Amyloid-β Fragment Research Articles

SKGQA, a Peptide Derived from the ANA/BTG3 Protein, Cleaves Amyloid-β with Proteolytic Activity.

Despite the extensive research conducted on Alzheimer's disease (AD) over the years, no effective drug for AD treatment has been found. Therefore, the development of new drugs for the treatment of AD is of the utmost importance. We recently reported the proteolytic activities of JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMA), synthetic peptides of nine amino acids each, derived from the Box A region of Tob1 and ANA/BTG3 proteins, respectively. Furthermore, two components of ANA-TA9, ANA-YA4 (YRMI) at the C-terminus end and ANA-SA5 (SKGQA) at the N-terminus end of ANA-TA9, exhibited proteolytic activity against amyloid-β (Aβ) fragment peptides. In this study, we identified the active center of ANA-SA5 using AEBSF, a serine protease inhibitor, and a peptide in which the Ser residue of ANA-SA5 was replaced with Leu. In addition, we demonstrate the proteolytic activity of ANA-SA5 against the soluble form Aβ42 (a-Aβ42) and solid insoluble form s-Aβ42. Furthermore, ANA-SA5 was not cytotoxic to A549 cells. These results indicate that ANA-SA5 is a promising Catalytide and a potential candidate for the development of new peptide drugs targeting Aβ42 for AD treatment.

Sulfonamido, amido heterocyclic adducts of tetrazole derivatives as BACE1 inhibitors: in silico exploration.

Alzheimer's disease is a neurodegenerative disorder accounting for 60-80% of dementia cases and is accompanied by a high mortality rate in patients above 70years of age. The formation of senile plaques composed of amyloid-β protein is a hallmark of Alzheimer's disease. Beta-site APP cleaving enzyme 1 (BACE1) is a proteolytic enzyme involved in the degradation of amyloid precursor protein, which further degrades to form toxic amyloid-β fragments. Hence, inhibition of BACE1 was stated to be an effective strategy for Alzheimer's therapeutics. Keeping in mind the structures of different BACE1 inhibitors that had reached the clinical trials, we designed a library of compounds (total 164) based on a substituted 5-amino tetrazole scaffold which was an isosteric replacement of the cyclic amidine moiety, a common component of the BACE1 inhibitors which reached the clinical trials. The scaffold was linked to different structural moieties with the aid of an amide or sulfonamide bond to design some novel molecules. Molecular docking was initially performed and the top 5 molecules were selected based on docking scores and protein-ligand interactions. Furthermore, molecular dynamic simulations were performed for these molecules (3g, 7k, 8n, 9d, 9g) for 100ns and MM-GBSA calculations were performed for each of these complexes. After critical evaluation of the obtained results, three potential molecules (9d, 8n, and 7k) were forwarded for prolonged stability studies by performing molecular dynamic simulations for 250ns and simultaneous MM-GBSA calculations. It was observed that the compounds (9d, 8n, and 7k) were forming good interactions with the amino acid residues of the catalytic site of the enzyme with multiple non-covalent interactions. In MD simulations, the compounds have shown better stability and better binding energy throughout the runtime.

Regulation of mitophagy and mitochondrial biogenesis by monocarbonyl analogues of curcumin in the cerebral cortex of rats in experimental Alzheimer's disease

To evaluate the effect of monocarbonyl analogues of curcumin on changes in the processes of mitophagy and mitochondrial biogenesis in the cerebral cortex of rats with experimental Alzheimer's disease. Alzheimer's disease was modeled in Wistar rats of both sexes by injection of β-amyloid fragments into the hippocampus of the animal. Compounds (1E, 4E)-1.5-bis (3.4.5-trimethoxyphenyl) penta-1.4-diene-3-one (AZBAX4 code) and (1E, 4E)-1.5-bis (2.4.6-trimethoxyphenyl) penta-1.4-diene-3-one (AZBAX6 code) at a dose of 20 mg/ kg (orally) and the reference drug donepezil at a dose of 50 mg/kg (orally) were administered for 30 days, after which changes in the activity of succinate dehydrogenase, cytochrome-c oxidase and citrate synthase as enzymatic biomarkers of mitochondrial biogenesis and mitophagy, respectively, were evaluated in the mitochondrial fraction of the cerebral cortex. The administration of AZBAX4 and AZBAX6 compounds led to an increase in the activity of succinate dehydrogenase; cytochrome-c oxidase, as well as citrate synthase in relation to the same indicators of the group of untreated animals. The use of the analyzed compounds was equally effective in both female and male rats. At the same time, it should be noted that the analyzed compounds significantly exceeded the activity level of the reference donepezil. AZBAX4 and AZBAX6 contribute to an increase in the intensity of mitochondrial biogenesis and mitophagy reactions in the cerebral cortex of rats with Alzheimer's disease, which makes them potentially effective neuroprotective compounds.

