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Background: Brain damage associated with Alzheimer's disease (AD) occurs even decades before the symptomatic onset, raising the need to investigate its progression from prodromal stages. In this context, animal models that progressively display AD pathological hallmarks (e.g. TgF344-AD) become crucial. Translational technologies, such as magnetic resonance spectroscopy (MRS), enable the longitudinal metabolic characterization of this disease. However, an integrative approach is required to unravel the complex metabolic changes underlying AD progression, from early to advanced stages.Methods: TgF344-AD and wild-type (WT) rats were studied in vivo on a 7 Tesla MRI scanner, for longitudinal quantitative assessment of brain metabolic profile changes using MRS. Disease progression was investigated at 4 time points, from 9 to 18 months of age, and in 4 regions: cortex, hippocampus, striatum, and thalamus.Results: Compared to WT, TgF344-AD rats replicated common findings in AD patients, including decreased N-acetylaspartate in the cortex, hippocampus and thalamus, and decreased glutamate in the thalamus and striatum. Different longitudinal evolution of metabolic concentration was observed between TgF344-AD and WT groups. Namely, age-dependent trajectories differed between groups for creatine in the cortex and thalamus and for taurine in cortex, with significant decreases in Tg344-AD animals; whereas myo-inositol in the thalamus and striatum showed greater increase along time in the WT group.Additional analysis revealed divergent intra- and inter-regional metabolic coupling in each group. Thus, in cortex, strong couplings of N-acetylaspartate and creatine with myo-inositol in WT, but with taurine in TgF344-AD rats were observed; whereas in the hippocampus, myo-inositol, taurine and choline compounds levels were highly correlated in WT but not in TgF344-AD animals. Furthermore, specific cortex-hippocampus-striatum metabolic crosstalks were found for for taurine levels in the WT group but for myo-inositol levels in the TgF344-AD rats.Conclusion: With a systems biology perspective of metabolic changes in AD pathology, our results shed light into the complex spatio-temporal metabolic rewiring in this disease, reported here for the first time. Age- and tissue-dependent imbalances between myo-inositol, taurine and other metabolites, such as creatine, unveil their role in disease progression, while pointing to the inadequacy of the latter as an internal reference for quantification.

Treballs Finals de Grau de Nutrició Humana i Dietètica, Facultat de Farmàcia i Ciències de l'Alimentació, Campus de l'Alimentació de Torribera, Universitat de Barcelona. Curs: 2020-2021. Tutor: Mónica Doménech, [eng] The popularity of dietary supplements is increasingly on the rise: people are turning to them both for the purpose of weight loss and to maintain good health, so that in recent months and with the influence of the Covid-19 lockdown, dietary supplement companies have multiplied their profits, with Herbalife®️ and Juice Plus+®️ among the biggest benefited, yet in both cases seems to be that the traders do not have the necessary knowledge on nutrition. The aim of this review is therefore to analyse the best-selling products of these companies in terms of their components and the available scientific evidence. The articles reviewed were obtained by a search through various databases, with specific selection criteria for each component and its supposed health benefits, giving preference to the most recent publications. Results show that some of these supplements’ ingredients do have scientific evidence supporting them (as is the case of coffee, green tea and, to a lesser extent, Aloe Vera), but others lack of significant evidence to attribute their claimed benefits (as is the case of substitute shakes, vitamin and omega supplements and glucomannan); moreover, adverse effects related to the consumption of these products have been reported and, in some cases, these had led to serious health consequences. In conclusion, it can be stated that the consumption of Herbalife®️ and Juice Plus+®️ supplements do not provide benefits in terms of health, and effective measures and policies are needed to promote healthy lifestyles as the main important measure to adopt by the whole population., [cat] L’ús de suplements dietètics està cada vegada més de moda: la gent confia en aquests productes tant amb l’objectiu de perdre pes com de mantenir una bona salut, de manera que durant els últims mesos i amb la influència del confiament per la Covid-19, les empreses dedicades a comercialitzar aquests suplements han multiplicat els seus beneficis, sent Herbalife® i Juice Plus+® dues de les més beneficiades, malgrat que en ambdós casos els comerciants no acostumen a disposar dels suficients coneixements necessaris en nutrició. Així doncs, l’objectiu d’aquesta revisió és analitzar els productes més venuts d’aquestes companyies en funció dels seus components i l’evidència científica disponible al respecte. Els articles revisats s’han obtingut mitjançant la cerca en diverses bases de dades, amb criteris de selecció específics per a cada component i els seus suposats beneficis, amb preferència de les publicacions més recents. Els resultats mostren que alguns dels ingredients d’aquests suplements sí que estan avalats per una sòlida evidència científica al darrere (com és el cas del cafè, el té verd i, en menor grau, l’Aloe Vera), però d’altres no compten amb una evidència prou significativa per atribuir-los els beneficis que asseguren (com succeeix amb els batuts substitutius, els suplements vitamínics i d’omegues i el glucomanan); inclús s’han reportat efectes adversos relatius al consum d’aquests productes que, en alguns casos, han resultat en greus conseqüències per la salut. En conclusió, podem afirmar que el consum dels productes d’Herbalife® i Juice Plus+® no aporten beneficis en termes de salut i, per tant, cal adoptar mesures i polítiques eficaces que promoguin per davant de tot, els estils de vida saludable com a pilar bàsic de salut a la població.

The regulation of AMPA-type receptor (AMPAR) abundance in the postsynaptic membrane is an important mechanism involved in learning and memory formation. Recent data suggest that one of the constituents of the AMPAR complex is carnitine palmitoyltransferase 1C (CPT1C), a brain-specific isoform located in the endoplasmic reticulum of neurons. Previous results had demonstrated that CPT1C deficiency disrupted spine maturation in hippocampal neurons and impaired spatial learning, but the role of CPT1C in AMPAR physiology had remained mostly unknown. In the present study, we show that CPT1C binds GluA1 and GluA2 and that the three proteins have the same expression profile during neuronal maturation. Moreover, in hippocampal neurons of CPT1C KO mice, AMPAR-mediated miniature excitatory postsynaptic currents and synaptic levels of AMPAR subunits GluA1 and GluA2 are significantly reduced. We show that AMPAR expression is dependent on CPT1C levels because total protein levels of GluA1 and GluA2 are decreased in CPT1C KO neurons and are increased in CPT1C-overexpressing neurons, whereas other synaptic proteins remain unaltered. Notably, mRNA levels of AMPARs remained unchanged in those cultures, indicating that CPT1C is post-transcriptionally involved. We demonstrate that CPT1C is directly involved in the de novo synthesis of GluA1 and not in protein degradation. Moreover, in CPT1C KO cultured neurons, GluA1 synthesis after chemical long term depression was clearly diminished, and brain-derived neurotrophic factor treatment was unable to phosphorylate the mammalian target of rapamycin (mTOR) and stimulate GluA1 protein synthesis. These data newly identify CPT1C as a regulator of AMPAR translation efficiency and therefore also synaptic function in the hippocampus.

