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31p.-7 fig. 1 graph. abst., Frontotemporal dementia is the second most prevalent type of early-onset dementia and up to 40% of cases are familial forms. One of the genes mutated in patients is CHMP2B, which encodes a protein found in a complex important for maturation of late endosomes, an essential process for recycling membrane proteins through the endolysosomal system. Here, we have generated a CHMP2B-mutated human embryonic stem cell line using genome editing with the purpose to create a human in vitro Frontotemporal dementia disease model. To date, most studies have focused on neuronal alterations; however, we present a new co-culture system in which neurons and astrocytes are independently generated from human embryonic stem cells and combined in co-cultures. With this approach, we have identified alterations in the endolysosomal system of Frontotemporal dementia astrocytes, a higher capacity of astrocytes to uptake and respond to glutamate, and a neuronal network hyperactivity as well as excessive synchronization. Overall, our data indicates that astrocyte alterations precede neuronal impairments and could potentially trigger neuronal network changes, indicating the important and specific role of astrocytes in disease development., This study was supported by funding from the Swedish Society for Medical Research (SSMF, S20-0003), Kockska, Segerfalk and Hardebo foundations to IC; the Swedish Research Council (VR:2018-02695), Swedish Alzheimer and Åhlen foundations to HA; the Swedish Research Council (VR:2016-01789) to AH; and the Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (RTI2018-097624-BI00) and European Regional Development Fund to DPS.

Impaired brain clearance mechanisms may result in the accumulation of aberrant proteins that define Alzheimer's disease (AD). The water channel protein astrocytic aquaporin 4 (AQP4) is essential for brain amyloid-β clearance, but it is known to be abnormally expressed in AD brains. The expression of AQPs is differentially regulated during diverse brain injuries, but, whereas AQP4 expression and function have been studied in AD, less is known about AQP5. AQP5 functions include not only water transport but also cell migration mediated by cytoskeleton regulation. Moreover, AQP5 has been reported to be expressed in astrocytes, which are regulated after ischemic and traumatic injury. Additionally, AQP5 is particularly abundant in the salivary glands suggesting that it may be a crucial factor in gland dysfunction associated with AD. Herein, we aim to determine whether AQP5 expression in submandibular glands and the brain was altered in AD. First, we demonstrated impaired AQP5 expression in submandibular glands in APP/PS1 mice and AD patients. Subsequently, we observed that AQP5 expression was upregulated in APP/PS1 cerebral cortex and confirmed its expression both in astrocytes and neurons. Our findings propose AQP5 as a significant role player in AD pathology, in addition to AQP4, representing a potential target for the treatment of AD. This study was supported by grants from Instituto de Salud Carlos III through the project PI2021/00679) and cofounded by the European Union, Hospital Universitario 12 de Octubre Research Institute (2022/0068), FEDER, Comunidad de Madrid (S2017/BMD-3700; NEUROMETAB-CM), and CIBERNED (CB07/502, PI2021/03). Sí

An in‐depth understanding of the neurodegenerative component of multiple sclerosis (MS) is crucial for the design of therapeutic approaches that may stop disease progression. Astrocytes have emerged as key contributors to the pathogenesis of MS. 1 However, the mechanisms underlying the regulation of maladaptive astrocytic responses remain unknown. In this report, we show that a high inflammatory activity in MS patients at disease onset induces a specific reactive astrocyte state that triggers synaptopathy and contributes to neuronal damage in vitro and ex vivo suggesting potential mechanisms that may ultimately lead to neurodegeneration. To investigate whether astrocytes are essential contributors to neuronal damage in MS, we cultured purified astrocytes with cerebrospinal fluid (CSF) samples from MS patients with high inflammatory activity at disease onset (MS‐High, Table S1). Then, we examined the effect of astrocytic secretomes on neurons (Figure 1A). Astrocytes became reactive upon high inflammatory CSF exposure (Figure 1B) and induced morphological alterations typically observed in neurodegenerative disorders, such as a less complex dendritic tree due to decreased arborisation (Figure 1C, D). Moreover, these abnormalities were accompanied with synaptic plasticity impairment (Figure 1E, F). Considering that a high lesion load at disease onset has been associated with an increased risk of neurological disability development, 2 we assessed whether the non‐cell‐autonomous effect on neuronal plasticity could be influenced by the degree of inflammatory activity of MS patients (Figure 2A and Table S1). Interestingly, we observed a direct correlation between the degree of inflammatory exposure and the extent of both astrocyte‐mediated synaptopathy (Figure 2B, C) and dendrite arborisation impairment.

