Neuroanatomical characterization of the G protein-coupled receptor activity evoked by galanin-related ligands

Galanin neuropeptide is distributed throughout the mammalian nervous system modulating a plethora of diverse physiological functions, including nociception, cognition and neuroendocrine regulation. The regulation of the galaninergic system is an interesting approach for the treatment of different diseases associated to those systems. Nevertheless, the pharmacological selectivity and activities of some galanin receptor (GalR) ligands are still in discussion and seem to depend on the dose, the receptor subtype and the second messengers to which they are coupled at different brain areas. The activity of different GalR ligands on Gi/o proteins, was evaluated by the guanosine 5′-(γ-[35S]thio)triphosphate ([35S]GTPγS) autoradiography in vitro assay applied to rat brain tissue slices in the presence of galanin, M15, M35, M40, gal(2−11) or galnon. The enhancement of the [35S]GTPγS binding induced by the chimerical peptides M15, M35 and M40 was similar to that produced by Gal in those brain areas showing the highest stimulations, such as dorsal part of the olfactory nucleus and ventral subiculum. In contrast to these peptides, using gal(2−11) no effect was measured on Gi/o protein coupling in areas of the rat brain with high GalR1 density such as posterior hypothalamic nucleus and amygdala, indicating low selectivity for GalR1 receptors. The effects evoked by the non-peptide ligand, galnon, were different from those induced by galanin, behaving as agonist or antagonist depending on the brain area, but the stimulations were always blocked by M35. Thus, the activity of most used GalR ligands on Gi/o protein mediated signalling is complex and depends on the brain area. More selective and potent GalR ligands are necessary to develop new treatments aimed to modulate the galaninergic system.Supported by grants from the regional Basque Government IT1454–22 awarded to the "Neurochemistry and Neurodegeneration" consolidated research group and ISCIII Spanish Ministry for Health PI20/00153 and co-funded by the European Union (ERDF "A way to make Europe"). Technical and human support provided by General Research Services SGIker [University of the Basque Country (UPV/EHU)]

Estudio de la correlación de marcadores lipídicos y Proteínas de Fase Aguda mediante técnicas moleculares

Los productores de leche y derivados se enfrentan desde hace un siglo al reto de responder a la demanda de la sociedad europea de disponer de garantías de una producción con bienestar animal. Durante los últimos años se han adoptado medidas para mejorar las condiciones de los animales en las explotaciones y desarrollado sistemas de evaluación que permiten certificarlas, como el protocolo Welfare Quality®. Sin embargo, se continúan buscando herramientas y determinaciones objetivas que garanticen el efecto positivo de las modificaciones adoptadas sobre los animales. Entre los marcadores que se utilizan para ello destacan las proteínas de fase aguda siendo la haptoglobina la más relevante en leche. Las muestras analizadas han presentado un veinticinco por ciento de animales sanos con niveles indicativos de estrés o fase aguda en la leche indicando una pérdida de bienestar en las explotaciones lecheras. Una de las estrategias para reducir esta ratio, es la prevención y la detección temprana. Dado que los lípidos son una fracción de gran importancia en este alimento, el estudio de su lipidoma mediante la espectrometría de masas resulta de gran interés. En este estudio se han identificado dos especies lipídicas que se presentan como potenciales biomarcadores del estado sanitario de las vacas lecheras, permitiendo discriminar tanto la leche de animales enfermos como aquella con una concentración de haptoglobina indicativa de estrés o fase aguda. Adicionalmente, mediante el estudio lipidómico de estas muestras se han podido determinar biomarcadores lipídicos que permiten identificar el origen de la muestra en función del grado de libertad del que disponen los animales en la granja. Este grado de libertad de los animales y su capacidad de movimiento repercute directamente en el bienestar animal, postulándose así la lipidómica como una potente herramienta en el apoyo de toma de decisiones para prevención y el control sanitario de las explotaciones ganaderas de producción láctea.<br /

Microarrays, Enzymatic Assays, and MALDI-MS for Determining Specific Alterations to Mitochondrial Electron Transport Chain Activity, ROS Formation, and Lipid Composition in a Monkey Model of Parkinson’s Disease