Poly(gamma-glutamic acid) (gamma-PGA) is one of the few bacterial polymers in nature; as such, it possesses the added value of a “green birth” (fermentation) and “green death” (biodegradability), which is ideal for sustainable macromolecular platforms. Efficient functionalization strategies are required in order to transform the native bacterial biopolymer into a material with tailored properties for bulk scale or biomedical applications. We report on a novel approach to the facile functionalization of bacterial gamma-PGA, realized with several levels of control: (a) the modulation of the molecular weight through sonication, and the solubilization in organic solvents through counterion exchange with suitable quaternary ammonium salts, (b) the introduction of reactive functionalities through reactions in homogeneous conditions in organic solvents, to afford homo- or copolymers; (c) a second tier functionalization using copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC) click chemistry.

Glutamate transporter solute carrier family 1, member 2 (GLT1/EAAT2), a major modulator of glutamate homeostasis in astrocytes, is assessed in post-mortem human brain samples of frontal cortex area 8 in advanced stages of Alzheimer disease (AD) and terminal stages of dementia with Lewy bodies (DLB) in order to gain understanding of astrogliopathy in diseases manifested by dementia. Glial fibrillary acidic protein (GFAP) mRNA expression is significantly increased in AD but not in DLB, whereas GLT1, vesicular glutamate transporter 1 (vGLUT1) and aldehyde dehydrogenase 1 family member 1 (ALDH1L1) are not modified in AD and DLB when compared with controls. GLT1 protein levels are not altered in AD and DLB but GFAP and ALDH1L1 are significantly increased in AD, and GFAP in DLB. As a result, a non-significant decrease in the ratio between GLT1 and GFAP, and between GLT1 and ALDH1L1, is found in both AD and DLB. Double-labeling immunofluorescence and confocal microscopy revealed no visible reduction of GLT1 immunoreactivity in relation to beta-amyloid plaques in AD. These data suggest a subtle imbalance between GLT1, and GFAP and ALDH1L1 expression, with limited consequences in glutamate transport.

Altres ajuts: CiberNed's intramural program (Ref. No. PI2016/02); Fundació la Marató de TV3 N -methyl--aspartate receptors (NMDARs) respond to glutamate to allow the influx of calcium ions and the signaling to the mitogen-activated protein kinase (MAPK) cascade. Both MAPK- and Ca 2+ -mediated events are important for both neurotransmission and neural cell function and fate. Using a heterologous expression system, we demonstrate that NMDAR may interact with the EF-hand calcium-binding proteins calmodulin, calneuron-1, and NCS1 but not with caldendrin. NMDARs were present in primary cultures of both neurons and microglia from cortex and hippocampus. Calmodulin in microglia, and calmodulin and NCS1 in neurons, are necessary for NMDA-induced MAP kinase pathway activation. Remarkably, signaling to the MAP kinase pathway was blunted in primary cultures of cortical and hippocampal neurons and microglia from wild-type animals by proteins involved in neurodegenerative diseases: α-synuclein, Tau, and p-Tau. A similar blockade by pathogenic proteins was found using samples from the APP transgenic Alzheimer's disease model. Interestingly, a very marked increase in NMDAR-NCS1 complexes was identified in neurons and a marked increase of both NMDAR-NCS1 and NMDAR-CaM complexes was identified in microglia from the transgenic mice. The results show that α-synuclein, Tau, and p-Tau disrupt the signaling of NMDAR to the MAPK pathway and that calcium sensors are important for NMDAR function both in neurons and microglia. Finally, it should be noted that the expression of receptor-calcium sensor complexes, specially those involving NCS1, is altered in neural cells from APP mouse embryos/pups.

The reduction of oxidative stress is suggested to be one of the main mechanisms to explain the benefits of subnormothermic perfusion against ischemic liver damage. In this study we investigated the early cellular mechanisms induced in isolated ratliversafter15minperfusionattemperaturesrangingfromnormothermia(37 ∘ C) to subnormothermia (26 ∘ Cand22 ∘ C). Subnormothermic perfusion was found to maintain hepatic viability. Perfusion at 22 ∘ C raised reduced glutathione levels and the activity of glutathione reductase; however, lipid and protein oxidation still occurred as determined by malondialdehyde, 4-hydroxynonenal-protein adducts, and advanced oxidation protein products. In livers perfused at 22 ∘ C the lysosomal and ubiquitin proteasome system (UPS) were both activated. The 26S chymotrypsin-like ( 훽 5) proteasome activity was significantly increased in the 26 ∘ C (46%) and 22 ∘ C (42%) groups. The increased proteasome activity may be due to increased Rpt6 Ser120 phosphorylation, which is known to enhance 26S proteasome activity. Together, our results indicate that the early events produced by subnormothermic perfusion in the liver can induce oxidative stress concomitantly with antioxidant glutathione preservation and enhanced function of the lysosomal and UPS systems. Thus, a brief hypothermia could trigger antioxidant mechanisms and may be functioning as a preconditioning stimulus.

