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Physiological aging is characterized by an imbalance of pro-inflammatory and anti-inflammatory mediators leading to neuroinflammation. Microglial cells, which are highly regulated by the local microenvironment, undergo specific changes depending upon the brain area during aging. The aim of this study was to evaluate the influence of age over microglial cells along different brain areas and microenvironments. For this purpose, transgenic mice with overproduction of either the anti-inflammatory IL-10 cytokine or the pro-inflammatory IL-6 cytokine were used. Our results show that, during aging, microglial cells located in white matter (WM) areas maintain their phagocytic capacity but present a specific phagocytic phenotype with receptors involved in myelin recognition, arguing for aging-derived myelin damage. Whereas IL-10 overproduction anticipates the age-related microglial phagocytic phenotype, maintaining it over time, IL-6 overproduction exacerbates this phenotype in aging. These modifications were linked with a higher efficiency of myelin engulfment by microglia in aged transgenic animals. Moreover, we show, in a novel way, lower lipid oxidation during aging in WM areas, regardless of the genotype. The novelty of the insights presented in this study open a window to deeply investigate myelin lipid oxidation and the role of microglial cells in its regulation during physiological aging.

Amyloid plaques are one of the principal hallmarks of Alzheimer's disease and are mainly composed of Aβ amyloid peptides together with other components such as lipids, cations, or glycosaminoglycans. The structure of amyloid peptide's aggregates is related to the peptide toxicity and highly depends on the aggregation conditions and the presence of cofactors. While fibrillary aggregates are nowadays considered nontoxic, oligomeric/granular (nonfibrillary) aggregates have been found to be toxic. In this work we have characterized in situ two different types of amyloid deposits analyzing sections of the cortex of patients in advanced stages of Alzheimer disease. By combining SR-μFTIR for the study of the secondary structure of the peptide and ThS fluorescence as an indicator of fibrillary structures, we found two types of plaques: ThS positive plaques with a clear infrared band at 1630 cm-1 that would correspond to fibrillary plaques and ThS negative plaques showing a mixture of nonfibrillar β-sheet and unordered aggregated structures that would correspond to the nonfibrillary plaques (plaques with increased unordered structure). The analysis of the FTIR spectra has allowed correlation of lipid oxidation with the presence of nonfibrillary plaques. The metal composition of the two types of plaques has been analyzed using SR-nano-XRF and XANES. The results have shown higher accumulation of iron (mainly Fe2+) in fibrillary plaques than in nonfibrillary ones. However, in nonfibrillary plaques Fe3+ has been found to predominate over Fe2+. The identification of different types of aggregated forms and the different composition of metals found in the different types of plaques could be of paramount importance for the understanding of the development of Alzheimer disease.

Amyloid deposition of WT human β2-microglobulin (WT-hβ2m) in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis. In vitro, WT-hβ2m does not form amyloid fibrils at physiological pH and temperature unless co-solvents or other reagents are added. Therefore, understanding how fibril formation is initiated and maintained in the joint space is important for elucidating WT-hβ2m aggregation and dialysis-related amyloidosis onset. Here, we investigated the roles of collagen I and the commonly administered anticoagulant, low-molecular-weight (LMW) heparin, in the initiation and subsequent aggregation phases of WT-hβ2m in physiologically relevant conditions. Using thioflavin T fluorescence to study the kinetics of amyloid formation, we analyzed how these two agents affect specific stages of WT-hβ2m assembly. Our results revealed that LMW-heparin strongly promotes WT-hβ2m fibrillogenesis during all stages of aggregation. However, collagen I affected WT-hβ2m amyloid formation in contrasting ways: decreasing the lag time of fibril formation in the presence of LMW-heparin and slowing the rate at higher concentrations. We found that in self-seeded reactions, interaction of collagen I with WT-hβ2m amyloid fibrils attenuates surface-mediated growth of WT-hβ2m fibrils, demonstrating a key role of secondary nucleation in WT-hβ2m amyloid formation. Interestingly, collagen I fibrils did not suppress surface-mediated assembly of WT-hβ2m monomers when cross-seeded with fibrils formed from the N-terminally truncated variant ΔN6-hβ2m. Together, these results provide detailed insights into how collagen I and LMW-heparin impact different stages in the aggregation of WT-hβ2m into amyloid, which lead to dramatic effects on the time course of assembly.