Multiple evidences suggest that mitochondrial dysfunction is implicated in the pathogenesis of Parkinson’s disease via the selective cell death of dopaminergic neurons, such as that which occurs after prolonged exposure to the mitochondrial electron transport chain (ETC) complex I inhibitor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP). However, the effects of chronic MPTP on the ETC complexes and on enzymes of lipid metabolism have not yet been thoroughly determined. To face these questions, the enzymatic activities of ETC complexes and the lipidomic profile of MPTP-treated non-human primate samples were determined using cell membrane microarrays from different brain areas and tissues. MPTP treatment induced an increase in complex II activity in the olfactory bulb, putamen, caudate, and substantia nigra, where a decrease in complex IV activity was observed. The lipidomic profile was also altered in these areas, with a reduction in the phosphatidylserine (38:1) content being especially relevant. Thus, MPTP treatment not only modulates ETC enzymes, but also seems to alter other mitochondrial enzymes that regulate the lipid metabolism. Moreover, these results show that a combination of cell membrane microarrays, enzymatic assays, and MALDI-MS provides a powerful tool for identifying and validating new therapeutic targets that might accelerate the drug discovery process.This research has been supported by grants from the regional Basque Government ITI1454-22 awarded to the “Neurochemistry and Neurodegeneration” consolidated research group and ISCIII Spanish Ministry for Health PI20/00153 and co-funded by the European Union (ERDF “A way to make Europe”), a grant from the Ministry of Economy and Competitiveness (IPT-2011-1205) and Scholarship Program for the Transition from Educational to Occupational Word (UPV-Basque Government)

Elevation of Tear MMP-9 Concentration as a Biomarker of Inflammation in Ocular Pathology by Antibody Microarray Immunodetection Assays

Matrix metalloproteinases are a family of enzymes fundamental in inflammatory processes. Between them, MMP-9 is up-regulated during inflammation; thus, its quantification in non-invasive fluids is a promising approach for inflammation identification. To this goal, a biomarker quantification test was developed for ocular inflammation detection using anti-MMP-9 antibody microarrays (AbMAs). After validation with eight healthy control tear samples characterized by ELISA, 20 samples were tested from individuals diagnosed with ocular inflammation due to: cataracts, glaucoma, meibomian gland dysfunction, allergy, or dry eye. Concentration values of tear MMP-9 were obtained for each sample, and 12 patients surpassed the pathological threshold (30 ng/mL). A significant elevation of MMP-9 concentration in the tears of glaucoma patients compared with healthy controls was observed. In order to evaluate the diagnostic ability, an ROC curve analysis was performed using our data, determining the optimal threshold for the test at 33.6 ng/mL of tear MMP-9. In addition, a confusion matrix was applied, estimating sensitivity at 60%, specificity at 88%, and accuracy at 68%. In conclusion, we demonstrated that the AbMAs system allows the quantification of MMP-9 in pathologies that involve inflammation of the ocular surface.This research was funded by Basque Government, BIKAINTEK, grant number 48-AF-W2-2019-00006; by the University of the Basque Country, PIFIND19/02, grant number 201900016247; by ELKARTEK, grant number (KK-2019/00086), by MINECO-Retos, grant number (PID2019-111139RBI00) to E.V.; and by FISS, grant number FISS-21-RD21/0002/0041, to I.R.-A. and A.A

Ketamina eta esketaminaren erabilera depresioerresistentea tratatzeko: etorkizunerako erronkak

Depressive disorders have become one of the major public health problems in recent years, not only because of their impact on quality of life, but also because of the partial response or lack of response to antidepressant treatment. Therefore, the rapid and potent antidepressant effect of (R,S)-ketamine (ketamine) is considered one of the most important psychiatric discoveries of the last decades. Although, intravenous administration of ketamine has prevailed in most clinical trials conducted so far, the intranasal route has been considered a useful and safe alternative. Thus, the FDA (Food and Drug Administration) and the EMA (European Medicines Agency) approved a esketamine nasal spray named Spravato for the use in treatment resistant depression, although clinical trials supporting its marketing are not long-term. In line with this, major long-term safety concerns, such as dependence and cognitive impairment, has not been assessed so far. Therefore, there is an urgent need to investigate the relevance ofrepeated administration protocols on the long-term antidepressant response and safety. In this regard, potential pharmacological interactions between ketamine and some antidepressant drugs and benzodiazepines may impact the antidepressant response. In fact, while in some clinical trials adjunctive antidepressant medication is allowed, in others it is specifically removed, and this pharmacological variability may alter the efficacy of the treatment. Consequently, it is undeniable that in addition to massive long-term clinical trials, post-marketing studies are necessary to ensure the safety and efficacy of long-term ketamine and esketamine use.; Nahasmendu depresiboak osasun publikoko arazo handienetako bat bilakatu dira azken urteotan, ez bakarrik bizi-kalitatean duten eraginagatik, baita tratamendu antidepresiboaren erantzun partzial edo erantzun ezagatik ere. Hori dela eta, (R,S)-ketaminaren (ketamina) efektu antidepresibo azkarra eta eraginkorra azken hamarkadetako aurkikuntza garrantzitsuenen artean dago psikiatria arloan. Orain arteko entseguetan ketaminaren zain barneko bidea gailendu den arren sudur bideko administrazioa aukera erabilgarri eta segurutzat jo da. Hori dela eta, FDAk (Food and Drug Administration) eta EMAk (European Medicines Agency) S-ketamina (esketamina) baimendu dute Spravato izen komertzialarekin merkaturatutako sudur-lainoztagailurako soluzio bezala, depresio erresistentea tratatzeko, farmako horren merkaturatze-baimena sostengatzen duten entsegu klinikoak epe luzekoak ez diren arren. Horregatik, epe luzeko segurtasun-profila ikertzea premiazkoa da, eta, testuinguru berean, baita administrazio-protokolo errepikatuen egokitasuna ere. Izan ere, ketaminak farmako antidepresibo batzuekin eta bentzodiazepinekin izan ditzakeen elkarrekintzak ebaluatzea gomendatzen da, tratamenduaren eragin antidepresiboa baldintza dezaketelako. Ildo horretan, orain arte egindako entseguen diseinuaren aldakortasuna kontuan hartzeko ezaugarria litzatekeela proposatu da. Izan ere, entsegu batzuetan, tratamendu antidepresibo gehigarria baztertzen da, eta beste batzuetan, berriz, mantendu egiten da; aldakortasun metodologiko horrek tratamendu antidepresiboaren eraginkortasuna alda dezake. Ondorioz, uka ezina da epe luzerako entsegu klinikoak egiteaz gain merkaturatze ondorengo farmakozaintza-azterlanak ere nahitaezkoak direla ketaminaren eta esketaminaren segurtasuna eta eraginkortasuna epe luzera bermatzeko

