Neurotoxic Oligomers Research Articles

β- N-Methylamino-l-alanine (BMAA) Not Involved in Alzheimer's Disease.

β- N-Methylamino-l-alanine (BMAA) is a neurotoxic agent implicated in ALS as well as Parkinson's and Alzheimer's diseases. It is produced by blue-green algae and could find its way via fish and seafood into the human food supply. Isolation from biological samples yields the compound in monomeric and protein-bound form. It has been suggested that the protein-bound fraction may result from genetic misincorporation into proteins in place of serine. Concomitant misfolding of the mutated proteins may be responsible for the neurological diseases. Recent reports that contradict the misincorporation theory leave unresolved the nature of the protein-bound form of BMAA. We have found from quantum mechanical calculations on model systems that it is possible to bind BMAA with high affinity in a noncovalent fashion to proteins. Because of our interest in Alzheimer's disease, molecular dynamics simulations were applied to search for such binding between BMAA and the β-amyloid peptide and to discover whether replacement of either of its two serine residues could affect its aggregation into neurotoxic oligomers. No stable noncovalently bound complex was found, and it was concluded that incorporation of BMAA in place of serine would not alter the conformational dynamics of the β-amyloid peptide.

Peptidomimetic-Based Multidomain Targeting Offers Critical Evaluation of Aβ Structure and Toxic Function.

The prevailing hypothesis stipulates that the preamyloid oligomers of Aβ are the main culprits associated with the onset and progression of Alzheimer's disease (AD), which has prompted efforts to search for therapeutic agents with the ability to inhibit Aβ oligomerization and amyloidogenesis. However, clinical progress is impeded by the limited structural information about the neurotoxic oligomers. To address this issue, we have adopted a synthetic approach, where a library of oligopyridylamide-based small molecules was tested against various microscopic events implicated in the self-assembly of Aβ. Two oligopyridylamides bind to different domains of Aβ and affect distinct microscopic events in Aβ self-assembly. The study lays the foundations for a dual recognition strategy to simultaneously target different domains of Aβ for further improvement in antiamyloidogenic activity. The data demonstrate that one of the most effective oligopyridylamides forms a high affinity complex with Aβ, which sustains the compound's activity in cellular milieu. The oligopyridylamide was able to rescue cells when introduced 24 h after the incubation of Aβ. The rescue of Aβ toxicity is potentially a consequence of the colocalization of the oligopyridylamide with Aβ. The synthetic tools utilized here provide a straightforward strategic framework to identify a range of potent antagonists of Aβ-mediated toxic functions. This approach could be a powerful route to the design of candidate drugs for various amyloid diseases that have so far proven to be "untargetable".

A new purified Lawsoniaside remodels amyloid-β42 fibrillation into a less toxic and non-amyloidogenic pathway

Mounting evidence indicates soluble Aβ42 oligomers as the most toxic species causing neuronal death which leads to the onset and progression of Alzheimer disease (AD). Recently, it has been found that neurotoxic Aβ42 oligomers grow from monomeric species or arise following secondary nucleation by preformed mature fibrils. Thus, the use of natural compounds such as polyphenols to hinder the growth or to remodel Aβ42 fibrils is one of the most promising strategies for AD treatment. In our previous study, we showed that 1, 2, 4-trihydroxynaphthalene-2-O-β-d-glucopyranoside (THNG) inhibits Aβ42 aggregation during the early steps of the aggregation process, inhibits its conformational change to a β-sheet-rich structure, decreases its polymerization, inhibits its fibrillogenisis and reduces oxidative stress and aggregate cytotoxicity. Here, we used different spectroscopic and cell culture methods to check the effect of THNG on fibrils disaggregation. We showed that THNG binds to mature Aβ42 fibrils, rearrange their secondary structure, and remodels them into non-amyloid, less toxic, species by inhibiting their interaction with the plasma membrane. Our findings reveal that THNG is a good agent to remodel amyloid fibrils and could be used as a starting molecular scaffold to design new anti-AD drugs.

Critical role of C-terminal residues of the Alzheimer's associated β-amyloid protein in mediating antiviral activity and modulating viral and bacterial interactions with neutrophils.

