A β‐amino acid modified heptapeptide containing a designed recognition element disrupts fibrillization of the amyloid β‐peptide

We study the complex formation of a peptide βAβAKLVFF, previously developed by our group, with Aβ(1–42) in aqueous solution. Circular dichroism spectroscopy is used to probe the interactions between βAβAKLVFF and Aβ(1–42), and to study the secondary structure of the species in solution. Thioflavin T fluorescence spectroscopy shows that the population of fibers is higher in βAβAKLVFF/Aβ(1–42) mixtures compared to pure Aβ(1–42) solutions. TEM and cryo‐TEM demonstrate that co‐incubation of βAβAKLVFF with Aβ(1–42) causes the formation of extended dense networks of branched fibrils, very different from the straight fibrils observed for Aβ(1–42) alone. Neurotoxicity assays show that although βAβAKLVFF alters the fibrillization of Aβ(1–42), it does not decrease the neurotoxicity, which suggests that toxic oligomeric Aβ(1–42) species are still present in the βAβAKLVFF/Aβ(1–42) mixtures. Our results show that our designed peptide binds to Aβ(1–42) and changes the amyloid fibril morphology. This is shown to not necessarily translate into reduced toxicity. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Mechanism of cholesterol‐assisted oligomeric channel formation by a short Alzheimer β‐amyloid peptide

Alzheimer β‐amyloid (Aβ) peptides can self‐organize into oligomeric ion channels with high neurotoxicity potential. Cholesterol is believed to play a key role in this process, but the molecular mechanisms linking cholesterol and amyloid channel formation have so far remained elusive. Here, we show that the short Aβ22‐35 peptide, which encompasses the cholesterol‐binding domain of Aβ, induces a specific increase of Ca²⁺ levels in neural cells. This effect is neither observed in calcium‐free medium nor in cholesterol‐depleted cells, and is inhibited by zinc, a blocker of amyloid channel activity. Double mutations V24G/K28G and N27R/K28R in Aβ22‐35 modify cholesterol binding and abrogate channel formation. Molecular dynamic simulations suggest that cholesterol induces a tilted α‐helical topology of Aβ22‐35. This facilitates the establishment of an inter‐peptide hydrogen bond network involving Asn‐27 and Lys‐28, a key step in the octamerization of Aβ22‐35 which proceeds gradually until the formation of a perfect annular channel in a phosphatidylcholine membrane. Overall, these data give mechanistic insights into the role of cholesterol in amyloid channel formation, opening up new therapeutic options for Alzheimer's disease.

     

Neurotoxicity of acetylcholinesterase amyloid β-peptide aggregates is dependent on the type of Aβ peptide and the AChE concentration present in the complexes

Alzheimer’s disease (AD) is a neurodegenerative disorder whose hallmark is the presence of senile plaques and neurofibrillary tangles. Senile plaques are mainly composed of amyloid β-peptide (Aβ) fibrils and several proteins including acetylcholinesterase (AChE). AChE has been previously shown to stimulate the aggregation of Aβ_1–40 into amyloid fibrils. In the present work, the neurotoxicity of different amyloid aggregates formed in the absence or presence of AChE was evaluated in rat pheochromocytoma PC12 cells. Stable AChE-Aβ complexes were found to be more toxic than those formed without the enzyme, for Aβ_1–40 and Aβ_1–42, but not for amyloid fibrils formed with Aβ_Val18→Ala, a synthetic variant of the Aβ_1–40 peptide. Of all the AChE-Aβ complexes tested the one containing the Aβ_1–40 peptide was the most toxic. When increasing concentrations of AChE were used to aggregate the Aβ_1–40 peptide, the neurotoxicity of the complexes increased as a function of the amount of enzyme bound to each complex. Our results show that AChE-Aβ_1–40 aggregates are more toxic than those of AChE-Aβ_1–42 and that the neurotoxicity depends on the amount of AChE bound to the complexes, suggesting that AChE may play a key role in the neurodegeneration observed in Alzheimer brain.     