Enzyme Therapy: Current Challenges and Future Perspectives

In recent years, enzymes have risen as promising therapeutic tools for different pathologies, from metabolic deficiencies, such as fibrosis conditions, ocular pathologies or joint problems, to cancer or cardiovascular diseases. Treatments based on the catalytic activity of enzymes are able to convert a wide range of target molecules to restore the correct physiological metabolism. These treatments present several advantages compared to established therapeutic approaches thanks to their affinity and specificity properties. However, enzymes present some challenges, such as short in vivo half-life, lack of targeted action and, in particular, patient immune system reaction against the enzyme. For this reason, it is important to monitor serum immune response during treatment. This can be achieved by conventional techniques (ELISA) but also by new promising tools such as microarrays. These assays have gained popularity due to their high-throughput analysis capacity, their simplicity, and their potential to monitor the immune response of patients during enzyme therapies. In this growing field, research is still ongoing to solve current health problems such as COVID-19. Currently, promising therapeutic alternatives using the angiotensin-converting enzyme 2 (ACE2) are being studied to treat COVID-19.This research was funded by the Basque Government, BIKAINTEK, grant number 48-AF-W2-2019-00006; and by University of the Basque Country, PIFIND19/02, grant number 201900016247

Upregulated phospholipase D2 expression and activity is related to the metastatic properties of melanoma

[EN] The incidence rates of melanoma have increased steadily in recent decades and nearly 25% of the patients diagnosed with early-stage melanoma will eventually develop metastasis, for which there is currently no fully effective treatment. The link between phospholipases and tumors has been studied extensively, particularly in breast and colon cancers. With the aim of finding new biomarkers and therapeutic options for melanoma, the expression of different phospholipases was assessed in 17 distinct cell lines in the present study, demonstrating that phospholipase D2 (PLD2) is upregulated in metastatic melanoma as compared to normal skin melanocytes. These results were corroborated by immunofluorescence and lipase activity assays. Upregulation of PLD2 expression and increased lipase activity were observed in metastatic melanoma relative to normal skin melanocytes. So far, the implication of PLD2 activity in melanoma malignancies has remained elusive. To the best of our knowledge, the present study was the first to demonstrate that the overexpression of PLD2 enhances lipase activity, and its effect to increase the proliferation, migration and invasion capacity of melanoma cells was assessed with XTT and Transwell assays. In addition, silencing of PLD2 in melanoma cells reduced the metastatic potential of these cells. The present study provided evidence that PLD2 is involved in melanoma malignancy and in particular, in its metastatic potential, and established a basis for future studies evaluating PLD2 blockade as a therapeutic strategy to manage this condition.This study was supported by grants from the University of the Basque Country/EHU (grant no. GIU17/066) and Ministerio de Economia y Competividad MINECO-ONCOFINDER of the Spanish Government (grant no. RTC.2015-3693-1)

Analysis of Acetylcholinesterase Activity in Cell Membrane Microarrays of Brain Areas as a Screening Tool to Identify Tissue Specific Inhibitors

