates in PBS. Cou-AIE-TPP+ (λem 604 nm) selectively targets and images neuronal cell mitochondria, is useful to monitor mitochondrial morphology alteration and damage during Aβ40-induced neurotoxicity, recognizes neurotoxic Aβ fibrils, and is highly colocalized with thioflavin T, showing a decent Pearson correlation coefficient of 0.91 in the human neuroblastoma SH-SY5Y cell line. These findings indicate that the mitochondria targeting, de novo designed, functional AIE-based solvatofluorochromic Cou-AIE-TPP+ probe is a promising switch on biomarkers for fluorescence imaging of Aβ aggregates and to monitor mitochondrial morphology change and dysfunction during Aβ-induced neurotoxicity, which may offer imperative direction for the advancement of compelling AIE biomarkers for targeted early stage Aβ diagnosis in the future.

Molecular engineering of a theranostic molecule that detects Aβ plaques, inhibits Iowa and Dutch mutation Aβ self-aggregation and promotes lysosomal biogenesis for Alzheimer's disease.

Extracellular clustering of amyloid-β (Aβ) and an impaired autophagy lysosomal pathway (ALP) are the hallmark features in the early stages of incurable Alzheimer's disease (AD). There is a pressing need to find or develop new small molecules for diagnostics and therapeutics for the early stages of AD. Herein, we report a small molecule, namely F-SLCOOH, which can bind and detect Aβ1-42, Iowa mutation Aβ, Dutch mutation Aβ fibrils and oligomers exhibiting enhanced emission with high affinity. Importantly, F-SLCOOH can readily pass through the blood-brain barrier and shows highly selective binding toward the extracellular Aβ aggregates in real-time in live animal imaging of a 5XFAD mice model. In addition, a high concentration of F-SLCOOH in both brain and plasma of wildtype mice after intraperitoneal administration was found. The ex vivo confocal imaging of hippocampal brain slices indicated excellent colocalization of F-SLCOOH with Aβ positive NU1, 4G8, 6E10 A11 antibodies and THS staining dye, affirming its excellent Aβ specificity and targetability. The molecular docking studies have provided insight into the unique and specific binding of F-SLCOOH with various Aβ species. Importantly, F-SLCOOH exhibits remarkable anti-fibrillation properties against toxic Aβ aggregate formation of Aβ1-42, Iowa mutation Aβ, and Dutch mutation Aβ. F-SLCOOH treatment also exerts high neuroprotective functions and promotes autophagy lysosomal biogenesis in neuronal AD cell models. In summary, the present results suggest that F-SLCOOH is a highly promising theranostic agent for diagnosis and therapeutics of AD.

Structural Impact of N-terminal Pyroglutamate in an Amyloid-β(3-42) Fibril Probed by Solid-State NMR Spectroscopy.

Extracellular amyloid-β (Aβ) plaques, primarily formed by Aβ(1-40) and Aβ(1-42) fibrils, are a hallmark of Alzheimer's disease. The Aβ peptide can undergo a high variety of different post-translational modifications including formation of a pyroglutamate (pGlu, pE) at N-terminal Glu3 or Glu11 of truncated Aβ(3-x) or Aβ(11-x), respectively. Here we studied structural similarities and differences between pEAβ(3-42) and LS-shaped Aβ(1-42) fibrils grown under identical conditions (pH 2) using solid-state NMR spectroscopy. We show that the central region of pEAβ(3-42) fibrils including the turn region around V24 is almost identical to Aβ(1-42) showing similar β-strands also at the N-terminus. The missing N-terminal residues D1-A2 along with pE3 formation in pEAβ(3-42) preclude a salt bridge between K28-D1' as in Aβ(1-42) fibrils. G37 and G38 act as highly sensitive internal sensors for the modified N-terminus, which remains rigid over ~five pH units.