Antibodies bound to Aβ oligomers potentiate the neurotoxicity of Aβ by activating microglia

Beta amyloid (Aβ) oligomers are thought to contribute to the pathogenesis of Alzheimer's disease. However, clinical trials using Aβ immunization were unsuccessful due to strong brain inflammation, the mechanisms of which are poorly understood. In this study we tested whether monoclonal antibodies to oligomeric Aβ would prevent the neurotoxicity of Aβ oligomers in primary neuronal‐glial cultures. However, surprisingly, the antibodies dramatically increased the neurotoxicity of Aβ. Antibodies bound to monomeric Aβ fragments were non‐toxic to cultured neurons, while antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein, human metapneumovirus nucleocapsid protein, and measles virus nucleocapsid protein, strongly potentiated the neurotoxicity of their antigens. The neurotoxicity of antibody‐oligomeric antigen complexes was abolished by removal of the Fc region from the antibodies or by removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. In conclusion, we find that immune complexes formed by Aβ oligomers or other oligomeric/multimeric antigens and their specific antibodies can cause death and loss of neurons in primary neuronal‐glial cultures via Fc‐dependent microglial activation. The results suggest that therapies resulting in antibodies to oligomeric Aβ or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation.

     

The N‐terminal tripeptide of insulin‐like growth factor‐I protects against β‐amyloid‐induced somatostatin depletion by calcium and glycogen synthase kinase 3β modulation

The protective effects of insulin‐like growth factor I on the somatostatin (SRIF) system in the temporal cortex after β‐amyloid (Aβ) injury may be mediated through its N‐terminal tripeptide glycine‐proline‐glutamate (GPE). GPE is cleaved to cyclo[Pro‐Gly] (cPG), a metabolite suggested to mediate in neuroprotective actions. We evaluated the effects of GPE and cPG in the temporal cortex of Aβ25–35‐treated rats on SRIF and SRIF receptor protein and mRNA levels, adenylyl cyclase activity, cell death, Aβ25–35 accumulation, cytosolic calcium levels ([Ca²⁺]_c) and the intracellular signaling mechanisms involved. GPE and cPG did not change Aβ25–35 levels, but GPE partially restored SRIF and SRIF receptor 2 protein content and mRNA levels and protected against cell death after Aβ25–35 insult, which was coincident with Akt activation and glycogen synthase kinase 3β inhibition. In addition, GPE displaced glutamate from NMDA receptors and blocked the glutamate induced rise in cytosolic calcium in isolated rat neurons and moderately increased Ca²⁺ influx per se. Our findings suggest that GPE, but not its metabolite, mimics insulin‐like growth factor I effects on the SRIF system through a mechanism independent of Aβ clearance that involves modulation of calcium and glycogen synthase kinase 3β signaling.     

N‐terminal truncated pyroglutamyl β amyloid peptide Aβpy3‐42 shows a faster aggregation kinetics than the full‐length Aβ1‐42

We tested directly the differences in the aggregation kinetics of three important β amyloid peptides, the full‐length Aβ1‐42, and the two N‐terminal truncated and pyroglutamil modified Aβpy3‐42 and Aβpy11‐42 found in different relative concentrations in the brains in normal aging and in Alzheimer disease. By following the circular dichroism signal and the ThT fluorescence of the solution in phosphate buffer, we found substantially faster aggregation kinetics for Aβpy3‐42. This behavior is due to the particular sequence of this peptide, which is also responsible for the specific oligomeric aggregation states, found by TEM, during the fibrillization process, which are very different from those of Aβ1‐42, more prone to fibril formation. In addition, Aβpy3‐42 is found here to have an inhibitory effect on Aβ1‐42 fibrillogenesis, coherently with its known greater infective power. This is an indication of the important role of this peptide in the aggregation process of β‐peptides in Alzheimer disease. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 861–873, 2009. This article was originally published online as an accepted preprint. The “Published Online“ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The effect of terminal groups and halogenation of KLVFF peptide on its activity as an inhibitor of β‐amyloid aggregation

The aggregation of Aβ peptide into amyloid fibrils in the brain is associated with Alzheimer's disease (AD). Inhibition of Aβ aggregation seemed a potential treatment for AD. It was previously shown that a short fragment of Aβ peptide (KLVFF, 16‐20) bound Aβ inhibited its aggregation. In this work, using KLVFF peptide, we synthesized two peptide families and then evaluated their inhibitory capacities by conventional assays such as thioflavin T (ThT) fluorescence spectroscopy, turbidity measurement, and the 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS). The effect of peptide terminal groups on its inhibitory activity was first studied. Subsequently, the influence of halogenated amino acids on peptide anti‐aggregation properties was investigated. We found that iodinated peptide with amine in the N and amide in the C termini, respectively, was the best inhibitor of Aβ fibers formation. Halogenated peptides seemed to decrease the number of Aβ fibrils; however, they did not reduce Aβ cytotoxicity. The data obtained in this work seemed promising in developing potential peptide drugs for treatment of AD.     