Acetylcholinesterase (AChE) is responsible for hydrolyzing the acetylcholine neurotransmitter, bringing an end point to cholinergic neurotransmission. Thus, AChE is the primary target of a wide spectrum of compounds used as pesticides, nerve agents or therapeutic drugs for neurodegenerative diseases such as Alzheimer’s disease (AD). This enzyme is heterogeneously distributed in the brain showing different activity depending on the nervous region. Therefore, the aim of this work is to report a novel technology that enables the simultaneous determination of tissue specific AChE activity, as well as the analysis and screening of specific inhibitors, by using cell membrane microarrays. These microarrays were composed of cell membranes, isolated from 41 tissues, organs and brain areas, that were immobilized over a slide, maintaining the functionality of membrane proteins. To validate this platform, demonstrating its usefulness in drug discovery as a high throughput screening tool, a colorimetric protocol to detect the membrane-bound AChE activity was optimized. Thus, rat cortical and striatal AChE activities were estimated in presence of increased concentrations of AChE inhibitors, and the donepezil effect was assessed simultaneously in 41 tissues and organs, demonstrating the major potential of this microarray’s technology.The Spanish Ministry of Economy and Competitiveness (Innpacto program: IPT-2011-1205-010000), and the Basque Government Department of Economic Development, sustainability and environment (Etorgai program: ER-2011/00015, Bikaintek program: 48-AF-W2-2019-7)

Review of Technological Challenges in Personalised Medicine and Early Diagnosis of Neurodegenerative Disorders

Neurodegenerative disorders are characterised by progressive neuron loss in specific brain areas. The most common are Alzheimer’s disease and Parkinson’s disease; in both cases, diagnosis is based on clinical tests with limited capability to discriminate between similar neurodegenerative disorders and detect the early stages of the disease. It is common that by the time a patient is diagnosed with the disease, the level of neurodegeneration is already severe. Thus, it is critical to find new diagnostic methods that allow earlier and more accurate disease detection. This study reviews the methods available for the clinical diagnosis of neurodegenerative diseases and potentially interesting new technologies. Neuroimaging techniques are the most widely used in clinical practice, and new techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) have significantly improved the diagnosis quality. Identifying biomarkers in peripheral samples such as blood or cerebrospinal fluid is a major focus of the current research on neurodegenerative diseases. The discovery of good markers could allow preventive screening to identify early or asymptomatic stages of the neurodegenerative process. These methods, in combination with artificial intelligence, could contribute to the generation of predictive models that will help clinicians in the early diagnosis, stratification, and prognostic assessment of patients, leading to improvements in patient treatment and quality of life.This publication is part of the Grant PID2 021-126434OB-I00 funded by MCIN/AEI/10.13039/501100011033 and ERDF A way of making Europe. It has also been funded by the Basque Government (IT1706-22 and PUE21-03) and the University of the Basque Country, UPV/EHU (GIU19/092 and COLAB20/07). This research was conducted in the scope of the Transborder Joint Laboratory (LTC) “non-motor Comorbidities in Parkinson’s Disease (CoMorPD)”

Spinal Inhibition of GABAB Receptors by the Extracellular Matrix Protein Fibulin-2 in Neuropathic Rats

In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein-coupled receptors (GPCRs). Receptor interaction with partner proteins has emerged as a novel mechanism to alter GPCR signaling in pathophysiological conditions. We propose here that GABAB activity is inhibited through the specific binding of fibulin-2, an extracellular matrix protein, to the B1a subunit in a rat model of neuropathic pain. We demonstrate that fibulin-2 hampers GABAB activation, presumably through decreasing agonist-induced conformational changes. Fibulin-2 regulates the GABAB-mediated presynaptic inhibition of neurotransmitter release and weakens the GABAB-mediated inhibitory effect in neuronal cell culture. In the dorsal spinal cord of neuropathic rats, fibulin-2 is overexpressed and colocalized with B1a. Fibulin-2 may thus interact with presynaptic GABAB receptors, including those on nociceptive afferents. By applying anti-fibulin-2 siRNAin vivo, we enhanced the antinociceptive effect of intrathecal baclofen in neuropathic rats, thus demonstrating that fibulin-2 limits the action of GABAB agonistsin vivo. Taken together, our data provide an example of an endogenous regulation of GABAB receptor by extracellular matrix proteins and demonstrate its functional impact on pathophysiological processes of pain sensitization.This work was funded by the ANR ImNet (ANR-07-NEURO015-01). Imaging was performed on the Bordeaux Imaging Center, member of the FranceBioImaging national infrastructure (ANR-10-INBS-04)