Cryo-EM Analysis of the Effect of Seeding with Brain-derived Aβ Amyloid Fibrils

Aβ amyloid fibrils from Alzheimer’s brain tissue are polymorphic and structurally different from typical in vitro formed Aβ fibrils. Here, we show that brain-derived (ex vivo) fibril structures can be proliferated by seeding in vitro. The proliferation reaction is only efficient for one of the three abundant ex vivo Aβ fibril morphologies, which consists of two peptide stacks, while the inefficiently proliferated fibril morphologies contain four or six peptide stacks. In addition to the seeded fibril structures, we find that de novo nucleated fibril structures can emerge in seeded samples if the seeding reaction is continued over multiple generations. These data imply a competition between de novo nucleation and seed extension and suggest further that seeding favours the outgrowth of fibril morphologies that contain fewer peptide stacks.

A Biomimetic Upconversion Nanobait-Based Near Infrared Light Guided Photodynamic Therapy Alleviates Alzheimer's Disease by Inhibiting β-Amyloid Aggregation.

Aberrant β-amyloid (Aβ) fibrillation is the key event in Alzheimer's disease (AD), the inhibition and degradation of which are recognized as a promising therapeutic strategy to alleviate the nerve damage of AD. Photodynamic therapy (PDT) holds great potential for modulation of Aβ self-assembly, which is nevertheless limited by the inefficient utilization of reactive oxygen species (ROS). Herein, an erythrocyte membrane (EM)-modified core-shell upconversion nanoparticle (UCNP/Cur@EM) is designed and fabricated as a biomimetic nanobait to improve the PDT efficiency in AD. The UCNP with the outlayer of mesoporous silica is synthesized to load a high amount of the photosensitizer (curcumin), the unique optical feature of which can trigger curcumin to generate ROS upon near-infrared light (NIR) irradiation. Integration of EM enables the biomimetic nanobait to attract Aβ peptides trapped in the phospholipid bilayer, restraining the growth of Aβ monomers to form aggregates and improving the utilization rate of ROS to degrade the preformed Aβ aggregates. In vivo studies demonstrate that UCNP/Cur@EM irradiated by NIR enables to decrease Aβ deposits, ameliorates memory deficits, and rescues cognitive functions in the APP/PS1 transgenic mouse model. A biocompatible and controllable way is provided here to inhibit the amyloid protein-associated pathological process of AD.

Amelioration of Amyloid-β Induced Alzheimer's Disease by Bacopa monnieri through Modulation of Mitochondrial Dysfunction and GSK-3β/Wnt/β-Catenin Signaling.

Alzheimer's disease (AD) is the most prevalent dementia, affecting a large number of populations. Despite being under scrutiny for decades, an effective therapeutic option is still not available. This study explores the therapeutic role of a nootropic herb Bacopa monnieri (BM) in AD-like pathological conditions produced by injecting preformed amyloid-β42 (Aβ42) fibril bilaterally into hippocampus of Wistar rats, and ethanolic extract of BM is orally administered for 4 weeks. Assessment of behavioral changes reveals that BM treatment ameliorates Aβ42-induced cognitive impairment and compromised explorative behavior. Supplementation of BM also reduces oxidative stress biomarkers, proinflammatory cytokines, and cholinesterase activity in the AD rats. Additionally, BM treatment restores Bcl-2-associated X protein (Bax)/ B-cell lymphoma 2 (Bcl-2) imbalance, increases neurotrophic factors expression, and prevents neurodegeneration validated by quantifying Nissl-positive hippocampal neurons. Interestingly, BM administration eliminates amyloid plaques in the hippocampal region and normalizes the Aβ42-induced increase in phospho-tau and total tau expression. Mechanistic investigations reveal that BM interacts with glycogen synthase kinase (GSK-3β) and restores Wnt/β-catenin signaling. BM has been used in diet as a nootropic herb for several centuries. This study highlights the anti-Alzheimer activity of BM from the behavioral to the molecular level by modulating mitochondrial dysfunction, and GSK-3β mediates the Wnt/β-catenin signaling pathway.

Hydrodynamic Control of Alzheimer Aβ Fibrillation with Glucosaminic Acid Containing Click-Cyclized β-Bodies.

