Spectroscopic investigation of local mechanical impedance of living cells

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.Comment: 8 pages, 8 figure

In situ identification and G4-PPI-His-Mal-dendrimer-induced reduction of early-stage amyloid aggregates in Alzheimer’s disease transgenic mice using synchrotron-based infrared imaging

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

Arrhythmic Effects Evaluated on <i>Caenorhabditis elegans</i>: The Case of Polypyrrole Nanoparticles

Experimental studies and clinical trials of nanoparticles for treating diseases are increasing continuously. However, the reach to the market does not correlate with these efforts due to the enormous cost, several years of development, and off-target effects like cardiotoxicity. Multicellular organisms such as the Caenorhabditis elegans (C. elegans) can bridge the gap between in vitro and vertebrate testing as they can provide extensive information on systemic toxicity and specific harmful effects through facile experimentation following 3R EU directives on animal use. Since the nematodes' pharynx shares similarities with the human heart, we assessed the general and pharyngeal effects of drugs and polypyrrole nanoparticles (Ppy NPs) using C. elegans. The evaluation of FDA-approved drugs, such as Propranolol and Racepinephrine reproduced the arrhythmic behavior reported in humans and supported the use of this small animal model. Consequently, Ppy NPs were evaluated due to their research interest in cardiac arrhythmia treatments. The NPs' biocompatibility was confirmed by assessing survival, growth and development, reproduction, and transgenerational toxicity in C. elegans. Interestingly, the NPs increased the pharyngeal pumping rate of C. elegans in two slow-pumping mutant strains, JD21 and DA464. Moreover, the NPs increased the pumping rate over time, which sustained up to a day post-excretion. By measuring pharyngeal calcium levels, we found that the impact of Ppy NPs on the pumping rate could be mediated through calcium signaling. Thus, evaluating arrhythmic effects in C. elegans offers a simple system to test drugs and nanoparticles, as elucidated through Ppy NPs

From Mouse To Human : Comparative Analysis Between Grey And White Matter By Synchrotron-Fourier Transformed Infrared Microspectroscopy

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

Poly(propylene imine) dendrimers with histidine-maltose shell as novel type of nanoparticles for synapse and memory protection

Poly(propylene imine) dendrimers have been shown to be promising 3-dimensional polymers for the use in the pharmaceutical andbiomedical applications. Our aims of this study were first, to synthesize a novel type of dendrimer with poly(propylene imine) core andmaltose-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 ofdendrimers. Moreover, G4HisMal demonstrated properties for synapse and memory protection when administered to Alzheimer diseasetransgenic mice. Therefore, G4HisMal can be considered as a promising drug candidate to prevent Alzheimer disease via synapse protection

Agregats amorfs d’alt pes molecular: membranes, pH i toxicitat del pèptid amiloide en la malaltia d’Alzheimer