Amyloid plaques composed of Aβ amyloid peptides and neurofibrillary tangles are a pathological hallmark of Alzheimer Disease. In situ identification of early-stage amyloid aggregates in Alzheimer’s disease is relevant for their importance as potential targets for effective drugs. Synchrotron-based infrared imaging is here used to identify early-stage oligomeric/granular aggregated amyloid species in situ in the brain of APP/PS1 transgenic mice for the first time. Also, APP/PS1 mice show fibrillary aggregates at 6 and 12 months. A significant decreased burden of early-stage aggregates and fibrillary aggregates is obtained following treatment with poly(propylene imine) dendrimers with histidine-maltose shell (a neurodegenerative protector) in 6-month-old APP/PS1 mice, thus demonstrating their putative therapeutic properties of in AD models. Identification, localization, and characterization using infrared imaging of these non-fibrillary species in the cerebral cortex at early stages of AD progression in transgenic mice point to their relevance as putative pharmacological targets. No less important, early detection of these structures may be useful in the search for markers for non-invasive diagnostic techniques.

Amyloid plaques composed of Aβ amyloid peptides and neurofibrillary tangles are a pathological hallmark of Alzheimer's disease. In situ identification of early-stage amyloid aggregates in Alzheimer's disease is relevant for their importance as potential targets for effective drugs. Synchrotron-based infrared imaging is here used to identify early-stage oligomeric/granular aggregated amyloid species in situ in the brain of APP/PS1 transgenic mice and Octodon degus for the first time. Also, APP/PS1 mice show fibrillary aggregates at 6 and 12 months whereas very little formation of fibrils is found in aged Octodon degus. Finally, significant decreased burden of early-stage aggregates and fibrillary aggregates is obtained following treatment with G4-His-Mal dendrimers (a neurodegenerative protector) in 6-month-old APP/PS1 mice, thus demonstrating putative therapeutic properties of G4-His-Mal dendrimers in AD models. Identification, localization, and characterization using infrared imaging of these non-fibrillary species in the cerebral cortex at early stages of AD progression in transgenic mice point to their relevance as putative pharmacological targets. No less important, early detection of these structures may be useful in the search for markers for non-invasive diagnostic techniques.

Amyloid plaques composed of Aβ amyloid peptides and neurofibrillary tangles are a pathological hallmark of Alzheimer’s disease. In situ identification of early-stage amyloid aggregates in Alzheimer’s disease is relevant for their importance as potential targets for effective drugs. Synchrotron-based infrared imaging is here used to identify early-stage oligomeric/granular aggregated amyloid species in situ in the brain of APP/PS1 transgenic mice and Octodon degus for the first time. Also, APP/PS1 mice show fibrillary aggregates at 6 and 12 months whereas very little formation of fibrils is found in aged Octodon degus. Finally, significant decreased burden of early-stage aggregates and fibrillary aggregates is obtained following treatment with G4-His-Mal dendrimers (a neurodegenerative protector) in 6-month-old APP/PS1 mice, thus demonstrating putative therapeutic properties of G4-His-Mal dendrimers in AD models. Identification, localization, and characterization using infrared imaging of these non-fibrillary species in the cerebral cortex at early stages of AD progression in transgenic mice point to their relevance as putative pharmacological targets. No less important, early detection of these structures may be useful in the search for markers for non-invasive diagnostic techniques.

Fourier Transform Infrared microspectroscopy (μFTIR) is a very useful method to analyze the biochemical properties of biological samples in situ. Many diseases affecting the central nervous system (CNS) have been studied using this method, to elucidate alterations in lipid oxidation or protein aggregation, among others. In this work, we describe in detail the characteristics between grey matter (GM) and white matter (WM) areas of the human brain by μFTIR, and we compare them with the mouse brain (strain C57BL/6), the most used animal model in neurological disorders. Our results show a clear different infrared profile between brain areas in the lipid region of both species. After applying a second derivative in the data, we established a 1.5 threshold value for the lipid/protein ratio to discriminate between GM and WM areas in non-pathological conditions. Furthermore, we demonstrated intrinsic differences of lipids and proteins by cerebral area. Lipids from GM present higher C=CH, C=O and CH3 functional groups compared to WM in humans and mice. Regarding proteins, GM present lower Amide II amounts and higher intramolecular β-sheet structure amounts with respect to WM in both species. However, the presence of intermolecular β-sheet structures, which is related to β-aggregation, was only observed in the GM of some human individuals. The present study defines the relevant biochemical properties of non-pathological human and mouse brains by μFTIR as a benchmark for future studies involving CNS pathological samples.

A new and convenient strategy for the preparation of metallosomes has been developed by mixing organometallic metallosurfactants and phospholipids. These aggregates show the characteristic properties of liposomes (stability upon dilution and low toxicity) and the toxicity is at least ten-fold lower than that of the metallosurfactant aggregates without phospholipids.

