Removal and identification of external protein corona members from RBC-derived extracellular vesicles by surface manipulating antimicrobial peptides.

In the last years, extracellular vesicles (EVs), secreted by various cells and body fluids have shown extreme potential in biomedical applications. Increasing number of studies suggest that a protein corona could adhere to the surface of EVs which can have a fundamental effect on their function, targeting and therapeutical efficacy. However, removing and identifying these corona members is currently a challenging task to achieve. In this study we have employed red blood cell-derived extracellular vesicles (REVs) as a model system and three membrane active antimicrobial peptides (AMPs), LL-37, FK-16 and CM15, to test whether they can be used to remove protein corona members from the surface of vesicles. These AMPs were reported to preferentially exert their membrane-related activity via one of the common helical surface-covering models and do not significantly affect the interior of lipid bilayer bodies. The interaction between the peptides and the REVs was followed by biophysical techniques, such as flow-linear dichroism spectroscopy which provided the effective applicable peptide concentration for protein removal. REV samples were then subjected to subsequent size exclusion chromatography and to proteomics analysis. Based on the comparison of control REVs with the peptide treated samples, seventeen proteins were identified as external protein corona members. From the three investigated AMPs, FK-16 can be considered as the best candidate to further optimize EV-related applicability of AMPs. Our results on the REV model system envisage that membrane active peptides may become a useful set of tools in engineering and modifying surfaces of EVs and other lipid-based natural particles.

Effects of sub-chronic, in vivo administration of sigma-1 receptor ligands on platelet and aortic arachidonate cascade in streptozotocin-induced diabetic rats.

BACKGROUND: Diabetes mellitus is a chronic metabolic disorder which induces endothelial dysfunction and platelet activation. Eicosanoids produced from arachidonic acid regulate cellular and vascular functions. Sigma-1 receptors (S1R) are expressed in platelets and endothelial cells and S1R expression is protective in diabetes. OBJECTIVES: Our aim was to examine the influence of sub-chronic, in vivo administered S1R ligands PRE-084, (S)-L1 (a new compound) and NE-100 on the ex vivo arachidonic acid metabolism of platelets and aorta in streptozotocin-induced diabetic rats. METHODS: The serum level of the S1R ligands was detected by LC-MS/MS before the ex vivo analysis. Sigma-1 receptor and cyclooxygenase gene expression in platelets were determined by RT-qPCR. The eicosanoid synthesis was examined with a radiolabelled arachidonic acid substrate and ELISA. RESULTS: One month after the onset of STZ-induced diabetes, in vehicle-treated, diabetic rat platelet TxB2 and aortic 6-k-PGF1α production dropped. Sub-chronic in vivo treatment of STZ-induced diabetes in rats for one week with PRE-084 enhanced vasoconstrictor and platelet aggregator and reduced vasodilator and anti-aggregator cyclooxygenase product formation. (S)-L1 reduced the synthesis of vasodilator and anti-aggregator cyclooxygenase metabolites and promoted the recovery of physiological platelet function in diabetic rats. The S1R antagonist NE-100 produced no significant changes in platelet arachidonic acid metabolism. (S)-L1 decreased the synthesis of vasoconstrictor and platelet aggregator cyclooxygenase metabolites, whereas NE-100 increased the quantity of aortic vasodilator and anti-aggregator cyclooxygenase products and promoted the recovery of diabetic endothelial dysfunction in the aorta. The novel S1R ligand, (S)-L1 had similar effects on eicosanoid synthesis in platelets as the agonist PRE-084 and in aortas as the antagonist NE-100. CONCLUSIONS: S1R ligands regulate cellular functions and local blood circulation by influencing arachidonic acid metabolism. In diabetes mellitus, the cell-specific effects of S1R ligands have a compensatory role and aid in restoring physiological balance between the platelet and vessel.

Peptide-based chemical models for lytic polysaccharide monooxygenases.

