Synthetic Aβ Peptides Research Articles

Inhibition of BACE1 affected both its Aβ producing and degrading activities and increased Aβ42 and Aβ40 levels at high-level BACE1 expression

The beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the predominant β-secretase, cleaving the amyloid precursor protein (APP) via the amyloidogenic pathway. In addition, BACE1 as an amyloid degrading enzyme (ADE), cleaves Aβ to produce the C-terminally truncated non-toxic Aβ fragment Aβ34 which is an indicator of amyloid clearance.Here, we analyzed effects of BACE1 inhibitors on its opposing enzymatic functions, i.e., amyloidogenic (Aβ producing) and amyloidolytic (Aβ degrading) activities, using cell culture models with varying BACE1/APP ratios. Under high level BACE1 expression, low-dose inhibition unexpectedly yielded a two-fold increase in Aβ42 and Aβ40 levels. The concomitant decrease in Aβ34 and secreted APPβ levels suggested that the elevated Aβ42 and Aβ40 levels were due to the attenuated Aβ degrading activity of BACE1. Notably, the amyloidolytic activity of BACE1 was impeded at lower BACE1 inhibitor concentrations compared to its amyloidogenic activity, thereby suggesting that the Aβ degrading activity of BACE1 was more sensitive to inhibition than its Aβ producing activity. Under endogenous BACE1 and APP levels, “low-dose” BACE1 inhibition affected both the Aβ producing and degrading activities of BACE1, i.e., significantly increased Aβ42/Aβ40 ratio and decreased Aβ34 levels, respectively. Further, we incubated recombinant BACE1 with synthetic Aβ peptides and found that BACE1 has higher affinity for Aβ substrates over APP. In summary, our results suggest that stimulating BACE1’s ADE activity and halting Aβ production without decreasing Aβ clearance could still be a promising therapeutic approach with new, yet to be developed, BACE1 modulators.

Structural polymorphism and cytotoxicity of brain-derived β-amyloid extracts.

To date, more than 37 amyloidogenic proteins have been found to form toxic aggregates that are implicated in the progression of numerous debilitating protein misfolding diseases including Alzheimer's disease (AD). Extensive literature highlights the role of β-amyloid (Aβ) aggregates in causing excessive neuronal cell loss in the brains of AD patients. In fact, major advances in our understanding of Aβ aggregation process, including kinetics, toxicity, and structures of fibrillar aggregates have been revealed by examining in vitro preparations of synthetic Aβ peptides. However, ongoing research shows that brain-derived Aβ aggregates have specific characteristics that distinguish them from in vitro prepared species. Notably, the molecular structures of amyloid fibrils grown in the human brain were found to be markedly different than synthetic Aβ fibrils. In addition, recent findings report the existence of heterogeneous Aβ proteoforms in AD brain tissue in contrast to synthetically produced full-length aggregates. Despite their high relevance to AD progression, brain-derived Aβ species are less well-characterized compared with synthetic aggregates. The aim of this review is to provide an overview of the literature on brain-derived Aβ aggregates with particular focus on recent studies that report their structures as well as pathological roles in AD progression. The main motivation of this review is to highlight the importance of utilizing brain-derived amyloids for characterizing the structural and toxic effects of amyloid species. With this knowledge, brain-derived aggregates can be adopted to identify more relevant drug targets and validate potent aggregation inhibitors toward designing highly effective therapeutic strategies against AD.

Methods for the isolation and analysis of Aβ from postmortem brain.

Amyloid β-protein (Aβ) plays an initiating role in Alzheimer's disease (AD), but only a small number of groups have studied Aβ extracted from human brain. Most prior studies have utilized synthetic Aβ peptides, but the relevance of these test tube experiments to the conditions that prevail in AD is uncertain. Here, we describe three distinct methods for studying Aβ from cortical tissue. Each method allows the analysis of different ranges of species thus enabling the examination of different questions. The first method allows the study of readily diffusible Aβ with a relatively high specific activity. The second enables the analysis of readily solubilized forms of Aβ the majority of which are inactive. The third details the isolation of true Aβ dimers which have disease-related activity. We also describe a bioassay to study the effects of Aβ on the neuritic integrity of iPSC-derived human neurons. The combined use of this bioassay and the described extraction procedures provides a platform to investigate the activity of different forms and mixtures of Aβ species, and offers a tractable system to identify strategies to mitigate Aβ mediated neurotoxicity.