Multistep molecular mechanisms of Aβ16-22 fibril formation revealed by lattice Monte Carlo simulations.

As a model of self-assembly from disordered monomers to fibrils, the amyloid-β fragment Aβ16-22 was subject to past numerous experimental and computational studies. Because dynamics information between milliseconds and seconds cannot be assessed by both studies, we lack a full understanding of its oligomerization. Lattice simulations are particularly well suited to capture pathways to fibrils. In this study, we explored the aggregation of 10 Aβ16-22 peptides using 65 lattice Monte Carlo simulations, each simulation consisting of 3 × 109 steps. Based on a total of 24 and 41 simulations that converge and do not converge to the fibril state, respectively, we are able to reveal the diversity of the pathways leading to fibril structure and the conformational traps slowing down the fibril formation.

The Role of MARK ERK1/2 and p38 in Regulation of Functions of Neural Stem Cells and Neuroglia under Conditions of β-Amyloid-Induced Neurodegeneration.

The role of ERK1/2 and p38 in the realization of the growth potential of neural stem cells and secretion of neurotrophic growth factors by glial cells was studied using in vitro model of β-amyloid-induced neurodegeneration. It was shown that amyloid-β fragment 25-35 significantly inhibits the cell cycle progression of neural stem cells against the background of stimulation of their differentiation and reduced production of growth factors by neuroglia. The inhibitory role of ERK1/2 and p38 in relation to the proliferative activity of neural stem cells and the secretory activity of glial elements was revealed. ERK1/2 and p38 inhibitors increased proliferation of progenitor cells of the nervous tissue and reduced the intensity of their specialization, as well as stimulated production of growth factors by neuroglial cells under conditions of simulated β-amyloid-induced neurodegeneration.

Linking Plasma Amyloid Beta and Neurofilament Light Chain to Intracortical Myelin Content in Cognitively Normal Older Adults

Evidence suggests that lightly myelinated cortical regions are vulnerable to aging and Alzheimer’s disease (AD). However, it remains unknown whether plasma markers of amyloid and neurodegeneration are related to deficits in intracortical myelin content, and whether this relationship, in turn, is associated with altered patterns of resting-state functional connectivity (rs-FC). To shed light into these questions, plasma levels of amyloid-β fragment 1–42 (Aβ1–42) and neurofilament light chain (NfL) were measured using ultra-sensitive single-molecule array (Simoa) assays, and the intracortical myelin content was estimated with the ratio T1-weigthed/T2-weighted (T1w/T2w) in 133 cognitively normal older adults. We assessed: (i) whether plasma Aβ1–42 and/or NfL levels were associated with intracortical myelin content at different cortical depths and (ii) whether cortical regions showing myelin reductions also exhibited altered rs-FC patterns. Surface-based multiple regression analyses revealed that lower plasma Aβ1–42 and higher plasma NfL were associated with lower myelin content in temporo-parietal-occipital regions and the insular cortex, respectively. Whereas the association with Aβ1–42 decreased with depth, the NfL-myelin relationship was most evident in the innermost layer. Older individuals with higher plasma NfL levels also exhibited altered rs-FC between the insula and medial orbitofrontal cortex. Together, these findings establish a link between plasma markers of amyloid/neurodegeneration and intracortical myelin content in cognitively normal older adults, and support the role of plasma NfL in boosting aberrant FC patterns of the insular cortex, a central brain hub highly vulnerable to aging and neurodegeneration.

Plasma biomarkers and genetics in the diagnosis and prediction of Alzheimer's disease.