Copper(II) complexes of HPH-NH2 (L1) and HPHPY-NH2 (L2) peptides have been studied as small molecular models of lytic polysaccharide monooxygenases by pH-potentiometry and UV-vis, CD and EPR spectroscopy. The coordination properties of these ligands are fundamentally different from those of other non-protected N-terminal HXH-sequences concerning the metal binding ability of amide nitrogens. The proline units prevent the formation of fused chelates with the participation of amide nitrogens; therefore, instead of ATCUN-type {NH2,2N-,Nim} coordination, dimer complexes (Cu2HxL2, where x = -1, -2, and -3 for L1 and 1, 0, and -1 for L2) are formed in equimolar systems above pH 5. Using H2O2 as the oxidant and PNPG as the activated substrate, these dimer complexes were proved to be relevant functional models of LPMOs, even at neutral pH. Although the tyrosine residue in L2 participates in the coordination at pH 7-9.6, it does not seem to play a role in the oxidation process. In the presence of H2O2, the dimer complexes partially dissociate to form mononuclear hydroperoxo complexes, which are stable for 1-2 hours in equimolar concentrations of H2O2. On the other hand, with excess H2O2 both their formation and their decomposition are faster. The decay of (hydro)peroxo complexes, after longer reaction times, results in the evolution of dioxygen bubbles and the formation of Cu(I) (probably through catalytic disproportionation). However, in the presence of PNPG, the formation of dioxygen bubbles was not observed. Therefore, we assumed that the formed Cu(I) complexes bind H2O2 and enter into a similar catalytic cycle as suggested recently for native LPMOs.

Examination of Longitudinal Alterations in Alzheimer's Disease-Related Neurogenesis in an APP/PS1 Transgenic Mouse Model, and the Effects of P33, a Putative Neuroprotective Agent Thereon.

Neurogenesis plays a crucial role in cognitive processes. During aging and in Alzheimer's disease (AD), altered neurogenesis and neuroinflammation are evident both in C57BL/6J, APPSwe/PS1dE9 (Tg) mice and humans. AD pathology may slow down upon drug treatment, for example, in a previous study of our group P33, a putative neuroprotective agent was found to exert advantageous effects on the elevated levels of APP, Aß, and neuroinflammation. In the present study, we aimed to examine longitudinal alterations in neurogenesis, neuroinflammation and AD pathology in a transgenic (Tg) mouse model, and assessed the putative beneficial effects of long-term P33 treatment on AD-specific neurological alterations. Hippocampal cell proliferation and differentiation were significantly reduced between 8 and 12 months of age. Regarding neuroinflammation, significantly elevated astrogliosis and microglial activation were observed in 6- to 7-month-old Tg animals. The amounts of the molecules involved in the amyloidogenic pathway were altered from 4 months of age in Tg animals. P33-treatment led to significantly increased neurogenesis in 9-month-old animals. Our data support the hypothesis that altered neurogenesis may be a consequence of AD pathology. Based on our findings in the transgenic animal model, early pharmacological treatment before the manifestation of AD symptoms might ameliorate neurological decline.

Effects of sub-chronic, in vivo administration of sigma non-opioid intracellular receptor 1 ligands on platelet and aortic arachidonate cascade in rats.

Platelets regulate cell-cell interactions and local circulation through eicosanoids from arachidonic acid. Sigma non-opioid intracellular receptor 1 (sigma-1 receptor) expressed in platelets and endothelial cells can regulate intracellular signalization. Our aim was to examine the influence of sub-chronic, in vivo-administered sigma-1 receptor ligands 2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate (PRE-084); N-benzyl-2-[(1S)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl]ethan-1-amine; dihydrochloride, a new compound ((S)-L1); and N-[2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethyl]-N-propylpropan-1-amine (NE-100) on the ex vivo arachidonic acid metabolism of the platelets and aorta of male rats. The serum level of sigma-1 receptor ligands was determined by liquid chromatography-mass spectrometry. Sigma-1 receptor and cyclooxygenase gene expression in the platelets were determined by a reverse transcription-coupled quantitative polymerase chain reaction. The eicosanoid synthesis was examined using a radiolabeled arachidonic acid substrate and enzyme-linked immunosorbent assay. We confirmed the absorption of sigma-1 receptor ligands and confirmed that the ligands were not present during the ex vivo studies, so their acute effect could be excluded. We detected no changes in either sigma-1 receptor or cyclooxygenase mRNA levels in the platelets. Nevertheless, (S)-L1 and NE-100 increased the quantity of cyclooxygenases there. Both platelet and aortic eicosanoid synthesis was modified by the ligands, although in different ways. The effect of the new sigma-1 receptor ligand, (S)-L1, was similar to that of PRE-084 in most of the parameters studied but was found to be more potent. Our results suggest that sigma-1 receptor ligands may act at multiple points in arachidonic acid metabolism and play an important role in the control of the microcirculation by modulating the eicosanoid synthesis of the platelets and vessels.