Electrochemical Analysis in Studying β-Amyloid Aggregation.

β-amyloid (Aβ) is comprised of a group of peptides formed as a result of cleavage of the amyloid precursor protein by secretases. Aβ aggregation is considered as a central event in pathogenesis of Alzheimer's disease, the most common human neurodegenerative disorder. Molecular mechanisms of Aβ aggregation have intensively being investigated using synthetic Aβ peptides by methods based on monitoring of aggregates, including determination of their size and structure. In this review, an orthogonal approach to the study of Aβ aggregation is considered, which relies on electrochemical registration of the loss of peptide monomers. Electrochemical analysis of Aβ (by voltammetry and amperometric flow injection analysis) is based on registration of the oxidation signal of electroactive amino acid residues of the peptide on an electrode surface. The Aβ oxidation signal disappears, when the peptide is included in the aggregate. The advantages and disadvantages of electrochemical analysis for the study of spontaneous and metal-induced aggregation of Aβ, comparative analysis of various peptide isoforms, and study of the process of complexation of metal ions with the metal-binding domain of Aβ are discussed. It is concluded that the combined use of the electrochemical method and the methods based on detection of Aβ aggregates makes it possible to obtain more complete information about the mechanisms of peptide aggregation.

Blood–brain barrier penetrating neprilysin degrades monomeric amyloid-beta in a mouse model of Alzheimer’s disease

BackgroundAggregation of the amyloid-β (Aβ) peptide in the brain is one of the key pathological events in Alzheimer’s disease (AD). Reducing Aβ levels in the brain by enhancing its degradation is one possible strategy to develop new therapies for AD. Neprilysin (NEP) is a membrane-bound metallopeptidase and one of the major Aβ-degrading enzymes. The secreted soluble form of NEP (sNEP) has been previously suggested as a potential protein-therapy degrading Aβ in AD. However, similar to other large molecules, peripherally administered sNEP is unable to reach the brain due to the presence of the blood–brain barrier (BBB).MethodsTo provide transcytosis across the BBB, we recombinantly fused the TfR binding moiety (scFv8D3) to either sNEP or a previously described variant of NEP (muNEP) suggested to have higher degradation efficiency of Aβ compared to other NEP substrates, but not per se to degrade Aβ more efficiently. To provide long blood half-life, an Fc-based antibody fragment (scFc) was added to the designs, forming sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3. The ability of the mentioned recombinant proteins to degrade Aβ was first evaluated in vitro using synthetic Aβ peptides followed by sandwich ELISA. For the in vivo studies, a single injection of 125-iodine-labelled sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3 was intravenously administered to a tg-ArcSwe mouse model of AD, using scFc-scFv8D3 protein that lacks NEP as a negative control. Different ELISA setups were applied to quantify Aβ concentration of different conformations, both in brain tissues and blood samples.ResultsWhen tested in vitro, sNEP-scFc-scFv8D3 retained sNEP enzymatic activity in degrading Aβ and both constructs efficiently degraded arctic Aβ. When intravenously injected, sNEP-scFc-scFv8D3 demonstrated 20 times higher brain uptake compared to sNEP. Both scFv8D3-fused NEP proteins significantly reduced aggregated Aβ levels in the blood of tg-ArcSwe mice, a transgenic mouse model of AD, following a single intravenous injection. In the brain, monomeric and oligomeric Aβ were significantly reduced. Both scFv8D3-fused NEP proteins displayed a fast clearance from the brain.ConclusionA one-time injection of a BBB-penetrating NEP shows the potential to reduce, the likely most toxic, Aβ oligomers in the brain in addition to monomers. Also, Aβ aggregates in the blood were reduced.

