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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).

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)].

Cannabis has been used for decades as a palliative therapy in the treatment of cancer. This is because of its beneficial effects on the pain and nausea that patients can experience as a result of chemo/radiotherapy. Tetrahydrocannabinol and cannabidiol are the main compounds present in Cannabis sativa, and both exert their actions through a receptor-mediated mechanism and through a non-receptor-mediated mechanism, which modulates the formation of reactive oxygen species. These oxidative stress conditions might trigger lipidic changes, which would compromise cell membrane stability and viability. In this sense, numerous pieces of evidence describe a potential antitumor effect of cannabinoid compounds in different types of cancer, although controversial results limit their implementation. In order to further investigate the possible mechanism involved in the antitumoral effects of cannabinoids, three extracts isolated from Cannabis sativa strains with high cannabidiol content were analyzed. Cell mortality, cytochrome c oxidase activity and the lipid composition of SH-SY5Y cells were determined in the absence and presence of specific cannabinoid ligands, with and without antioxidant pre-treatment. The cell mortality induced by the extracts in this study appeared to be related to the inhibition of the cytochrome c oxidase activity and to the THC concentration. This effect on cell viability was similar to that observed with the cannabinoid agonist WIN55,212-2. The effect was partially blocked by the selective CB1 antagonist AM281, and the antioxidant α-tocopherol. Moreover, certain membrane lipids were affected by the extracts, which demonstrated the importance of oxidative stress in the potential antitumoral effects of cannabinoids. This work has been partially supported by a grant from the Ministry of Economy and Competitiveness (DIN2019-010902 and DIN2020-011349) and the Basque Government Department of Economic Development, Sustainability and Environment (Bikaintek program: 005-B2/2021).

Melanoma is a carcinoma known to evade the host immune defenses via the downregulation of the immune response. One of the molecules involved in this mechanism is programmed cell death ligand 1 (PD-L1), which interacts with its receptor, programmed cell death protein 1 (PD-1), expressed on T cells, leading to a reduction in cytokine release and cytotoxic activity, as well as a halt in T-cell proliferation. The approved therapeutic monoclonal antibodies, such as pembrolizumab, target the PD-1/PD-L1 interaction and are revolutionizing cancer treatments. We developed an assay that provides a quantitative readout of PD-1/PD-L1 interactive states between cell membranes of human immune cells (peripheral blood mononuclear cells, PBMCs) and PD-L1-expressing samples. For this purpose, cell membrane microarrays (CMMAs) were developed from membranes isolated from a HT144 cell line and melanoma samples, and PD-L1 expression was quantified using immunofluorescence (IF). CMMAs were incubated with cell membranes of PBMCs expressing PD-1, and the interaction with PD-L1 was quantified by time-resolved Förster resonance energy transfer, in the presence and absence of pembrolizumab as a blocking drug. The developed assay was able to quantify the PD-1/PD-L1 interaction, and this engagement was disrupted in the presence of the blocking antibody. This demonstrates the potential of the method to analyze monoclonal antibody drugs, as well as the functional states of immune checkpoint regulators. Furthermore, our findings provide evidence to support the future implementation of this methodology for both drug discovery and immune system monitoring in cancer, transplantation, and inflammatory and autoimmune diseases.

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.

Increasing evidence supports a relationship between lipid metabolism and mental health. In particular, the biostatus of polyunsaturated fatty acids (PUFAs) correlates with some symptoms of psychiatric disorders, as well as the efficacy of pharmacological treatments. Recent findings highlight a direct association between brain PUFA levels and dopamine transmission, a major neuromodulatory system implicated in the etiology of psychiatric symptoms. However, the mechanisms underlying this relationship are still unknown. Here we demonstrate that membrane enrichment in the n-3 PUFA docosahexaenoic acid (DHA), potentiates ligand binding to the dopamine D2 receptor (D2R), suggesting that DHA acts as an allosteric modulator of this receptor. Molecular dynamics simulations confirm that DHA has a high preference for interaction with the D2R and show that membrane unsaturation selectively enhances the conformational dynamics of the receptor around its second intracellular loop. We find that membrane unsaturation spares G protein activity but potentiates the recruitment of β-arrestin in cells. Furthermore, in vivo n-3 PUFA deficiency blunts the behavioral effects of two D2R ligands, quinpirole and aripiprazole. These results highlight the importance of membrane unsaturation for D2R activity and provide a putative mechanism for the ability of PUFAs to enhance antipsychotic efficacy.

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)”.