Impact of Two Neuronal Sigma-1 Receptor Modulators, PRE084 and DMT, on Neurogenesis and Neuroinflammation in an Aß1-42-Injected, Wild-Type Mouse Model of AD.

Alzheimer's disease (AD) is the most common form of dementia characterized by cognitive dysfunctions. Pharmacological interventions to slow the progression of AD are intensively studied. A potential direction targets neuronal sigma-1 receptors (S1Rs). S1R ligands are recognized as promising therapeutic agents that may alleviate symptom severity of AD, possibly via preventing amyloid-ß-(Aß-) induced neurotoxicity on the endoplasmic reticulum stress-associated pathways. Furthermore, S1Rs may also modulate adult neurogenesis, and the impairment of this process is reported to be associated with AD. We aimed to investigate the effects of two S1R agonists, dimethyltryptamine (DMT) and PRE084, in an Aß-induced in vivo mouse model characterizing neurogenic and anti-neuroinflammatory symptoms of AD, and the modulatory effects of S1R agonists were analyzed by immunohistochemical methods and western blotting. DMT, binding moderately to S1R but with high affinity to 5-HT receptors, negatively influenced neurogenesis, possibly as a result of activating both receptors differently. In contrast, the highly selective S1R agonist PRE084 stimulated hippocampal cell proliferation and differentiation. Regarding neuroinflammation, DMT and PRE084 significantly reduced Aß1-42-induced astrogliosis, but neither had remarkable effects on microglial activation. In summary, the highly selective S1R agonist PRE084 may be a promising therapeutic agent for AD. Further studies are required to clarify the multifaceted neurogenic and anti-neuroinflammatory roles of these agonists.

Novel Therapeutic Target for Prevention of Neurodegenerative Diseases: Modulation of Neuroinflammation with Sig-1R Ligands.

Neurodegenerative diseases (NDDs) are characterized by progressive deterioration of the structure and function of cells and their networks in the nervous system. There are currently no drugs or other treatments that can stop the progression of NDDs. NDDs have many similarities and common pathways, e.g., formation of misfolded amyloid proteins, intra- and extracellular amyloid deposits, and chronic inflammation. Initially, the inflammation process has a cytoprotective function; however, an elevated and prolonged immune response has damaging effects and causes cell death. Neuroinflammation has been a target of drug development for treating and curing NDDs. Treatment of different NDDs with non-steroid anti-inflammatory drugs (NSAIDs) has failed or has given inconsistent results. The use of NSAIDs in diagnosed Alzheimer's disease is currently not recommended. Sigma-1 receptor (Sig-1R) is a novel target for NDD drug development. Sig-1R plays a key role in cellular stress signaling, and it regulates endoplasmic reticulum stress and unfolded protein response. Activation of Sig-1R provides neuroprotection in cell cultures and animal studies. Clinical trials demonstrated that several Sig-1R agonists (pridopidine, ANAVEX3-71, fluvoxamine, dextrometorphan) and their combinations have a neuroprotective effect and slow down the progression of distinct NDDs.

Novel High Affinity Sigma-1 Receptor Ligands from Minimal Ensemble Docking-Based Virtual Screening.