Recent studies have shown that the Alzheimer’s associated β-amyloid protein (βA) can inhibit growth of bacteria, fungi and viruses. We reported that the 42 amino acid βA protein inhibits replication of seasonal and pandemic strains of H3N2 and H1N1 influenza A virus (IAV) in vitro and modulates activation of neutrophils and monocytes exposed IAV. We here show that fragments composed of the N and C terminal domain of βA42, including βA22-42 and the 8 amino acid βA35-42, retain viral neutralizing and viral aggregating activity, whereas fragments lacking the C-terminal amino acids 41 and 42 (e.g. βA1-40, βA1-34, βA1-28, βA22-40 or βA33-40) have markedly diminished activities on these assays. βA22-42 also increased viral uptake, and virus induced respiratory burst responses, by human neutrophils, while peptides lacking residues 41 and 42 did not. Similar results were obtained with regard to bacterial aggregation, or promotion of bacterial uptake by neutrophils. Published structural studies have shown that βA1-42 has a greater propensity to form neurotoxic oligomers than βA1-40 due to a molecular interaction between Met35 and Ala42. Our findings suggest that there is a relationship between neurotoxic and antimicrobial activities of βA1-42. Truncated peptides containing the last 8 C-terminal amino acids of βA1-42 retain antimicrobial and opsonizing activities likely resulting from their ability to induce viral or bacterial aggregation.

Weaker N-Terminal Interactions for the Protective over the Causative Aβ Peptide Dimer Mutants.

Knowing that abeta amyloid peptide (Aβ42) dimers are the smallest and most abundant neurotoxic oligomers for Alzheimer's disease (AD), we used molecular simulations with advanced sampling methods (replica-exchange) to characterize and compare interactions between the N-termini (residues 1-16) of wild type (WT-WT) and five mutant dimers under constrained and unconstrained conditions. The number of contacts and distances between the N-termini, and contact maps of their conformational landscape illustrate substantial differences for a single residue change. The N-terminal contacts are significantly diminished for the dimers containing the monomers that protect against (WT-A2T) as compared with those that predispose toward (A2V-A2V) AD and for the control WT-WT dimers. The reduced number of N-terminal contacts not only occurs at or near the second residue mutations but also is distributed through to the 10th residue. These findings provide added support to the accumulating evidence for the "N-terminal hypothesis of AD" and offer an alternate mechanism for the cause of protection from the A2T mutant.

Quantitative Hydroxyl Radical Footprinting Study Reveals Structural Details of the Disorder-to-Order Transition in Amyloid-Beta (1-42) Oligomerization

This work presents a novel integrated analysis approach to characterize the intrinsically disordered 42 amino acid long β-amyloid peptide (Aβ42) and to elucidate mechanisms driving its misfolding and aggregation into neurotoxic oligomers. We describe this approach, which uses synchrotron based hydroxyl radical footprinting (HRF) results to determine residue specific solvent accessibilities for each of seventeen individual amino acids in monomer, multimer (2-4mer), and oligomer (∼150 kDa) Aβ42 sample preparations. Results complement published solid state NMR constraints with solution state structural details for the oligomers, and provide much needed experimental support for molecular dynamics (MD) computational studies of the monomer and 2-4mer species, where solid state NMR cannot be used. The pattern of protection from hydroxyl radical attack in the Aβ42 monomer (relative to the same residues in the fragmented unstructured peptide) shows partial folding in the monomer. The sequence location of this partial folding is in agreement with MD predictions of transient β-strand formation in the hydrophobic core and C-terminal regions. Sequence-specific protection (i.e. reduced side chain solvent accessibility) upon further assembly into the 2-4mer and oligomer species suggest specific conformers that are selected from the intrinsically disordered monomer ensemble to drive oligomerization. Results of the current study are also consistent with general models developed from previously published solid state NMR oligomer studies from this and other laboratories.

Out-of-Register Aβ42 Assemblies as Models for Neurotoxic Oligomers and Fibrils.

We propose a variant of the recently found S-shaped Aβ1-42-motif that is characterized by out-of-register C-terminal β-strands. We show that chains with this structure can form not only fibrils that are compatible with the NMR signals but also barrel-shaped oligomers that resemble the ones formed by the much smaller cylindrin peptides. By running long all-atom molecular dynamics simulations at physiological temperatures with an explicit solvent, we study the stability of these constructs and show that they are plausible models for neurotoxic oligomers. After analyzing the transitions between different assemblies, we suggest a mechanism for amyloid formation in Alzheimer's disease.