The production of reactive oxygen species (ROS) increases considerably in situations of cellular stress, inducing lipid peroxidation and multiple alterations in proteins and nucleic acids. However, sensitivity to oxidative damage varies between organs and tissues depending on the triggering process. Certain drugs used in the treatment of diverse diseases such as malaria have side effects similar to those produced by oxidative damage, although no specific study has been conducted. For this purpose, cell membrane microarrays were developed and the superoxide production evoked by the mitochondrial activity was assayed in the presence of specific inhibitors: rotenone, antimycin A and azide. Once the protocol was set up on cell membrane isolated from rat brain areas, the effect of six antimalarial drugs (atovaquone, quinidine, doxycycline, mefloquine, artemisinin, and tafenoquine) and two essential oils (Rosmarinus officinalis and Origanum majoricum) were evaluated in multiple human samples. The basal activity was different depending on the type of tissue, the liver, jejunum and adrenal gland being the ones with the highest amount of superoxide. The antimalarial drugs studied showed specific behavior according to the type of human tissue analyzed, with atovaquone and quinidine producing the highest percentage of superoxide formation, and doxycycline the lowest. In conclusion, the analysis of superoxide production evaluated in cell membranes of a collection of human tissues allowed for the characterization of the safety profile of these antimalarial drugs against toxicity mediated by oxidative stress This work has been partially supported by grant Ministry of Economy and Competitiveness PID2019-106222RB-C31/SRA (State Research Agency, 10.13039/501100011033), the Basque Government Department of Economic Development, Sustainability and Environment (PUE21-03 and Bikaintek program: 48-AF-W2-2019-7 and 007-B2/2020) and University of the Basque Country (price to the best Master Thesis in collaboration with Institutions and Business).

International audience; The recent developments in mass spectrometry have revealed the importance of lipids as biomarkers in the context of different diseases and as indicators of the cell's homeostasis. However, further advances are required to unveil the complex relationships between lipid classes and lipid species with proteins. Here, we present a new methodology that combines microarrays with mass spectrometry to obtain the lipid fingerprint of samples of a different nature in a standardized and fast way, with minimal sample consumption. As a proof of concept, we use the methodology to obtain the lipid fingerprint of 20 rat tissues and to create a lipid library for tissue classification. Then, we combine those results with immunohistochemistry and enzymatic assays to unveil the relationship between some lipid species and two enzymes. Finally, we demonstrate the performance of the methodology to explore changes in lipid composition of the nucleus accumbens from mice subjected to two lipid diets.

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.

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells., Ministerio Espanol de Economia y Competitividad (MINECO), grant from the Fundacion Ramon Areces and Leonardo Grant from fBBVA to Maria Yanez-Mo

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.

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).

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).

SUMMARYThe idea that the effects of drugs largely depend on subcellular target location is emerging as a novel predictive factor of their beneficial or adverse outcomes. G protein-coupled type-1 cannabinoid receptors (CB1) are regulators of several brain functions as well as the main targets of cannabinoid-based medicines.Besides their classical location at plasma membranes, CB1receptors are present at different locations within cells, including in association to mitochondrial membranes (mtCB1). Here we report the generation and characterization of a mutant mouse line, which lack mtCB1receptors.

Melanoma is the deadliest form of skin cancer due to its ability to colonize distant sites and initiate metastasis. Although these processes largely depend on the lipid-based cell membrane scaffold, our understanding of the melanoma lipid phenotype lags behind most other aspects of this tumor cell. Here, we examined a panel of normal human epidermal and nevus melanocytes and primary and metastatic melanoma cell lines to determine whether distinctive cell-intrinsic lipidomes can discern non-neoplastic from neoplastic melanocytes and define their metastatic potential. Lipidome profiles were obtained by UHPLC-ESI mass-spectrometry, and differences in the signatures were analyzed by multivariate statistical analyses. Significant and highly specific changes in more than 30 lipid species were annotated in the initiation of melanoma, whereas less numerous changes were associated with melanoma progression and the non-malignant transformation of nevus melanocytes. Notably, the “malignancy lipid signature” features marked drops in pivotal membrane lipids, like sphingomyelins, and aberrant elevation of ether-type lipids and phosphatidylglycerol and phosphatidylinositol variants, suggesting a previously undefined remodeling of sphingolipid and glycerophospholipid metabolism. Besides broadening the molecular definition of this neoplasm, the different lipid profiles identified may help improve the clinical diagnosis/prognosis and facilitate therapeutic interventions for cutaneous melanoma. This research was funded in part by grants from the Ministry of Economy; Industry and Competitiveness (RTC-2015-3693-1); Ministry of Science and Innovation (RTI-2018-095134-B-I00); Basque Government (IT971-16; IT1162-19; KK2016-036; KK2017-041 and KK2018-00090) and UPV/EHU (GIU17/066).