Amyloid deposition of WT human β2-microglobulin (WT-hβ2m) in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis. In vitro, WT-hβ2m does not form amyloid fibrils at physiological pH and temperature unless co-solvents or other reagents are added. Therefore, understanding how fibril formation is initiated and maintained in the joint space is important for elucidating WT-hβ2m aggregation and dialysis-related amyloidosis onset. Here, we investigated the roles of collagen I and the commonly administered anticoagulant, low-molecular-weight (LMW) heparin, in the initiation and subsequent aggregation phases of WT-hβ2m in physiologically relevant conditions. Using thioflavin T fluorescence to study the kinetics of amyloid formation, we analyzed how these two agents affect specific stages of WT-hβ2m assembly. Our results revealed that LMW-heparin strongly promotes WT-hβ2m fibrillogenesis during all stages of aggregation. However, collagen I affected WT-hβ2m amyloid formation in contrasting ways: decreasing the lag time of fibril formation in the presence of LMW-heparin and slowing the rate at higher concentrations. We found that in self-seeded reactions, interaction of collagen I with WT-hβ2m amyloid fibrils attenuates surface-mediated growth of WT-hβ2m fibrils, demonstrating a key role of secondary nucleation in WT-hβ2m amyloid formation. Interestingly, collagen I fibrils did not suppress surface-mediated assembly of WT-hβ2m monomers when cross-seeded with fibrils formed from the N-terminally truncated variant ΔN6-hβ2m. Together, these results provide detailed insights into how collagen I and LMW-heparin impact different stages in the aggregation of WT-hβ2m into amyloid, which lead to dramatic effects on the time course of assembly.

Myoglobin, encoded by MB, is a small cytoplasmic globular hemoprotein highly expressed in cardiac myocytes and oxidative skeletal myofibers. Myoglobin binds O-2, facilitates its intracellular transport and serves as a controller of nitric oxide and reactive oxygen species. Here, we identify a recurrent c.292C>T ( p.His98Tyr) substitution in MB in fourteen members of six European families suffering from an autosomal dominant progressive myopathy with highly characteristic sarcoplasmic inclusions in skeletal and cardiac muscle. Myoglobinopathy manifests in adulthood with proximal and axial weakness that progresses to involve distal muscles and causes respiratory and cardiac failure. Biochemical characterization reveals that the mutant myoglobin has altered O-2 binding, exhibits a faster heme dissociation rate and has a lower reduction potential compared to wild-type myoglobin. Preliminary studies show that mutant myoglobin may result in elevated superoxide levels at the cellular level. These data define a recognizable muscle disease associated with MB mutation., We thank the patients and their families for their collaboration. This study has been funded by Instituto de Salud Carlos III through the project PI14/00738 (co-funded by European Regional Development Fund. ERDF, a way to build Europe) and from a grant from Genzyme Foundation to MO. We thank CERCA Programme/Generalitat de Catalunya for institutional support. GR, NGL, and ARRF are supported by Australian National Health and Medical Research Council Fellowships APP1002147, APP1035955, APP1154524, and Project Grants APP1080587 and APP1146321. ARRF was also supported by a Senior Research Fellowship from the Cancer Research Trust, the MACA Ride to Conquer Cancer and an Australian Research Council Discovery Project Grant (DP160101960). MC is supported by ISCIII (JR15/00042). AWr is a Ragnar Soderberg Fellow in Medicine (M77/13) and is supported by the Swedish Research Council (AW (VR2016-02179). AWe is supported by the Swedish Research Council (2016-01082), Stockholm County Council (20170022), the Swedish Brain Foundation (FO2015-0146), and the Knut & Alice Wallenberg Foundation (KAW 2013.0026 and KAW 2014.0293). KGC is supported by the KOOR, UZ Leuven, Belgium. AG is supported by the Research Council KU Leuven (C24/16/045). JK is a recipient of the Royal Society Wolfson Research Merit Award. TS is supported by Stockholm County Council (20170831), Stiftelsen Frimurare Foundation, and Promobilia. LH is supported by an Australian National Health and Medical Research Council Fellowship (APP1117366). HV is supported by a National Heart Foundation of Australia Fellowship (101930). mu FTIR microscope analysis was performed at MIRAS beamline at ALBA Synchrotron with the collaboration of ALBA staff. We thank professor Josep Garcia Valero from the University of Barcelona for helpful comments on the ultrastructural features. We thank Dolores Moreno, Susana Arnedo, Guy Brochier, and Benjamin Torrejon-Escribano for excellent technical assistance and to the Audiovisual Department from Hospital Universitari de Bellvitge for their help with the preparation of the figures. KCG wants to thank Klara Mallants for technical support and to Dr. Sjoerd G. van Duinen (Leiden), and professsor Joachim Weis (Aachen). Muscle sample and associated data from Family 5 were obtained from Cardiobiotec Biobank (CRB-HCL Hospices Civils de Lyon BB-0033-00046).