Sigma-1 receptor (S1R) is an intracellular, multi-functional, ligand operated protein that also acts as a chaperone. It is considered as a pluripotent drug target in several pathologies. The publication of agonist and antagonist bound receptor structures has paved the way for receptor-based in silico drug design. However, recent studies on this subject payed no attention to the structural differences of agonist and antagonist binding. In this work, we have developed a new ensemble docking-based virtual screening protocol utilizing both agonist and antagonist bound S1R structures. This protocol was used to screen our in-house compound library. The S1R binding affinities of the 40 highest ranked compounds were measured in competitive radioligand binding assays and the sigma-2 receptor (S2R) affinities of the best S1R binders were also determined. This way three novel high affinity S1R ligands were identified and one of them exhibited a notable S1R/S2R selectivity.

Membrane Association Modes of Natural Anticancer Peptides: Mechanistic Details on Helicity, Orientation, and Surface Coverage.

Anticancer peptides (ACPs) could potentially offer many advantages over other cancer therapies. ACPs often target cell membranes, where their surface mechanism is coupled to a conformational change into helical structures. However, details on their binding are still unclear, which would be crucial to reach progress in connecting structural aspects to ACP action and to therapeutic developments. Here we investigated natural helical ACPs, Lasioglossin LL-III, Macropin 1, Temporin-La, FK-16, and LL-37, on model liposomes, and also on extracellular vesicles (EVs), with an outer leaflet composition similar to cancer cells. The combined simulations and experiments identified three distinct binding modes to the membranes. Firstly, a highly helical structure, lying mainly on the membrane surface; secondly, a similar, yet only partially helical structure with disordered regions; and thirdly, a helical monomeric form with a non-inserted perpendicular orientation relative to the membrane surface. The latter allows large swings of the helix while the N-terminal is anchored to the headgroup region. These results indicate that subtle differences in sequence and charge can result in altered binding modes. The first two modes could be part of the well-known carpet model mechanism, whereas the newly identified third mode could be an intermediate state, existing prior to membrane insertion.

New short cationic antibacterial peptides. Synthesis, biological activity and mechanism of action.

We report a theoretical and experimental study on a new series of small-sized antibacterial peptides. Synthesis and bioassays for these peptides are reported here. In addition, we evaluated different physicochemical parameters that modulate antimicrobial activity (charge, secondary structure, amphipathicity, hydrophobicity and polarity). We also performed molecular dynamic simulations to assess the interaction between these peptides and their molecular target (the membrane). Biophysical characterization of the peptides was carried out with different techniques, such as circular dichroism (CD), linear dichroism (LD), infrared spectroscopy (IR), dynamic light scattering (DLS), fluorescence spectroscopy and TEM studies using model systems (liposomes) for mammalian and bacterial membranes. The results of this study allow us to draw important conclusions on three different aspects. Theoretical and experimental results indicate that small-sized peptides have a particular mechanism of action that is different to that of large peptides. These results provide additional support for a previously proposed four-step mechanism of action. The possible pharmacophoric requirement for these small-sized peptides is discussed. Furthermore, our results indicate that a net +4 charge is the adequate for 9 amino acid long peptides to produce antibacterial activity. The information reported here is very important for designing new antibacterial peptides with these structural characteristics.

Decreased pH in the aging brain and Alzheimer's disease.

Using publicly available data sets, we compared pH in the human brain and the cerebrospinal fluid (CSF) of postmortem control and Alzheimer's disease cases. We further investigated the effects of long-term acidosis in vivo in the APP-PS1 mouse model of Alzheimer's disease. We finally examined in vitro whether low pH exposure could modulate the release of proinflammatory cytokines and the uptake of amyloid beta by microglia. In the human brain, pH decreased with aging. Similarly, we observed a reduction of pH in the brain of C57BL/6 mice with age. In addition, independent database analyses revealed that postmortem brain and CSF pH is further reduced in Alzheimer's disease cases compared with controls. Moreover, in vivo experiments showed that low pH CSF infusion increased amyloid beta plaque load in APP-PS1 mice. We further observed that mild acidosis reduced the amyloid beta 42-induced release of tumor necrosis factor-alpha by microglia and their capacity to uptake this peptide. Brain acidosis is associated with aging and might affect pathophysiological processes such as amyloid beta aggregation or inflammation in Alzheimer's disease.

Neuroinflammatory processes are augmented in mice overexpressing human heat-shock protein B1 following ethanol-induced brain injury.