It is well established that the dynamic hydration shell plays a vital role in macromolecular functions such as protein-ligand, protein-protein, protein-DNA, and protein-lipid interactions. Here we investigate how the water modality affects conformational changes, solubility, and motion of fibrillar proteins. The hypothesis is that the introduction of a poly hydroxyl amino acid would increase solvation of the fibril forming peptides, preventing their misfolding and aggregation. For the amyloid β (Aβ) peptide, which is considered to be connected with nervous system diseases, including dementia and cognitive decline in Alzheimer's disease, the formation of β-sheet fibrils always occurs with a conformational change and a decrease in the dynamic hydration shell around Aβ(1-42). We present novel cyclic d-amino acid peptides that effectively inhibit fibrillation through affecting the dynamic hydration shell of Aβ(1-42) in vitro. Using de novo design within the software Molecular Operating Environment (MOE), five different peptides that recognize Alzheimer's fibrils were designed and synthesized. Three of them were cyclic all-d-amino acid peptides incorporating the same polyhydroxy building block derived from d-glucosaminic acid (GA). One peptide was the parent cyclic all d-amino acid inhibitor with no GA incorporated, and another was an all l-amino acid linear fibrillation inhibitor. The GA-containing peptides were found to show significantly improved inhibition of Aβ(1-42) aggregation. The inhibition was dramatically improved by the synergistic application of two GA peptides targeting each end of the growing fibril. The present study may facilitate future developments of intervention strategies for Alzheimer's disease and similar neurodegenerative diseases.

Characterizing cannabis-prevalent terpenes for neuroprotection reveal a role for α and β-pinenes in mitigating amyloid β-evoked neurotoxicity and aggregation in vitro

BackgroundCannabis sativa L. (C. sativa) can efficiently synthesize of over 200 terpenes, including monoterpenes, sesquiterpenes and triterpenes that may contribute to the known biological activities of phytocannabinoids of relevance for the burgeoning access to medicinal cannabis formulations globally; however, to date have been uncharacterized. We assessed twelve predominant terpenes in C. sativa for neuroprotective and anti-aggregative properties in semi-differentiated PC12 neuronal cell line that is robust and validated as a cell model responsive to amyloid β (Aβ1–42) protein exposure and oxidative stress. MethodsCell viability was assessed biochemically using the MTT assay in the presence of myrcene, β-caryophyllene, terpinolene, limonene, linalool, humulene, α-pinene, nerolidol, β-pinene, terpineol, citronellol and friedelin (1–200 μM) for 24 hr. Sub-toxic threshold test concentrations of each terpene were then applied to cells, alone or with concomitant incubation with the lipid peroxidant tert-butyl hyrdroperoxide (t-BHP; 0–250 μM) or amyloid β (Aβ1–42; 0–1 μM) to assess neuroprotective effects. Direct effects of each terpene on Aβ fibril formation and aggregation were also evaluated using the Thioflavin T (ThT) fluorometric kinetic assay and transmission electron microscopy (TEM) to visualize fibril and aggregate morphology. ResultsTerpenes were intrinsically benign to PC12 cells up to 50 μM, with higher concentrations of β-caryophyllene, humulene and nerolidol inducing some loss of PC12 cell viability. No significant protective effects of terpenes were observed following t-BHP (0–200 µM) administration, with some enhanced toxicity instead demonstrated from both β-caryophyllene and humulene treatment (each at 50 µM). α-pinene and β-pinene demonstrated a significant neuroprotective effect against amyloid β exposure. α-pinene, β-pinene, terpineol, terpinolene and friedelin were associated with a variable inhibition of Aβ1–42 fibril and aggregate density. ConclusionsThe outcomes of this study underline a neuroprotective role of α-pinene and β-pinene against Aβ-mediated neurotoxicity associated with an inhibition of Aβ1–42 fibrilization and density. This demonstrates the bioactive potential of selected terpenes for consideration in the development of medicinal cannabis formulations targeting neurodegenerative diseases.

Ganglioside Micelles Affect Amyloid β Aggregation by Coassembly.

Amyloid β peptide (Aβ) is the crucial protein component of extracellular plaques in Alzheimer's disease. The plaques also contain gangliosides lipids, which are abundant in membranes of neuronal cells and in cell-derived vesicles and exosomes. When present at concentrations above its critical micelle concentration (cmc), gangliosides can occur as mixed micelles. Here, we study the coassembly of the ganglioside GM1 and the Aβ peptides Aβ40 and 42 by means of microfluidic diffusional sizing, confocal microscopy, and cryogenic transmission electron microscopy. We also study the effects of lipid-peptide interactions on the amyloid aggregation process by fluorescence spectroscopy. Our results reveal coassembly of GM1 lipids with both Aβ monomers and Aβ fibrils. The results of the nonseeded kinetics experiments show that Aβ40 aggregation is delayed with increasing GM1 concentration, while that of Aβ42 is accelerated. In seeded aggregation reactions, the addition of GM1 leads to a retardation of the aggregation process of both peptides. Thus, while the effect on nucleation differs between the two peptides, GM1 may inhibit the elongation of both types of fibrils. These results shed light on glycolipid-peptide interactions that may play an important role in Alzheimer's pathology.