Background-Glutamate metabolism may play a role in the pathophysiology of cardiometabolic disorders. However, there is limited evidence of an association between glutamate-related metabolites and, moreover, changes in these metabolites, and risk of cardiovascular disease (CVD). Methods and Results-Plasma levels of glutamate and glutamine were measured at baseline and 1-year follow-up in a case-cohort study including 980 participants (mean age 68 years; 46% male) from the PREvencion con DIeta MEDiterranea (PREDIMED) randomized trial, which assessed a Mediterranean diet intervention in the primary prevention of CVD. During median 4.8 years of follow-up, there were 229 incident CVD events (nonfatal stroke, nonfatal myocardial infarction, or CVD death). In fully adjusted models, per 1-SD, baseline glutamate was associated with 43% (95% CI: 16% to 76%) and 81% (39% to 137%) increased risk of composite CVD and stroke alone, respectively, and baseline glutamine-to-glutamate ratio with 25% (6% to 40%) and 44% (25% to 58%) decreased risk of composite CVD and stroke alone, respectively. Associations appeared linear for stroke (both P-linear trend, This work was supported by the National Institutes of Health, 1R01HL118264. The PREDIMED trial was supported by the official funding agency for Biomedical Research of the Spanish Government, Instituto de Salud Carlos III, through grants provided to research networks specifically developed for the trial: RTIC G03/140 (Period 2003-2006, Coordinator: R. Estruch, MD, PhD), and RTIC RD 06/0045 (Period 2007-2013, Coordinator: M. A. Martinez-Gonzalez, MD, PhD). We also acknowledge the grants from Centro Nacional de Investigaciones Cardiovasculares CNIC06/2007, Fondo de Investigacion Sanitaria-Fondo Europeo de Desarrollo Regional (PI04-2239, PI05/2584, CP06/00100, PI07/0240, PI07/1138, PI07/0954, PI 07/0473, PI08/1259, PI10/01407, PI11/01647, PI11/01791), and Consejeria de Salud de la Junta de Andalucia (PI0105/2007). Ministerio de Ciencia e Innovacion (AGL-2009-13906-C02, AGL2010-22319-C03), Fundacion Mapfre 2010, Consejeria de Salud de la Junta de Andalucia (PI0105/2007), Agencia Canaria de Investigacion, Innovacion y Sociedad de la Informacion-EU FEDER (PI 2007/050), Public Health Division of the Department of Health of the Autonomous Government of Catalonia and Generalitat Valenciana (ACOMP06109, GVA-COMP2010-181, GVACOMP2011-151, CS2010-AP-111, CS2011-AP-042, AP-042/11 and BEST11-263) and Ministerio de Economia (PI07-0954, CNIC-06, AGL2010-22319-C03-03, PI11/02505). CIBEROBN is an initiative of ISCIII, Spain.

A subgroup of breast cancers has several metabolic compartments. The mechanisms by which metabolic compartmentalization develop in tumors are poorly characterized. TP53 inducible glycolysis and apoptosis regulator (TIGAR) is a bisphosphatase that reduces glycolysis and is highly expressed in carcinoma cells in the majority of human breast cancers. Hence we set out to determine the effects of TIGAR expression on breast carcinoma and fibroblast glycolytic phenotype and tumor growth. The overexpression of this bisphosphatase in carcinoma cells induces expression of enzymes and transporters involved in the catabolism of lactate and glutamine. Carcinoma cells overexpressing TIGAR have higher oxygen consumption rates and ATP levels when exposed to glutamine, lactate, or the combination of glutamine and lactate. Coculture of TIGAR overexpressing carcinoma cells and fibroblasts compared with control cocultures induce more pronounced glycolytic differences between carcinoma and fibroblast cells. Carcinoma cells overexpressing TIGAR have reduced glucose uptake and lactate production. Conversely, fibroblasts in coculture with TIGAR overexpressing carcinoma cells induce HIF (hypoxia-inducible factor) activation with increased glucose uptake, increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), and lactate dehydrogenase-A expression. We also studied the effect of this enzyme on tumor growth. TIGAR overexpression in carcinoma cells increases tumor growth in vivo with increased proliferation rates. However, a catalytically inactive variant of TIGAR did not induce tumor growth. Therefore, TIGAR expression in breast carcinoma cells promotes metabolic compartmentalization and tumor growth with a mitochondrial metabolic phenotype with lactate and glutamine catabolism. Targeting TIGAR warrants consideration as a potential therapy for breast cancer.