Role of Fragile X Mental Retardation protein in Amyloid Beta 1‐42 Induced Translational Suppression

AbstractBackgroundAlzheimer’s Disease (AD) is the most common cause of dementia where the accumulation of the peptide Amyloid‐Beta (Aβ) is the main theory linked to the pathogenesis of the disease. Fragile X Mental Retardation Protein (FMRP) is an RNA‐binding protein that functions to suppress global translation. Because Aβ is known to be a toxic peptide capable of inducing the Endoplasmic Reticulum (ER) stress response and because augmented translational suppression is one of the main molecular phenotypes in ER stress response, we ask whether and how FMRP is involved in Aß induced translational suppression.MethodPrimary mouse cortical neuronal cultures from wild type (WT) or Fmr1 KO pups were treated with Aβ (Aβ1–42) during 24 hours. For protein expression identification and quantification we used western blot and ImageJ software respectively. Protein translation was assessed by inhibition with puromycin. Neuronal networking assessment was made with multielectrode array system (MEA).ResultIn this study, we observed that treatments of synthetic Aβ peptides in culture primary mouse cortical neurons induces the expression of FMRP and FMRP‐dependent ER stress response and subsequent translational suppression. Also Aβ induces FMRP‐dependent neuronal hyposynchronicity . We further characterized that two translational regulators, eukaryotic translation initiation factor 2α (eIF2α) and eukaryotic translation elongation factor 2 (eEF2), are involved in Aβ induced translational suppression and are dysregulated in the absence of FMRP. Mechanistically we found that FMRP is responsible for regulating the levels of Protein Phosphatase 2 (PP2A) and Protein Phosphatase 1 (PP1) that differentially modulate the phosphorylation status and thereby activity of eIF2α and eEF2 following treatment of Aβ. Using a pharmacological approach to inhibit PP2A and PP1 with Okadaic Acid, we were able to rescue the impairment of eIF2α and eEF2 as well as the translational suppression and lack of neuronal hyposynchrony in the absence of FMRP.ConclusionThese results suggest a novel function of FMRP in regulating translational suppression and neuronal networking function following Aβ treatment by fine‐tuning two distinct signaling pathways involved in global translation.

Biophysical characterization as a tool to predict amyloidogenic and toxic properties of amyloid-β42 peptides.

Amyloid-β42 (Aβ42) peptides are central to the amyloid pathology in Alzheimer's disease (AD). As biological mimetics, properties of synthetic Aβ peptides usually vary between vendors and batches, thus impacting the reproducibility of experimental studies. Here, we tested recombinantly expressed Aβ42 (Asp1 to Ala42) against synthetic Aβ42 from different suppliers using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), circular dichroism (CD) spectroscopy, thioflavin T aggregation, surface plasmon resonance, and MTT cell viability assays. Overall, our recombinant Aβ42 provided a reproducible mimetic of desired properties. Across experimental approaches, the combined detection of Aβ42 dimers and random coil to β-sheet transition only correlated with aggregation-prone and cytotoxic peptides. Conclusively, combining MALDI-MS with CD appears to provide a rapid, reliable means to predict the 'bioactivity' of Aβ42.

Amyloid beta induces Fmr1-dependent translational suppression and hyposynchrony of neural activity via phosphorylation of eIF2α and eEF2.

Alzheimer's disease (AD) is the most common cause of dementia, with the accumulation of amyloid beta peptide (Aβ) being one of the main causes of the disease. Fragile X mental retardation protein (FMRP), encoded by fragile X mental retardation 1 (Fmr1), is an RNA‐binding protein that represses translation of its bound mRNAs or exerts other indirect mechanisms that result in translational suppression. Because the accumulation of Aβ has been shown to cause translational suppression resulting from the elevated cellular stress response, in this study we asked whether and how Fmr1 is involved in Aβ‐induced translational regulation. Our data first showed that the application of synthetic Aβ peptide induces the expression of Fmr1 in cultured primary neurons. We followed by showing that Fmr1 is required for Aβ‐induced translational suppression, hyposynchrony of neuronal firing activity, and loss of excitatory synapses. Mechanistically, we revealed that Fmr1 functions to repress the expression of phosphatases including protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), leading to elevated phosphorylation of eukaryotic initiation factor 2‐α (eIF2α) and eukaryotic elongation factor 2 (eEF2), and subsequent translational suppression. Finally, our data suggest that such translational suppression is critical to Aβ‐induced hyposynchrony of firing activity, but not the loss of synapses. Altogether, our study uncovers a novel mechanism by which Aβ triggers translational suppression and we reveal the participation of Fmr1 in altered neural plasticity associated with Aβ pathology. Our study may also provide information for a better understanding of Aβ‐induced cellular stress responses in AD.