Plasma biomarkers for Alzheimer's disease-related pathologies have undergone rapid developments during the past few years, and there are now well-validated blood tests for amyloid and tau pathology, as well as neurodegeneration and astrocytic activation. To define Alzheimer's disease with biomarkers rather than clinical assessment, we assessed prediction of research-diagnosed disease status using these biomarkers and tested genetic variants associated with the biomarkers that may reflect more accurately the risk of biochemically defined Alzheimer's disease instead of the risk of dementia. In a cohort of Alzheimer's disease cases [n = 1439, mean age 68 years (standard deviation = 8.2)] and screened controls [n = 508, mean age 82 years (standard deviation = 6.8)], we measured plasma concentrations of the 40 and 42 amino acid-long amyloid-β (Aβ) fragments (Aβ40 and Aβ42, respectively), tau phosphorylated at amino acid 181 (P-tau181), neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) using state-of-the-art Single molecule array (Simoa) technology. We tested the relationships between the biomarkers and Alzheimer's disease genetic risk, age at onset and disease duration. We also conducted a genome-wide association study for association of disease risk genes with these biomarkers. The prediction accuracy of Alzheimer's disease clinical diagnosis by the combination of all biomarkers, APOE and polygenic risk score reached area under receiver operating characteristic curve (AUC) = 0.81, with the most significant contributors being ε4, Aβ40 or Aβ42, GFAP and NfL. All biomarkers were significantly associated with age in cases and controls (P < 4.3 × 10-5). Concentrations of the Aβ-related biomarkers in plasma were significantly lower in cases compared with controls, whereas other biomarker levels were significantly higher in cases. In the case-control genome-wide analyses, APOE-ε4 was associated with all biomarkers (P = 0.011-4.78 × 10-8), except NfL. No novel genome-wide significant single nucleotide polymorphisms were found in the case-control design; however, in a case-only analysis, we found two independent genome-wide significant associations between the Aβ42/Aβ40 ratio and WWOX and COPG2 genes. Disease prediction modelling by the combination of all biomarkers indicates that the variance attributed to P-tau181 is mostly captured by APOE-ε4, whereas Aβ40, Aβ42, GFAP and NfL biomarkers explain additional variation over and above APOE. We identified novel plausible genome wide-significant genes associated with Aβ42/Aβ40 ratio in a sample which is 50 times smaller than current genome-wide association studies in Alzheimer's disease.

Effect of amyloid-β on the redox system activity in SH-SY5Y cells preincubated with lipopolysaccharide or co-cultured with microglia cells

Amyloid deposits and hyperphosphorylation of the tau protein are still believed to be the two main causes of Alzheimer’s disease. However, newer studies show the beneficial (including antiradical and antimicrobial) effects of amyloid at physiological concentrations. Therefore, this study aimed to investigate the impact of three amyloid fragments – 25–35, 1–40, and 1–42 at concentrations close to physiological levels on the oxidative stress induced by the administration of lipopolysaccharide (LPS) or co-culturing with microglia cells. Differentiated SH-SY5Y cells were used, constituting a model of neuronal cells that were preincubated with LPS or supernatant collected from THP-1 cell culture. The cells were treated with amyloid-β fragments at concentrations of 0.001, 0.1, and 1.0 µM, and then biological assays were carried out. The results of the study support the antioxidant properties of Aβ, which may protect neurons from the damaging effects of neuroinflammation. All tested amyloid-β fragments reduced oxidative stress and increased the levels of enzymatic stress parameters – the activity of SOD, GPx and catalase. In addition, the administration of amyloid-β at low physiological concentrations also increased reduced glutathione (GSH) levels and the ratio between reduced and oxidized glutathione (GSH/GSSG), which is considered a good indicator of maintaining cellular redox balance. Furthermore, a stronger antioxidant effect of 1–40 fragment was observed, occurring in a wider range of concentrations, compared to the other tested fragments 25–35 and 1–42.

Polymorphism Rs2421943 of the Insulin-Degrading Enzyme Gene and the Risk of Late-Onset Alzheimer's Disease.