Amyloid models for quantitative x‐ray brain amyloid imaging

AbstractBackgroundA range of human neurological disorders are related to amyloid fibrillar deposits including Alzheimer’s disease (AD). AD is linked to the presence of amyloid‐β (Aβ) fibrils and those are comprised of β‐sheet motifs. The precise mechanism through which Aβ leads to AD are not completely understood. The scarcity of human derived Aβ fibrils and high cost of recombinant Aβ fibrils further restrict the research in this area. However, intrinsically disordered proteins (IDPs) have been reported as amyloid model proteins. Even though, these models qualitatively mimic β‐sheet structure of Aβ fibrils but often fail to quantitatively match the β‐sheet content. Therefore, this study aimed to develop model proteins to closely mimic Aβ fibrils structure and produce them in a large quantity to evaluate emerging X‐ray methods to detect and quantify amyloid plaques.MethodsWe used a commercially available β‐lactoglobulin A and B genetic mixture (LGAB) protein as our amyloid model to mimic the structural properties of Aβ fibrils. Here, the alteration in the protein conformation was induced by altering the net charge on the polypeptide chain by changing the pH from 2 to 7. Additionally, we studied the fibrillization of these proteins in presence of more cooperative transitions at 90°C. The secondary structural compositions of LGAB were studied using attenuated total reflectance Fourier transform infrared (ATR‐FTIR) spectroscopy. The nanostructures of LGAB fibrils were characterized using small angle X‐ray scattering (SAXS). We further varied the mass fraction of LGAB fibrils (amyloid model) and quantitatively measured their amyloid content using a SAXS based method.ResultsATR‐FTIR (Fig.1) and SAXS (Fig.2) studies confirm that thermally aggregated LGAB fibrils exhibited parallel β‐sheet secondary structure similar to Aβ fibrils. Systematic variation in LGAB fibril concentration in the tissue mimicking environment showed a linear correlation between protein mass fraction and area under the curve of a peak (∼ 6 nm−1) corresponding to inter β‐sheet spacing (Fig.3).ConclusionOur study suggests that the amyloid model based on LGAB fibrils can likely be used to simulate Aβ fibrils in phantom studies for the evaluation of emerging X‐ray methods to detect and quantify amyloid plaques.

The Potential of Dendrimers as Alzheimer’s Disease Nanotherapeutics – Evidence of Antiamyloidogenic Properties Against Aβ42

AbstractBackgroundToday, available therapies for Alzheimer’s disease (AD) fail to modify the disease progression for the major AD clinical stages [1]. Therefore, new therapeutic approaches are an urgent need. Within nanomedicine, the use of nanomaterials, and dendrimers in particular, pose as promising AD therapies [2]. Dendrimers are globular macromolecules, with a well‐defined and hyperbranched structure, controllable nanosize (<15 nm) and multivalent surface. Due to their intrinsic characteristics, they are first‐in‐class for drug delivery as they can act as efficient and multivalent carriers of different therapeutic molecules. In addition, dendrimers can be used as a drug per se. Different types of dendrimers display antioxidant, anti‐inflammatory, anti‐prion and anti‐amyloidogenic properties [2]. With that in mind, a proprietary family of dendrimers was explored to hamper Aβ (1‐42) peptide aggregation.MethodThe dendrimer was synthesized following a divergent strategy up to generation 3 and characterized by nuclear magnetic resonance and Fourier‐transform infrared spectroscopy. To analyse the dendrimer’s effect on fibrillation, the aggregation kinetics of the Aβ (1‐42) peptide was studied by thioflavin T (ThT) in the presence of different dendrimer concentrations. The aggregates’ morphology was assessed by transmission electron microscopy. The cytotoxicity of dendrimers was evaluated in SH‐SY5Y cells by metabolic assays (resazurin).ResultThe dendrimers showed to interfere with Aβ fibrillation in a concentration‐dependent manner. Their behaviour was similar to fibrillation inhibitors that can break fibrils as well [3]. When at a lower molar ratio than Aβ, dendrimers hampered fibrillation and small/nonfibrillar aggregates were present. For a higher molar ratio (dendrimer/peptide ratio >1), they promoted Aβ fibrillation as more fibrils were present. More importantly, they were shown non‐toxic for neuroblastoma cells.ConclusionIn conclusion, these results show that these dendrimers can interfere with Aβ fibrillation. Future studies will be performed to understand the mechanism of action of this new family of dendrimers.