Presence of endogenous Aβ in plasma of patient with Alzheimer’s disease differentiates Aβ oligomerization tendencies caused by synthetic Aβ

AbstractBackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia. A number of studies have presented accumulating evidence that oligomeric amyloid‐β (Aβ) is the major factor in the AD pathogenesis. To detect oligomeric Aβ, Multimer Detection System‐Oligomeric Amyloid‐β (MDS‐OAβ), a technique for measuring Aβ oligomerization in plasma to diagnose AD was previously developed. In this study, In this study, we aim to evaluate the role of the seeding effect in plasma in differentiating between AD and individuals without AD pathology.MethodPlasma was spiked with synthetic Aβ peptides and incubated to confirm the tendency of Aβ oligomerization in AD samples and samples without AD pathology. AlphaLISA assay was performed in comparison with MDS‐OAβ to confirm the relationship between endogenous Aβ and spiked synthetic Aβ peptides in plasma. In addition, Aβ immuno‐depletion in plasma confirmed the effect of synthetic Aβ peptides on the Multimer Detection System (MDS) signals.ResultThe difference in the seeding effect between AD plasma and normal control plasma was confirmed. AlphaLISA assay presented that synthetic Aβ peptide recruits endogenous Aβ in plasma and the seeding effect is significantly increased in AD plasma than normal control plasma. In addition, the absence of the MDS signals in Aβ‐depleted plasma supported that the increased oligomerization tendency in AD plasma is dependent on the presence of endogenous Aβ in plasma. MDS‐OAβ can differentiate between AD patients and non‐AD controls by measuring the degree of Aβ oligomerization after spiking synthetic Aβ into plasma samples with incubation.ConclusionSpiking of synthetic Aβ peptides had a greater seeding effect in AD plasma than in normal control plasma. Therefore, the measurement of Aβ oligomerization in plasma using MDS‐OAβ has high utility in AD diagnosis.

Endogenous Aβ peptide promote Aβ oligomerization tendency of spiked synthetic Aβ in Alzheimer's disease plasma

Alzheimer's disease (AD) is the most common form of age-associated dementia. Several studies have predicted that AD is caused by the production and deposition of the β-amyloid peptide (Aβ) in the brain, which is one of pathologic hallmarks of AD. In particular, Aβ oligomers are reportedly the most toxic and pathogenic of other peptide forms. We previously developed Multimer Detection System-Oligomeric Amyloid-β (MDS-OAβ), a technique for measuring Aβ oligomerization in plasma to diagnose AD. Here, we clarified the molecular sizes of oligomers that can be detected by the MDS and investigated differences in plasma spiking with a synthetic Aβ peptide in the plasma of AD patients and individuals with non-AD neurological conditions. To determine Aβ oligomer sizes detectable by MDS, size exclusion chromatography (SEC) was first performed on incubated samples of synthetic Aβ42 peptides. As a result, no MDS signals were observed for the Aβ42 monomer fractions, but strong signals were found for oligomers of 7–35-mers long. Also, an amplified luminescent proximity homogeneous assay-linked immunoassay (AlphaLISA) was used to confirm that synthetic Aβ peptides not only recruited endogenous Aβ in plasma but also induced significantly stronger seeding in AD plasma than in healthy control plasma. In addition, the absence of the MDS signals in Aβ-depleted plasma confirmed that the increased oligomerization tendency in AD plasma is dependent on the presence of endogenous Aβ in plasma. Therefore, the MDS-OAβ measurement of oligomerization differences in plasma after incubation with spiked synthetic Aβ peptides has significant potential in AD diagnosis.

A Cationic Gallium Phthalocyanine Inhibits Amyloid β Peptide Fibril Formation.