In order to better understand retinal physiology, alterations to which underlie some ocular diseases, we set out to establish the lipid signature of two fundamental cell types in the retina, Muller Glia and Retinal Ganglion Cells (RGCs). Moreover, we compared the lipid signature of these cells in sections (in situ), as well as after culturing the cells and isolating their cell membranes (in vitro). The lipidome of Muller glia and RGCs was analyzed in porcine retinal sections using Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS). Isolated membranes, as well as whole cells from primary cell cultures of RGCs and Muller glia, were printed onto glass slides using a non-contact microarrayer (Nano Plotter), and a LTQ-Orbitrap XL analyzer was used to scan the samples in negative ion mode, thereafter identifying the RGCs and Muller cells immunohistochemically. The spectra acquired were aligned and normalized against the total ion current, and a statistical analysis was carried out to select the lipids specific to each cell type in the retinal sections and microarrays. The peaks of interest were identified by MS/MS analysis. A cluster analysis of the MS spectra obtained from the retinal sections identified regions containing RGCs and Muller glia, as confirmed by immunohistochemistry in the same sections. The relative density of certain lipids differed significantly (p-value

International audience; Recent advances in neuroscience have positioned brain circuits as key units in controlling behavior, implying that their positive or negative modulation necessarily leads to specific behavioral outcomes. However, emerging evidence suggests that the activation or inhibition of specific brain circuits can actually produce multimodal behavioral outcomes. This study shows that activation of a receptor at different subcellular locations in the same neuronal circuit can determine distinct behaviors. Pharmacological activation of type 1 cannabinoid (CB1) receptors in the striatonigral circuit elicits both antinociception and catalepsy in mice. The decrease in nociception depends on the activation of plasma membrane-residing CB1 receptors (pmCB1), leading to the inhibition of cytosolic PKA activity and substance P release. By contrast, mitochondrial-associated CB1 receptors (mtCB1) located at the same terminals mediate cannabinoid-induced catalepsy through the decrease in intra-mitochondrial PKA-dependent cellular respiration and synaptic transmission. Thus, subcellular-specific CB1 receptor signaling within striatonigral circuits determines multimodal control of behavior.

International audience; Reward-processing impairment is a common symptomatic dimension of several psychiatric disorders. However, whether the underlying pathological mechanisms are common is unknown. Herein, we asked if the decrease in the n-3 polyunsaturated fatty acid (PUFA) lipid species, consistently described in these pathologies, could underlie reward-processing deficits. We show that reduced n-3 PUFA biostatus in mice leads to selective motivational impairments. Electrophysiological recordings revealed increased collateral inhibition of dopamine D2 receptor-expressing medium spiny neurons (D2-MSNs) onto dopamine D1 receptor-expressing MSNs in the nucleus accumbens, a main brain region for the modulation of motivation. Strikingly, transgenically preventing n-3 PUFA deficiency selectively in D2-expressing neurons normalizes MSN collateral inhibition and enhances motivation. These results constitute the first demonstration of a causal link between a behavioral deficit and n-3 PUFA decrease in a discrete neuronal population and suggest that lower n-3 PUFA biostatus in psychopathologies could participate in the etiology of reward-related symptoms.

This paper reports the first validation results of a promising point of care device based on an injected plastic microfluidic cartridge and commercial off the shelf components. An 8x8 spotted array is immobilized in the incubation chamber inside the cartridge, and once the chemical reactions have been finished, the colorimetric results are captured by a CMOS camera. The captured image of the array is analysed in terms of intensity levels at each spot location. This device is intended to detect allergen specific IgE in veterinary applications, but it can also be applied to any application based on ELISA like protocols.

Cellular activity in the brain depends on the high energetic support provided by mitochondria, the cell organelles which use energy sources to generate ATP. Acute cannabinoid intoxication induces amnesia in humans and animals, and the activation of type-1 cannabinoid receptors present at brain mitochondria membranes (mtCB1) can directly alter mitochondrial energetic activity. Although the pathological impact of chronic mitochondrial dysfunctions in the brain is well established, the involvement of acute modulation of mitochondrial activity in high brain functions, including learning and memory, is unknown. Here, we show that acute cannabinoid-induced memory impairment in mice requires activation of hippocampal mtCB1 receptors. Genetic exclusion of CB1 receptors from hippocampal mitochondria prevents cannabinoid-induced reduction of mitochondrial mobility, synaptic transmission and memory formation. mtCB1 receptors signal through intra-mitochondrial Gαi protein activation and consequent inhibition of soluble-adenylyl cyclase (sAC). The resulting inhibition of protein kinase A (PKA)-dependent phosphorylation of specific subunits of the mitochondrial electron transport system eventually leads to decreased cellular respiration. Hippocampal inhibition of sAC activity or manipulation of intra-mitochondrial PKA signalling or phosphorylation of the Complex I subunit NDUFS2 inhibit bioenergetic and amnesic effects of cannabinoids. Thus, the G protein-coupled mtCB1 receptors regulate memory processes via modulation of mitochondrial energy metabolism. By directly linking mitochondrial activity to memory formation, these data reveal that bioenergetic processes are primary acute regulators of cognitive functions.