Design of nonapeptide LVFFARKHH: A bifunctional agent against Cu2+‐mediated amyloid β‐protein aggregation and cytotoxicity

Dysfunctional accumulation of amyloid β‐protein (Aβ) mediated by Cu²⁺ exhibits higher neurotoxicity and accelerates the progress of Alzheimer's disease, so inhibition of Cu²⁺‐mediated Aβ aggregation and cytotoxicity has been considered as a therapeutic strategy for the disease. Herein, a nonapeptide was designed by linking HH to the C‐terminus of a peptide inhibitor of Aβ aggregation, LVFFARK (LK7). We found that the nonapeptide, LK7‐HH, possessed dual functionality, including enhanced inhibition capability on Aβ aggregation as compared to LK7, and chelating Cu²⁺ with a dissociation constant of 5.50 μM. This enabled LK7‐HH to arrest the generation of reactive oxygen species catalyzed by Cu²⁺ or Cu²⁺‐Aβ complex, and to inhibit Cu²⁺‐induced Aβ aggregation. Moreover, in contrast with the cytotoxicity of LK7 aggregates, LK7‐HH was biocompatible because HH conjugation made its aggregation behavior different from LK7. Thus, LK7‐HH efficiently suppressed Cu²⁺‐mediated Aβ aggregation and cytotoxicity. An equimolar concentration of LK7‐HH increased cell viability from 50% to 90% when treating Aβ₄₀‐Cu²⁺ complexes. The results provided insights into the roles of HH in enhancing the inhibition of Aβ and Cu²⁺‐induced Aβ aggregations, in eliminating Cu²⁺‐induced cytotoxicities by arresting generation of reactive oxygen species, and in making the peptide biocompatible. Therefore, this work would contribute to the design of potent peptide‐based inhibitors of Cu²⁺‐mediated Aβ aggregation and cytotoxicity.     

Syntheses and anti‐tubercular activity of β‐substituted and α,β‐disubstituted peptidyl β‐aminoboronates and boronic acids

Tuberculosis is still affecting millions of people worldwide, and new resistant strains of Mycobacterium tuberculosis are being found. It is therefore necessary to find new compounds for treatment. In this paper, we report the synthesis and in vitro testing of peptidyl β‐aminoboronic acids and β‐aminoboronates with anti‐tubercular activity. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Differential physiologic responses of α7 nicotinic acetylcholine receptors to β‐amyloid1–40 and β‐amyloid1–42

The β‐amyloid peptides (Aβ), Aβ_1–40 and Aβ_1–42, have been implicated in Alzheimer's disease (AD) pathology. Although Aβ_1–42 is generally considered to be the pathological peptide in AD, both Aβ_1–40 and Aβ_1–42 have been used in a variety of experimental models without discrimination. Here we show that monomeric or oligomeric forms of the two Aβ peptides, when interact with the neuronal cation channel, α7 nicotinic acetylcholine receptors (α7nAChR), would result in distinct physiologic responses as measured by acetylcholine release and calcium influx experiments. While Aβ_1–42 effectively attenuated these α7nAChR‐dependent physiology to an extent that was apparently irreversible, Aβ_1–40 showed a lower inhibitory activity that could be restored upon washings with physiologic buffers or treatment with α7nAChR antagonists. Our data suggest a clear pharmacological distinction between Aβ_1–40 and Aβ_1–42. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 25–30, 2003     

Characteristics of C‐terminal, β‐amyloid peptide binding fragment of neuroprotective protease inhibitor,cystatin C