Single domain antibodies as promising tools for the selective detection and neutralization of neurotoxic Aβ42 oligomers in the cerebrospinal fluid of Alzheimer’s disease patients

AbstractBackgroundExtracellular amyloid β (Aβ) plaques and intracellular neurofibrillary tangles of the hyperphosphorylated tau protein are the main hallmarks of Alzheimer’s disease (AD). Small, soluble oligomers, rather than mature fibrils, are the major neurotoxic agents. The heterogeneous structures and the transient nature of these oligomers make their isolation and characterization very challenging. Single domain Abs (sdAbs), composed only of a variable domain of the heavy chain with high specificity and affinity, appear as promising tools for an early diagnosis and therapy for AD.MethodsPerforming an in vitro and in vivo screening of different sdAbs, we selected those targeting Aβ42 oligomers or fibrils with high specificity using dot‐blot, ELISA assay and the super resolution stimulated emission depletion (STED) microscopy. The potential of sdAbs was also investigated in preventing Aβ42 oligomer‐induced cytotoxicity in cultured neuronal cells. Then, the sdAbs were used to selectively detect Aβ42 species in the cerebrospinal fluid (CSF) of AD patients and control subjects and to neutralize their associated harmful effects following cell exposure to CSF samples.ResultssdAbs can selectively detect Aβ42 oligomers or fibrils both in vitro and in cultured cells, preventing the neurotoxicity induced by small oligomers. Moreover, sdAbs can significantly identify Aβ42 oligomers in the CSF of AD patients counteracting their induced toxicity in our cell models.ConclusionsAll these data provide a solid foundation for the development of sdAbs‐based immunodiagnostic tools that can selectively detect toxic aggregates in human body fluids for an early differential diagnosis of protein deposition diseases, such as AD. Furthermore, our results contribute to the improvement of the current therapeutic approaches against AD.The study was supported by Airalzh (AGYR 2020 to R.C.).

Discovery of high‐affinity amyloid ligands using a ligand‐based virtual screening pipeline

AbstractBackgroundA ligand‐based virtual screening pipeline was developed to discover structurally novel ligands for Aβ(1‐42) fibrils.MethodA database of ligands that bind to Aβ(1‐42) fibrils was used to develop a two‐step ligand‐based virtual screening pipeline. The first step involved developing machine learnings models that use relatively simple chemical descriptors to predict ligand dissociation constants (Kd ). The second step involved constructing 3D models based on field points that describe the surface, shape, and electronic properties of ligands. The combined pipeline was then used to screen 63 million compounds from the ZINC15 database.1 The most promising compounds were experimentally evaluated using binding assays.ResultFor the first chemical descriptor model, a support vector machine model was found to predict the ‐log(Kd /M) of Aβ(1‐42) fibril ligands with a mean absolute error of only 0.41. An ensemble of four 3D models were then developed that each predicted ‐log(Kd /M) with a cross‐validation regression coefficient of 0.48–0.56. The ZINC15 database compounds were screened through the first chemical descriptor model, and the 10,000 compounds with the highest predicted affinities were then screened through the 3D models. The 46 highest‐ranked ligands were then evaluated using Thioflavin T competition assays, resulting in five new Aβ(1‐42) ligands with novel structures that exhibited nanomolar binding.ConclusionA ligand‐based virtual screening pipeline has been developed to discover new fibril‐binding ligands. Using Aβ(1‐42) fibrils as a target, five new structurally diverse ligands were found that exhibit nanomolar binding affinities.1. Sterling and J. J. Irwin, J. Chem. Inf. Model. 2015, 55, 2324‐2337.