Droplets with colloidal biological suspensions evaporating on substrates with defined wetting properties generate confined environments for initiating aggregation and self-assembly processes. We describe smart micro- and nanostructured surfaces, optimized for probing single droplets and residues by synchrotron radiation micro- and nanobeam diffraction techniques. Applications are presented for Ac-IVD and β-amyloid (1–42) peptides capable of forming cross-β sheet structures. Complementary synchrotron radiation FTIR microspectroscopy addresses secondary structure formation. The high synchrotron radiation source brilliance enables fast raster-scan experiments.

IntroductionSince the time of the installation of the first IR beamline, built in the early 1980s by Yarwood and colleagues [1] at Daresbury in the UK, the number of IR beamlines worldwide has cons...

Poly(propylene imine) dendrimers have been shown to be promising 3-dimensional polymers for the use in the pharmaceutical and biomedical applications. Our aims of this study were first, to synthesize a novel type of dendrimer with poly(propylene imine) core and maltose-histidine shell (G4HisMal) assessing if maltose-histidine shell can improve the biocompatibility and the ability to cross the blood-brain barrier, and second, to investigate the potential of G4HisMal to protect Alzheimer disease transgenic mice from memory impairment. Our data demonstrate that G4HisMal has significantly improved biocompatibility and ability to cross the blood-brain barrier in vivo. Therefore, we suggest that a maltose-histidine shell can be used to improve biocompatibility and ability to cross the blood-brain barrier of dendrimers. Moreover, G4HisMal demonstrated properties for synapse and memory protection when administered to Alzheimer disease transgenic mice. Therefore, G4HisMal can be considered as a promising drug candidate to prevent Alzheimer disease via synapse protection.

New supramolecular systems have been prepared by mixing molybdenum organometallic metallosurfactants M(CO)5L and M(CO)4L2 {L = Ph2P(CH2)6SO3Na} with the phospholipid phosphatidylcholine. The analysis of the resulting supramolecular structures using dynamic light scattering and cryo-transmission electron microscopy has shown the formation of different aggregates depending on the metallosurfactant/phospholipid ratio, as well as a significantly different behaviour between the two studied metallosurfactants. Mixed vesicles, with properties very similar to liposomes, can be obtained with both compounds, and are called metallosomes. The formation of the mixed aggregates has also been studied by microfluidics using MeOH and EtOH as organic solvents, and it has been observed that the size of the aggregates is strongly dependent on the organic solvent used. In order to analyse the viability of these mixed systems as CO Releasing Molecules (CORMs) for biomedical applications, the CO release was studied by FT-IR spectroscopy, showing that they behave as photo-CORMs with visible and ultraviolet light. Toxicity studies of the different mixed aggregate systems have shown that metallosomes exhibit a very low toxicity, similar to liposomes that do not contain metallosurfactants, which is well below the results observed for pure metallosurfactants. Micro-FTIR microscopy using synchrotron radiation has shown the presence of metallosurfactants in cells. The results of this study show the influence of the length of the hydrocarbon chain on the properties of these mixed systems, compared with previously reported data.

Amyloid plaques made of aggregated Aβ amyloid peptide are a pathological hallmark in brains affected by Alzheimer's disease (AD). Moreover, the amyloid peptide may play a major role in the onset and development of the disease in association to other factors such as oxidative stress. Although the molecular nature of the amyloid toxic species is still unknown, there is experimental evidence pointing to their nonfibrillar nature. In the present paper, we report the use of synchrotron Fourier transform infrared microspectroscopy (μFTIR) for the study of the effect of two different types of Alzheimer's Aβ(1-40) aggregates (amyloid fibrils and granular nonfibrillar aggregates) on PC12 cultured cells. The principal component analysis (PCA) of the infrared spectra has been complemented with a correlation analysis, which permits one to study different spectroscopic parameters as a function of peptide aggregation. The results show that the treatment of PC12 cells with amorphous aggregates generates a higher degree of oxidation in the vicinity of the amyloid aggregates than the treatment with preformed amyloid fibrils. These results, which permit, for the first time, the in situ colocalization of amyloid aggregates and oxidized macromolecules in cell culture, are in agreement with previous data from our group, showing that oxidation was higher in regions surrounding amyloid plaques in human brain samples affected by AD.