Insulin-degrading enzyme (IDE) is a widely distributed Zn2+-binding metalloprotease that cleaves multiple short and medium-sized peptides prone to form β-structures. These include insulin and amyloid-β peptides. Accumulation and fibrillation of amyloid-β peptides leading to the formation of amyloid plaques is a characteristic sign of Alzheimer's disease (AD) pathology. The study investigated the rs2421943 single nucleotide polymorphism (SNP) of the IDE gene as a risk factor for MCI (mild cognitive impairment) and AD. Two independent groups of 1670 patients and controls were included. The AD group consisted of 595 patients and 400 controls; the MCI group involved 135 patients and 540 matched controls. PCR and restriction fragment length analysis were used to analyze the rs2421943 polymorphism. Using the miRBase and RNA22 prediction tools in silico indicated that the rs2421943 polymorphism is a potential target for a specific miRNA (hsa-miR-7110-5p). AG and GG genotypes of rs2421943 significantly increased the risk of AD, and the AG genotype increased the risk of MCI. It seems the G allele both increases the risk of AD and accelerates the transition through the MCI phase. In silico study revealed that rs2421943 is inside the sequence binding miRNA hsa-miR-7110-5p. The polymorphism could affect the rate of IDE pre-RNA (heterogeneous nuclear RNA, hnRNA) processing, resulting in slower translation, lower levels of IDE, deficient removal of amyloid-β fragments, and greater risk of and/or accelerated progression of AD. GG and AG genotypes of the single nucleotide polymorphism rs2421943 of insulindegrading enzyme gene increase the risk of AD and MCI.

Replica permutation with solute tempering for molecular dynamics simulation and its application to the dimerization of amyloid-β fragments.

We propose the replica permutation with solute tempering (RPST) by combining the replica-permutation method (RPM) and the replica exchange with solute tempering (REST). Temperature permutations are performed among more than two replicas in RPM, whereas temperature exchanges are performed between two replicas in the replica-exchange method (REM). The temperature transition in RPM occurs more efficiently than in REM. In REST, only the temperatures of the solute region, the solute temperatures, are exchanged to reduce the number of replicas compared to REM. Therefore, RPST is expected to be an improved method taking advantage of these methods. For comparison, we applied RPST, REST, RPM, and REM to two amyloid-β(16-22) peptides in explicit water. We calculated the transition ratio and the number of tunneling events in the temperature space and the number of dimerization events of amyloid-β(16-22) peptides. The results indicate that, in RPST, the number of replicas necessary for frequent random walks in the temperature and conformational spaces is reduced compared to the other three methods. In addition, we focused on the dimerization process of amyloid-β(16-22) peptides. The RPST simulation with a relatively small number of replicas shows that the two amyloid-β(16-22) peptides form the intermolecular antiparallel β-bridges due to the hydrophilic side-chain contact between Lys and Glu and hydrophobic side-chain contact between Leu, Val, and Phe, which stabilizes the dimer of the peptides.

Modulation of γ-Secretase Activity by a Carborane-Based Flurbiprofen Analogue.

All over the world, societies are facing rapidly aging populations combined with a growing number of patients suffering from Alzheimer’s disease (AD). One focus in pharmaceutical research to address this issue is on the reduction of the longer amyloid-β (Aβ) fragments in the brain by modulation of γ-secretase, a membrane-bound protease. R-Flurbiprofen (tarenflurbil) was studied in this regard but failed to show significant improvement in AD patients in a phase 3 clinical trial. This was mainly attributed to its low ability to cross the blood–brain barrier (BBB). Here, we present the synthesis and in vitro evaluation of a racemic meta-carborane analogue of flurbiprofen. By introducing the carborane moiety, the hydrophobicity could be shifted into a more favourable range for the penetration of the blood–brain barrier, evident by a logD7.4 value of 2.0. Furthermore, our analogue retained γ-secretase modulator activity in comparison to racemic flurbiprofen in a cell-based assay. These findings demonstrate the potential of carboranes as phenyl mimetics also in AD research.

