Amyloid-ß Effects on Peripheral Nerve: A New Model System.

Although there are many biochemical methods to measure amyloid-ß (Aß)42 concentration, one of the critical issues in the study of the effects of Aß42 on the nervous system is a simple physiological measurement. The in vitro rat sciatic nerve model is employed and the nerve action potential (NAP) is quantified with different stimuli while exposed to different concentrations of Aß42. Aß42 predominantly reduces the NAP amplitude with minimal effects on other parameters except at low stimulus currents and short inter-stimulus intervals. The effects of Aß42 are significantly concentration-dependent, with a maximum reduction in NAP amplitude at a concentration of 70 nM and smaller effects on the NAP amplitude at higher and lower concentrations. However, even physiologic concentrations in the range of 70 pM did reduce the NAP amplitude. The effects of Aß42 became maximal 5-8 h after exposure and did not reverse during a 30 min washout period. The in vitro rat sciatic nerve model is sensitive to the effects of physiologic concentrations of Aß42. These experiments suggest that the effect of Aß42 is a very complex function of concentration that may be the result of amyloid-related changes in membrane properties or sodium channels.

A Nanoenzyme Constructed from Manganese and Strandberg-Type Phosphomolybdate with Versatility in Antioxidant and Modulating Conformation of Aß Protein Misfolding Aggregates In Vitro.

Amyloid ß-peptide (Aß) misfolding aggregates with ß-sheet structures and surplus reactive oxygen species (ROS) are both considered to be the culprit of neuronal toxicity in Alzheimer's disease (AD). Therefore, modulating the misfolding mode of Aß and inhibiting ROS simultaneous has become an important method for anti-AD. Herein, a nanoscale manganese-substituted polyphosphomolybdate (H2en)3[Mn(H2O)4][Mn(H2O)3]2[P2Mo5O23]2·14.5H2O (abbreviated as MnPM) (en = ethanediamine) was designed and synthesized by single crystal to single crystal transformation method. MnPM can modulate the ß-sheet rich conformation of Aß aggregates, and thus reduce the formation of toxic species. Moreover, MnPM also possesses the ability to eliminate the free radicals produced by Cu2+-Aß aggregates. It can inhibit the cytotoxicity of ß-sheet-rich species and protect synapses of PC12 cells. MnPM combines the conformation modulating ability of Aß and anti-oxidation ability, which makes a promising multi-funcational molecular with a composite mechanism for the new conceptual designing in treatment of such protein-misfolding diseases.

Substrate recruitment by γ-secretase.

γ-Secretase is a membrane-embedded protease complex that is crucial for many physiological processes throughout life. Due to its pivotal role in the etiology of Alzheimer's disease (AD), in particular the familial forms of the disease, the enzyme is one of the most studied intramembrane proteases and an important drug target. By cleaving a C-terminal fragment of the ß-amyloid precursor protein (APP), γ-secretase generates several amyloid ß-peptide (Aß) species including longer, neurotoxic forms such as Aß42 that are a widely believed to trigger AD. Besides APP, γ-secretase cleaves numerous other substrates including most prominently Notch1, whose cleavage by γ-secretase is essential for cell differentiation and affected in certain types of cancer. In this review, we will describe the exciting progress made in our understanding of how the γ-secretase complex recognizes and recruits its substrates to its catalytic subunit presenilin for their intramembrane proteolytic cleavage. This complicated process is not well understood and only recently insights from biochemical studies and structural biology are beginning to reveal this secret of γ-secretase.

Deep neural network based CEST and AREX processing: Application in imaging a model of Alzheimer's disease at 3 T.