El pèptid amiloide Aβ(1-40) és un dels principals components de les plaques dels cervells afectats per la malaltia d’Alzheimer, on normalment es troba associat als lípids de la membrana cel·lular. Les membranes poden tenir un rol important en la toxicitat del pèptid, tant com a possible diana com influenciant la formació d’agregats peptídics. Un punt particularment interessant de la influència de les membranes en el procés d’agregació, del que existeixen evidències experimentals, és la peroxidació lipídica com a factor important en la neurodegeneració. L’objectiu general d’aquest treball ha estat la caracterització molecular de la interacció del pèptid amiloide amb les membranes biològiques, parant especial esment en el rol de la peroxidació lipídica. Els resultats han mostrat que en presència de membranes de lípid d’extracte de cervell s’observa una clara acceleració de formació de fibres pel pèptid Aβ(1-40) i una major formació d’aquestes. Per altra banda, l’agregació del pèptid en presència de membranes no oxidades mostra un alentiment i una menor formació de fibres. S’ha observat que aquesta diferència en l’agregació del pèptid segons el grau d’oxidació de les membranes, és deguda a l’aparició de càrregues negatives a la superfície de les membranes durant el procés d’agregació. L’aparició de càrregues negatives a la superfície de la membrana implica un augment de la concentració de protons i per tant una davallada del pH a la superfície de la membrana. Aquesta baixada de pH afecta al plegament del pèptid Aβ(1-40). A pH neutre, aquest pèptid tendeix a formar fibres, passant per una sèrie d’agregats oligomèrics. A pH inferior a 6, quan les histidines del pèptid es troben protonades, aquest pèptid forma agregats amorfs d’alt pes molecular. En aquesta tesi, s’ha vist com aquests agregats tenen una capacitat més gran de desestabilitzar la membrana que no els agregats que es formen a pH neutre. La comparació amb els sistemes vius s’ha dut a terme utilitzant dues línies cel·lulars amb característiques diferents. S’ha vist com el pèptid és més tòxic en cèl·lules PC12 que en cèl·lules SH-5YSY. Les cèl·lules PC12 són crònicament apoptòtiques, la qual cosa implica un augment de fosfatidilserina a la superfície, lípid amb cap polar negatiu, que aporta càrregues negatives a la superfície, i que, per tant, implica una davallada del pH a la superfície cel·lular d’aquestes cèl·lules. El treball conclou que aquests agregats amorfs que es formen a pH baix podrien ser els responsables de la toxicitat del pèptid. Els resultats són rellevants ja que donen un possible mecanisme molecular a la relació que els processos oxidatius i les malalties vasculars en la malaltia d’Alzheimer. Tant els processos oxidatius, que implicarien una acidificació a la superfície cel·lular degut a la peroxidació lipídica, com les malalties vasculars, que poden implicar una acidificació local del teixit, podrien afavorir la formació d’aquests agregats amorfs d’alt pes molecular augmentant la toxicitat del pèptid.Aβ peptides are the main components of the characteristic fibrilar amyloid plaques found in central nervous systems affected by Alzheimer’s disease. Amyloid plaques are usually found associated to lipids from the plasma cell membrane. Membranes may play a role in amyloid toxicity, either as a peptide’s target or by influencing the peptide aggregation state. A particularly interesting aspect of the possible influence of lipid bilayers is the existence of an important amount of experimental evidence pointing to lipid peroxidation as an important factor in neurodegeneration. The general objective of this thesis was the molecular characterization of the peptide interaction with biological membranes. 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

Toxicogenomics of iron oxide nanoparticles in the nematode C. elegans

<p>We present a mechanistic study of the effect of iron oxide nanoparticles (SPIONs) in <i>Caenorhabditis elegans</i> combining a genome-wide analysis with the investigation of specific molecular markers frequently linked to nanotoxicity. The effects of two different coatings were explored: citrate, an anionic stabilizer, and bovine serum albumin, as a pre-formed protein corona. The transcriptomic study identified differentially expressed genes following an exposure to SPIONs. The expression of genes involved in oxidative stress, metal detoxification response, endocytosis, intestinal integrity and iron homeostasis was quantitatively evaluated. The role of oxidative stress was confirmed by gene expression analysis and by synchrotron Fourier Transform infrared microscopy based on the higher tissue oxidation of NP-treated animals. The observed transcriptional modulation of key signaling pathways such as MAPK and Wnt suggests that SPIONs might be endocytosed by clathrin-mediated processes, a putative mechanism of nanotoxicity which deserves further mechanistic investigations.</p

Agregats amorfs d'alt pes molecular: membranes, pH i toxicitat del pèptid amiloide en la malaltia d'Alzheimer