Influence of Epigallocatechin gallate stabilized Gold nanoparticles in amyloid beta fibril inhibition

AbstractBackgroundA pathological Hallmark of Alzheimer’s Disease (AD) is the significant deposition of amyloid beta (Aβ) plaques in the brain. Several studies were reported to inhibit the Aβ fibrillation using nanoparticles, nanoclusters and bioconjugate nanomaterials for Alzheimer’s treatment. In recent years, natural compound (phytochemicals) have been largely used to study and identified as promising agents for the prevention and treatment of neurodegenerative diseases including AD.MethodGold nanoparticle with appropriate surface conjugates with higher affinity has been studied and acknowledge the interaction between Bio‐Nano interfaces. The surface covering or anchoring conjugates will play a major role in vitro and in vivo. Herein, we reported Epigallocatechin gallate (EGCG) stabilized gold nanoparticle (AuNps) were synthesised as a Aβ inhibitor.ResultThe synthesised EGCG‐AuNps Characterized using UV‐Vis Spectroscopy, DLS, XRD, SEM (EDAX) and TEM (SAED) and the biocompatibility were studied by using MTT and scratch assay. The synthesised EGCG‐AuNPs inhibiting Aβ aggregation, dissociation Aβ fibrils which lead to prevent plaque formation, which confirmed using Thioflavin T‐assay and TEM studies.ConclusionThis work provides new insights into the synthesised AuNps as functional therapeutic agent for AD.

Correlations between [18F]flutemetamol PET and region‐matched postmortem integrated density measures of cyano‐flutemetamol signal in Aβ plaques

AbstractBackgroundThe binding substrates of [18F]flutemetamol and [11C]PiB PET consist of fibrillar amyloid‐β (Aβ) aggregates in morphologically and structurally distinct diffuse plaques (DP) and cored plaques (CP) which are present at different proportions across brain regions in Alzheimer’s disease (AD). To account for this heterogeneity, an unbiased quantitative measure integrating plaque size and density of amyloid‐β (Aβ) fibrils in plaques could help avoid false positive results, improve imaging‐to‐autopsy correlations, and further advance our understanding of pathological substrates for binding of amyloid PET tracers.MethodSixteen subjects underwent [18F]flutemetamol PET imaging in the pivotal Phase III end‐of‐life study (Study GE067‐007 and GE067‐026) and later came to autopsy (mean imaging‐to‐autopsy interval was 111 ± 87.6 days). [18F]flutemetamol SUVRpons values in four neocortical regions were correlated with region‐matched postmortem measures of cyano‐flutemetamol labeling of all plaques, as well as CP and DP separately, using percent area coverage, mean grayscale intensity of fluorescence, or a combination of the two measures referred to as integrated density.ResultIn the analysis of all cyano‐flutemetamol‐labeled plaques, [18F]flutemetamol SUVRpons values were more closely associated with integrated density measures than percent area coverage and mean grayscale values. Similarly, when CP and DP were analyzed separately, the integrated density values showed the closest association with neocortical region‐matched [18F]flutemetamol SUVRpons values. When area coverage of CP and DP were each expressed as a fraction of total plaque area coverage with or without integrating fluorescence intensity values, [18F]flutemetamol SUVRpons values within each region of interest were associated with the DP, but not the CP, area coverage measure.ConclusionThe association of antemortem [18F]flutemetamol PET and region‐matched postmortem integrated density measurements of cyano‐flutemetamol labeled plaques further supports the hypothesis that amyloid PET signal is influenced both by plaque size and Aβ fibril density within DP and CP. Further elucidating the relative contributions of each plaque type to the retention of amyloid PET radioligands could influence how a positive amyloid PET signal is interpreted in relation to postmortem pathology data.