International audience; The systemic or local administration of a photosensitizer for photodynamic therapy is highly limited by poor selectivity, rapid deactivation and long-lasting skin toxicity due to unfavorable biodistribution. Drug delivery systems based on nanocarriers may help specific and effective delivery of photosensitizers. In the present paper, the interaction of two photosensitizers, methylene blue and rose bengal, with phosphorous cationic and anionic dendrimers as potential nanocarriers, has been characterized. A novel method is presented based on the analysis of the infrared spectra of mixtures of photosensitizer and dendrimer. The capacity of dendrimers to bind the photosensitizers has been evaluated by obtaining the corresponding binding curves. It is shown that methylene blue interacts with both cationic and anionic dendrimers, whereas rose bengal only binds to the cationic ones. Dendrimers are shown to be potential nanocarriers for a specific delivery of both photosensitizers.

Dendrimers have been shown to be capable of interfering in vitro with the formation of the amyloid fibrillar structures typically related to the onset and development of the so-called conformational diseases, such as Alzheimer's disease and prion diseases. This makes dendrimers potentially useful as compounds that could prevent or inhibit the action of the cytotoxic amyloid species. In the present paper we summarise the works on the interaction of dendrimers with amyloid peptides related to Alzheimer and prion diseases and take it as the basis from which to focus on some developments that will contribute to enhance the potential of dendrimers as antiamyloidogenic agents. An important first set of works was dedicated to the characterization of the effects of positively charged PAMAM and phosphorous dendrimers on the polymerization processes of different amyloid peptides. However, due to the inherent toxicity of positively charged dendrimers, the research has moved towards the design of more biocompatible dendrimers such as glycodendrimers. This sugar-decorated dendrimers have shown as well their capacity to interact with amyloids, a low level of cell toxicity has been measured for different cell lines and they have shown interesting properties as antiamyloidogenic agents. Other surface-decorated dendrimeric structures are finally also taken into account.

Dendrimers have been proved to interact with amyloids, although most of dendrimers assayed in amyloidogenic systems are toxic to cells. The development of glycodendrimers, poly(propyleneimine) (PPI) dendrimers decorated with maltose (Mal), represents the possibility of using dendrimers with a low intrinsic toxicity. In the present paper we show that fourth (PPI-G4-Mal) and fifth (PPI-G5-Mal) generation glycodendrimers have the capacity to interfere with Alzheimer's amyloid peptide Aβ(1-40) fibrilization. The interaction is generation dependent: PPI-G5-Mal blocks amyloid fibril formation generating granular nonfibrillar amorphous aggregates, whereas PPI-G4-Mal generates clumped fibrils at low dendrimer-peptide ratios and amorphous aggregates at high ratios. Both PPI-G4-Mal and PPI-G5-Mal are nontoxic to PC12 and SH-SY5Y cells. PPI-G4-Mal reduces amyloid toxicity by clumping fibrils together, whereas amorphous aggregates are toxic to PC12 cells. The results show that glycodendrimers are promising nontoxic agents in the search for anti-amyloidogenic compounds. Fibril clumping may be an anti-amyloid toxicity strategy.

Amyloid peptides are the main component of one of the characteristic pathological hallmarks of Alzheimer's disease (AD): senile plaques. According to the amyloid cascade hypothesis, amyloid peptides may play a central role in the sequence of events that leads to neurodegeneration. However, there are other factors, such as oxidative stress, that may be crucial for the development of the disease. In the present paper, we show that it is possible, by using Fourier tranform infrared (FTIR) microscopy, to co-localize amyloid deposits and lipid peroxidation in tissue slides from patients affected by Alzheimer's disease. Plaques and lipids can be analyzed in the same sample, making use of the characteristic infrared bands for peptide aggregation and lipid oxidation. The results show that, in samples from patients diagnosed with AD, the plaques and their immediate surroundings are always characterized by the presence of oxidized lipids. As for samples from non-AD individuals, those without amyloid plaques show a lower level of lipid oxidation than AD individuals. However, it is known that plaques can be detected in the brains of some non-AD individuals. Our results show that, in such cases, the lipid in the plaques and their surroundings display oxidation levels that are similar to those of tissues with no plaques. These results point to lipid oxidation as a possible key factor in the path that goes from showing the typical neurophatological hallmarks to suffering from dementia. In this process, the oxidative power of the amyloid peptide, possibly in the form of nonfibrillar aggregates, could play a central role.