PURPOSE: To optimize and apply deep neural network based CEST (deepCEST) and apparent exchange dependent-relaxation (deepAREX) for imaging the mouse brain with Alzheimer's disease (AD) at 3T MRI. METHODS: CEST and T1 data of central and anterior brain slices of 10 AD mice and 10 age-matched wild type (WT) mice were acquired at a 3T animal MRI scanner. The networks of deepCEST/deepAREX were optimized and trained on the WT data. The CEST/AREX contrasts of AD and WT mice predicted by the networks were analyzed and further validated by immunohistochemistry. RESULTS: After optimization and training on CEST data of WT mice, deepCEST/deepAREX could rapidly (~1 s) generate precise CEST and AREX results for unseen CEST data of AD mice, indicating the accuracy and generalization of the networks. Significant lower amide weighted (3.5 ppm) signal related to amyloid ß-peptide (Aß) plaque depositions, which was validated by immunohistochemistry results, was detected in both central and anterior brain slices of AD mice compared to WT mice. Decreased magnetization transfer (MT) signal was also found in AD mice especially in the anterior slice. CONCLUSION: DeepCEST/deepAREX could rapidly generate accurate CEST/AREX contrasts in animal study. The well-optimized deepCEST/deepAREX have potential for AD differentiation at 3T MRI.

Aß43-producing PS1 FAD mutants cause altered substrate interactions and respond to γ-secretase modulation.

Abnormal generation of neurotoxic amyloid-ß peptide (Aß) 42/43 species due to mutations in the catalytic presenilin 1 (PS1) subunit of γ-secretase is the major cause of familial Alzheimer's disease (FAD). Deeper mechanistic insight on the generation of Aß43 is still lacking, and it is unclear whether γ-secretase modulators (GSMs) can reduce the levels of this Aß species. By comparing several types of Aß43-generating FAD mutants, we observe that very high levels of Aß43 are often produced when presenilin function is severely impaired. Altered interactions of C99, the precursor of Aß, are found for all mutants and are independent of their particular effect on Aß production. Furthermore, unlike previously described GSMs, the novel compound RO7019009 can effectively lower Aß43 production of all mutants. Finally, substrate-binding competition experiments suggest that RO7019009 acts mechanistically after initial C99 binding. We conclude that altered C99 interactions are a common feature of diverse types of PS1 FAD mutants and that also patients with Aß43-generating FAD mutations could in principle be treated by GSMs.

Dysfunction of Mitochondria in Alzheimer's Disease: ANT and VDAC Interact with Toxic Proteins and Aid to Determine the Fate of Brain Cells.

Alzheimer's disease (AD), certainly the most widespread proteinopathy, has as classical neuropathological hallmarks, two groups of protein aggregates: senile plaques and neurofibrillary tangles. However, the research interest is rapidly gaining ground in a better understanding of other pathological features, first, of all the mitochondrial dysfunctions. Several pieces of evidence support the hypothesis that abnormal mitochondrial function may trigger aberrant processing of amyloid progenitor protein or tau and thus neurodegeneration. Here, our aim is to emphasize the role played by two 'bioenergetic' proteins inserted in the mitochondrial membranes, inner and outer, respectively, that is, the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC), in the progression of AD. To perform this, we will magnify the ANT and VDAC defects, which are measurable hallmarks of mitochondrial dysfunction, and collect all the existing information on their interaction with toxic Alzheimer's proteins. The pathological convergence of tau and amyloid ß-peptide (Aß) on mitochondria may finally explain why the therapeutic strategies used against the toxic forms of Aß or tau have not given promising results separately. Furthermore, the crucial role of ANT-1 and VDAC impairment in the onset/progression of AD opens a window for new therapeutic strategies aimed at preserving/improving mitochondrial function, which is suspected to be the driving force leading to plaque and tangle deposition in AD.