Metabotropic glutamate receptors (mGlu receptors) are class C GPCRs widely distributed through the CNS and considered pharmacological targets for neurologic disorders, such as neuropathic pain and Parkinson’s disease, between others. Classical allosteric modulators receptors might be useful to treat these disorders, but they the control over of the compound, once it is administered, is lost. Photoswitchable allosteric modulators have an azo-­‐moiety in their chemical scaffold and might offer a powerful alternative to these classical drugs, since their pharmacological activity can be “switched on and off”, depending on the wavelength of illumination. First, we designed and synthesised alloswitch-­‐1 using an azo-­‐replacement strategy, using previously described mGlu4 PAM with two diarylamides. We replaced one of these amides with an azo-­‐bond to obtain the first GPCR photoswitchable allosteric modulator with activity in-­‐vivo. Alloswitch-­‐1 selectively performed NAM activity in mGlu5 in the trans-­‐isomer, whether in the cis disposition it was inactive. This behaviour was easily controlled by illumination with 380 nm and 500 nm of wavelength and was consistent and reversible in transfected cells and native cultures. Moreover, it allowed the possibility to control the natatorial motility of native Zebrafish larvae or Xenopus Tropicalis with light, and it was also effective in the reduction of pain-­‐like behaviour in rodents, showing a clear light-­‐ dependent activity. We next designed and synthesised new related phenylazopyiridnes not only to study the structure -­‐ pharmacological activity relationship, but also the efficiency of photoisomerisation from trans to cis isomers and the rate of thermal relaxing from cis to trans isomers. Thus, we obtained many compounds with a very similar potency to alloswitch-­‐1, but other with an enhanced potency from 4-­‐ to 10-­‐fold shift, improved photoswitching properties or other ones that requires a wider wavelength for photoisomerisating, which is less harmful for in-­‐vivo tissues. Furthermore, we demonstrated that if we adjust the wavelength of illumination, we are able to control the rate of activation of the receptor, something that only can be afforded with dose-­‐adjustment with conventional pharmacology. We also synthesised optogluram with the previous azo-­‐replacement approach used with alloswitch-­‐1, but replacing a second amide with an azo-­‐bond. It resulted in a photoswitchable positive allosteric modulator (PAM) of mGlu4, with similar photoswitching properties to alloswitch-­‐1 and also effective in the reduction of pain-­‐like behaviour in rodents. Additionally we also designed and synthesised two non-­‐photoswitchable bioisosters of alloswitch-­‐1 and optogluram, which did not have an azo-­‐moiety in their scaffold but conserved the previous pharmacological properties as mGlu ligands. We also synthesised a series of 12 compounds, six of them photoisomerisable, to study the relationships between mGlu4 PAMs and mGlu5 NAMs. Among them, we found two compounds having both pharmacological activities in the low-­‐micromolar range of potency. Concerning the mGlu5 NAMs of this family, we discovered the chemical particularities that make them inverse agonists or partial antagonists. Finally, we tried to design and synthesise photoswitchable mGlu4 PAMs and mGlu5 NAMs with a trans isomer inactive and a cis isomer active (cis-­‐on). To do that, we designed new modulators and computationally verified the suitability of the cis-­‐on with molecular dockings. Despite the efforts made, we were not successful to find some cis-­‐on without unspecific effects. Overall, we found the molecular characteristics that relate mGlu4 PAMs and mGlu5 NAMs, and how to obtain a molecular switch between both pharmacological activities. We make a success of azo-­‐ replacing approaches of previous active compounds for obtaining photoswitchable compounds. We additionally proved that optopharmacology might be more advantageous than optogenetics or conventional pharmacology, because of the possibility to perform assays with native tissues or in-­‐ vivo with no genetic modification and adjust the activation of the receptor with light with a single dose of compound, instead of changing doses to find the optimal one., Els receptors de glutamat metabotròpics (mGlu) són GPCRs distribuïts a través del CNS i es consideren dianes farmacològiques per a trastorns neurològics, com ara el dolor neuropàtic i la malaltia de Parkinson, entre d'altres. En primer lloc, vam dissenyar i sintetitzar l’alloswitch-­‐1 utilitzant una estratègia de azo-­‐ reemplaçament, per obtenir el primer modulador al·∙lostèric de GPCR fotocommutable amb activitat in vivo. L’alloswitch-­‐1 té activitat de NAM en mGlu5 en l'isòmer trans, mentre que en la disposició cis és inactiu. Aquest comportament es controla fàcilment amb il·∙luminació amb 380 nm i 500 nm i és reversible in-­‐vitro. D'altra banda, va permtre la possibilitat de controlar la motilitat de larves de peix zebra o capgrossos amb llum. També vam poder controlar la sensació de dolor en rosegadors depenent de la llum. Seguidament, vam realitzar el disseny i síntesi de noves feniliazopiridines, no només per l'estudi de la relació SAR, sinó també per l'eficiència de fotoisomerizació. Vam obtenir compostos amb bona potència farmacològica i vam millorar les propietats de fotoisomerització. També vam sintetitzar l’optogluram amb l’estratègia azo-­‐reemplaçament anterior. El resultat va ser un modulador al·∙lostèric positiu (PAM) de mGlu4 fotoisomerable, amb propietats òptiques similars a l’alloswitch-­‐1 i també és eficaç en el control de la sensació de dolor en rosegadors. A més també vam dissenyar i sintetitzar dos bioisòsters no fotisomeritzables que conservaven les propietats farmacològiques anteriors. Vam sintetitzar una sèrie per trobar compostos amb activitat dual com a PAM de mGlu4 i NAM de mGlu5. Dos dels compostos ho va arribar a ser i amb els que van resultar NAMs de mGlu5, en vam descobrir les particularitats químiques que els fan agonistes inversos o antagonistes parcials. Finalment, vam intentar de dissenyar i sintetitzar PAMs de mGlu4 i NAMs de mGlu5 amb un isòmer trans inactiu i un isòmer cis actiu, però sense èxit. En resum, vam trobar les característiques moleculars que relacionen PAMS de mGlu4 i NAMS mGlu5, i com obtenir un interruptor molecular entre ambdues activitats farmacològiques. A més, vam demostrar que l’optofarmacologia presenta avantatges respecte la farmacologia convencional, ja que permet ajustar l'activació del receptor amb llum.

Glutamate metabolism may play a role in the pathophysiology of cardiometabolic disorders. However, there is limited evidence of an association between glutamate-related metabolites and, moreover, changes in these metabolites, and risk of cardiovascular disease (CVD). METHODS AND RESULTS: Plasma levels of glutamate and glutamine were measured at baseline and 1-year follow-up in a case-cohort study including 980 participants (mean age 68 years; 46% male) from the PREvención con DIeta MEDiterránea (PREDIMED) randomized trial, which assessed a Mediterranean diet intervention in the primary prevention of CVD. During median 4.8 years of follow-up, there were 229 incident CVD events (nonfatal stroke, nonfatal myocardial infarction, or CVD death). In fully adjusted models, per 1-SD, baseline glutamate was associated with 43% (95% CI: 16% to 76%) and 81% (39% to 137%) increased risk of composite CVD and stroke alone, respectively, and baseline glutamine-to-glutamate ratio with 25% (6% to 40%) and 44% (25% to 58%) decreased risk of composite CVD and stroke alone, respectively. Associations appeared linear for stroke (both Plinear trend≤0.005). Among participants with high baseline glutamate, the interventions lowered CVD risk by 37% compared to the control diet; the intervention effects were not significant when baseline glutamate was low (Pinteraction=0.02). No significant effect of the intervention on year-1 changes in metabolites was observed, and no effect of changes themselves on CVD risk was apparent. CONCLUSIONS: Baseline glutamate was associated with increased CVD risk, particularly stroke, and glutamine-to-glutamate ratio was associated with decreased risk. Participants with high glutamate levels may obtain greater benefits from the Mediterranean diet than those with