Complexation of NAC-Derived Peptide Ligands with the C-Terminus of α-Synuclein Accelerates Its Aggregation.

Aggregation of α-synuclein (α-Syn) into neurotoxic oligomers and amyloid fibrils is suggested to be the pathogenic mechanism for Parkinson's disease (PD). Recent studies have indicated that oligomeric species of α-Syn are more cytotoxic than their mature fibrillar counterparts, which are responsible for dopaminergic neuronal cell death in PD. Therefore, the effective therapeutic strategies for tackling aggregation-associated diseases would be either to prevent aggregation or to modulate the aggregation process to minimize the formation of toxic oligomers during aggregation. In this work, we showed that arginine-substituted α-Syn ligands, based on the most aggregation-prone sequence of α-Syn, accelerate the protein aggregation in a concentration-dependent manner. To elucidate the mechanism by which Arg-substituted peptides could modulate α-Syn aggregation kinetics, we performed surface plasmon resonance (SPR) spectroscopy, nuclear magnetic resonance (NMR) studies, and all-atom molecular dynamics (MD) simulation. The SPR analysis showed a high binding potency of these peptides with α-Syn but one that was nonspecific in nature. The two-dimensional NMR studies suggest that a large stretch within the C-terminus of α-Syn displays a chemical shift perturbation upon interacting with Arg-substituted peptides, indicating C-terminal residues of α-Syn might be responsible for this class of peptide binding. This is further supported by MD simulation studies in which the Arg-substituted peptide showed the strongest interaction with the C-terminus of α-Syn. Overall, our results suggest that the binding of Arg-substituted ligands to the highly acidic C-terminus of α-Syn leads to reduced charge density and flexibility, resulting in accelerated aggregation kinetics. This may be a potentially useful strategy while designing peptides, which act as α-Syn aggregation modulators.

Oil Palm Phenolics Inhibit the In Vitro Aggregation of β-Amyloid Peptide into Oligomeric Complexes.

Alzheimer's disease is a severe neurodegenerative disease characterized by the aggregation of amyloid-β peptide (Aβ) into toxic oligomers which activate microglia and astrocytes causing acute neuroinflammation. Multiple studies show that the soluble oligomers of Aβ42 are neurotoxic and proinflammatory, whereas the monomers and insoluble fibrils are relatively nontoxic. We show that Aβ42 aggregation is inhibited in vitro by oil palm phenolics (OPP), an aqueous extract from the oil palm tree (Elaeis guineensis). The data shows that OPP inhibits stacking of β-pleated sheets, which is essential for oligomerization. We demonstrate the inhibition of Aβ42 aggregation by (1) mass spectrometry; (2) Congo Red dye binding; (3) 2D-IR spectroscopy; (4) dynamic light scattering; (5) transmission electron microscopy; and (6) transgenic yeast rescue assay. In the yeast rescue assay, OPP significantly reduces the cytotoxicity of aggregating neuropeptides in yeast genetically engineered to overexpress these peptides. The data shows that OPP inhibits (1) the aggregation of Aβ into oligomers; (2) stacking of β-pleated sheets; and (3) fibrillar growth and coalescence. These inhibitory effects prevent the formation of neurotoxic oligomers and hold potential as a means to reduce neuroinflammation and neuronal death and thereby may play some role in the prevention or treatment of Alzheimer's disease.

Imbalances in the Hsp90 Chaperone Machinery: Implications for Tauopathies.