Lorella Pascolo,1 Barbara Bortot,1 Nuria Benseny-Cases,2 Alessandra Gianoncelli,3 Giovanni Tosi,4 Barbara Ruozi,4 Clara Rizzardi,5 Eleonora De Martino,1 Maria Angela Vandelli,4 Giovanni Maria Severini11Institute for Maternal and Child Health, Istituto di Ricovero e Cura a Carattere Scientifico Burlo Garofolo, Trieste, Italy; 2European Synchrotron Radiation Facility, Polygone Scientifique Louis Néel, Grenoble, France; 3Elettra-Sincrotrone Trieste, Area Science Park, Basovizza, Trieste, Italy; 4Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 5Department of Anatomical Pathology, Department of Pathology and Forensic Medicine, University of Trieste, Trieste, ItalyAbstract: Poly-lactide-co-glycolide (PLGA) is one of the few polymers approved by the US Food and Drug Administration as a carrier for drug administration in humans; therefore, it is one of the most used materials in the formulation of polymeric nanoparticles (NPs) for therapeutic purposes. Because the cellular uptake of polymeric NPs is a hot topic in the nanomedicine field, the development of techniques able to ensure incontrovertible evidence of the presence of NPs in the cells plays a key role in gaining understanding of their therapeutic potential. On the strength of this premise, this article aims to evaluate the application of synchrotron radiation-based Fourier transform infrared spectroscopy (SR-FTIR) spectromicroscopy and SR X-ray fluorescence (SR-XRF) microscopy in the study of the in vitro interaction of PLGA NPs with cells. To reach this goal, we used PLGA NPs, sized around200nm and loaded with superparamagnetic iron oxide NPs (PLGA-IO-NPs; Fe3O4; size,10–15nm). After exposing human mesothelial (MeT5A) cells to PLGA-IO-NPs (0.1mg/mL), the cells were analyzed after fixation both by SR-FTIR spectromicroscopy and SR-XRF microscopy setups. SR-FTIR-SM enabled the detection of PLGA NPs at single-cell level, allowing polymer detection inside the biological matrix by the characteristic band in the1,700–2,000cm-1region. The precise PLGA IR-signature (1,750cm-1centered pick) also was clearly evident within an area of high amide density. SR-XRF microscopy performed on the same cells investigated under SR-FTIR microscopy allowed us to put in evidence the Fe presence in the cells and to emphasize the intracellular localization of the PLGA-IO-NPs. These findings suggest that SR-FTIR and SR-XRF techniques could be two valuable tools to follow the PLGA NPs’ fate in in vitro studies on cell cultures.Keywords: PLGA-NPs, cell targeting, SR-FTIR, SR-XRF, imaging

Poly(propylene imine) (PPI) glycodendrimers are promising candidates as drug carriers and antiamyloidogenic and antiprionic agents. In this study the anti-β-amyloid capacity of PPI glycodendrimers of the fourth and fifth generations was investigated in vitro and in vivo. We assessed distinct PPI glycodendrimers including G4mDS and G5mDS, with electroneutral maltose shell, and G4mOS and G4m-IIIOS, with cationic maltose or maltotriose shell. Our results show that in vitro PPI maltose dendrimers reduce the toxicity of Aβ(1-42). However, only the electroneutral maltose dendrimers G4mDS and G5mDS reduce the toxicity of Alzheimer's disease brain extracts in SH-SY5Y neuroblastoma cells. PPI maltose dendrimers with electroneutral or cationic surface penetrate the cytoplasm of cultured cells, and they reach the brain when administered intranasally. Both cationic G4mOS and electroneutral G4mDS are able to modify the total burden of β-amyloid in APP/PS1 mice. The studied dendrimers did not reverse memory impairment in APP/PS1 mice following chronic administration; moreover, cationic G4mOS caused cognitive decline in nontransgenic mice. In spite of the capacity of G4mDS and G4mOS to cross the blood-brain barrier and modulate Aβ aggregation in APP/PS1 mice, further studies are needed to learn how to reduce the harmful effects of maltose dendrimers in vivo.

The amyloid beta Ab(1-40) and Ab(1-42) peptides are the main components of the fibrillar plaques characteristically found in the brains affected by Alzheimer's disease. Fibril formation has been thoroughly studied in vitro using synthetic amyloid peptides and has been described to be a nucleation dependent polymerization process. During this process, defined by a slow nucleation phase followed by a rapid exponential elongation reaction, a whole range of aggregated species (low and high molecular weight aggregates) precede fibril formation. Toxic species related to the onset and development of Alzheimer's disease are thought to be found among these prefibrillar aggregates. Two main procedures are used to experimentally monitor fibril formation kinetics: through the measurement of the light scattered by the different peptide aggregates and using the fluorescent dye thioflavin T, which fluorescence increases when specifically interacting with amyloid fibrils. Reproducibility may, however, be difficult to achieve when measuring and characterizing fibril formation kinetics. This fact is mainly due to the difficulty in experimentally handling amyloid peptides, which is directly related to the difficulty of having them in a monomeric form at the beginning of the polymerization process. This has to do mainly with the type of solvent used for the preparation of the peptide stock solutions (water, DMSO, TFE, HFIP) and the control of determinant physicochemical parameters such as pH. Moreover, kinetic progression turns out to be highly dependent on the type of peptide counter-ion used, which will basically determine the duration of the nucleation phase and the rate at which high molecular weight oligomers are formed. Centrifugation and filtration procedures used in the preparation of the peptide stock solutions will also greatly influence the duration of the fibril formation process. In this chapter, a survey of the alluded experimental procedures is provided and a general frame is proposed for the interpretation of the fibril formation kinetics, intended to integrate the results from the different experimental approaches. The significance of the different aggregated species in terms of cell toxicity will be discussed. Special emphasis will be given to the influence of pH on the structural and toxic characteristics of amyloid aggregates, an aspect that may be particularly relevant in some specific physiological conditions.