BACKGROUND: Heat-shock protein B1 (HSPB1) is among the most well-known and versatile member of the evolutionarily conserved family of small heat-shock proteins. It has been implicated to serve a neuroprotective role against various neurological disorders via its modulatory activity on inflammation, yet its exact role in neuroinflammation is poorly understood. In order to shed light on the exact mechanism of inflammation modulation by HSPB1, we investigated the effect of HSPB1 on neuroinflammatory processes in an in vivo and in vitro model of acute brain injury. METHODS: In this study, we used a transgenic mouse strain overexpressing the human HSPB1 protein. In the in vivo experiments, 7-day-old transgenic and wild-type mice were treated with ethanol. Apoptotic cells were detected using TUNEL assay. The mRNA and protein levels of cytokines and glial cell markers were examined using RT-PCR and immunohistochemistry in the brain. We also established primary neuronal, astrocyte, and microglial cultures which were subjected to cytokine and ethanol treatments. TNFα and hHSPB1 levels were measured from the supernates by ELISA, and intracellular hHSPB1 expression was analyzed using fluorescent immunohistochemistry. RESULTS: Following ethanol treatment, the brains of hHSPB1-overexpressing mice showed a significantly higher mRNA level of pro-inflammatory cytokines (Tnf, Il1b), microglia (Cd68, Arg1), and astrocyte (Gfap) markers compared to wild-type brains. Microglial activation, and 1 week later, reactive astrogliosis was higher in certain brain areas of ethanol-treated transgenic mice compared to those of wild-types. Despite the remarkably high expression of pro-apoptotic Tnf, hHSPB1-overexpressing mice did not exhibit higher level of apoptosis. Our data suggest that intracellular hHSPB1, showing the highest level in primary astrocytes, was responsible for the inflammation-regulating effects. Microglia cells were the main source of TNFα in our model. Microglia isolated from hHSPB1-overexpressing mice showed a significantly higher release of TNFα compared to wild-type cells under inflammatory conditions. CONCLUSIONS: Our work provides novel in vivo evidence that hHSPB1 overexpression has a regulating effect on acute neuroinflammation by intensifying the expression of pro-inflammatory cytokines and enhancing glial cell activation, but not increasing neuronal apoptosis. These results suggest that hHSPB1 may play a complex role in the modulation of the ethanol-induced neuroinflammatory response.

The interaction of half-sandwich (η5-Cp*)Rh(III) cation with histidine containing peptides and their ternary species with (N,N) bidentate ligands.

Our goal was to explore the possible interactions of the potential metallodrug (η5-Cp*)Rh(III) complexes with histidine containing biomolecules (peptides/proteins) in order to understand the most important thermodynamic factors influencing the biospeciation and biotransformation of (η5-Cp*)Rh(III) complexes. To this end, here we report systematic solution thermodynamic and solution structural study on the interaction of (η5-Cp*)Rh(III) cation with histidine containing peptides and their constituents ((N-methyl)imidazole, GGA-OH, GGH-OH, histidine-amide, HGG-OH, GHG-NH2), based on extensive 1H NMR, ESI-MS and potentiometric investigations. The comparative evaluation of our data indicated that (η5-Cp*)Rh(III) cation is able to induce the deprotonation of amide nitrogen well below pH 7. Consequently, at physiological pH the peptides are coordinated to Rh(III) by tridentate manner, with the participation of amide nitrogen. At pH 7.4 the (η5-Cp*)Rh(III) binding affinity of peptides follow the order GGA-OH < < GGH-OH < < histidine-amide < HGG-OH < GHG-NH2, i.e. the observed binding strength essentially depends on the presence and position of histidine within the peptide sequence. We also performed computational study on the possible solution structures of complexes present at near physiological pH. At pH 7.4 all histidine containing peptides form ternary complexes with strongly coordinating (N,N) bidentate ligands (ethylenediamine or bipyridyl), in which the peptides are monodentately coordinated to Rh(III) through their imidazole N1­nitrogens. In addition, the strongest chelators histidine-amide, HGG-OH and GHG-NH2 are also able to displace these powerful bidentate ligands from the coordination sphere of Rh(III).