Alzheimer’s Disease (AD): Vitamin B12 deficiency is associated with lower Mini‐Mental State Exam (MMSE) scores

AbstractBackgroundAlzheimer’s Disease (AD) is the leading cause of dementia in the elderly and several lines of evidence indicate that B12 hypovitaminosis is linked to AD. The biochemical pathways involved in AD that are affected by vitamin B12 are APP processing, Aβ fibrillization, Aβ‐induced oxidative damage as well as tau hyperphosphorylation and tau aggregation.Method103 outpatients with the diagnosis of AD conforming to criteria proposed by NINCDS‐ADRDA (2011), followed‐up at a public university hospital, had their vitamin B12 levels assessed and MMSE scores evaluated.Result91 patients (88,35%) presented vitamin B12 normal range (200 – 800 pg/mL) and 12 patients (11,65%) presented vitamin B12 deficiency (≤ 200 pg/mL). From the normal range group, 13 patients (14,29%) presented severe cognitive impairment (MMSE < 10), 33 patients (36,26%) presented moderate cognitive impairment (MMSE = 10 – 20) and 45 patients (49,45%) presented mild cognitive impairment (MMSE > 20). From the deficiency group, 2 patients (16,8%) presented severe cognitive impairment, 5 patients (41,6%) presented moderate cognitive impairment and 5 patients (41,6%) presented mild cognitive impairment. Both groups, vitamin B12 normal range and deficiency, had on average the same educational level: 2,91 and 3,42 years, respectively.ConclusionIn this sample of patients with AD, B12 hypovitaminosis is associated with lower MMSE scores when compared to vitamin B12 normal range.

Distribution of APP in endo‐lysosomal system induced by Aβ is modulated by Gβγ signaling

AbstractBackgroundAlzheimer´s disease is characterized by the deposition of aggregated species of amyloid beta (Aβ) in the brain, which leads to progressive cognitive deficits and dementia. We recently found that Aβ assemblies, oligomers and fibrils, increase APP and BACE1 interaction in recycling endosomes and accumulation of intracellular Aβ42 in human neurons derived from iPSCs by a mechanism dependent of APP/Go/Gβγ signaling (Antonino et al 2022, https://doi.org/10.3389/fcell.2022.852738). Now, we are interested on deepen in the APP trafficking in the endo‐lysosomal pathway in order to understand the mechanism underlying such effect.MethodN2a and human neurons derived from iPSc cultures were transfected with fluorescently tagged proteins (APP‐YFP, BACE‐CH, Rab11‐RFP, LAMP1‐CH, Rab7‐CH, APP‐VN, BACE‐VC). Cultures were treated with vehicle or the Gβγ inhibitor gallein and 30 minutes later with vehicle or 1 µM of Aβ oligomeric or fibrillar. After 24 h of treatment quantitative colocalization analysis and bimolecular fluorescence complementation assays were performed.ResultWe found that Aβ induced an enrichment of APP in recycling endosomes at the expense of a decrement of its levels in lysosomes. This change in APP intracellular distribution is drive by Gβγ signaling. Moreover, we found that the changes on APP distribution correlate with an increase in its interaction with BACE1 and with an increase in the number of recycling endosomes, both events modulated by Gβγ signaling (Figure 1,2).ConclusionTogether, these results suggest that Aβ pathological assemblies induce a re‐direction of APP from its physiological route to lysosomes, to recycling endosomes which favors its encounter with BACE1 and its amyloidogenic processing. These finding elucidate the intracellular process which sustain the feed‐forward mechanisms implicated in the amyloidogenesis induced by pathological assemblies of Aβ.

Safety, tolerability, pharmacokinetics and pharmacodynamics of a single intravenous dose of SHR‐1707 in healthy adult subjects: two randomized, double‐blind, phase 1 studies