The ATP-dependent 90 kDa heat shock protein, Hsp90, is a major regulator of protein triage, from assisting in nascent protein folding to refolding or degrading aberrant proteins. Tau, a microtubule associated protein, aberrantly accumulates in Alzheimer's disease (AD) and other neurodegenerative diseases, deemed tauopathies. Hsp90 binds to and regulates tau fate in coordination with a diverse group of co-chaperones. Imbalances in chaperone levels and activity, as found in the aging brain, can contribute to disease onset and progression. For example, the levels of the Hsp90 co-chaperone, FK506-binding protein 51 kDa (FKBP51), progressively increase with age. In vitro and in vivo tau models demonstrated that FKBP51 synergizes with Hsp90 to increase neurotoxic tau oligomer production. Inversely, protein phosphatase 5 (PP5), which dephosphorylates tau to restore microtubule-binding function, is repressed with aging and activity is further repressed in AD. Similarly, levels of cyclophilin 40 (CyP40) are reduced in the aged brain and further repressed in AD. Interestingly, CyP40 was shown to breakup tau aggregates in vitro and prevent tau-induced neurotoxicity in vivo. Moreover, the only known stimulator of Hsp90 ATPase activity, Aha1, increases tau aggregation and toxicity. While the levels of Aha1 are not significantly altered with aging, increased levels have been found in AD brains. Overall, these changes in the Hsp90 heterocomplex could drive tau deposition and neurotoxicity. While the relationship of tau and Hsp90 in coordination with these co-chaperones is still under investigation, it is clear that imbalances in these proteins with aging can contribute to disease onset and progression. This review highlights the current understanding of how the Hsp90 family of molecular chaperones regulates tau or other misfolded proteins in neurodegenerative diseases with a particular emphasis on the impact of aging.

Нейропротекторные эффекты антител к глутамату при нарушениях памяти, индуцированных олигомерами провоспалительного белка S100A9, у стареющих животных

Цель исследования - изучение эффектов хронического интраназального введения антител к глутамату совместно с полученными in vitro олигомерами провоспалительного белка S100A9 на процесс воспроизведения пространственной памяти, а также на содержание нейромедиаторных аминокислот и биогенных аминов в релевантных структурах мозга - гиппокампе и префронтальной коре у 12-месячных мышей С57Bl/6. Методика. В поведенческих экспериментах у всех животных проводили выработку условного рефлекса пассивного избегания и тестировали воспроизведение памятного следа, после этого в нейрохимическом исследовании в гиппокампе и префронтальной коре методом ВЖХ проводили определение концентрации нейромедиаторных аминокислот и биогенных аминов. Результаты. Показано, что введение белка S100A9 олигомеров индивидуально приводило к нарушению воспроизведения памятного следа, а в сочетании с антителами к глутамату данного эффекта выявлено не было. Обнаружено значительное повышение концентрации глутамата в обеих церебральных структурах стареющих животных при действии S100A9 олигомеров и снижение содержания аминокислоты при совместном введении с антителами к глутамату до нормы. Показано существенное снижение содержания дофамина в гиппокампе и префронтальной коре в условиях влияния S100A9 олигомеров, а также повышение уровня его метаболитов в гиппокампе с нормализацией обмена дофамина в присутствии антелел к глутамату. Заключение. Выявленные антиамнестические эффекты антител к глутамату и нормализация нейрохимического профиля в условиях вызванной центральной токсичностью S100A9 олигомерами могут быть использованы в разработке подходов нейропротективной коррекции в том числе при болезни Альцгеймера. The aim of this study was to investigate effects of chronic intranasal administration of glutamate antibodies with in vitro -generated proinflammatory S100A9 protein oligomers, on spatial memory of 12-month old C57Bl/6 mice. In addition, the brain content of neurotransmitter amino acids and biogenic amines was monitored in memory-relevant brain structures (hippocampus and prefrontal cortex) of these animals. Methods. In behavioral experiments, all animals were conditioned in a passive avoidance reflex test and the memory trace was evaluated. In a simultaneous neurochemical study, HPLC-electrochemical detection analysis was performed to measure concentrations of neurotransmitter amino acids and biogenic amines in the hippocampus and prefrontal cortex. Results. Administration of S100A9 oligomers alone resulted in disruption of the memory trace retrieval whereas their combination with glutamate antibodies abolished this memory disorder. Significant increases in glutamate concentration were observed in both of the cerebral structures of ageing animals in response to S100A9 oligomers alone, and there was a reduction of the amino acid levels when coadministered with glutamate antibodies. S100A9 oligomers also evoked a decrease in hippocampal and prefrontal cortical dopamine and synchronously elevated dopamine metabolite concentrations, both of these actions being normalized by glutamate antibody coadministration. Conclusion. Disclosure of the antiamnesic effects of glutamate antibodies, along with their neurochemical stabilizing activity to S100A9 oligomer neurotoxicity might be utilized in the development of neuroprotective approaches in Alzheimer’s disease.