The Moringin/α-CD Pretreatment Induces Neuroprotection in an In Vitro Model of Alzheimer's Disease: A Transcriptomic Study.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and represents the most common form of senile dementia. Autophagy and mitophagy are cellular processes that play a key role in the aggregation of ß-amyloid (Aß) and tau phosphorylation. As a consequence, impairment of these processes leads to the progression of AD. Thus, interest is growing in the search for new natural compounds, such as Moringin (MOR), with neuroprotective, anti-amyloidogenic, antioxidative, and anti-inflammatory properties that could be used for AD prevention. However, MOR appears to be poorly soluble and stable in water. To increase its solubility MOR was conjugated with α-cyclodextrin (MOR/α-CD). In this work, it was evaluated if MOR/α-CD pretreatment was able to exert neuroprotective effects in an AD in vitro model through the evaluation of the transcriptional profile by next-generation sequencing (NGS). To induce the AD model, retinoic acid-differentiated SH-SY5Y cells were exposed to Aß1-42. The MOR/α-CD pretreatment reduced the expression of the genes which encode proteins involved in senescence, autophagy, and mitophagy processes. Additionally, MOR/α-CD was able to induce neuronal remodeling modulating the axon guidance, principally downregulating the Slit/Robo signaling pathway. Noteworthy, MOR/α-CD, modulating these important pathways, may induce neuronal protection against Aß1-42 toxicity as demonstrated also by the reduction of cleaved caspase 3. These data indicated that MOR/α-CD could attenuate the progression of the disease and promote neuronal repair.

Studies of transmembrane peptides by pulse dipolar spectroscopy with semi-rigid TOPP spin labels.

Electron paramagnetic resonance (EPR)-based pulsed dipolar spectroscopy measures the dipolar interaction between paramagnetic centers that are separated by distances in the range of about 1.5-10 nm. Its application to transmembrane (TM) peptides in combination with modern spin labelling techniques provides a valuable tool to study peptide-to-lipid interactions at a molecular level, which permits access to key parameters characterizing the structural adaptation of model peptides incorporated in natural membranes. In this mini-review, we summarize our approach for distance and orientation measurements in lipid environment using novel semi-rigid TOPP [4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-L-phenylglycine] labels specifically designed for incorporation in TM peptides. TOPP labels can report single peak distance distributions with sub-angstrom resolution, thus offering new capabilities for a variety of TM peptide investigations, such as monitoring of various helix conformations or measuring of tilt angles in membranes.

Transmembrane ß-peptide helices as molecular rulers at the membrane surface.

ß-Peptides are known to form 14-helices with high conformational rigidity, helical persistence length, and well-defined spacing and orientation regularity of amino acid side chains. Therefore, ß-peptides are well suited to serve as backbone structures for molecular rulers. On the one hand, they can be functionalized in a site-specific manner with molecular probes or fluorophores, and on the other hand, the ß-peptide helices can be recognized and anchored in a biological environment of interest. In this study, the ß-peptide helices were anchored in lipid bilayer membranes, and the helices were elongated in the outer membrane environment. The distances of the covalently bound probes to the membrane surface were determined using graphene-induced energy transfer (GIET) spectroscopy, a method based on the distance-dependent quenching of a fluorescent molecule by a nearby single graphene sheet. As a proof of principle, the predicted distances were determined for two fluorophores bound to the membrane-anchored ß-peptide molecular ruler.

Live Cell FRET Imaging Reveals Amyloid ß-Peptide Oligomerization in Hippocampal Neurons.

Amyloid ß-peptide (Aß) oligomerization is believed to contribute to the neuronal dysfunction in Alzheimer disease (AD). Despite decades of research, many details of Aß oligomerization in neurons still need to be revealed. Förster resonance energy transfer (FRET) is a simple but effective way to study molecular interactions. Here, we used a confocal microscope with a sensitive Airyscan detector for FRET detection. By live cell FRET imaging, we detected Aß42 oligomerization in primary neurons. The neurons were incubated with fluorescently labeled Aß42 in the cell culture medium for 24 h. Aß42 were internalized and oligomerized in the lysosomes/late endosomes in a concentration-dependent manner. Both the cellular uptake and intracellular oligomerization of Aß42 were significantly higher than for Aß40. These findings provide a better understanding of Aß42 oligomerization in neurons.