[eng] AMPA receptors (AMPARs) are responsible for the 90% of synaptic transmission and are involved in plasticity, developmental and neurological processes. Their function depends on the proteins interacting with the AMPAR complex, which determine their specific gating and trafficking properties and hence their specific roles. In addition to well-known interacting proteins of AMPARs, recent studies have described the CPT1C as an associated partner in the outer core of the AMPAR complex in the hippocampus, cortex and cerebellum. CPT1C is a neuron specific homologue of the carnitine acyltransferase family of enzymes, which are involved in fatty acid oxidation at the mitochondria. Contrary to the rest of the CPT1 family, CPT1C localizes at the endoplasmic reticulum and apparently is not related with the functions that other CPT1s carry out. To elucidate the physiological role of CPT1C isoform different studies have been performed, showing that CPT1C is involved in energy homeostasis, control of body weight and motor function as well as behavioral learning mechanisms. Of note, CPT1C KO animals display impairments in spatial learning along with immature dendritic spines. Despite all these studies with CPT1C, including its interaction with AMPARs, it is unknown the CPT1C physiological relevance and role, particularly in the regulation of the AMPAR function and its implication in neurological diseases. In this thesis we have described a novel role of CPT1C in AMPA receptor function regulation. More specifically, the results show that glutamate-evoked currents of the recombinant GluA1 receptors are increased when CPT1C is present and this effect is specific of the CPT1 isoform CPT1C, since CPT1A does not share the same pattern. The ER location of CPT1C seems to be crucial to modulate AMPAR surface expression since mislocalization of CPT1C avoids its AMPAR modulation. Co-localization studies confirmed that GluA1-CPT1C interaction happens at ER level but not at the cell surface. On the other hand, no changes in current density have been found in cells expressing GluA2 subunit along with CPT1C indicating AMPA subunit specificity. Additionally, electrophysiological experiments have determined that GluA1 channel properties are no altered, thus indicating that the increased current is probably due to a rise in AMPAR number at the cell surface. Indeed, this hypothesis is corroborated by the findings from the immunofluorescence experiments, where surface expression of GluA1 is increased in the presence of CPT1C in heterologous systems and cortical neurons. In agreement with a putative role of CPT1C in determining the AMPAR content at synapse level, we have demonstrated that synaptic transmission is altered in CPT1C KO neurons. In parallel, we have studied the molecular mechanisms of CPT1C effect on AMPARs. We have shown that the palmitoylable cysteine 585 of the GluA1 subunit is crucial for the CPT1C enhancement of AMPA receptors trafficking by using immunofluorescence and electrophysiological techniques. However, the palmitoylation state of this residue does not determine AMPAR-CPT1C interaction. We have found evidences for a supposed depalmitoylation activity by CPT1C studying the role of the C-terminus of CPT1C, which contains the residue His469 with catalytic activity. Specifically we have found that CPT1C H469A mutant form does not alter GluA1 induced whole-cell currents as the wild type CPT1C does indicating that the C-terminal catalytic domain plays a crucial role in GluA1 modulation. This is supported by the fact that Palmostatin B – a newly described palmitoyl thioesterase inhibitor – decreases the CPT1C effect on GluA1 induced currents, most likely by inhibiting its PTE activity. In summary, this thesis unravels a novel regulation of AMPA receptor function by the interacting protein CPT1C, which modulates AMPA receptor trafficking and this effect depends on the catalytic domain of CPT1C C-terminal acting on the cysteine 585 of GluA1 AMPAR subunit., [spa] Los receptores de glutamato tipo AMPA son fundamentales en la transmisión excitatoria rápida que se da en el sistema nervioso central. A parte de su papel crucial en la comunicación neuronal, los receptores AMPA son responsables de ciertos tipos de plasticidad sináptica, siendo importantes en el desarrollo del sistema nervioso central además de estar involucrados en multitud de procesos patológicos y enfermedades neurodegenerativas. La función de los receptores AMPA depende principalmente de dos factores: la composición de las subunidades que lo conforman (el receptor propiamente dicho) y la presencia de proteínas que interactúan con el receptor y actúan como subunidades auxiliares. Estos dos factores establecen las características intrínsecas del canal así como las interacciones de los receptores AMPA con otras proteínas intracelulares que determinarán sus propiedades de tráfico (ensamblaje, exocitosis, endocitosis y anclaje sináptico) y en último término sus funciones en la neurona. Entre la gran cantidad de proteínas que interaccionan con los receptores AMPA, recientes estudios proteómicos han demostrado que la proteína CPT1C forma parte del conjunto macromolecular de los receptores AMPA en el tejido neuronal. El objetivo principal de esta tesis se ha focalizado en el estudio esta proteína (CPT1C) en el contexto de la función de los receptores AMPA debido a su interacción. Para ello se han utilizado técnicas electrofisiológicas, de inmunofluorescencia y de biología molecular y celular tanto en sistemas de expresión heterólogos como en cultivos de células neuronales. Los experimentos llevados al cabo durante la tesis han confirmado la interacción entre las dos proteínas y han atribuido un papel modulador de CPT1C en el trafico de los receptores AMPA. El efecto regulador de la CPT1C es dependiente de la composición del receptor, afectando de manera diferencial subtipos distintos de receptores AMPA. La proteína CPT1C aumenta el tráfico de los receptores AMPA a la superficie celular sin alterar sus propiedades biofísicas y sin interaccionar a nivel de membrana plasmática ambas proteínas. Además en esta tesis se han descrito los mecanismos implicados en la modulación de dichos receptores por parte de CPT1C, desentrañando un residuo cisteína concreto determinante para la modulación de los receptores AMPA por la CPT1C. Finalmente, los experimentos llevados a cabo en esta tesis nos indican que los efectos observados se deben a una posible depalmitoilación del receptor, lo cual lleva a un aumento de su capacidad de acumulación en la membrana. En resumen, los resultados obtenidos durante esta tesis demuestran que el número de receptores AMPA en las sinapsis está aumentado en presencia de CPT1C y este efecto es debido a la actividad catalítica de CPT1C.

Receptors for neurotransmitters require scaffolding proteins for membrane microdomain targeting and for regulating receptor function. Using a yeast two-hybrid screen, alpha-actinin-1, a major F-actin cross-linking protein, was identified as a binding partner for the C-terminal domain of metabotropic glutamate receptor type 5b (mGlu(5b) receptor). Co-expression, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between mGlu(5b) receptor and alpha-actinin-1 in both transfected HEK-293 cells and rat striatum. The interaction of alpha-actinin-1 with mGlu(5b) receptor modulated the cell surface expression of the receptor. This was dependent on the binding of alpha-actinin-1 to the actin cytoskeleton. In addition, the alpha-actinin-1/mGlu(5b) receptor interaction regulated receptor-mediated activation of the mitogen-activated protein kinase pathway. Together, these findings indicate that there is an alpha-actinin-1-dependent mGlu(5b) receptor association with the actin cytoskeleton modulating receptor cell surface expression and functioning.