Bri2 BRICHOS client specificity and chaperone activity are governed by assembly state

Protein misfolding and aggregation is increasingly being recognized as a cause of disease. In Alzheimer’s disease the amyloid-β peptide (Aβ) misfolds into neurotoxic oligomers and assembles into amyloid fibrils. The Bri2 protein associated with Familial British and Danish dementias contains a BRICHOS domain, which reduces Aβ fibrillization as well as neurotoxicity in vitro and in a Drosophila model, but also rescues proteins from irreversible non-fibrillar aggregation. How these different activities are mediated is not known. Here we show that Bri2 BRICHOS monomers potently prevent neuronal network toxicity of Aβ, while dimers strongly suppress Aβ fibril formation. The dimers assemble into high-molecular-weight oligomers with an apparent two-fold symmetry, which are efficient inhibitors of non-fibrillar protein aggregation. These results indicate that Bri2 BRICHOS affects qualitatively different aspects of protein misfolding and toxicity via different quaternary structures, suggesting a means to generate molecular chaperone diversity.

Foldamer-Mediated Structural Rearrangement Attenuates Aβ Oligomerization and Cytotoxicity.

The conversion of the native random coil amyloid beta (Aβ) into amyloid fibers is thought to be a key event in the progression of Alzheimer's disease (AD). A significant body of evidence suggests that the highly dynamic Aβ oligomers are the main causal agent associated with the onset of AD. Among many potential therapeutic approaches, one is the modulation of Aβ conformation into off-pathway structures to avoid the formation of the putative neurotoxic Aβ oligomers. A library of oligoquinolines was screened to identify antagonists of Aβ oligomerization, amyloid formation, and cytotoxicity. A dianionic tetraquinoline, denoted as 5, was one of the most potent antagonists of Aβ fibrillation. Biophysical assays including amyloid kinetics, dot blot, ELISA, and TEM show that 5 effectively inhibits both Aβ oligomerization and fibrillation. The antagonist activity of 5 toward Aβ aggregation diminishes with sequence and positional changes in the surface functionalities. 5 binds to the central discordant α-helical region and induces a unique α-helical conformation in Aβ. Interestingly, 5 adjusts its conformation to optimize the antagonist activity against Aβ. 5 effectively rescues neuroblastoma cells from Aβ-mediated cytotoxicity and antagonizes fibrillation and cytotoxicity pathways of secondary nucleation induced by seeding. 5 is also equally effective in inhibiting preformed oligomer-mediated processes. Collectively, 5 induces strong secondary structure in Aβ and inhibits its functions including oligomerization, fibrillation, and cytotoxicity.

Initial Structural Models of the Aβ42 Dimer from Replica Exchange Molecular Dynamics Simulations.

Experimental characterization of the molecular structure of small amyloid (A)β oligomers that are currently considered as toxic agents in Alzheimer’s disease is a formidably difficult task due to their transient nature and tendency to aggregate. Such structural information is of importance because it can help in developing diagnostics and an effective therapy for the disease. In this study, molecular simulations and protein–protein docking are employed to explore a possible connection between the structure of Aβ monomers and the properties of the intermonomer interface in the Aβ42 dimer. A structurally diverse ensemble of conformations of the monomer was sampled in microsecond timescale implicit solvent replica exchange molecular dynamics simulations. Representative structures with different solvent exposure of hydrophobic residues and secondary structure content were selected to build structural models of the dimer. Analysis of these models reveals that formation of an intramonomer salt bridge (SB) between Asp23 and Lys28 residues can prevent the building of a hydrophobic interface between the central hydrophobic clusters (CHCs) of monomers upon dimerization. This structural feature of the Aβ42 dimer is related to the difference in packing of hydrophobic residues in monomers with the Asp23–Lys28 SB in on and off states, in particular, to a lower propensity to form hydrophobic contacts between the CHC domain and C-terminal residues in monomers with a formed SB. These findings could have important implications for understanding the difference between aggregation pathways of Aβ monomers leading to neurotoxic oligomers or inert fibrillar structures.

Transient increase in sAPPα secretion in response to Aβ1–42 oligomers: an attempt of neuronal self-defense?