International audience; The ID21 Scanning X-ray Microscope (SXM) is optimized for micro-spectroscopy with submicron resolution in the 2 to 9.5 keV energy range. After a brief description of the microscope setup, we present here recent developments, in particular, the latest version of the compact Wavelength Dispersive Spectrometer and the refurbished cryo-stage

Dendrimers have been shown to be capable of interfering in vitro with the formation of the amyloid fibrillar structures typically related to the onset and development of the so-called conformational diseases, such as Alzheimer's disease and prion diseases. This makes dendrimers potentially useful as compounds that could prevent or inhibit the action of the cytotoxic amyloid species. In the present paper we summarise the works on the interaction of dendrimers with amyloid peptides related to Alzheimer and prion diseases and take it as the basis from which to focus on some developments that will contribute to enhance the potential of dendrimers as antiamyloidogenic agents. An important first set of works was dedicated to the characterization of the effects of positively charged PAMAM and phosphorous dendrimers on the polymerization processes of different amyloid peptides. However, due to the inherent toxicity of positively charged dendrimers, the research has moved towards the design of more biocompatible dendrimers such as glycodendrimers. This sugar-decorated dendrimers have shown as well their capacity to interact with amyloids, a low level of cell toxicity has been measured for different cell lines and they have shown interesting properties as antiamyloidogenic agents. Other surface-decorated dendrimeric structures are finally also taken into account.

The amyloid beta Ab(1–40) and Ab(1–42) peptides are the main components of the fibrillar plaques characteristically found in the brains affected by Alzheimer’s disease. Fibril formation has been thoroughly studied in vitro using synthetic amyloid peptides and has been described to be a nucleation dependent polymerization process. During this process, defined by a slow nucleation phase followed by a rapid exponential elongation reaction, a whole range of aggregated species (low and high molecular weight aggregates) precede fibril formation. Toxic species related to the onset and development of Alzheimer’s disease are thought to be found among these prefibrillar aggregates. Two main procedures are used to experimentally monitor fibril formation kinetics: through the measurement of the light scattered by the different peptide aggregates and using the fluorescent dye thioflavin T, which fluorescence increases when specifically interacting with amyloid fibrils. Reproducibility may, however, be difficult to achieve when measuring and characterizing fibril formation kinetics. This fact is mainly due to the difficulty in experimentally handling amyloid peptides, which is directly related to the difficulty of having them in a monomeric form at the beginning of the polymerization process. This has to do mainly with the type of solvent used for the preparation of the peptide stock solutions (water, DMSO, TFE, HFIP) and the control of determinant physicochemical parameters such as pH. Moreover, kinetic progression turns out to be highly dependent on the type of peptide counter-ion used, which will basically determine the duration of the nucleation phase and the rate at which high molecular weight oligomers are formed. Centrifugation and filtration procedures used in the preparation of the peptide stock solutions will also greatly influence the duration of the fibril formation process. In this chapter, a survey of the alluded experimental procedures is provided and a general frame is proposed for the interpretation of the fibril formation kinetics, intended to integrate the results from the different experimental approaches. The significance of the different aggregated species in terms of cell toxicity will be discussed. Special emphasis will be given to the influence of pH on the structural and toxic characteristics of amyloid aggregates, an aspect that may be particularly relevant in some specific physiological conditions.

Granular non-fibrillar aggregates (GNAs) are identified as possible toxic species in Alzheimer's disease. GNAs form on the surface of negatively charged biological membranes and as a consequence of an acidic environment, off the polymerization pathway at neutral pH. Aβ (1-40) GNAs disturb the bilayer structure of model membranes and seem to be more toxic to cells with negatively charged membranes (consequence of chronic pre-apoptosis). GNAs may be relevant in physiological situations associated to Alzheimer's disease: a local acidic pH at the cell surface (consequence of lipid oxidation or other cell insults) and acidification as a consequence of vascular events causing hypoxia. Together with previous descriptions of granular aggregates with poly-glutamine peptides related to Huntington's disease and the SH3 domain of PI3, GNAs related to Alzheimer's disease are a further example of a possible common aggregation and toxicity mechanism in conformational diseases. GNAs may represent a new pharmacological target in Alzheimer's disease.