INTRODUCCIÓN Los mecanismos celulares y moleculares a través de los cuales produce sus efectos son, por ahora, mal conocidos. En los últimos años se está valorando con gran interés la interacción existente entre el etanol y el glutamato. Se han realizado múltiples experimentos con animales de laboratorio que han demostrado que el etanol es un potente inhibidor de la acción del glutamato sobre los receptores NDMA, surgiendo la hipótesis de que la relación etanol-NDMA condiciona parte de los efectos lesivos del tóxico sobre el sistema nervioso central (SNC). OBJETIVOS 1.- Estudiar, mediante la técnica de la autorradiografía de receptores, la distribución y la densidad de los receptores NMDA del glutamato en diferentes áreas del SNC de un grupo de personas consumidoras regulares de diferentes dosis de alcohol, y comparar los resultados con los obtenidos en controles, pacientes no consumidores de alcohol 2.- Valorar los cambios en la densidad de receptores NMDA entre los consumidores crónicos de alcohol no abstinentes (consumidores de alcohol hasta horas antes de su defunción) y abstinentes 3.- Analizar las posibles diferencias en la densidad de receptores NMDA entre los consumidores crónicos de alcohol según la intensidad de la ingesta habitual, diferenciando entre consumidores moderados (40-80g/dia) y consumidores de dosis altas de alcohol (>80g/dia) 4.- Estudiar las posibles diferencias en la densidad de receptores entre consumidores de alcohol atribuible a las circunstancias de la defunción (fallecidos por muerte súbita o no) PACIENTES Y MÉTODOS Fueron seleccionados para el estudio un total de 16 pacientes alcohólicos y 16 controles. Las áreas cerebrales incluidas en los diferentes experimentos fueron las siguientes: Córtex frontal, núcleo estriado, hipocampo, y vermis y hemiferio cerebeloso Los experimentos de autorradiografía de receptores se realizaron en el Departament de Neuroquímica del Consell Superior d'Investigacions Científiques (CSIC). RESULTADOS Y CONCLUSIONES 1.- El consumo prolongado de alcohol no modifica significativamente la densidad de receptores NMDA en las áreas estudiadas. A la luz de los recientes estudios sobre las subunidades del receptor NMDA, el etanol probablemente actúa modificando la composición y proporción de las mismas, y a través de estos cambios provoca las alteraciones funcionales en el sistema glutamatérgico, sin que se observen cambios en la densidad global de estos receptores. 3.- No se han detectado diferencias significativas entre la densidad de receptores NMDA en pacientes consumidores abstinentes y no abstinentes. Debido a los fenómenos asociados de sobre-regulación adaptativa e inhibición funcional de los receptores NDMA bajo los efectos del etanol, la densidad global de los mismos puede mantenerse estable mientras el individuo siga manteniendo la ingesta de forma regular. Durante la fase de abstinencia aguda, la liberación brusca de receptores inhibidos hasta ese momento, y el incremento real del número de los mismos (sobre-regulación) provocarían la hiperestimulación glutamatérgica y la excitotoxicidad. En ese momento probablemente sí se detectarían alteraciones en la densidad de receptores, durante un período de varios días. Al cabo de unas semanas en ausencia del tóxico en el medio, los receptores NDMA retornarían a sus niveles previos. 4.- No se han detectado diferencias en la densidad de receptores NMDA entre las muestras de tejido provenientes de alcohólicos fallecidos por muerte súbita y no súbita., Specific binding of [3H]MK801 to N-methyl-D-aspartate (NMDA) receptors in the frontal cortex, cerebellum, striatum and hippocampus (CA1 and gyrus dentatus) was measured by receptor autoradiography in 16 Caucasian chronic alcohol consumers free of clinical manifestations of alcoholism, and compared to 16 Caucasian control subjects. Binding densities were not significantly different between heavy and moderate drinkers, neither between alcohol consumers that were abstinent or non-abstinent before death, nor between ethanol drinkers and controls. Continued alcohol consumption, in the absence of hepatic, neurologic or psychiatric disorders related to alcoholism, does not alter the binding properties of NMDA receptors in the brain areas studied.

Metabotropic glutamate receptors (mGluR) are present in cells of the nervous system, where they are activated by one of the main neurotransmitters, glutamate. They are also expressed in cells outside the nervous system. We identified and characterized two receptors belonging to group I mGluR, mGlu1R and mGlu5R, in human cell lines of lymphoid origin and in resting and activated lymphocytes from human peripheral blood. Both are highly expressed in the human Jurkat T cell line, whereas mGlu5R is expressed only in the human B cell line SKW6.4. In blood lymphocytes, mGlu5R is expressed constitutively, whereas mGlu1R is expressed only upon activation via the T cell receptor-CD3 complex. Group I receptors in the central nervous system are coupled to phospholipase C, whereas in blood lymphocytes, activation of mGlu5R does not trigger this signaling pathway, but instead activates adenylate cyclase. On the other hand, mGlu5R does not mediate ERK1/2 activation, whereas mGlu1R, which is coupled neither to phospholipase C nor to calcium channels and whose activation does not increase cAMP, activates the mitogen-activated protein kinase cascade. The differential expression of mGluR in resting and activated lymphocytes and the different signaling pathways that are triggered when mGlu1Rs or mGlu5Rs are activated point to a key role of glutamate in the regulation of T cell physiological function. The study of the signaling pathways (cAMP production and ERK1/2 phosphorylation) and the proliferative response obtained in the presence of glutamate analogs suggests that mGlu1R and mGlu5R have distinct functions. mGlu5R mediates the reported inhibition of cell proliferation evoked by glutamate, which is reverted by the activation of inducible mGlu1R. This is a novel non-inhibitory action mechanism for glutamate in lymphocyte activation. mGlu1R and mGlu5R thus mediate opposite glutamate effects in human lymphocytes.