Amyloid precursor protein (APP), a key molecule of Alzheimer disease, is metabolized in 2 antagonist pathways generating the soluble APP alpha (sAPPα) having neuroprotective properties and the beta amyloid (Aβ) peptide at the origin of neurotoxic oligomers, particularly Aβ1–42. Whether extracellular Aβ1–42 oligomers modulate the formation and secretion of sAPPα is not known. We report here that the addition of Aβ1–42 oligomers to primary cortical neurons induced a transient increase in α-secretase activity and secreted sAPPα 6–9 hours later. Preventing the generation of sAPPα by using small interfering RNAs (siRNAs) for the α-secretases ADAM10 and ADAM17 or for APP led to increased Aβ1–42 oligomer–induced cell death after 24 hours. Neuronal injuries due to oxidative stress or growth factor deprivation also generated sAPPα 7 hours later. Finally, acute injection of Aβ1–42 oligomers into wild-type mouse hippocampi induced transient secretion of sAPPα 48–72 hours later. Altogether, these data suggest that neurons respond to stress by generating sAPPα for their survival. These data must be taken into account when interpreting sAPPα levels as a biomarker in neurological disorders.

Dietary arachidonic acid increases deleterious effects of amyloid-\u03b2 oligomers on learning abilities and expression of AMPA receptors: putative role of the ACSL4-cPLA2 balance

BackgroundPolyunsaturated fatty acids play a crucial role in neuronal function, and the modification of these compounds in the brain could have an impact on neurodegenerative diseases such as Alzheimer’s disease. Despite the fact that arachidonic acid is the second foremost polyunsaturated fatty acid besides docosahexaenoic acid, its role and the regulation of its transfer and mobilization in the brain are poorly known.MethodsTwo groups of 39 adult male BALB/c mice were fed with an arachidonic acid-enriched diet or an oleic acid-enriched diet, respectively, for 12 weeks. After 10 weeks on the diet, mice received intracerebroventricular injections of either NaCl solution or amyloid-β peptide (Aβ) oligomers. Y-maze and Morris water maze tests were used to evaluate short- and long-term memory. At 12 weeks on the diet, mice were killed, and blood, liver, and brain samples were collected for lipid and protein analyses.ResultsWe found that the administration of an arachidonic acid-enriched diet for 12 weeks induced short-term memory impairment and increased deleterious effects of Aβ oligomers on learning abilities. These cognitive alterations were associated with modifications of expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, postsynaptic density protein 95, and glial fibrillary acidic protein in mouse cortex or hippocampus by the arachidonic acid-enriched diet and Aβ oligomer administration. This diet also led to an imbalance between the main ω-6 fatty acids and the ω-3 fatty acids in favor of the first one in erythrocytes and the liver as well as in the hippocampal and cortical brain structures. In the cortex, the dietary arachidonic acid also induced an increase of arachidonic acid-containing phospholipid species in phosphatidylserine class, whereas intracerebroventricular injections modified several arachidonic acid- and docosahexaenoic acid-containing species in the four phospholipid classes. Finally, we observed that dietary arachidonic acid decreased the expression of the neuronal form of acyl-coenzyme A synthetase 4 in the hippocampus and increased the cytosolic phospholipase A2 activation level in the cortices of the mice.ConclusionsDietary arachidonic acid could amplify Aβ oligomer neurotoxicity. Its consumption could constitute a risk factor for Alzheimer’s disease in humans and should be taken into account in future preventive strategies. Its deleterious effect on cognitive capacity could be linked to the balance between arachidonic acid-mobilizing enzymes.

Development and comprehensive comparison of two on-line capillary electrophoretic methods for β-secretase inhibitor screening

Alzheimer’s disease is the most common cause of dementia, afflicting over 34 million patients worldwide. Since β-secretase is a rate-limiting enzyme of the production of neurotoxic β-amyloid peptide oligomers abnormally accumulated in the affected brain tissue, its specific inhibition appears to be a promising approach to slowing down or even stopping the progression of the disease. Hence two on-line capillary electrophoretic methods for studies of β-secretase activity based on the principles of transverse diffusion of laminar flow profiles and electrophoretically mediated microanalysis were developed, both using a simple unlabeled peptide substrate and UV detection. The optimized procedures were thoroughly validated and applied for determining the enzyme’s kinetic parameters and the inhibition characteristics of two potent probe inhibitors. The resulting values were found to be comparable to literature data obtained with other analytical techniques. The suitability of the employed methodologies for different experimental designs is discussed on the basis of a statistical evaluation of the experimental data. The presented methods constitute a miniaturized and fully automated tool, which should be suitable for kinetic and inhibition studies of β-secretase as a target for Alzheimer’s disease drug discovery in the early stages of the development of a new drug.