Inhibition of Aß aggregates in Alzheimer's disease by epigallocatechin and epicatechin-3-gallate from green tea.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive accumulation of senile plaques, which are primarily composed of misfolded amyloid ß-peptide (Aß). Aß aggregates are believed to be a key factor in the pathogenesis of AD, affecting the nervous system in human body. The therapeutic potential of tea-derived polyphenolic compounds, (-)-epigallocatechin (EGC) and (-)-epicatechin-3-gallate (ECG), for AD was investigated by assessing their effects on the Cu2+/Zn2+-induced or self-assembled Aß40 aggregation using thioflavine T fluorescent spectrometry, inductively coupled plasma mass spectrometry, UV-Vis spectroscopy, transmission electron microscope, silver staining, immunohistochemistry, and immunofluorescence assays. EGC and ECG mildly bind to Cu2+ and Zn2+, and diminish the Cu2+- or Zn2+-induced or self-assembled Aß aggregates; they also modulate the Cu2+/Zn2+-Aß40 induced neurotoxicity on mouse neuroblastoma Neuro-2a cells by reducing the production of ROS. Metal chelating, hydrogen bonding or Van Der Waals force may drive the interaction between the polyphenolic compounds and Aß. The results demonstrate that green tea catechins EGC and ECG are able to alleviate the toxicity of Aß oligomers and fibrils. Particularly, ECG can cross the blood-brain barrier to reduce the Aß plaques in the brain of APP/PS1 mice, thereby protecting neurons from injuries. The results manifest the potential of green tea for preventing or ameliorating the symptoms of AD.

Redox proteomics and amyloid ß-peptide: insights into Alzheimer disease.

Alzheimer disease (AD) is a progressive neurodegenerative disorder associated with aging and characterized pathologically by the presence of senile plaques, neurofibrillary tangles, and neurite and synapse loss. Amyloid beta-peptide (1-42) [Aß(1-42)], a major component of senile plaques, is neurotoxic and induces oxidative stress in vitro and in vivo. Redox proteomics has been used to identify proteins oxidatively modified by Aß(1-42) in vitro and in vivo. In this review, we discuss these proteins in the context of those identified to be oxidatively modified in animal models of AD, and human studies including familial AD, pre-clinical AD (PCAD), mild cognitive impairment (MCI), early AD, late AD, Down syndrome (DS), and DS with AD (DS/AD). These redox proteomics studies indicate that Aß(1-42)-mediated oxidative stress occurs early in AD pathogenesis and results in altered antioxidant and cellular detoxification defenses, decreased energy yielding metabolism and mitochondrial dysfunction, excitotoxicity, loss of synaptic plasticity and cell structure, neuroinflammation, impaired protein folding and degradation, and altered signal transduction. Improved access to biomarker imaging and the identification of lifestyle interventions or treatments to reduce Aß production could be beneficial in preventing or delaying the progression of AD. This article is part of the special issue "Proteomics".

Piezoelectric property of bundled peptide nanotubes stapled by bis-cyclic-ß-peptide.

Cyclic tetra-ß-peptides (CP4s) and a bis-CP4 were synthesized to prepare peptide nanotubes (PNTs) by molecular stacking of cyclic peptides. The addition of bis-CP4 to the PNT preparation afforded PNT bundles increasing the direct and converse piezoelectiric coefficients, which is ascribable to bis-CP4 stapling PNTs into the parallel alignment of PNT dipoles.

Ion mobility spectrometry combined with multivariate statistical analysis: revealing the effects of a drug candidate for Alzheimer's disease on Aß1-40 peptide early assembly.

Inhibition of the initial stages of amyloid-ß peptide self-assembly is a key approach in drug development for Alzheimer's disease, in which soluble and highly neurotoxic low molecular weight oligomers are produced and aggregate in the brain over time. Here we report a high-throughput method based on ion mobility mass spectrometry and multivariate statistical analysis to rapidly select statistically significant early-stage species of amyloid-ß1-40 whose formation is inhibited by a candidate theranostic agent. Using this method, we have confirmed the inhibition of a Zn-porphyrin-peptide conjugate in the early self-assembly of Aß40 peptide. The MS/MS fragmentation patterns of the species detected in the samples containing the Zn-porphyrin-peptide conjugate suggested a porphyrin-catalyzed oxidation at Met-35(O) of Aß40. We introduce ion mobility MS combined with multivariate statistics as a systematic approach to perform data analytics in drug discovery/amyloid research that aims at the evaluation of the inhibitory effect on the Aß early assembly in vitro models at very low concentration levels of Aß peptides.