Astrocytes have emerged as crucial regulators of neuronal network activity, synapse formation, and underlying behavioral and cognitive processes. Despite some pathways have been identified, the communication between astrocytes and neurons remains to be completely elucidated. Unraveling this communication is crucial to design potential treatments for neurological disorders like temporal lobe epilepsy (TLE). The BDNF and TrkB molecules have emerged as very promising therapeutic targets. However, their modulation can be accompanied by several off-target effects such as excitotoxicity in case of uncontrolled upregulation or dementia, amnesia, and other memory disorders in case of downregulation. Here, we show that BDNF and TrkB from astrocytes modulate neuronal dysfunction in TLE models. First, conditional overexpression of BDNF from astrocytes worsened the phenotype in the lithium-pilocarpine mouse model. Our evidences pointed out to the astrocytic pro-BDNF isoform as a major player of this altered phenotype. Conversely, specific genetic deletion of BDNF in astrocytes prevented the increase in the number of firing neurons and the global firing rate in an in vitro model of TLE. Regarding to the TrkB, we generated mice with a genetic deletion of TrkB specifically in hippocampal neurons or astrocytes. Interestingly, both lines displayed neuroprotection in the lithium-pilocarpine model but only the mice with genetic deletion of TrkB in astrocytes showed significantly preserved spatial learning skills. These data identify the astrocytic BDNF and TrkB molecules as promising therapeutic targets for the treatment of TLE.

[cat] La Leucoencefalopatia Megalencefàlica amb Quists subcorticals (de l’anglès Megalencephalic Leukoencephalopathy with subcortical Cysts, MLC) és un tipus rar de leucodistròfia caracteritzada per la presència de vacuoles en la substància blanca del sistema nerviós. Es suggereix que la patogènesi de la malaltia és causada pel mal funcionament de les cèl·lules glials en el control de la homeòstasi iònica i el fluid cerebral. Els gens responsables de la malaltia són MLC1 i GLIALCAM que codifiquen per dues proteïnes que reben el mateix nom i que actualment es desconeix la seva funció biològica i la seva estructura. Ambdues proteïnes formen un complex a la membrana plasmàtica que s’expressa principalment en els astròcits que envolten la barrera hematoencefàlica i a la glia de Bergmann del cerebel. Es coneix que aquest complex regula de manera directa a varis canals iònics i transportadors involucrats en el control del flux de ions i de l’aigua en les cèl·lules glials, afectant la seva localització i funció; i indirectament a través de vies de senyalització. El principal objectiu d’aquesta Tesi és augmentar el coneixement estructural d’ambdues proteïnes amb la finalitat d’entendre la seva funció biològica i el mecanisme patològic de la malaltia., [eng] Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a rare genetic disorder characterized by macrocephaly and white matter vacuolization, whose pathogenesis is suggested to rely on a dysfunction on the control of water and ionic homeostasis by glial cells. MLC disease is caused by mutations in either MLC1 or GLIALCAM genes, which codify for membrane proteins that form a complex with an unknown function mainly expressed in astrocytes. GlialCAM, an Ig-like cell adhesion molecule, has also been described as an auxiliary subunit of the chloride channel ClC-2. The main objective of this thesis was increasing the structural knowledge of GlialCAM, proposing a structural model, based on bioinformatic and biochemical evidences, of GlialCAM dimerization through its extracellular domains; and MLC1 protein, characterising a system to express and purify MLC1 to obtain his 3D structure.

