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Antibodies against phosphorylated alpha-synuclein (aSyn) at S129 have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues of patients with Parkinson’s disease and other neurodegenerative diseases. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal post-translational modifications (PTMs) (e.g., phosphorylation at Tyrosine 125 or truncation at residue 133 or 135) differentially influences the detection of pS129-aSyn species by pS129-aSyn antibodies. These observations prompted us to systematically reassess the specificity of the most commonly used pS129 antibodies against monomeric and aggregated forms of pS129-aSyn in mouse brain slices, primary neurons, mammalian cells and seeding models of aSyn pathology formation. We identified two antibodies that are insensitive to pS129 neighboring PTMs. Although most pS129 antibodies showed good performance in detecting aSyn aggregates in cells, neurons and mouse brain tissue containing abundant aSyn pathology, they also showed cross-reactivity towards other proteins and often detected non-specific low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or high molecular weight aSyn species. Our observations suggest that not all pS129 antibodies capture the biochemical and morphological diversity of aSyn pathology, and all should be used with the appropriate protein standards and controls when investigating aSyn under physiological conditions. Finally, our work underscores the need for more pS129 antibodies that are not sensitive to neighboring PTMs and more thorough characterization and validation of existing and new antibodies.

ObjectiveWe used magnetization transfer imaging to assess white matter tissue integrity in migraine, to explore whether white matter microstructure was more diffusely affected beyond visible white matter hyperintensities (WMHs), and to explore whether focal invisible microstructural changes precede visible focal WMHs in migraineurs.MethodsWe included 137 migraineurs (79 with aura, 58 without aura) and 74 controls from the Cerebral Abnormalities in Migraine, an Epidemiological Risk Analysis (CAMERA) study, a longitudinal population-based study on structural brain lesions in migraine patients, who were scanned at baseline and at a 9-year follow-up. To assess microstructural brain tissue integrity, baseline magnetization transfer ratio (MTR) values were calculated for whole brain white matter. Baseline MTR values were determined for areas of normal-appearing white matter (NAWM) that had progressed into MRI-detectable WMHs at follow-up and compared to MTR values of contralateral NAWM.ResultsMTR values for whole brain white matter did not differ between migraineurs and controls. In migraineurs, but not in controls, NAWM that later progressed to WMHs at follow-up had lower mean MTR (mean [SD] 0.354 [0.009] vs 0.356 [0.008], p = 0.047) at baseline as compared to contralateral white matter.ConclusionsWe did not find evidence for widespread microstructural white matter changes in migraineurs compared to controls. However, our findings suggest that a gradual or stepwise process might be responsible for evolution of focal invisible microstructural changes into focal migraine-related visible WMHs.

Neurotransmitters are signaling molecules, playing key roles in neuronal communications in the brain. Drug induced changes in neurotransmitters and other brain metabolite concentration may be used to characterize drugs according to their targeted metabolomics profile. Here, we report the development and validation of a straightforward liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 16 endogenous small polar compounds in rat plasma and brain homogenates. The method enables the quantification of the neurotransmitters γ-aminobutyric acid, glutamate, acetylcholine and adenosine, as well as choline, glutamine, acetylcarnitine, carnitine, creatine, creatinine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan. After optimizing the sample preparation, chromatographic and spectrometric conditions, the method was successfully validated using the standard addition approach and a hydrophilic interaction chromatography (HILIC) with an amide column. The method was shown to be linear (r > 0.99) as all the compounds were within the ±25% values of intra and inter-day precision and accuracy acceptance. A matrix effect was corrected with the use of 10 isotopically labelled internal standards and the compound stability was evaluated for all compounds. Relevant exaltation of choline (in plasma) and creatinine (in brain) were solved with −20 °C conditions. The applicability of the method was tested by evaluating brain alterations in the concentrations of neurotransmitters and related compounds after the administration of two psychostimulant drugs of abuse (cocaine and methylenedioxypyrovalerone) to rats. A neuro-metabolic fingerprint of each drug was obtained that reflected their pharmacological profile. Altogether, this methodology presents a valuable targeted metabolomics tool for basic and clinical research studies. This work was supported by the European Commission (Drugs Policy Initiatives, Justice Programme 2014–2020, contract n°. HOME/2014/JDRU/AG/DRUG/7082, Predicting Risk of Emerging Drugs with In silico and Clinical Toxicology). JRM is acknowledged for the EU's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant No. 712949 (TECNIOspring PLUS) from the Agency for Business Competitiveness of the Government of Catalonia. The Spanish Health National System is also acknowledged for the contract of OJP (CPII16/00027)