Amyloid β (Aβ) peptide deposition is considered as the main cause of Alzheimer's disease (AD). Previously, we have shown that a Zn containing neutral phthalocyanine (Zn-Pc) inhibits Aβ fibril formation. The objective of this study is to investigate the effects of a cationic gallium containing Pc (GaCl-Pc) on Aβ fibril formation process. Aβ fibril formation was induced by incubating synthetic Aβ peptides in a fibril forming buffer, and the amount of fibril was evaluated by ThT fluorescence assay. GaCl-Pc dosedependently inhibited both Aβ1-40 and Aβ1-42 fibril formation. It mainly inhibited the elongation phase of Aβ1-42 fibril formation kinetics, but not the lag phase. Western blotting results showed that it did not inhibit its oligomerization process, rather increased it. Additionally, GaCl-Pc destabilized preformed Aβ1- 42 fibrils dose-dependently in vitro condition, and decreased Aβ levels in the brain slice culture of APP transgenic AD model mice (J20 strain). Near-infrared scanning results showed that GaCl-Pc had the ability to bind to Aβ1-42. MTT assay demonstrated that GaCl-Pc did not have toxicity towards a neuronal cell line (A1) in culture rather, showed protective effects on Aβ-induced toxicity. Moreover, it dosedependently decreased Aβ-induced reactive oxygen species levels in A1 culture. Thus, our result demonstrated that GaCl-Pc decreased Aβ aggregation and destabilized the preformed fibrils. Since cationic molecules show a better ability to cross the blood-brain barrier, cationic GaCl-Pc could be important for the therapy of AD.

N-terminal heterogeneity of parenchymal and vascular amyloid-β deposits in Alzheimer's disease.

Aims:The deposition of amyloid-β (Aβ) peptides in the form of extracellular plaques in the brain represents one of the classical hallmarks of Alzheimer’s disease (AD). In addition to ‘full-length’ Aβ starting with aspartic acid (Asp-1), considerable amounts of various shorter, N-terminally truncated Aβ peptides have been identified by mass spectrometry in autopsy samples from individuals with AD.Methods:Selectivity of several antibodies detecting full-length, total or N-terminally truncated Aβ species has been characterized with capillary isoelectric focusing assays using a set of synthetic Aβ peptides comprising different N-termini. We further assessed the N-terminal heterogeneity of extracellular and vascular Aβ peptide deposits in the human brain by performing immunohistochemical analyses using sporadic AD cases with antibodies targeting different N-terminal residues, including the biosimilar antibodies Bapineuzumab and Crenezumab.Results:While antibodies selectively recognizing Aβ1–x showed a much weaker staining of extracellular plaques and tended to accentuate cerebrovascular amyloid deposits, antibodies detecting Aβ starting with phenylalanine at position 4 of the Aβ sequence showed abundant amyloid plaque immunoreactivity in the brain parenchyma. The biosimilar antibody Bapineuzumab recognized Aβ starting at Asp-1 and demonstrated abundant immunoreactivity in AD brains.Discussion:In contrast to other studied Aβ1–x-specific antibodies, Bapineuzumab displayed stronger immunoreactivity on fixed tissue samples than with sodium dodecyl sulfate-denatured samples on Western blots. This suggests conformational preferences of this antibody. The diverse composition of plaques and vascular deposits stresses the importance of understanding the roles of various Aβ variants during disease development and progression in order to generate appropriate target-developed therapies.

A mechanistic hypothesis for the impairment of synaptic plasticity by soluble Aβ oligomers from Alzheimer's brain.

It is increasingly accepted that early cognitive impairment in Alzheimer's disease results in considerable part from synaptic dysfunction caused by the accumulation of a range of oligomeric assemblies of amyloid β-protein (Aβ). Most studies have used synthetic Aβ peptides to explore the mechanisms of memory deficits in rodent models, but recent work suggests that Aβ assemblies isolated from human (AD) brain tissue are far more potent and disease-relevant. Although reductionist experiments show Aβ oligomers to impair synaptic plasticity and neuronal viability, the responsible mechanisms are only partly understood. Glutamatergic receptors, GABAergic receptors, nicotinic receptors, insulin receptors, the cellular prion protein, inflammatory mediators, and diverse signaling pathways have all been suggested. Studies using AD brain-derived soluble Aβ oligomers suggest that only certain bioactive forms (principally small, diffusible oligomers) can disrupt synaptic plasticity, including by binding to plasma membranes and changing excitatory-inhibitory balance, perturbing mGluR, PrP, and other neuronal surface proteins, down-regulating glutamate transporters, causing glutamate spillover, and activating extrasynaptic GluN2B-containing NMDA receptors. We synthesize these emerging data into a mechanistic hypothesis for synaptic failure in Alzheimer's disease that can be modified as new knowledge is added and specific therapeutics are developed.