Computational Study on the Assembly of Amyloid ß-Peptides in the Hydrophobic Environment.

Fibrillated aggregation of amyloid ß (Aß) peptides is a potential factor causing toxic amyloid deposition in neurodegenerative diseases. A toxic fibril formation of Aß is known to be enhanced on the ganglioside-rich lipid membrane containing some amounts of cholesterol and sphingomyelin. This ganglioside-rich membrane is supposed to provide a hydrophobic environment that promotes the formation of Aß fibrils. Molecular dynamics simulations were carried out to investigate the structure of Aß complex in the hydrophobic solution composed of dioxane and water molecules. The Aß conformation was contrasted to that in the aqueous condition by executing multiple computational trials with the calculation models containing one, four, or six Aß peptides. The conformation was also compared between the calculations with the 42-mer (Aß42) and 40-mer (Aß40) peptides. The simulations for Aß42 demonstrated that Aß peptides had a tendency to stretch out in the hydrophobic environment. In contrast, Aß peptides were closely packed in the aqueous solution, and the motions of Aß peptides were suppressed significantly. The N-terminal polar domains of Aß peptides tended to be positioned at the inside of the Aß complex in the hydrophobic environment, which supported the C-terminal domains in expanding outward for inter-molecular interaction. Since Aß peptides were not tightly packed in the hydrophobic environment, the total surface area of the Aß complex in the hydrophobic solution was larger than that in the aqueous one. The simulation for Aß40 peptides also showed a difference between the hydrophobic and aqueous solutions. The difference was compatible with the results of Aß42 in the structure of the Aß complex, while the C-terminal outward expansion was not so distinct as Aß42 peptides.

Production of the herb Ruta chalepensis L. expressing amyloid ß-GFP fusion protein.

The herb Ruta chalepensis L. exhibits medical effects, such as anti-inflammatory, central nervous system depressant, and antipyretic activities. However, a genetic transformation method has not yet been developed for this species. In this paper, a simple and efficient tissue culture and genetic transformation system for R. chalepensis is reported. An amyloid ß-peptide (Aß) gene, which is considered to be a causative agent of Alzheimer's disease (AD), fused with green-fluorescent protein (GFP), was introduced into R. chalepensis. When the leaves of R. chalepensis expressing Aß-GFP were administered orally to C57BL/6J mice, serum anti-Aß antibody titers of several mice were elevated without the use of an adjuvant. These results indicated that an oral vaccine against AD using R. chalepensis may be feasible. R. chalepensis is rich in bioactive compounds that may have synergistic effects with the vaccine for AD. Plant-derived vaccines are safer and cheaper than those produced from animal cells or microbes, because plants can serve as biofactories at low cost and with high biosynthetic capacity.

Pelargonidin exhibits restoring effects against amyloid ß-induced deficits in the hippocampus of male rats.

Background: Alzheimer's disease (AD) is characterized by amyloid-beta plaques, neuronal loss, and cognitive dysfunction. Oxidative stress plays a key role in the pathophysiology of AD, and it has been suggested that antioxidants may slow the progress of the disease. In this study, the possible protective effects of pelargonidin (a natural flavonoid) against amyloid ß (Aß)-induced behavioral deficits was investigated in rats. Methods: Adult Wistar male rats were treated with intrahippocampal injections of the Aß (aa 25-35) and intraperitoneal injection of pelargonidin. Learning and spatial memory were tested using the Morris water maze (MWM) task. The antioxidant activity was evaluated using the ferric reducing/antioxidant power assay (FRAP assay). Data were analyzed using SPSS 20, and value of p≤0.05 was considered significant. Results: The results of this study showed that Aß significantly increased escape latency and the distance traveled in the MWM, and pelargonidin attenuated these behavioral changes. Aß induced a significant decrease in the total thiol content of hippocampus, and pelargonidin restored the hippocampal antioxidant capacity. Conclusion: The results of this study suggest that pelargonidin can improve Aß-induced behavioral changes in rats.