Alzheimer risk factors age and female sex induce cortical Aß aggregation by raising extracellular zinc.

Aging and female sex are the major risk factors for Alzheimer's disease and its associated brain amyloid-ß (Aß) neuropathology, but the mechanisms mediating these risk factors remain uncertain. Evidence indicates that Aß aggregation by Zn2+ released from glutamatergic neurons contributes to amyloid neuropathology, so we tested whether aging and sex adversely influences this neurophysiology. Using acute hippocampal slices, we found that extracellular Zn2+-elevation induced by high K+ stimulation was significantly greater with older (65 weeks vs 10 weeks old) rats, and was exaggerated in females. This was driven by slower reuptake of extracellular Zn2+, which could be recapitulated by mitochondrial intoxication. Zn2+:Aß aggregates were toxic to the slices, but Aß alone was not. Accordingly, high K+ caused synthetic human Aß added to the slices to form soluble oligomers as detected by bis-ANS, attaching to neurons and inducing toxicity, with older slices being more vulnerable. Age-dependent energy failure impairing Zn2+ reuptake, and a higher maximal capacity for Zn2+ release by females, could contribute to age and sex being major risk factors for Alzheimer's disease.

Effects of the Pentapeptide P33 on Memory and Synaptic Plasticity in APP/PS1 Transgenic Mice: A Novel Mechanism Presenting the Protein Fe65 as a Target.

Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) leads to the formation of fragments, among which the intracellular domain of APP (AICD) was also identified to be a causative of early pathological events. AICD-counteracting proteins, such as Fe65, may serve as alternative therapeutic targets of Alzheimer's disease (AD). The detection of elevated levels of Fe65 in the brains of both human patients and APP transgenic mice may further strengthen the hypothesis that influencing the interaction between Fe65 and APP may have a beneficial effect on the course of AD. Based on a PXP motif, proven to bind to the WW domain of Fe65, a new pentapeptide was designed and tested. The impedimental effect of P33 on the production of beta amyloid (Aß) (soluble fraction and aggregated plaques) and on the typical features of the AD pathology (decreased dendritic spine density, synaptic markers, elevated inflammatory reactions) was also demonstrated. Significant enhancements of both learning ability and memory function were observed in a Morris water maze paradigm. The results led us to formulate the theory that P33 acts by altering the conformation of Fe65 via binding to its WW domain, consequently hindering any interactions between Fe65 and key members involved in APP processing.

Dual Action of the PN159/KLAL/MAP Peptide: Increase of Drug Penetration across Caco-2 Intestinal Barrier Model by Modulation of Tight Junctions and Plasma Membrane Permeability.

The absorption of drugs is limited by the epithelial barriers of the gastrointestinal tract. One of the strategies to improve drug delivery is the modulation of barrier function by the targeted opening of epithelial tight junctions. In our previous study the 18-mer amphiphilic PN159 peptide was found to be an effective tight junction modulator on intestinal epithelial and blood⁻brain barrier models. PN159, also known as KLAL or MAP, was described to interact with biological membranes as a cell-penetrating peptide. In the present work we demonstrated that the PN159 peptide as a penetration enhancer has a dual action on intestinal epithelial cells. The peptide safely and reversibly enhanced the permeability of Caco-2 monolayers by opening the intercellular junctions. The penetration of dextran molecules with different size and four efflux pump substrate drugs was increased several folds. We identified claudin-4 and -7 junctional proteins by docking studies as potential binding partners and targets of PN159 in the opening of the paracellular pathway. In addition to the tight junction modulator action, the peptide showed cell membrane permeabilizing and antimicrobial effects. This dual action is not general for cell-penetrating peptides (CPPs), since the other three CPPs tested did not show barrier opening effects.

Mechanisms Associated with Type 2 Diabetes as a Risk Factor for Alzheimer-Related Pathology.