Amyloids are proteinaceous aggregates related to the so-called conformational diseases, such as Alzheimer's and prion diseases. The cytotoxicity of amyloids may be related to the interaction of the amiloidogenic peptides or proteins with the cell membrane. In order to gain information on the physico-chemical effects of amyloids on membranes, we have studied the interaction of the human prion amyloidogenic fragment PrP 185–206 with negatively charged model membranes. The results show that the peptide causes the destabilization of the membrane, making it permeable to potassium ions and to charged organic compounds. This effect correlates with the interaction of the peptide with the membrane, causing a variation in the magnitude of the electrostatic surface and dipole membrane potentials. This effect on the electrostatic properties of the membranes may help explaining the observed permeability: a neutralization of the surface negative charge and a decrease of the inside-positive dipole potential would facilitate the translocation of positive ions. The structural analysis of the peptide in the presence of model membranes reveals that it adopts a predominantly unordered structure without any signs of amyloid formation. The results may be relevant in relation to the recently described cell toxic capacity of the peptide. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.

In previous chapters, the basis of SAXS for the study of biological systems like proteins in solution have been presented. The SAXS patterns of proteins in solution present, in general, broad dependences with the scattering vector, and the interpretation requires a huge component of modelling. In this chapter and in the following one, it is shown how SAXS technique can be used to study biological systems that are partially crystalline and with a large crystalline cells. This is done by analysing the diffraction obtained from these systems at small angles. In this chapter, a new approach to the application of small-angle X-ray scattering (SAXS) for diagnosis using the diffraction pattern of collagen is presented. This chapter shows the development of a new strategy in the preventive diagnosis of breast cancer following changes on collagen from breast connective tissue. SAXS profiles are related to different features in cutaneous preparations and to the supra-molecular arrangement of skin layers (stratum corneum, epidermis and dermis), in order to introduce objective values on the diagnosis of different skin pathologies. Working parameters (size, thickness) and methods (freezing, paraffin embedment) have been established. The results suggest that collagen diffraction patterns could be used as diagnostic indicators; especially for breast cancer and preliminary results obtained with skin collagen are promising too.

The mechanical properties of PC12 living cells have been studied at the nanoscale with a Force Feedback Microscope using two experimental approaches. Firstly, the local mechanical impedance of the cell membrane has been mapped simultaneously to the cell morphology at constant force. As the force of the interaction is gradually increased, we observed the appearance of the sub-membrane cytoskeleton. We shall compare the results obtained with this method with the measurement of other existing techniques. Secondly, a spectroscopic investigation has been performed varying the indentation of the tip in the cell membrane and consequently the force applied on it. In contrast with conventional dynamic atomic force microscopy techniques, here the small oscillation amplitude of the tip is not necessarily imposed at the cantilever first eigenmode. This allows the user to arbitrarily choose the excitation frequency in developing spectroscopic AFM techniques. The mechanical response of the PC12 cell membrane is found to be frequency dependent in the 1 kHz - 10 kHz range. The damping coefficient is reproducibly observed to decrease when the excitation frequency is increased., 8 pages, 8 figures

Small angle X-ray scattering (SAXS) using synchrotron radiation (SR) is an excellent tool for studying non-crystalline tissue materials. This work introduces this powerful technique to study the supra-molecular arrangement of collagen and the lipid lamellar structure from skin samples under several conditions. Healthy skin presented a characteristic collagen pattern with a series of narrow rings associated with a repeat distance of 63.4 nm in both the whole skin and the dermis layer. In the epidermis, only the lipid pattern from the organisation of the lamellar stratum corneum (SC) was visible. The SAXS patterns of samples from young subjects presented a high number of diffraction orders and marked anisotropy, most likely due to a higher degree of collagen orientation than from older subjects. The collagen peaks from skin samples with stretch marks showed a lower intensity than those from normal skin. In dermal tissue invaded by melanoma, the collagen peaks disappeared, leaving only the underlying diffuse scattering; the absence of the narrow rings indicated degradation of ordered collagen fibrils in the invaded tissue. The evaluation of collagen arrangement provides information about ageing, dysfunctions and pathology of tissue, and the systematic study of different samples results in scattering signatures characteristic of each skin layer. All in all, the potential of this methodology as a diagnostic tool for skin conditions should be considered further.