Gabriel Barreda-Gómez

Profiling and Imaging of Lipids on Brain and Liver Tissue by Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry Using 2-Mercaptobenzothiazole as a Matrix

2-Mercaptobenzothiazole (MBT) is employed for the first time as a matrix for the analysis of lipids from tissue extracts using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We demonstrate that the performance of MBT is superior to that of the matrixes commonly employed for lipids, due to its low vapor pressure, its low acidity, and the formation of small crystals, although because of the strong background at low m/z, it precludes detection of species below approximately 500 Da. This inconvenience can be partly overcome with the formation of Cs adducts. Using a polymer-based dual calibration, a mass accuracy of approximately 10 ppm in lipid extracts and of approximately 80 ppm in tissues is achieved. We present spectra from liver and brain lipid extracts where a large amount of lipid species is identified, in both positive and negative ion modes, with high reproducibility. In addition, the above-mentioned special properties of MBT allow its employment for imaging mass spectrometry. In the present work, images of brain and liver tissues showing different lipid species are presented, demonstrating the advantages of the employment of MBT.

Distribution of lipids in human brain

The enormous abundance of lipid molecules in the central nervous system (CNS) suggests that their role is not limited to be structural and energetic components of cells. Over the last decades, some lipids in the CNS have been identified as intracellular signalers, while others are known to act as neuromodulators of neurotransmission through binding to specific receptors. Neurotransmitters of lipidic nature, currently known as neurolipids, are synthesized during the metabolism of phospholipid precursors present in cell membranes. Therefore, the anatomical identification of each of the different lipid species in human CNS by imaging mass spectrometry (IMS), in association with other biochemical techniques with spatial resolution, can increase our knowledge on the precise metabolic routes that synthesize these neurolipids and their localization. The present study shows the lipid distribution obtained by MALDI-TOF IMS in human frontal cortex, hippocampus, and striatal area, together with functional autoradiography of cannabinoid and LPA receptors. The combined application of these methods to postmortem human brain samples may be envisioned as critical to further understand neurological diseases, in general, and particularly, the neurodegeneration that accompanies Alzheimer’s disease.

Development of New Drugs that Act Through Membrane Receptors and Involve an Action of Inverse Agonism

Synaptic transmission and, consequently, neurological processes are regulated by neurotransmitters and other neuro-modulators that recognize specific receptors. There are two main families of receptors that are targets for most of the compounds which act in the central nervous system (CNS); ion channel coupled receptors and G-protein coupled receptors (GPCR). The drugs that act through these receptors and are used for the treatment of different diseases related with the CNS, have traditionally been classified as agonists or antagonists. However, since the discovery of the constitutive activity of some neurotransmitter receptors during the eighties, the inverse agonist drugs have emerged as a new group of bioactive compounds with the ability to decrease receptor basal activity. New experimental evidence indicates that pathologies associated with different diseases that affect the CNS physiology could involve constitutively active receptors. Therefore, different methods and systems have been patented to explore the receptors that show high basal activity and to test the decrease of the receptor activity produced by inverse agonist compounds. In recent years some inverse agonist drugs and their targets have been patented which are capable of treating CNS related disorders. These include inverse agonists that are selective for serotonin or histamine receptors aimed at treating neuropsychiatric disorders, cannabinoids with an anorexigenic effect and inverse agonists selective for gabaergic receptors for the treatment of neurodegenerative or cognitive disorders.

Galanin activated Gi/o-proteins in human and rat central nervous systems.

The neuropeptide galanin (GAL) is involved in the control of hormone secretion, nociception, feeding behavior, attention, learning and memory. The anatomical localization of galanin receptors in the brain has been described using autoradiography and immunohistochemistry, but both techniques are limited by the availability of specific radioligands or antibodies. Functional autoradiography provides an alternative method by combining anatomical resolution and information of the activity mediated by G-protein coupled receptors. The present study analyzes the functional GAL receptors coupled to Gi/o-proteins in human and rat brain nuclei using [(35)S]GTPγS autoradiography. The results show the anatomical distribution of Gi/o-proteins activated by GAL receptors that trigger intracellular signaling mechanisms. The activity mediated by GAL receptors in human and rat brain showed a good correlation of the net stimulation in areas such as spinal cord, periaqueductal gray, putamen, CA3 layers of hippocampus, substantia nigra and diverse thalamic nuclei. The functional GAL receptors coupled to Gi/o-proteins showed a similar pattern for both species in most of the areas analyzed, but some discrete nuclei showed differences in the activity mediated by GAL, such as the ventroposteromedial thalamic nucleus, or areas that regulate learning and memory processes in the hippocampus. Taken into consideration the present results, the rat could be used as an experimental model for the study of the physiological role of GAL-mediated neurotransmission and the modulation of GAL receptors activity in the human CNS.

Neurotransmitter receptor localization: from autoradiography to imaging mass spectrometry.