Protofibrils of Amyloid-β are Important Targets of a Disease-Modifying Approach for Alzheimer's Disease.

Worldwide, Alzheimer’s disease (AD) is the most common age-related neurodegenerative disease and is characterized by unique pathological hallmarks in the brain, including plaques composed of amyloid β-protein (Aβ) and neurofibrillary tangles of tau protein. Genetic studies, biochemical data, and animal models have suggested that Aβ is responsible for the pathogenesis of AD (i.e., the amyloid hypothesis). Indeed, Aβ molecules tend to aggregate, forming oligomers, protofibrils, and mature fibrils. However, while these Aβ species form amyloid plaques of the type implicated in AD neurodegeneration, recent clinical trials designed to reduce the production of Aβ and/or the plaque burden have not demonstrated clinical efficacy. In addition, recent studies using synthetic Aβ peptides, cell culture models, Arctic transgenic mice, and human samples of AD brain tissues have suggested that the pre-fibrillar forms of Aβ, particularly Aβ protofibrils, may be the most critical species, compared with extracellular fibrillar forms. We recently reported that protofibrils of Aβ1-42 disturbed membrane integrity by inducing reactive oxygen species generation and lipid peroxidation, resulting in decreased membrane fluidity, intracellular calcium dysregulation, depolarization, and synaptic toxicity. Therefore, the therapeutic reduction of protofibrils may prevent the progression of AD by ameliorating neuronal damage and cognitive dysfunction through multiple mechanisms.

Associative Interactions among Zinc, Apolipoprotein E, and Amyloid-β in the Amyloid Pathology.

Zinc and apolipoprotein E (apoE) are reportedly involved in the pathology of Alzheimer’s disease. To investigate the associative interaction among zinc, apoE, and amyloid-β (Aβ) and its role in amyloid pathogenesis, we performed various biochemical and immunoreactive analyses using brain tissues of Tg2576 mice and synthetic Aβ and apoE peptides. On amyloid plaques or in brain lysates of Tg2576 mice, apoE and Aβ immunoreactivities increased after zinc chelation and were restored by its subsequent replacement. Zinc depletion dissociated apoE/Aβ complexes or larger-molecular sizes of Aβ oligomers/aggregates into smaller-molecular sizes of apoE and/or Aβ monomers/complexes. In the presence of zinc, synthetic apoE and/or Aβ peptides aggregated into larger-molecular sizes of oligomers or complexes. Endogenous proteases or plasmin in brain lysates degraded apoE and/or Aβ complexes, and their proteolytic activity increased with zinc depletion. These biochemical findings suggest that zinc associates with apoE and Aβ to encourage the formation of apoE/Aβ complexes or large aggregates, raising the deposition of zinc-rich amyloid plaques. In turn, the presence of abundant zinc around and within apoE/Aβ complexes may block the access or activity of Aβ-degrading antibodies or proteases. These results support the plausibility of chelation strategy aiming at reducing amyloid pathology in Alzheimer’s disease.

ROLE OF RADIATION AS EFFECTIVE INTERVENTION IN Aβ INDUCED OXIDATIVE STRESS IN ANIMAL MODEL OF ALZHEIMER’S DISEASE

Objective: The present study was undertaken to study the therapeutic effects of low dose fractionated cranial X-irradiation on reducing the amyloid-beta (Aβ) induced oxidative stress burden in an animal model of Alzheimer’s disease (AD).Methods: S.D. female rats received an intracerebroventricular injection of Aβ peptide at stereotaxically defined points. Experimental sessions were conducted by randomly dividing animals into four groups, namely sham-operated, Aβ-injected, and Aβ injection followed by cranial X-irradiation and only cranial X-irradiated. Anesthetized animals received 5 μl synthetic Aβ peptide injection with a 10 μl Hamilton microsyringe with the needle kept in place for a period of 2min following injection. Sham-operated group received 5 μl of bidistilled water instead of Aβ peptide. Animals were treated 6 weeks post-surgery with fractionated radiation of 2Gy for 5 days. Neurobehavior studies were undertaken to confirm memory impairment along with biochemical indices involved in the antioxidant defense system.Results: Fractionated cranial X-irradiation proved effective in restoration of activity of enzymes involved in the antioxidant defense system; the lipid peroxidation and catalase levels that showed a significant increase in Aβ-treated group decreased on subsequent X-irradiation. Moreover, the decrease in the superoxide dismutase, glutathione, glutathione-S-transferase, and glutathione reductase levels witnessed an increase post-irradiation, implicating the X-irradiation to be an effective intervention to restore the redox status of the oxidatively stressed brain cells in AD condition.Conclusion: The present study evaluated the therapeutic potential of low dose fractionated cranial X- irradiation by mitigating the amyloid-induced oxidative stress suggesting a novel treatment for AD-associated pathologies.