[cat] L’estudi de la neurotransmissió requereix noves eines moleculars, i els fotocommutadors ofereixen grans possibilitats. Aquesta tesi està centrada en l’ús de receptors de glutamat activables per llum (LiGluRs) pel control de l’activitat neuronal i dels processos de neurosecreció. Al primer bloc de resultats, la permeabilitat a Ca2+ dels receptors de glutamat s’aprofita per manipular de manera directa -independentment del voltatge de membrana- i reversible la concentració intracel•lular de Ca2+ amb llum. Així, és possible desencadenar els processos de secreció en cèl•lules cromafins i de neurotransmissió en neurones hipocampals. A la segona part dels resultats de la tesi, s’investiga l’estimulacio multifotó del receptor de glutamat modificat químicament amb fotocommutadors basats en l’azobenzè. Els resultats mostren l’estimulació per dos fotons del LiGluR, incloent-hi dos fotocommutadors nous, que milloren l’absorció multifotó del commutador azobenzè. Finalment, s’aplica aquesta tècnica per estimular neurones i astròcits amb una resolució a l’escala d’una cèl•lula o de compartiment subcel•lular. Al tercer capítol dels resultats, es descriu un nou mètode per aconseguir el fotocontrol de receptors neuronals endògens, utilitzant lligands covalents. Amb l’aplicació d’aquest mètode es pot fotocommutar l’activitat del receptor de kainat subtipus 1 (GluK1) no mutant, quan se sobreexpressa el receptor en cèl•lules de mamífer. Aquesta estratègia també permet obtenir fotocorrents en cultius de neurones dels ganglis de l’arrel dorsal, on GluK1 és la subunitat de glutamat endògenament més expressada. Els nous mètodes desenvolupats en aquesta tesi milloren la utilització dels LiGluRs, encaminant l’ús dels fotocommutadors cap al control òptic dels receptors endògens, amb la possibilitat d’estimular cèl•lules individuals o estructures subneuronals, fet que situaria els fotocomutados com a eines indispensables per l’estudi del cervell, des de la fisiologia fins als circuits neuronals., [eng] Optical tools to control neuronal activity include synthetic photoswitchable ligands of receptors and ion channels. Photoswitches can act either as soluble molecules (photochromic ligands, PCLs) or tethered to the protein (photoswitchable tethered ligands, PTLs), and they have been used to photocontrol many ion channels and receptors such as voltage-gated potassium channels, acetylcholine or glutamate receptors. Recognizing both the need for new optical tools in neuroscience and the opportunities offered by photoswitches, this work is focused on the use of light gated glutamate receptors to control neuronal activity and neurotransmission. In the first chapter of results of the thesis, we demonstrate that the Ca2+-permeable LiGluR can be used as a tool to reversibly control neurosecretion by directly affecting the intracellular [Ca2+]. To achieve this goal, LiGluR was expressed in cultured bovine chromaffin cells and cultured hippocampal neurons. We measured secretion in chromaffin cells using two techniques, amperometry and membrane capacitance, and current-clamp recordings to assess neurotransmission in cultured neurons. The results indicated that the magnitude of LiGluR-mediated Ca2+ influx is sufficiently large to trigger regulated exocytosis in chromaffin cells and neurons. In addition, LiGluR induced secretion can be modulated with the wavelength of illumination. This new application of LiGluR opens the possibility to reversibly control the activity of individual synapses, which might help to understand the computational properties of neurons and to unravel how brain circuits work. To use LiGluR as an effective method to interrogate the neuronal function it should support high-spatial 3D resolution and tissue penetration. Multiphoton excitation with near-infrared light enables stimulation in intact tissue with cellular and subcellular resolution, and it has been extensively applied to optical actuators such as caged compounds and more recently to optogenetics. However, two-photon stimulation of synthetic photoswitches had not been explored before. In the second section of the results, the two-photon stimulation of LiGluR is investigated. Two new photoswitches were designed (MAG2p and MAGA2p) based on the structure of the original photoswitch (MAG) and intended to enhance the two-photon absorption ability of the azobenzene switch. The three PTLs, including MAG, successfully activate LiGluR under two-photon stimulation, suggesting that multiphoton excitation can be applied to other azobenzene-based molecules. Interestingly, the rationally designed photoswitches were more efficient in opening LiGluR as lower power and shorter simulation time were required. Finally we validated MAG2p and MAG as new tools to control the activation of neurons and astrocytes with cellular and subcellular resolution. In the last chapter, a new method based on the affinity labeling approach is presented in order to confer light sensitivity to endogenous receptors. Glutamate-azobenzene-reactive PTLs with different lengths and reactive groups were tested on kainate receptors. These reactive PTLs successfully and functionally conjugate to the ionotropic glutamate receptor subunit GluK1, thus enabling to photoswitch its activity, as evidenced from photocurrent recordings of mammalian cells overexpressing the non-mutated receptor. These results are also supported by the photocontrol of GluK1 currents in dorsal root ganglion neurons, where GluK1 is the main glutamate subunit that is endogenously expressed. The new strategy proposed is versatile and we suggest that it can be extended to label other endogenous receptors, giving rise to novel optpharmacological therapies. The new methods here developed improve the LiGluR performance, and address photoswitches to use endogenous neuronal receptors to optically control neuronal activity, being able to stimulate them in small volumes corresponding to the neuronal functional unit (i.e. synaptic terminals). In this way, light would emerge as a unique tool to dissect neuronal physiology and to understand the function of neuronal circuits.

El projecte en el qual s’emmarca aquest treball de recerca es centra en el desenvolupament d’interruptors moleculars que permeten el control del funcionament d’un canal iònic de les cèl·lules del sistema nerviós central. En aquest treball de Màster en Experimentació Química, s’ha treballat, més concretament, en la síntesi del derivat del glutamat. S’ha dissenyat i desenvolupat una ruta sintètica del fragment de glutamat en 8 passos amb un 7% de rendiment global.

At synaptic boutons, metabotropic glutamate receptor 7 (mGlu7 receptor) serves as an autoreceptor, inhibiting glutamate release. In this response, mGlu7 receptor triggers pertussis toxin-sensitive G protein activation, reducing presynaptic Ca(2+) influx and the subsequent depolarization evoked release. Here we report that receptor coupling to signaling pathways that potentiate release can be seen following prolonged exposure of nerve terminals to the agonist l-(+)-phosphonobutyrate, l-AP4. This novel mGlu7 receptor response involves an increase in the release induced by the Ca(2+) ionophore ionomycin, suggesting a mechanism that is independent of Ca(2+) channel activity, but dependent on the downstream exocytotic release machinery. The mGlu7 receptor-mediated potentiation resists exposure to pertussis toxin, but is dependent on phospholipase C, and increased phosphatidylinositol (4,5)-bisphosphate hydrolysis. Furthermore, the potentiation of release does not depend on protein kinase C, although it is blocked by the diacylglycerol-binding site antagonist calphostin C. We also found that activation of mGlu7 receptors translocate the active zone protein essential for synaptic vesicle priming, munc13-1, from soluble to particulate fractions. We propose that the mGlu7 receptor can facilitate or inhibit glutamate release through multiple pathways, thereby exerting homeostatic control of presynaptic function.