International audience; Absence of the astrocyte-specific membrane protein MLC1 is responsible for megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare type of leukodystrophy characterized by early-onset macrocephaly and progressive white matter vacuolation that lead to ataxia, spasticity, and cognitive decline. During postnatal development (from P5 to P15 in the mouse), MLC1 forms a membrane complex with GlialCAM (another astrocytic transmembrane protein) at the junctions between perivascular astrocytic processes. Perivascular astrocytic processes along with blood vessels form the gliovascular unit. It was not previously known how MLC1 influences the physiology of the gliovascular unit. Here, using the Mlc1 knock-out (KO) mouse model of MLC, we demonstrated that MLC1 controls the postnatal development and organization of perivascular astrocytic processes, vascular smooth muscle cell contractility, neurovascular coupling and intraparenchymal interstitial fluid clearance. Our data suggest that MLC is a developmental disorder of the gliovascular unit, and perivascular astrocytic processes and vascular smooth muscle cell maturation defects are primary events in the pathogenesis of MLC and therapeutic targets for this disease.

International audience; Glial vulnerability to prions is assessed in murine Creutzfeldt-Jakob disease (CJD) using the tg340 mouse line expressing four-fold human PrP M129 levels on a mouse PrP null background at different days following intracerebral inoculation of sCJD MM1 brain tissues homogenates. The mRNA expression of several astrocyte markers, including glial fibrillary acidic protein (gfap), aquaporin-4 (aqp4), solute carrier family 16, member 4 (mct4), mitochondrial pyruvate carrier 1 (mpc1) and solute carrier family 1, member 2 (glial high-affinity glutamate transporter, slc1a2) increases at 120 and 180 dpi. In contrast, the mRNA expression of oligodendrocyte and myelin markers oligodendrocyte transcription factor 1 (olig1), olig2, neural/glial antigen 2 (cspg), solute carrier family 16, member 1 (mct1), myelin basic protein (mbp), myelin oligodendrocyte glycoprotein (mog) and proteolipid protein 1 (plp1) is preserved. Yet, myelin regulatory factor (myrf) mRNA is increased at 180 dpi. In the striatum, a non-significant increase in the number of GFAP-positive astrocytes and Iba1-immunoreactive microglia occurs at 160 dpi; a significant increase in the number of astrocytes and microglia, and a significant reduction in the number of Olig2-immunoreactive oligodendrocytes occur at 180 dpi. A decrease of MBP, but not PLP1, immunoreactivity is also observed in the striatal fascicles. These observations confirm the vulnerability and the reactive responses of astrocytes, together with the microgliosis at middle stages of prion diseases. More importantly, these findings show oligodendrocyte vulnerability and myelin alterations at advanced stages of murine CJD. They confirm oligodendrocyte involvement in the pathogenesis of CJD.

International audience; Neuroinflammation has been correlated with the progress of neurodegeneration in many neuropathologies. Although glial cells have traditionally been considered to be protective, the concept of them as neurotoxic cells has recently emerged. Thus, a major unsolved question is the exact role of astroglia and microglia in neurodegenerative disorders. On the other hand, it is well known that glucocorticoids are the first choice to regulate inflammation and, consequently, neuroglial inflammatory activity. The objective of this study was to determine how chronic dexamethasone treatment influences the host immune response and to characterize the beneficial or detrimental role of glial cells. To date, this has not been examined using a natural neurodegenerative model of scrapie. With this aim, immunohistochemical expression of glial markers, prion protein accumulation, histopathological lesions and clinical evolution were compared with those in a control group. The results demonstrated how the complex interaction between glial populations failed to compensate for brain damage in natural conditions, emphasizing the need for using natural models. Additionally, the data showed that modulation of neuroinflammation by anti-inflammatory drugs might become a research focus as a potential therapeutic target for prion diseases, similar to that considered previously for other neurodegenerative disorders classified as prion-like diseases.