ROLE OF RADIATION AS EFFECTIVE INTERVENTION IN Aβ INDUCED OXIDATIVE STRESS IN ANIMAL MODEL OF ALZHEIMER’S DISEASE

Objective: The present study was undertaken to study the therapeutic effects of low dose fractionated cranial X-irradiation on reducing the amyloid-beta (Aβ) induced oxidative stress burden in an animal model of Alzheimer’s disease (AD).Methods: S.D. female rats received an intracerebroventricular injection of Aβ peptide at stereotaxically defined points. Experimental sessions were conducted by randomly dividing animals into four groups, namely sham-operated, Aβ-injected, and Aβ injection followed by cranial X-irradiation and only cranial X-irradiated. Anesthetized animals received 5 μl synthetic Aβ peptide injection with a 10 μl Hamilton microsyringe with the needle kept in place for a period of 2min following injection. Sham-operated group received 5 μl of bidistilled water instead of Aβ peptide. Animals were treated 6 weeks post-surgery with fractionated radiation of 2Gy for 5 days. Neurobehavior studies were undertaken to confirm memory impairment along with biochemical indices involved in the antioxidant defense system.Results: Fractionated cranial X-irradiation proved effective in restoration of activity of enzymes involved in the antioxidant defense system; the lipid peroxidation and catalase levels that showed a significant increase in Aβ-treated group decreased on subsequent X-irradiation. Moreover, the decrease in the superoxide dismutase, glutathione, glutathione-S-transferase, and glutathione reductase levels witnessed an increase post-irradiation, implicating the X-irradiation to be an effective intervention to restore the redox status of the oxidatively stressed brain cells in AD condition.Conclusion: The present study evaluated the therapeutic potential of low dose fractionated cranial X- irradiation by mitigating the amyloid-induced oxidative stress suggesting a novel treatment for AD-associated pathologies.

Time-dependent effect of oligomeric amyloid-β (1–42)-induced hippocampal neurodegeneration in rat model of Alzheimer’s disease

ABSTRACTObjective: Alzheimer's disease (AD) is characterized with an abnormal deposition of insoluble amyloid-beta (Aβ) peptide plaques, tangles formation and synaptic dysfunction. These result in impaired functioning of neuronal circuits and alter the behavioral response owing to activation of neurotransmitter receptors. Recently, it has been implicated that Aβ influences N-methyl d-aspartate (NMDA) receptor activation in AD; however, the molecular mechanism underlying remains unclear. Thus, emerged specific aim to study the time-course effect of oligomeric Aβ(1–42) (oAβ1–42) on the mRNA expression of genes encoding NMDA and acetylcholine receptors in the rat model of AD.Methods: Aggregated forms of synthetic Aβ peptides were injected bilaterally into the intrahippocampal region of rat brain using stereotaxic surgery. Behavioral analysis was performed using eight-arm Radial Arm Maze task at the end of experimental period. Euthanized rat brain hippocampal tissue was used to study the mRNA expression of glutamatergic and cholinergic receptor using semiquantitative reverse transcription–polymerase chain reaction.Results: oAβ1–42 decreased the gene expression level of α7-nicotinic acetylcholine receptor and increased the mRNA expression of NMDA receptor 2A, and -2B subunits. In particular, oAβ1–42 aggregates increased the retention time and altered the behavioral response in rats after 15 days of injection. Further, amyloid-β1–42 are highly expressed in 15 days after postinjection in hippocampus of adult rats.Conclusion: Acute exposure of oAβ1–42 modulated differential gene expression of glutamatergic and cholinergic receptors in hippocampus of adult rats and is duration dependent reflecting changes in hippocampal circuitry system underlying learning and memory impairments.Abbreviations: AD: Alzheimer’s disease, Aβ: amyloid-β; oAβ1–42: oligomeric amyloid-β 1–42 full length peptide; CAM: calmodulin; CNS: central nervous system; CR: Congo red; DG: dentate gyrus; EC: entorhinal cortex; HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol; IBO: ibotenic acid; NMDA: N-methyl d-aspartate; NMDAR: N-methyl d-aspartate receptor; NR2A: N-methyl d-aspartate receptor 2A; NR2B: N-methyl d-aspartate receptor 2B; ACh: acetylcholine; α7-nAChR: α7-nicotinic acetylcholine receptor; PBS: phosphate buffered saline; RAM: Radial Arm Maze; ThT: thioflavin T.