Protective effects of ginsenoside Rg2 against memory impairment and neuronal death induced by Aβ25-35 in rats

Ethnopharmacological relevanceGinsenoside Rg2 is an important ingredient of Panax ginseng which often appears in ancient prescriptions for forgetfulness. Ginsenoside Rg2 exert neuroprotective effects and could be a new potential medicine to treat AD. In our previous study, we reported that ginsenoside Rg2 appears protect PC12 cells against Amyloid β-fragment (25–35) (Aβ25-35)-induced apoptosis via the PI3K/Akt pathway. However, there are no in vivo studies on the protective effects of ginsenoside Rg2 on Aβ-induced neurotoxicity. Aim of the studyThe present study was performed to investigate the protective effects of ginsenoside Rg2 against Aβ25-35-induced memory impairment, and its underlying mechanisms in rats. Materials and methodsAn Alzheimer's Disease (AD) rat model was established by injecting the rats with Aβ25-35 (1 μg/μl). Cognitive performance was evaluated by the Morris Water Maze test (MWM). The brain sections were processed and neuronal apoptosis in the hippocampus was evaluated by Hematoxylin and Eosin staining (H&E). To explore the anti-neuronal apoptosis mechanism of ginsenoside Rg2, we analyzed the protein expression of Bcl-2/Bax, caspase-3, and phospho-protein kinase B/protein kinase B (p-Akt/Akt) via western blot. ResultsA significant improvement in cognitive function was observed in administrated ginsenoside Rg2 AD rats. The histological injury in hippocampus CA1 induced by Aβ25-35 was inhibited following administration of the ginsenoside Rg2. Ginsenoside Rg2 increase the Bcl-2/Bax ratio, attenuate the cleavage of caspase-3, and enhance the phosphorylation of Akt. ConclusionsThese findings suggest that ginsenoside Rg2 could ameliorate Aβ25-35-induced cognitive dysfunction by activating the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway.

Real-time in-situ 1H NMR of reactions in peptide solution: preaggregation of amyloid-β fragments prior to fibril formation

Abstract In-situ analytical methods are essential for the reliable observation of peptide reactions without perturbation of the system. In this work, a real-time in-situ NMR analysis was performed to gain insight into the initial stage of the aggregation of amyloid-beta (Aβ) 8–25 monomers, S8GY10EVHHQKLVFF20AEDVG25, in solution prior to the fibril formation. NMR chemical shift and intensity changes in combination with the CD spectra revealed no changes in Aβ secondary structure, but the presence of soluble, oligomeric intermediates followed by the appearance of insoluble and non-structured aggregates before β-fibril formation. Molecular views of intermediates and aggregation mechanisms were proposed in comparison with NMR spectral changes in wild-type Aβ 8–25 and its two mutants, A21G and E22G. The mutation of just one amino acid modified the aggregation properties of Aβ 8–25; it slowed or accelerated the fibril formation by controlling the progress of conversion from monomer to aggregate via a soluble, small oligomer.

Dysfunction of the Blood-Brain Barrier-A Key Step in Neurodegeneration and Dementia.

The vascular endothelium in the brain is an essential part of the blood-brain-barrier (BBB) because of its very tight structure to secure a functional and molecular separation of the brain from the rest of the body and to protect neurons from pathogens and toxins. Impaired transport of metabolites across the BBB due to its increasing dysfunction affects brain health and cognitive functioning, thus providing a starting point of neurodegenerative diseases. The term “cerebral metabolic syndrome” is proposed to highlight the importance of lifestyle factors in neurodegeneration and to describe the impact of increasing BBB dysfunction on neurodegeneration and dementia, especially in elderly patients. If untreated, the cerebral metabolic syndrome may evolve into dementia. Due to the high energy demand of the brain, impaired glucose transport across the BBB via glucose transporters as GLUT1 renders the brain increasingly susceptible to neurodegeneration. Apoptotic processes are further supported by the lack of essential metabolites of the phosphocholine synthesis. In Alzheimer’s disease (AD), inflammatory and infectious processes at the BBB increase the dysfunction and might be pace-making events. At this point, the potentially highly relevant role of the thrombocytic amyloid precursor protein (APP) in endothelial inflammation of the BBB is discussed. Chronic inflammatory processes of the BBB transmitted to an increasing number of brain areas might cause a lasting build-up of spreading, pore-forming β-amyloid fragments explaining the dramatic progression of the disease. In the view of the essential requirement of an early diagnosis to investigate and implement causal therapeutic strategies against dementia, brain imaging methods are of great importance. Therefore, status and opportunities in the field of diagnostic imaging of the living human brain will be portrayed, comprising diverse techniques such as positron emissions tomography (PET) and functional magnetic resonance imaging (fMRI) to uncover the patterns of atrophy, protein deposits, hypometabolism, and molecular as well as functional alterations in AD.