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.

Molecular mass images of tissues will be biased if differences in the physicochemical properties of the microenvironment affect the intensity of the spectra. To address this issue, we have performed-by means of MALDI-TOF mass spectrometry-imaging on slices and lipidomic analysis in extracts of frontal cortex, both from the same postmortem tissue samples of human brain. An external calibration was used to achieve a mass accuracy of 10 ppm (1σ) in the spectra of the extracts, although the final assignment was based on a comparison with previously reported species. The spectra recorded directly from tissue slices (imaging) show excellent s/n ratios, almost comparable to those obtained from the extracts. In addition, they retain the information about the anatomical distribution of the molecular species present in autopsied frozen tissue. Further comparison between the spectra from lipid extracts devoid of proteins and those recorded directly from the tissue unambiguously show that the differences in lipid composition between gray and white matter observed in the mass images are not an artifact due to microenvironmental influences of each anatomical area on the signal intensity, but real variations in the lipid composition.

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.

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.

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.

The action of galanin in the central nervous system is mediated by at least three galanin receptor subtypes (GalR1, GalR2 and GalR3) which belong to the family of G protein-coupled receptors. GalR1 and GalR2 are coupled to G(i/o) proteins, although the latter may also be coupled to G(q/11) proteins. The aim of the present study was to identify the anatomical distribution and quantify the density of GalRs coupled to G proteins. The galanin (10(-6) M) stimulated guanosine 5'-(gamma-[35S] thio)triphosphate binding assay was used in tissue sections from the rat brain. Maximal percentages of stimulation over basal levels were found in the anterior olfactory nucleus and in the lateral olfactory tract nucleus ( approximately 54%). High levels of stimulation were recorded in diverse hypothalamic nuclei (16-28%), in the amygdala (central amygdaloid nucleus, 40%), in the spinal trigeminal tract (23%) and in layers 1-2 of the spinal cord (26%). Moderate binding stimulation (5-13%) was observed in thalamus, substantia nigra pars compacta, parabrachial nucleus, locus coeruleus and dorsal raphe nucleus. The lowest stimulation induced by galanin was recorded in diverse areas of the cortex, striatum, hippocampus and substantia nigra pars reticulata. The results show an anatomical distribution similar to that described for GalR1. However, in diverse brain areas, in which a high density of these receptors has previously been reported, only a moderate coupling to G proteins was found. These findings would suggest that the efficacy of galanin to induce an effective coupling of its receptors to G proteins could be different depending on the brain area.

The cholinergic pathways, which originate in the basal forebrain and are responsible for the control of different cognitive processes including learning and memory, are also regulated by some neuropeptides. One of these neuropeptides, galanin (GAL), is involved in both neurotrophic and neuroprotective actions. The present study has evaluated in rats the effects on cognition induced by a subchronic treatment with GAL by analyzing the passive avoidance response, and the modulation of muscarinic cholinergic receptor densities and activities. [3H]-N-methyl-scopolamine, [3H]-oxotremorine, and [3H]-pirenzepine were used to quantify the density of muscarinic receptors (MRs) and the stimulation of the binding of guanosine 5′-(γ-[35S]thio)triphosphate by the muscarinic agonist, carbachol, to determine their functionality. Some cognitive deficits that were induced by the administration of artificial cerebrospinal fluid (aCSF) (i.c.v. aCSF 2 μl/min, once a day for 6 days) were not observed in the animals also treated with GAL (i.c.v. 1.5 mmol in aCSF, 2 μl/min, once a day for 6 days). GAL modulates the changes in M1 and M2 MR densities observed in the rats treated with aCSF, and also increased their activity mediated by Gi/o proteins in specific areas of the dorsal and ventral hippocampus. The subchronic administration of the vehicle was also accompanied by an increased number of positive fibers and cells for GAL around the cortical tract of the cannula used, but that was not the case in GAL-treated rats. In addition, the increase of GAL receptor density in the ventral hippocampus and entorhinal cortex in the aCSF group was avoided when GAL was administered. The number of acetylcholinesterase (AChE)-positive neurons was decreased in the nucleus basalis of Meynert of both GAL- and aCSF-treated animals. In summary, GAL improves memory-related abilities probably through the modulation of MR density and/or efficacy in hippocampal areas.

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 G i/o -proteins in human and rat brain nuclei using [ 35 S]GTPγS autoradiography. The results show the anatomical distribution of G i/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 G i/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.

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).