Cystatin C originally identified as a cysteine proteases inhibitor has a broad spectrum of biological roles ranging from inhibition of extracellular cysteine protease activities, bone resorption, and modulation of inflammatory responses to stimulation of fibroblasts proliferation. There is an increasing number of evidence to suggest that human cystatin C (hCC) might play a protective role in the pathophysiology of sporadic Alzheimer's disease. In vivo and in vitro results well documented the association of hCC with Aβ and the hCC‐induced inhibition of Aβ fibril formation. In our earlier work, using a combination of selective proteolytic methods and MS spectroscopy, C‐terminal fragment hCC(101‐117) was identified as the Aβ‐binding region. The fragment of Aβ peptide responsible for the complex formation with hCC was found in the middle, highly hydrophobic part, Aβ(17‐24). Structures and affinities of both Aβ and hCC binding sites were characterized by the enzyme‐linked immunosorbent assay‐like assay, by surface plasmon resonance, and by nano‐ESI‐FTICR MS of the hCC–Aβ‐ binding peptide complexes. In the in vitro inhibition studies, the binding cystatin sequence, hCC(101‐117), revealed the highest relative inhibitory effect toward Aβ‐fibril formation. Herein, we present further studies on molecular details of the hCC‐Aβ complex. With Ala substitution, affinity experiments, and enzyme‐linked immunosorbent assay‐like assays for the Aβ‐binding fragment, hCC(101‐117), and its variants, the importance of individual amino acid residues for the protein interaction was evaluated. The results were analyzed using hCC(101‐117) nuclear magnetic resonance structural data with molecular dynamics calculations and molecular modeling of the complexes. The results point to conformational requirements and special importance of some amino acid residues for the protein interaction. The obtained results might be helpful for the design of low molecular compounds modulating the biological role of both proteins. Copyright © 2016 John Wiley & Sons, Ltd.     

Multiple mechanisms of dimethyl fumarate in amyloid β‐induced neurotoxicity in human neuronal cells

Alzheimer disease (AD) is characterized by a complex heterogeneity of pathological changes, and any therapeutic approach categorically requires a multi‐targeted way. It has been demonstrated that together with the hallmarks of the disease such as neurofibrillary tangles and senile plaques, oxidative and inflammatory stress covered an important role. Dimethyl fumarate (DMF) is an orally bioavailable methyl ester of fumaric acid and activator of Nrf2 with potential neuroprotective and immunomodulating activities. Therefore, the aim of the present work was to evaluate the potential beneficial effects of DMF, compared with its active metabolite monomethyl fumarate (MMF) (both at 30 μM) in an in vitro Alzheimer's model using SH‐SY5Y human neuroblastoma cell lines stimulated with amyloid‐beta (Aβ). Moreover, the effect of DMF, compared with MMF, was evaluate by an ex vivo model using organotypic hippocampal slice cultures stimulated with Aβ_1‐42 (1 μg/ml), to better understand its action in a pathological setting. In both models, DMF pre‐treatment (30 μM) preserved cellular viability from Aβ stimulation, reducing tau hyper‐phosphorylation, much more efficiently then MMF (30 μM). Moreover, DMF was able to induce an activation of manganese superoxide dismutase (MnSOD) and heme‐oxygenase‐1 (HO‐1), decreasing the severity of oxidative stress. Our results showed important multi‐protective effects of DMF pre‐treatment from Aβ stimulation both in in vitro and ex vivo models, highlighting an Nrf2/NF‐κB‐dependent mechanism, which could provide a valuable support to the therapies for neurodegenerative diseases today.     

Interactions of amyloid Aβ(1–42) peptide with self‐assembled peptide nanospheres

In this work we have probed the interactions of the amyloid Aβ(1–42) peptide with self‐assembled nanospheres. The nanospheres were formed by self‐assembly of a newly developed bolaamphiphile bis(N‐alpha‐amido‐methionine)‐1,8 octane dicarboxylate under aqueous conditions. It was found that the interactions of the Aβ(1–42) peptide with the nanospheres were concentration as well as pH dependent and the peptide largely adopts a random coil structure upon interacting with the nanospheres. Further, upon incorporation with the nanospheres, we observed a relative diminution in the aggregation of Aβ(1–42) at low concentrations of Aβ(1–42). The interactions between the nanospheres and the Aβ(1–42) peptide were investigated by atomic force microscopy, transmission electron microscopy, circular dichroism, FTIR and fluorescence spectroscopy, and the degree of fibrillation in the presence and absence of nanospheres was monitored by the Thioflavine T assay. We believe that the outcome from this work will help further elucidate the binding properties of Aβ peptide as well as designing nanostructures as templates for further investigating the nucleation and fibrillation process of Aβ‐like peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