The Importance of Mitophagy in Maintaining Mitochondrial Function in U373MG Cells. Bafilomycin A1 Restores Aminochrome-Induced Mitochondrial Damage.

Aminochrome, an orthoquinone formed during the dopamine oxidation of neuromelanin, is neurotoxic because it induces mitochondria dysfunction, protein degradation dysfunction (both autophagy and proteasomal systems), α-synuclein aggregation to neurotoxic oligomers, neuroinflammation, and oxidative and endoplasmic reticulum stress. In this study, we investigated the relationship between aminochrome-induced autophagy/lysosome dysfunction and mitochondrial dysfunction in U373MGsiGST6 cells. Aminochrome (75 μM) induces mitochondrial dysfunction as determined by (i) a significant decrease in ATP levels (70%; P < 0.001) and (ii) a significant decrease in mitochondrial membrane potential (P < 0.001). Interestingly, the pretreatment of U373MGsiGST6 cells with 100 nM bafilomycin-A1, an inhibitor of lysosomal vacuolar-type H+-ATPase, restores ATP levels, mitochondrial membrane potential, and mitophagy, and decreases cell death. These results reveal (i) the importance of macroautophagy/the lysosomal degradation system for the normal functioning of mitochondria and for cell survival, and (ii) aminochrome-induced lysosomal dysfunction depends on the aminochrome-dependent inactivation of the vacuolar-type H+-ATPase, which pumps protons into the lysosomes. This study also supports the proposed protective role of glutathione transferase mu2-2 (GSTM2) in astrocytes against aminochrome toxicity, mediated by mitochondrial and lysosomal dysfunction.

Surprisingly high stability of the Aβ oligomer eliminating all-d-enantiomeric peptide D3 in media simulating the route of orally administered drugs

The aggregation of the amyloid β protein (Aβ) plays an important role in the pathology of Alzheimer's disease. Previously, we have developed the all-d-enantiomeric peptide D3, which is able to eliminate neurotoxic Aβ oligomers in vitro and improve cognition in a transgenic Alzheimer's disease mouse model in vivo even after oral administration. d-Peptides are expected to be more resistant against enzymatic proteolysis compared to their l-enantiomeric equivalents, and indeed, a pharmacokinetic study with tritiated D3 revealed the oral bioavailability to be about 58%. To further investigate the underlying properties, we examined the stability of D3 in comparison to its corresponding all-l-enantiomeric mirror image l-D3 in media simulating the gastrointestinal tract, blood and liver. Potential metabolization was followed by reversed-phase high-performance liquid chromatography. In simulated gastric fluid, D3 remained almost completely stable (89%) within 24h, while 70% of l-D3 was degraded within the same time period. Notably, in simulated intestinal fluid, D3 also remained stable (96%) for 24h, whereas l-D3 was completely metabolized within seconds. In human plasma and human liver microsomes, l-D3 was metabolized several hundred times faster than D3. The remarkably high stability may explain the high oral bioavailability seen in previous studies allowing oral administration of the drug candidate. Thus, all-d-enantiomeric peptides may represent a promising new compound class for drug development.

INT131 increases dendritic arborization and protects against Aβ toxicity by inducing mitochondrial changes in hippocampal neurons

In previous studies, we have demonstrated the beneficial effects of classic PPARγ agonists on neuroprotection against Aβ oligomer neurotoxicity in a double transgenic mouse model of Alzheimer' disease (AD). INT-131, a novel, non-thiazolidinedione compound that belongs to a new family of drugs, selective PPARγ modulators (SPPARMs), has provided an emerging opportunity for the treatment of type 2 diabetes mellitus and metabolic syndrome. However, its role in the central nervous system has not been studied. The aim of this study was to evaluate the putative neuroprotective role of INT131 in hippocampal neurons. We found that INT131 increased dendritic branching, promoted neuronal survival against Aβ amyloid, increased expression of PGC1-1α and modulated neuronal mitochondrial dynamics. Our results suggest that INT131, a drug that has been shown to be safe and effective in metabolic disorders, may constitute a new therapeutic alternative for AD.