Structure Analysis of Amyloid Aggregates at Lipid Bilayers by Supercritical Angle Raman Microscopy.

The amyloid-β peptide is correlated with Alzheimer's disease and is assumed to cause toxicity by its interaction with the neuron membrane. A custom-made microscope objective based on the supercritical angle technique was developed by our group, which allows investigation of interfacial events by performing surface-sensitive and low-invasive spectroscopy. Applied to Raman spectroscopy, this technique was used to collect information about the structure of polypeptides that interact with a supported lipid bilayer. Notably, the conformation used by amyloid-β(1-40) and amyloid-β(1-42) when interacting directly with or next to the supported lipid bilayer was characterized. We observed two distinct secondary structures, α-helix and β-sheet, which were exhibited by the peptide. These two structures were detected simultaneously. The propensity of the peptide to fold into these structures seemed dependent on both their number of amino acids and their proximity with the supported lipid bilayer. The α-helix structure was observed for amyloid-β(1-42) fragments that were closer to the lipid bilayer. Peptides that were located further away from the bilayer favored the β-sheet structure. Amyloid-β(1-40) was less prone to adopt the α-helix secondary structure.

The Ubiquitin System in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia, most prevalent in the elderly population and has a significant impact on individuals and their family as well as the health care system and the economy. While the number of patients affected by various forms of dementia including AD is on the increase, there is currently no cure. Although genome-wide association studies have identified genetic markers for familial AD, the molecular mechanisms underlying the initiation and development of both familial and sporadic AD remain poorly understood. Most neurodegenerative diseases and in particular those associated with dementia have been defined as proteinopathies due to the presence of intra- and/or extracellular protein aggregates in the brain of affected individuals. Although loss of proteostasis in AD has been known for decades, it is only in recent years that we have come to appreciate the role of ubiquitin-dependent mechanisms in brain homeostasis and in brain diseases. Ubiquitin is a highly versatile post-translational modification which regulates many aspects of protein fate and function, including protein degradation by the Ubiquitin-Proteasome System (UPS), autophagy-mediated removal of damaged organelles and proteins, lysosomal turnover of membrane proteins and of extracellular molecules brought inside the cell through endocytosis. Amyloid-β (Aβ) fragments as well as hyperphosphorylation of Tau are hallmarks of AD, and these are found in extracellular plaques and intracellular fibrils in the brain of individuals with AD, respectively. Yet, whether it is the oligomeric or the soluble species of Aβ and Tau that mediate toxicity is still unclear. These proteins impact on mitochondrial energy metabolism, inflammation, as well as a number of housekeeping processes including protein degradation through the UPS and autophagy. In this chapter, we will discuss the role of ubiquitin in neuronal homeostasis as well as in AD; summarise crosstalks between the enzymes that regulate protein ubiquitination and the toxic proteins Tau and Aβ; highlight emerging molecular mechanisms in AD as well as future strategies which aim to exploit the ubiquitin system as a source for next-generation therapeutics.

Activity Rhythms Are Largely Intact in APPNL-G-F Alzheimer's Disease Mice.

Circadian rhythm dysfunction is present in Alzheimer's disease. Animal models of Alzheimer's disease have been employed to investigate whether this dysfunction is a risk factor or symptom of the disease. The circadian phenotype in mouse models of Alzheimer's disease is very disparate in terms of the degree and timing of the dysfunction. This is likely a result of some models elevating amyloid-β protein precursor instead of just the amyloid-β fragment present in human Alzheimer's disease. We characterized activity rhythms in a novel knock-in mouse model (APPNL-G-F) of Alzheimer's disease that elevates amyloid-β without overexpressing amyloid-β protein precursor. Despite increased rhythm amplitude, total activity, and a shortening of free-running period at 15 months of age, all other aspects of the activity rhythm were similar to controls from three to fifteen months of age. At two months of age, these mice were also able to entrain to a light-dark cycle with a period right on the edge of entrainment, which further suggests a healthy functioning circadian system. These data open the possibility that circadian rhythm disruptions in transgenic models of Alzheimer's disease could be a result of these models having an artificial phenotype caused by overexpression of amyloid-β protein precursor.