Near-Infrared Switchable Fullerene-Based Synergy Therapy for Alzheimer's Disease.

C60 has a special dual function; it can act as both a powerful reactive oxygen species (ROS) producer under UV or visible light and an ROS scavenger in the dark. However, ROS has double-edged effects in living systems. It is still a great challenge for biomedical application to switch and adjust the two opposite properties of C60 in one system. Herein, UCNP@C60 -pep (UCNP: upconversion nanoparticle, pep: Aß-target peptide KLVFF) is designed as a near-infrared-switchable nanoplatform for synergy therapy of Alzheimer's disease (AD). Under near-infrared (NIR) light, the Aß-targeting hybrid nanoparticles produce ROS and result in Aß photooxygenation, which can hinder Aß aggregation and mitigate the attendant cytotoxicity. In the dark, UCNP@C60 -pep shows protective effects against the increased oxidative stress. The ROS-generating and ROS-quenching abilities of UCNP@C60 -pep are both beneficial for decreasing Aß-induced neurotoxicity and extending the longevity of the commonly used transgenic AD model Caenorhabditis elegans CL2006. Moreover, UCNP@C60-pep can also be used for upconversion luminescence (UCL) and magnetic resonance imaging (MRI), which has benefits for "image-guided therapy." This study may offer a new perspective for the biological applications of C60 .

12/10-Helix in Mixed ß-Peptides Alternating Bicyclic and Acyclic ß-Amino Acids: Probing the Relationship between Bicyclic Side Chain and Helix Stability.

12/10-Helices constitute suitable templates that can be used to design original structures. Nevertheless, they often suffer from a weak stability in polar solvents because they exhibit a mixed hydrogen-bond network resulting in a small macrodipole. In this work, stable and functionalizable 12/10-helices were developed by alternating a highly constrained ß2, 3, 3 -trisubstituted bicyclic amino acid (S)-1-aminobicyclo[2.2.2]octane-2-carboxylic acid ((S)-ABOC) and an acyclic substituted ß-homologated proteinogenic amino acid (l-ß3 -hAA). Based on NMR spectroscopic analysis, it was shown that such mixed ß-peptides display well-defined right-handed 12/10-helices in polar, apolar, and chaotropic solvents; that are, CD3 OH, CDCl3 , and [D6 ]DMSO, respectively. The stability of the hydrogen bonds forming the C10 and C12 pseudocycles as well as the benefit provided by the use of the constrained bicyclic ABOC versus typical acyclic ß-amino acids sequences when designing 12/10-helix were investigated using NH/ND NMR exchange experiments and DFT calculations in various solvents. These studies showed that the ß3 -hAA/(S)-ABOC helix displayed a more stable hydrogen-bond network through specific stabilization of the C10 pseudocycles involving the bridgehead NH of the ABOC bicyclic scaffold.

Searching for improved mimetic peptides inhibitors preventing conformational transition of amyloid-ß42 monomer.

A series of novel mimetic peptides were designed, synthesised and biologically evaluated as inhibitors of Aß42 aggregation. One of the synthesised peptidic compounds, termed compound 7 modulated Aß42 aggregation as demonstrated by thioflavin T fluorescence, acting also as an inhibitor of the cytotoxicity exerted by Aß42 aggregates. The early stage interaction between compound 7 and the Aß42 monomer was investigated by replica exchange molecular dynamics (REMD) simulations and docking studies. Our theoretical results revealed that compound 7 can elongate the helical conformation state of an early stage Aß42 monomer and it helps preventing the formation of ß-sheet structures by interacting with key residues in the central hydrophobic cluster (CHC). This strategy where early "on-pathway" events are monitored by small molecules will help the development of new therapeutic strategies for Alzheimer's disease.