Autoradiography is used to determine the anatomical distribution of biological molecules in human tissue and experimental animal models. This method is based on the analysis of the specific binding of radiolabeled compounds to locate neurotransmitter receptors or transporters in fresh frozen tissue slices. The anatomical resolution obtained by quantification of the radioligands has allowed the density of receptor proteins to be mapped over the last 40 years. The data yielded by autoradiography identify the receptors at their specific microscopic localization in the tissues and also in their native microenvironment, the intact cell membrane. Furthermore, in functional autoradiography, the effects of small molecules on the activity of G protein-coupled receptors are evaluated. More recently, autoradiography has been combined with membrane microarrays to improve the high-throughput screening of compounds. These technical advances have made autoradiography an essential analytical method for the progress of drug discovery. We include the future prospects and some preliminary results for imaging mass spectrometry (IMS) as a useful new method in pharmacodynamic and pharmacokinetic studies, complementing autoradiographic studies. IMS results could also be presented as density maps of molecules, proteins, and metabolites in tissue sections that can be identified, localized, and quantified, with the advantage of avoiding any labeling of marker molecules. The limitations and future developments of these techniques are discussed here.

Methods to measure g-protein-coupled receptor activity for the identification of inverse agonists.

Before the concept of constitutive or intrinsic activity of the biological systems, which was formulated about thirty years ago, it was thought that agonist compounds were the only drugs capable of activating physiological responses, while antagonists were the ones capable of blocking them. However, this basic classification of drugs in pharmacology started to change only at the end of the eighties, when bioactive ligands, with negative efficacy, were developed. The G-protein-coupled receptors (GPCR) were promptly selected as one of the most useful types of pharmacological targets to study this inverse efficacy. This family of receptors is responsible for the signaling and control of many physiological processes, from the peripheral nervous system to the central. Therefore, the GPCR have become the most studied family of receptors in drug discovery. It has been estimated that around a third of the drugs actually used act via the GPCR, nevertheless there are still many orphan GPCR encoded by the human genome. During the last decade, reports and patents have described new methods to detect GPCR inverse agonist compounds. The detection of the G-protein constitutive activity and the quantification of the positive or negative efficacies induced by agonists or inverse agonists respectively has been studied by analyzing the binding of the nonhydrolyzable GTP analog, [(³⁵S]GTPγS. The present chapter describes an optimized method to detect GPCR inverse agonist ligands such as cannabinoid compounds, in both membrane homogenates and tissue sections (autoradiography).

Descifrando la anatomía molecular de un tejido mediante análisis lipidómico por espectrometría de masas

Resumen La lipidomica esta emergiendo como una ciencia imprescindible para la comprension del funcionamiento de celulas y tejidos, dado que complementa el conocimiento obtenido mediante la genomica y la proteomica. La caracterizacion de los lipidos que componen una muestra biologica es una tarea compleja, debido a su enorme variedad y variabilidad, y por tanto necesita del empleo de tecnicas de alto rendimiento para su consecucion. La espectrometria de masas esta demostrando ser la tecnica ideal para llevar a cabo dicha tarea, especialmente desde el desarrollo de la imagen por espectrometria de masas. En el presente trabajo se presentan los resultados del empleo de una nueva matriz, 2-mercaptobenzotiazol, en el analisis lipidomico de extractos de higado quiescente y regenerante de roedor mediante espectrometria de masas MALDI-TOF. La buena reproducibilidad entre individuos, permite la observacion de diferencias en las especies lipidicas mayoritarias del higado normal y el afectado de esteatosis. Se muestran ademas imagenes de la disposicion anatomica de ciertas moleculas en el cerebro de rata, obtenidas representando la distribucion de las concentraciones de diversas especies de lipidos a lo largo de los cortes de tejido. Por ultimo, se discute la aplicacion de estas tecnicas a la investigacion experimental y clinica.

G protein coupling of galanin, muscarinic and cannabinoid receptors in areas related to the control of memory and cognitive functions of Alzheimer patients

was to determine the effects of beta amyloid 1-42 treatment on VDR expression in primary cortical neuron cultures. Methods: Cerebral cortex dissected from brains of Sprague Dawley rat embryos on the embryonic day 16 and cultured. The groups including 48 hours of 6 uM beta amyloid 1-42 treated group and control groups, were established. mRNA isolation and cDNA synthesis performed. The levels of VDR expressions were determined by RTqPCR. Localization of VDR was identified by immunofluorescent immunocytochemistry. Results: Expression of VDR in beta amyloid 1-42 treated group was found decreased when compared with control group. The immunolocalization of VDR was observed in both nucleus and cytoplasm of the primary cultured neurons of cerebral cortex. Conclusions: Relative attenuation in the levels of VDR expression by beta amyloid treatment in primary cortical neurons, might indicate potential role of beta amyloid to prevent probable neuroprotective effects of vitamin D in brain.