El pèptid amiloide Aβ(1-40) és un dels principals components de les plaques dels cervells afectats per la malaltia d'Alzheimer, on normalment es troba associat als lípids de la membrana cel·lular. Les membranes poden tenir un rol important en la toxicitat del pèptid, tant com a possible diana com influenciant la formació d'agregats peptídics. Un punt particularment interessant de la influència de les membranes en el procés d'agregació, del que existeixen evidències experimentals, és la peroxidació lipídica com a factor important en la neurodegeneració. L'objectiu general d'aquest treball ha estat la caracterització molecular de la interacció del pèptid amiloide amb les membranes biològiques, parant especial esment en el rol de la peroxidació lipídica. Els resultats han mostrat que en presència de membranes de lípid d'extracte de cervell s'observa una clara acceleració de formació de fibres pel pèptid Aβ(1-40) i una major formació d'aquestes. Per altra banda, l'agregació del pèptid en presència de membranes no oxidades mostra un alentiment i una menor formació de fibres. S'ha observat que aquesta diferència en l'agregació del pèptid segons el grau d'oxidació de les membranes, és deguda a l'aparició de càrregues negatives a la superfície de les membranes durant el procés d'agregació. L'aparició de càrregues negatives a la superfície de la membrana implica un augment de la concentració de protons i per tant una davallada del pH a la superfície de la membrana. Aquesta baixada de pH afecta al plegament del pèptid Aβ(1-40). A pH neutre, aquest pèptid tendeix a formar fibres, passant per una sèrie d'agregats oligomèrics. A pH inferior a 6, quan les histidines del pèptid es troben protonades, aquest pèptid forma agregats amorfs d'alt pes molecular. En aquesta tesi, s'ha vist com aquests agregats tenen una capacitat més gran de desestabilitzar la membrana que no els agregats que es formen a pH neutre. La comparació amb els sistemes vius s'ha dut a terme utilitzant dues línies cel·lulars amb característiques diferents. S'ha vist com el pèptid és més tòxic en cèl·lules PC12 que en cèl·lules SH-5YSY. Les cèl·lules PC12 són crònicament apoptòtiques, la qual cosa implica un augment de fosfatidilserina a la superfície, lípid amb cap polar negatiu, que aporta càrregues negatives a la superfície, i que, per tant, implica una davallada del pH a la superfície cel·lular d'aquestes cèl·lules. El treball conclou que aquests agregats amorfs que es formen a pH baix podrien ser els responsables de la toxicitat del pèptid. Els resultats són rellevants ja que donen un possible mecanisme molecular a la relació que els processos oxidatius i les malalties vasculars en la malaltia d'Alzheimer. Tant els processos oxidatius, que implicarien una acidificació a la superfície cel·lular degut a la peroxidació lipídica, com les malalties vasculars, que poden implicar una acidificació local del teixit, podrien afavorir la formació d'aquests agregats amorfs d'alt pes molecular augmentant la toxicitat del pèptid.Aβ peptides are the main components of the characteristic fibrilar amyloid plaques found in central nervous systems affected by Alzheimer's disease. Amyloid plaques are usually found associated to lipids from the plasma cell membrane. Membranes may play a role in amyloid toxicity, either as a peptide's target or by influencing the peptide aggregation state. A particularly interesting aspect of the possible influence of lipid bilayers is the existence of an important amount of experimental evidence pointing to lipid peroxidation as an important factor in neurodegeneration. The general objective of this thesis was the molecular characterization of the peptide interaction with biological membranes. 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

Aβ40 Aggregation under Changeable Conditions

Homeostasis is crucial for cell function, and disturbances in homeostasis can lead to health disorders. Under normal conditions, intracellular pH is maintained between 7.35 and 7.45. Altered endosomal and lysosomal pH together with a general drop in brain pH are associated with the aggregation of amyloid-β-peptide (Aβ) and the development of Alzheimer's disease. Under acidic conditions, close to the Aβ isoelectric point, the absence of charges favors the formation of intermolecular contacts and promotes aggregation. Here, we analyzed how pH levels affect the aggregation of Aβ40 considering the variations in brain pH and the coexistence of different aggregated conformations. Our results suggest that different macromolecular conformations can interact with each other and influence the aggregation process. In addition, we showed that neutral pH and physiological salt concentrations favor a slow aggregation, resulting in ordered, stable fibrils, with low cytotoxic effects. Overall, we highlight the complexity of the aggregation processes occurring in different physiological and pathological environments