AbstractBackgroundSHR‐1707 is a novel humanized anti‐Aβ IgG1 monoclonal antibody that binds to Aβ fibrils and monomers to block the formation of Aβ plaques and to promote the microglial phagocytosis of Aβ. Preclinical studies in 5xFAD transgenic mice showed that SHR‐1707 reduced brain Aβ deposition and ameliorated memory deficits. Herein, we conducted two phase 1 studies to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of a single intravenous dose of SHR‐1707 in healthy adult subjects.MethodTwo randomized, double‐blind, single‐ascending‐dose, phase 1 studies were conducted in China (Study CHN; NCT04973189) and Australia (Study AUS; NCT04745104). Study CHN consisted of 2 parts. In part 1, eligible healthy young adults (18‐45 years) were sequentially randomized 8:2 to receive SHR‐1707 (5 cohorts: 2, 6, 20, 40, and 60 mg/kg) or placebo in each cohort; in part 2, elderly subjects (55‐80 years) were randomized 8:4 to receive SHR‐1707 (20 mg/kg) or placebo. A similar design was used in Study AUS, but with only healthy young adults enrolled across 3 dosing cohorts (2, 20, and 60 mg/kg).Result62 (part 1/2, n = 50/12; mean age, 27.0/57.2 years) and 30 subjects (mean age, 26.1 years) were treated in Study CHN and Study AUS respectively. In Study CHN, across all dose levels, all TRAEs were mild in severity, with the most common being laboratory abnormalities. In Study AUS, TRAEs were mostly mild (1 moderate event each with SHR‐1707/placebo); the most common TRAEs with SHR‐1707 were dysgeusia and fatigue (8.3% each). In both studies, the exposure of SHR‐1707 increased in a slightly greater than dose‐proportional manner over the dose range of 2‐60 mg/kg in young adults; there was a dose‐dependent increase in plasma Aβ42 concentration following SHR‐1707 administration compared with the placebo group. The safety and PK/PD profiles of SHR‐1707 in the elderly subjects were consistent with the younger counterpart at the same dose level. No ethnic difference in safety and PK/PD of SHR‐1707 was observed.ConclusionA single intravenous dose of SHR‐1707 at 2‐60 mg/kg was safe and well tolerated in healthy young adult and elderly subjects. The PK/PD profiles are supportive of further clinical development.

A randomized, double‐blind, placebo‐controlled phase 1b study to evaluate the safety, tolerability, and pharmacodynamics of an anti‐amyloid‐β antibody SHR‐1707 in patients with mild cognitive impairment and mild Alzheimer’s disease

AbstractBackgroundSHR‐1707 is a humanized monoclonal antibody against amyloid‐β (Aβ). Pharmacodynamic studies in vitro showed that SHR‐1707 is more potent than lecanemab. SHR‐1707 had similar affinity to Aβ fibrils and protofibrils and relatively higher affinity to Aβ monomers than lecanemab. In 5xFAD transgenic mouse model of Alzheimer’s disease (AD), SHR‐1707 safely reduced brain Aβ deposition and ameliorated memory deficits. Phase 1 studies in China (NCT04973189) and Australia (NCT04745104) had demonstrated the safety and tolerability of SHR‐1707 in healthy young adults and elderly subjects at up to 60 mg/kg. The preclinical and clinical evidence support further development of SHR‐1707. Hence, we designed a phase 1b trial of SHR‐1707 to evaluate the safety, effective dosage, and effects on brain Aβ deposition in patients with mild cognitive impairment (MCI) due to AD or mild AD.MethodThis is a randomized, double‐blind, dose‐escalation, placebo‐controlled phase 1b study (NCT05681819; Figure 1). Patients with MCI due to AD or mild AD aged 55‐85 years will be randomized to placebo or intravenous SHR‐1707 at escalated doses of 5, 10, 20, and 40 mg/kg every 2 weeks for 26 weeks. Randomization will be based on the treatment to placebo ratio of 3:2 for 5 mg/kg dose group (N = 5, ≥3 ApoE ε4 carriers) and 10:2 for the remaining dose groups (for each group: N = 12, ≥5 ApoE ε4 carriers). In the following open‐label extension study, all patients will be assigned to SHR‐1707 for 52 weeks. The primary objective is to assess the safety and tolerability of 26‐week treatment of SHR‐1707. Secondary objectives are to assess effects on brain Aβ deposition by positron‐emission tomography, pharmacokinetic and pharmacodynamic profiles in blood and cerebrospinal fluid, immunogenicity, and clinical efficacy with 26‐week treatment of SHR‐1707; and long‐term safety, tolerability, and effects on brain Aβ deposition with 78‐week treatment. Figure 2 lists all study endpoints.ResultThe study is ongoing in China. Topline results are expected in Q1 2024.ConclusionThis trial will obtain safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity, and clinical efficacy data of SHR‐1707 to support advancement to confirmatory phase 2 or 3 studies in patients with MCI due to AD and mild AD.