Current evidence suggests dementia and pathology in Alzheimer's Disease (AD) are both dependent and independent of amyloid processing and can be induced by multiple 'hits' on vital neuronal functions. Type 2 diabetes (T2D) poses the most important risk factor for developing AD after ageing and dysfunctional IR/PI3K/Akt signalling is a major contributor in both diseases. We developed a model of T2D, coupling subdiabetogenic doses of streptozotocin (STZ) with a human junk food (HJF) diet to more closely mimic the human condition. Over 35 weeks, this induced classic signs of T2D (hyperglycemia and insulin dysfunction) and a modest, but stable deficit in spatial recognition memory, with very little long-term modification of proteins in or associated with IR/PI3K/Akt signalling in CA1 of the hippocampus. Intracerebroventricular infusion of soluble amyloid beta 42 (Aß42) to mimic the early preclinical rise in Aß alone induced a more severe, but short-lasting deficits in memory and deregulation of proteins. Infusion of Aß on the T2D phenotype exacerbated and prolonged the memory deficits over approximately 4 months, and induced more severe aberrant regulation of proteins associated with autophagy, inflammation and glucose uptake from the periphery. A mild form of environmental enrichment transiently rescued memory deficits and could reverse the regulation of some, but not all protein changes. Together, these data identify mechanisms by which T2D could create a modest dysfunctional neuronal milieu via multiple and parallel inputs that permits the development of pathological events identified in AD and memory deficits when Aß levels are transiently effective in the brain.

Key Peptides and Proteins in Alzheimer's Disease.

Alzheimer's Disease (AD) is a form of progressive dementia involving cognitive impairment, loss of learning and memory. Different proteins (such as amyloid precursor protein (APP), ß- amyloid (Aß) and tau protein) play a key role in the initiation and progression of AD. We review the role of the most important proteins and peptides in AD pathogenesis. The structure, biosynthesis and physiological role of APP are shortly summarized. The details of trafficking and processing of APP to Aß, the cytosolic intracellular Aß domain (AICD) and small soluble proteins are shown, together with other amyloid-forming proteins such as tau and α-synuclein (α-syn). Hypothetic physiological functions of Aß are summarized. The mechanism of conformational change, the formation and the role of neurotoxic amyloid oligomeric (oAß) are shown. The fibril formation process and the co-existence of different steric structures (U-shaped and S-shaped) of Aß monomers in mature fibrils are demonstrated. We summarize the known pathogenic and non-pathogenic mutations and show the toxic interactions of Aß species after binding to cellular receptors. Tau phosphorylation, fibrillation, the molecular structure of tau filaments and their toxic effect on microtubules are shown. Development of Aß and tau imaging in AD brain and CSF as well as blood biomarkers is shortly summarized. The most probable pathomechanisms of AD including the toxic effects of oAß and tau; the three (biochemical, cellular and clinical) phases of AD are shown. Finally, the last section summarizes the present state of Aß- and tau-directed therapies and future directions of AD research and drug development.

Structural Optimization of Foldamer-Dendrimer Conjugates as Multivalent Agents against the Toxic Effects of Amyloid Beta Oligomers.

Alzheimer's disease is one of the most common chronic neurodegenerative disorders. Despite several in vivo and clinical studies, the cause of the disease is poorly understood. Currently, amyloid ß (Aß) peptide and its tendency to assemble into soluble oligomers are known as a main pathogenic event leading to the interruption of synapses and brain degeneration. Targeting neurotoxic Aß oligomers can help recognize the disease at an early stage or it can be a potential therapeutic approach. Unnatural ß-peptidic foldamers are successfully used against many different protein targets due to their favorable structural and pharmacokinetic properties compared to small molecule or protein-like drug candidates. We have previously reported a tetravalent foldamer-dendrimer conjugate which can selectively bind Aß oligomers. Taking advantage of multivalency and foldamers, we synthesized different multivalent foldamer-based conjugates to optimize the geometry of the ligand. Isothermal titration calorimetry (ITC) was used to measure binding affinity to Aß, thereafter 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based tissue viability assay and impedance-based viability assay on SH-SY5Y cells were applied to monitor Aß toxicity and protective effects of the compounds. Important factors for high binding affinity were determined and a good correlation was found between influencing the valence and the capability of the conjugates for Aß binding.

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.