Catalytides derived from the Box A region in the ANA/BTG3 protein cleave amyloid-β fragment peptide

We have recently reported about shorter proteolytic peptides termed Catalytide as general name. JAL-TA9 (YKGSGFRMI), a fragment peptide derived from Box A region of Tob1 protein, is the first Catalytide and cleaves Aβ42 and its fragment peptides.Herein, we demonstrate the enzymatic properties of ANA-TA9 corresponding region to JAL-TA9 in ANA/BTG3 protein. ANA-TA9 showed the auto-proteolytic activity and cleaved 3 kinds of synthetic fragment peptides derived from Aβ42, especially on the central region of Aβ42 with a serine protease like activity. Interestingly, 2 kinds of components, ANA-SA5 (SKGQA) and ANA-YA4 (YRMI), also showed similar proteolytic activity. These results indicate that ANA-TA9 is composed of two different Catalytides.

Identification of a BACE1 Binding Peptide Candidate for the Prevention of Amyloid Beta in Alzheimer’s Disease

Amyloid plaques, generated during the progression of Alzheimer's disease, cause major neurological deficits due to substantial cell toxicity and death. The underlying cause of plaque generation stems from cleavage of the amyloid precursor protein (APP) by β-secretase (BACE1). A resulting amyloid-β (Aβ) fragment forms aggregates to produce the main constituent of a plaque. Phage display and biopanning techniques were used to identify a 12-mer peptide that had a natural affinity for the BACE1 enzyme. The peptide was translated from phage DNA and synthetically produced. The peptide, at concentrations of 1nM, 10nM and 100nM, was used to confirm binding by direct assay. Non-specific binding to BACE2, renin and cathepsin D was tested by direct binding assay. A BACE1 activity assay was used to determine the peptide effect on cleavage of an APP substrate. Treatment of SY5Y cells with the peptide was used to determine toxicity and prevention of Aβ40 and Aβ42 production. After identification and synthetic production, the peptide exhibited a strong affinity for BACE1 at nanomolar concentrations in the direct assay. In case of non-specific binding to homologous BACE2, renin and cathepsin D, the peptide showed minor binding but was nullified when in solution with BACE1. The peptide addition to a BACE1 activity assay was able to significantly reduce the amount of substrate cleavage. SY5Y cells, when treated with the peptide, did not show any detrimental morphological changes while being able to reduce the production of natural Aβ40 and Aβ42. Even under stressed conditions (H2O2 treatment) where the Aβ production was higher, the peptide was still able to significantly reduce the effect of BACE1 while not effecting cell viability. The identified peptide exhibited strong binding to BACE1 in vitro and was able to reduce production of Aβ, suggesting a favourable BACE1 inhibitor for future refining and characterisation.

Fabrication of Amyloid-β-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease.

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer's disease (AD) is the accumulation of toxic amyloid-β (Aβ) fragments, eventually leading to the classical features of the disease: amyloid plaques, neurofibrillary tangles and synaptic and neuronal loss. The lack of relevant non-transgenic preclinical models reflective of disease progression is one of the main factors hindering the discovery of effective drug treatments. To this end, we have developed a protocol for the fabrication of alginate microbeads containing amyloid-secreting cells useful for the study of the effects of chronic Aβ production. Chinese hamster ovary cells previously transfected with a human APP gene, secreting Aβ (i.e., 7PA2 cells), were used in this study. A three-dimensional (3D) in vitro model for the sustained release of Aβ was fabricated by encapsulation of 7PA2 cells in alginate. The process was optimized to target a bead diameter of 500-600 μm for further in vivo studies. Optimization of 7PA2 cell encapsulation in alginate was performed altering fabrication parameters, e.g., alginate concentration, gel flow rate, electrostatic potential, head vibration frequency, gelling solution. Levels of secreted Aβ were analyzed over time and compared between alginate beads and standard cell culture methods (up to 96 h). A concentration of 1.5 x 106 7PA2 cells/mL and an alginate concentration of 2% (w/v) buffered with HEPES and subsequent gelation in 0.5 M calcium chloride for 5 min were found to fabricate the most stable microbeads. Fabricated microbeads were 1) of uniform size, 2) with an average diameter of 550 μm, 3) containing about 100-150 cells per microbead and 4) able to secrete Aβ. In conclusion, our optimized method for the production of stable alginate microbeads containing amyloid-producing 7PA2 cells might enable the modeling of important aspects of AD both in vitro and in vivo.