IL‐1β and TNF‐α induce neurotoxicity through glutamate production: a potential role for neuronal glutaminase

Glutaminase 1 is the main enzyme responsible for glutamate production in mammalian cells. The roles of macrophage and microglia glutaminases in brain injury, infection, and inflammation are well documented. However, little is known about the regulation of neuronal glutaminase, despite neurons being a predominant cell type of glutaminase expression. Using primary rat and human neuronal cultures, we confirmed that interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), two pro‐inflammatory cytokines that are typically elevated in neurodegenerative disease states, induced neuronal death and apoptosis in vitro. Furthermore, both intracellular and extracellular glutamate levels were significantly elevated following IL‐1β and/or TNF‐α treatment. Pre‐treatment with N‐Methyl‐d ‐aspartate (NMDA) receptor antagonist MK‐801 blocked cytokine‐induced glutamate production and alleviated the neurotoxicity, indicating that IL‐1β and/or TNF‐α induce neurotoxicity through glutamate. To determine the potential source of excess glutamate production in the culture during inflammation, we investigated the neuronal glutaminase and found that treatment with IL‐1β or TNF‐α significantly upregulated the kidney‐type glutaminase (KGA), a glutaminase 1 isoform, in primary human neurons. The up‐regulation of neuronal glutaminase was also demonstrated in situ in a murine model of HIV‐1 encephalitis. In addition, IL‐1β or TNF‐α treatment increased the levels of KGA in cytosol and TNF‐α specifically increased KGA levels in the extracellular fluid, away from its main residence in mitochondria. Together, these findings support neuronal glutaminase as a potential component of neurotoxicity during inflammation and that modulation of glutaminase may provide therapeutic avenues for neurodegenerative diseases.     

Leisure Activity,Brain β‐amyloid,and Episodic Memory in Adults with Down Syndrome

The present study provided an investigation of associations between leisure activity and early Alzheimer’s disease neuropathology (i.e., brain β‐amyloid) and episodic memory in a sample of 65 adults with Down syndrome (aged 30–53 years), at baseline and follow‐up, approximately three years apart. Findings indicated that leisure activity at baseline was not associated with brain β‐amyloid at baseline or change in brain β‐amyloid from baseline to follow‐up. Greater cognitively stimulating leisure activity at baseline was associated with better episodic memory at baseline, and greater social leisure activity at baseline was associated with less decline in episodic memory from baseline to follow‐up. High (as opposed to low) levels of social and overall leisure activity at baseline moderated the association between increase in brain β‐amyloid and decline in episodic memory, from baseline to follow‐up. Findings suggest that cognitively stimulating and social leisure activity could protect against the effect of Alzheimer’s disease neuropathology on episodic memory in adults with Down syndrome.     

β‐amyloid model core peptides: Effects of hydrophobes and disulfides

The mechanism by which a disordered peptide nucleates and forms amyloid is incompletely understood. A central domain of β‐amyloid (Aβ21–30) has been proposed to have intrinsic structural propensities that guide the limited formation of structure in the process of fibrillization. In order to test this hypothesis, we examine several internal fragments of Aβ, and variants of these either cyclized or with an N‐terminal Cys. While Aβ21–30 and variants were always monomeric and unstructured (circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMRS)), we found that the addition of flanking hydrophobic residues in Aβ16–34 led to formation of typical amyloid fibrils. NMR showed no long‐range nuclear overhauser effect (nOes) in Aβ21–30, Aβ16–34, or their variants, however. Serial ¹H‐¹⁵N‐heteronuclear single quantum coherence spectroscopy, ¹H‐¹H nuclear overhauser effect spectroscopy, and ¹H‐¹H total correlational spectroscopy spectra were used to follow aggregation of Aβ16–34 and Cys‐Aβ16–34 at a site‐specific level. The addition of an N‐terminal Cys residue (in Cys‐Aβ16–34) increased the rate of fibrillization which was attributable to disulfide bond formation. We propose a scheme comparing the aggregation pathways for Aβ16–34 and Cys‐Aβ16–34, according to which Cys‐Aβ16–34 dimerizes, which accelerates fibril formation. In this context, cysteine residues form a focal point that guides fibrillization, a role which, in native peptides, can be assumed by heterogeneous nucleators of aggregation.