Accurate tissue phantoms are difficult to design due to the complex non-linear viscoelastic properties of real soft tissues. A composite hydrogel, resulting from a mix of poly(vinyl) alcohol and phytagel, is able to reproduce the viscoelastic responses of different soft tissues due to its compositional tunability. The aim of this work is to demonstrate the flexibility of the composite hydrogel in mimicking the interactions between surgical tools and various soft tissues, such as brain, lung and liver. Therefore compressive stiffness, insertion forces and frictional forces were used as matching criteria to determine the hydrogel compositions for each soft tissue. A full map of the behaviour of the synthetic material is provided for these three characteristics and the compositions found to best match the mechanical response of brain, lung and liver are reported. The optimised hydrogel samples are then tested and shown to mimic the behaviour of the three tissues with unprecedented fidelity. The effect of each hydrogel constituent on the compressive stiffness, needle insertion and frictional forces is also detailed in this work to explain their individual contributions and synergistic effects. This study opens important opportunities for the realisation of surgical planning and training devices and tools for in-vitro tissue testing. Keywords: Composite hydrogel, Soft tissue, Tissue mimic, Surgery, Material properties

Introduction: Amyloid imaging has been integrated into diagnostic criteria for Alzheimer's disease (AD). How amyloid tracers binding differ for different tracer structures and amyloid-β aggregates in autosomal dominant AD (ADAD) and sporadic AD is unclear. Methods: Binding properties of different amyloid tracers were examined in brain homogenates from six ADAD with APPswe, PS1 M146V, and PS1 EΔ9 mutations, 13 sporadic AD, and 14 control cases. Results: 3H-PIB, 3H-florbetaben, 3H-AZD2184, and BTA-1 shared a high- and a varying low-affinity binding site in the frontal cortex of sporadic AD. AZD2184 detected another binding site (affinity 33nM) in the frontal cortex of ADAD. The 3H-AZD2184 and 3H-PIB binding were significantly higher in the striatum of ADAD compared to sporadic AD and control. Polyphenol resveratrol showed strongest inhibition on 3H-AZD84 binding followed by 3H-florbetaben and minimal on 3H-PIB. Discussion: This study implies amyloid tracers of different structures detect different sites on amyloid-β fibrils or conformations.

In the quest for chronically reliable and bio-tolerable brain interfaces there has been a steady evolution towards the use of highly flexible, polymer-based electrode arrays. The reduced mechanical mismatch between implant and brain tissue has shown to reduce the evoked immune response, which in turn has a positive effect on signal stability and noise. Unfortunately, the low stiffness of the implants also has practical repercussions, making surgical insertion extremely difficult. In this work we explore the use of dextran as a coating material that temporarily stiffens the implant, preventing buckling during insertion. The mechanical properties of dextran coated neural probes are characterized, as well as the different parameters which influence the dissolution rate. Tuning parameters, such as coating thickness and molecular weight of the used dextran, allows customization of the stiffness and dissolution time to precisely match the user&rsquo, s needs. Finally, the immunological response to the coated electrodes was analyzed by performing a histological examination after four months of in vivo testing. The results indicated that a very limited amount of glial scar tissue was formed. Neurons have also infiltrated the area that was initially occupied by the dissolving dextran coating. There was no noticeable drop in neuron density around the site of implantation, confirming the suitability of the coating as a temporary aid during implantation of highly flexible polymer-based neural probes.