Recently, evidence has emerged that seven transmembrane G protein-coupled receptors may be present as homo- and heteromers in the plasma membrane. Here we describe a new molecular and functional interaction between two functionally unrelated types of G protein-coupled receptors, namely the metabotropic glutamate type 1alpha (mGlu(1alpha) receptor) and the adenosine A1 receptors in cerebellum, primary cortical neurons, and heterologous transfected cells. Co-immunoprecipitation experiments showed a close and subtype-specific interaction between mGlu(1alpha) and A1 receptors in both rat cerebellar synaptosomes and co-transfected HEK-293 cells. By using transiently transfected HEK-293 cells a synergy between mGlu(1alpha) and A1 receptors in receptor-evoked [Ca(2+)](i) signaling has been shown. In primary cultures of cortical neurons we observed a high degree of co-localization of the two receptors, and excitotoxicity experiments in these cultures also indicate that mGlu(1alpha) and A1 receptors are functionally related. Our results provide a molecular basis for adenosine/glutamate receptors cross-talk and open new perspectives for the development of novel agents to treat neuropsychiatric disorders in which abnormal glutamatergic neurotransmission is involved.

Positive and negative reinforcing systems are part of the mechanism of drug dependence. Drugs with abuse potential may change the manner of response to negative emotional stimuli, activate positive emotional reactions and possess primary reinforcing properties. Catecholaminergic and peptidergic processes are of importance in these mechanisms. Current research needs to understand the types of adaptations that underlie the particularly long-lived aspects of addiction. Presently, glutamate is candidate to play a role in the enduring effects of drugs of abuse. For example, it participates in the chronic pathological changes of corticostriatal terminals produced by methamphetamine. At the synaptic level, a link between over-activation of glutamate receptors, [C(a2+)](i) increase and neuronal damage has been clearly established leading to neurodegeneration. Thus, neurodegeneration can start after an acute over-stimulation whose immediate effects depend on a diversity of calcium-activated mechanisms. If sufficient, the initial insult results in calcification and activation of a chronic on-going process with a progressive loss of neurons. At present, long-term effects of drug dependence underlie an excitotoxicity process linked to a polysynaptic pathway that dynamically regulates synaptic glutamate. Retaliatory mechanisms include energy capability of the neurons, inhibitory systems and cytoplasmic calcium precipitation as part of the neuron-glia interactions. This paper presents an integrated view of these molecular and cellular mechanisms to help understand their relationship and interdependence in a chronic pathological process that suggest new targets for therapeutic intervention.

The kinetics of the aqueous reaction of Cr(III) with either L-glutamic acid or sodium hydrogen L-glutamate at pH 2.46-5.87 have been followed by means of absorbance readings. The rate of formation of the reaction products showed acceleration-deceleration periods, caused by the accumulation and posterior decay of an intermediate in nonnegligible concentration. A double-exponential integrated rate law allowed obtaining two rate constants for each absorbance-time experimental series, associated with the appearance (k1) and decay (k2) of the long-lived intermediate. An increase of the initial concentrations of either hydrogen L-glutamate (apparent kinetic orders < 1) or hydroxide (kinetic orders = 1) ions resulted in an increase of both k1 and k2, but addition of an inert electrolyte (KNO3) resulted in opposite effects on k1 (decrease) and k2 (increase). The experimental activation energies were 83 ± 10 (for k1) and 95 ± 5 (for k2) kJ mol−1. The electronic spectrum of the low reactivity detected intermediate resembled more closely to that of the blue/green reactant than that of the violet reaction product. The low number of protons set free by the complexating hydrogen L-glutamate ligand seems to suggest that some polymerization of the coordinated amino acid (to form a di- or tripeptide) might take place. The available experimental data indicate that the coordination of the organic ligand must be preceded by the breakdown of a strong Cr(III)-H2O chemical bond in the slow steps of the mechanism.

Poly (y-glutamic acid), a novel polyanionic and multifunctional macromolecule synthesized by Bacillus species, has attracted considerable attention because of its eco-friendly, biodegradable and biocompatible characteristics. Recently, its application in a wide range of fields such as food, agriculture, medicine, hygiene, cosmetics and the environment has been explored. This book discusses the chemistry, food sources and health benefits of glutamic acid.

Hypothermia may attenuate the progression of ischemia-induced damage in liver. Here, we determined the effects of a brief cycle of hypothermic preconditioning applied before an ischemic/reperfusion (I/R) episode in isolated perfused rat liver (IPRL) on tissue damage and oxidative stress. Rats (male, 200-250 g) were anaesthetised with sodium pentobarbital (60 mg·kg-1 i.p) and underwent laparatomy. The liver was removed and perfused in a temperature-regulated non-recirculating system. Livers were randomly divided into two groups (n = 6 each group). In the hypothermia-preconditioned group, livers were perfused with hypothermic buffer (cycle of 10 min at 22 °C plus 10 min at 37 °C) and the other group was perfused at 37 °C. Both groups were then submitted to 40 min of warm ischemia and 20 min of warm reperfusion. The level of tissue-damage indicators (alanine amino transferase, ALT; lactate dehydrogenase, LDH; and proteins), oxidative stress markers (thiobarbituric acid-reactive substances, TBARS; advanced oxidation protein products, AOPP; and glutathione, GSH) were measured in aliquots of perfusate sampled at different time intervals. Histological determinations and oxidative stress biomarkers in homogenized liver (AOPP; TBARS; nitric oxide derivatives, NOx; GSH and glutathione disulphide, GSSG) were also made in the tissue at the end. Results showed that both damage and oxidant indicators significantly decreased while antioxidant increased in hypothermic preconditioned livers. In addition, homogenized liver determinations and histological observations at the end of the protocol corroborate the results in the perfusate, confirming the utility of the perfusate as a non-invasive method. In conclusion, hypothermic preconditioning attenuates oxidative damage and appears to be a promising strategy to protect the liver against IR injury.

Catalysis of the aldol reaction between cyclohexanone and 4-nitrobenzaldehyde by mixtures of L-Arg and of L-Glu in wet DMSO takes place with higher enantioselectivity (up to a seven-fold enhancement in the anti- aldol for the 1:1 mixture) than that observed when either L-Glu or L-Arg alone are used as the catalysts. These results can be explained by the formation of a catalytically active hydrogen-bonded complex between both amino acids, and demonstrate the possibility of positive cooperative effects in catalysis by two different α-amino acids. Chirality