Neurotoxic Effects of Aβ6-42 Peptides Mimicking Putative Products Formed by the Angiotensin Converting Enzyme.

Angiotensin converting enzyme (ACE) is involved in proteolytic processing of the amyloid-β(Aβ) peptide implicated in the development of Alzheimer's disease (AD) and known products of ACE-based processing of Aβ42 are characterized by reduced aggregability and cytotoxicity. Recently it has been demonstrated that ACE can act as an arginine specific endopeptidase cleaving the N-terminal pentapeptide (Aβ1-5) from synthetic Aβ peptide analogues. In the context of proteolytic processing of full length Aβ42, this suggests possible formation of Aβ6-42 species. The aim of this study was to test a hypothesis that some N-terminally truncated Aβ peptide(s) could retain aggregability and neurotoxic properties typical for Aβ42. We have investigated aggregability of two amyloid-β peptides, Aβ6-42 and isoD7-Aβ6-42, mimicking potential proteolytic products of Aβ42 and isoD7-Aβ42, and evaluated their effects on the repertoire of brain Aβ binding proteins, and cytotoxicity towards neuroblastoma SH-SY5Y cells. Aggregability of isoD7-Aβ6-42 and Aβ6-42 was higher than that of full-length peptides Aβ42 and isoD7-Aβ42, while the repertoire of mouse brain Aβ binding proteins dramatically decreased. Aβ6-42 and isoD7-Aβ6-42 exhibited higher neurotoxicity towards SH-SY5Y cells than Aβ42 and isoD7-Aβ42, respectively. They effectively stimulated production of ROS and NO, and also TNFα secretion by cells. Thus, our results suggest that ACE-dependent processing of full-length Aβs could result in formation of more pathogenic peptides.

Diffusible, highly bioactive oligomers represent a critical minority of soluble Aβ in Alzheimer's disease brain.

Significant data suggest that soluble Aβ oligomers play an important role in Alzheimer's disease (AD), but there is great confusion over what exactly constitutes an Aβ oligomer and which oligomers are toxic. Most studies have utilized synthetic Aβ peptides, but the relevance of these test tube experiments to the conditions that prevail in AD is uncertain. A few groups have studied Aβ extracted from human brain, but they employed vigorous tissue homogenization which is likely to release insoluble Aβ that was sequestered in plaques during life. Several studies have found such extracts to possess disease-relevant activity and considerable efforts are being made to purify and better understand the forms of Aβ therein. Here, we compared the abundance of Aβ in AD extracts prepared by traditional homogenization versus using a far gentler extraction, and assessed their bioactivity via real-time imaging of iPSC-derived human neurons plus the sensitive functional assay of long-term potentiation. Surprisingly, the amount of Aβ retrieved by gentle extraction constituted only a small portion of that released by traditional homogenization, but this readily diffusible fraction retained all of the Aβ-dependent neurotoxic activity. Thus, the bulk of Aβ extractable from AD brain was innocuous, and only the small portion that was aqueously diffusible caused toxicity. This unexpected finding predicts that generic anti-oligomer therapies, including Aβ antibodies now in trials, may be bound up by the large pool of inactive oligomers, whereas agents that specifically target the small pool of diffusible, bioactive Aβ would be more useful. Furthermore, our results indicate that efforts to purify and target toxic Aβ must employ assays of disease-relevant activity. The approaches described here should enable these efforts, and may assist the study of other disease-associated aggregation-prone proteins.