Imaging mass spectrometry (IMS) has become a powerful tool to characterize the spatial distribution of biomolecules in thin tissue sections. In the case of matrix-assisted laser desorption ionization (MALDI) IMS, homogeneous matrix deposition is critical to produce high-quality ion images, and sublimation in particular has shown to be an excellent matrix deposition method for the imaging of lipids. Matrix deposition by sublimation is, however, a completely solvent-free system, which ought to prevent the mixing of matrix and analytes thought to be necessary for successful MALDI. Using 3D time-of-flight secondary ion imaging mass spectrometry, we have studied the matrix tissue interface in 3D with high resolution to understand the MALDI process of lipids after matrix deposition by sublimation. There is a strong indication that diffusion is the process by which lipids migrate from the tissue to the matrix layer. We show that triacylglycerols and phospholipids have a delayed migratory trend as compared to diacylglycerols and monoacylglycerols, which is dependent on time and matrix thickness. Additional experiments show that a pure lipid's capacity to migrate into the matrix is dependent on its fluidity at room temperature. Furthermore, it is shown that cholesterol can only migrate in the presence of a (fluid) lipid and appears to fluidize lipids, which could explain its colocalization with the diacylglycerols and monoacylglycerols in the matrix.

Amperometric enzyme-based biosensors, increasingly used for in vivo brain biomonitoring, typically suffer from electrochemical interference. At the potential (>500 mV) necessary to oxidize the target analyte, often hydrogen peroxide (H2O2), non-specific electroactive species are easily oxidizable, resulting in poor biosensor selectivity. The use of permselective membranes, alone or in combination, is an efficient method to improve biosensor selectivity. These membranes are thin-film polymers (nm to pin thick), able to reduce electrochemical interference. However, the exact mechanism by which these membranes reduce interference is not entirely understood. As membrane assembly is a surface dependent process, we explored the putative role of surface availability in membrane-induced selectivity. We modified the surface of microelectrodes with the most effective permselective membrane configurations available. Microelectrodes surface was characterized by electrochemical methods and scanning electron microscopy. All membranes reduced non-specific oxidation for all non-specific electroactive species. However, only PmPD (poly-m-phenylenediamine) (alone and combined with Nafion) and OPPy (overoxidized polypyrrole) were selective against both cations and anions. Besides reducing electrochemical interference, all membranes also reduced the sensitivity for the target analyte, H2O2. The use of membrane combinations resulted in an additional decrease in non-specific oxidation without an increase in selectivity. This additional decrease was highly correlated with a loss in H2O2 sensitivity, suggesting a reduction of active electrode surface. Additionally, microscopic evaluation indicated an intriguing "inner polymerization" process in microelectrodes coated with membrane combinations. Our results point to a significant role of surface availability in the mechanisms underlying membrane-induced selectivity, crucial for the design and performance of enzyme-based amperometric biosensors. (C) 2015 Elsevier B.V. All rights reserved.

International audience; Polarization resolved nonlinear microscopy (PRNM) is a powerful technique to gain microscopic structural information in biological media. However, deep imaging in a variety of biological specimens is hindered by light scattering phenomena, which not only degrades the image quality but also affects the polarization state purity. In order to quantify this phenomenon and give a framework for polarization resolved microscopy in thick scattering tissues, we develop a characterization methodology based on four wave mixing (FWM) process. More specifically, we take advantage of two unique features of FWM, meaning its ability to produce an intrinsic in-depth local coherent source and its capacity to quantify the presence of light depolarization in isotropic regions inside a sample. By exploring diverse experimental layouts in phantoms with different scattering properties, we study systematically the influence of scattering on the nonlinear excitation and emission processes. The results show that depolarization mechanisms for the nonlinearly generated photons are highly dependent on the scattering center size, the geometry used (epi/forward) and, most importantly, on the thickness of the sample. We show that the use of an un-analyzed detection makes the polarization-dependence read-out highly robust to scattering effects, even in regimes where imaging might be degraded. The effects are illustrated in polarization resolved imaging of myelin lipid organization in mouse spinal cords