Using an in vitro wound healing assay, De Pascalis et al. show that intermediate filaments (IFs) participate in the dynamics of the acto-myosin network and its association with adhesions in astrocytes during collective migration. Glial IFs control the distribution of forces and the interactions between neighboring cells, ultimately determining the speed and direction of collective migration., Mesenchymal cell migration relies on the coordinated regulation of the actin and microtubule networks that participate in polarized cell protrusion, adhesion, and contraction. During collective migration, most of the traction forces are generated by the acto-myosin network linked to focal adhesions at the front of leader cells, which transmit these pulling forces to the followers. Here, using an in vitro wound healing assay to induce polarization and collective directed migration of primary astrocytes, we show that the intermediate filament (IF) network composed of vimentin, glial fibrillary acidic protein, and nestin contributes to directed collective movement by controlling the distribution of forces in the migrating cell monolayer. Together with the cytoskeletal linker plectin, these IFs control the organization and dynamics of the acto-myosin network, promoting the actin-driven treadmilling of adherens junctions, thereby facilitating the polarization of leader cells. Independently of their effect on adherens junctions, IFs influence the dynamics and localization of focal adhesions and limit their mechanical coupling to the acto-myosin network. We thus conclude that IFs promote collective directed migration in astrocytes by restricting the generation of traction forces to the front of leader cells, preventing aberrant tractions in the followers, and by contributing to the maintenance of lateral cell–cell interactions.

It has been well documented that neurotrophins, including brain-derived neurotrophic factor (BDNF), are severely affected in Alzheimer's disease (AD), but their administration faces a myriad of technical challenges. Here we took advantage of the early astrogliosis observed in an amyloid mouse model of AD (5xFAD) and used it as an internal sensor to administer BDNF conditionally and locally. We first demonstrate the relevance of BDNF release from astrocytes by evaluating the effects of coculturing WT neurons and BDNF-deficient astrocytes. Next, we crossed 5xFAD mice with pGFAP:BDNF mice (only males were used) to create 5xFAD mice that overexpress BDNF when and where astrogliosis is initiated (5xF:pGB mice). We evaluated the behavioral phenotype of these mice. We first found that BDNF from astrocytes is crucial for dendrite outgrowth and spine number in cultured WT neurons. Double-mutant 5xF:pGB mice displayed improvements in cognitive tasks compared with 5xFAD littermates. In these mice, there was a rescue of BDNF/TrkB downstream signaling activity associated with an improvement of dendritic spine density and morphology. Clusters of synaptic markers, PSD-95 and synaptophysin, were also recovered in 5xF:pGB compared with 5xFAD mice as well as the number of presynaptic vesicles at excitatory synapses. Additionally, experimentally evoked LTP in vivo was increased in 5xF:pGB mice. The beneficial effects of conditional BDNF production and local delivery at the location of active neuropathology highlight the potential to use endogenous biomarkers with early onset, such as astrogliosis, as regulators of neurotrophic therapy in AD. SIGNIFICANCE STATEMENT Recent evidence places astrocytes as pivotal players during synaptic plasticity and memory processes. In the present work, we first provide evidence that astrocytes are essential for neuronal morphology via BDNF release. We then crossed transgenic mice (5xFAD mice) with the transgenic pGFAP-BDNF mice, which express BDNF under the GFAP promoter. The resultant double-mutant mice 5xF:pGB mice displayed a full rescue of hippocampal BDNF loss and related signaling compared with 5xFAD mice and a significant and specific improvement in all the evaluated cognitive tasks. These improvements did not correlate with amelioration of β amyloid load or hippocampal adult neurogenesis rate but were accompanied by a dramatic recovery of structural and functional synaptic plasticity.

Astrocytes are the most abundant cell type in the CNS (central nervous system). They exert multiple functions during development and in the adult CNS that are essential for brain homeostasis. Both cation and anion channel activities have been identified in astrocytes and it is believed that they play key roles in astrocyte function. Whereas the proteins and the physiological roles assigned to cation channels are becoming very clear, the study of astrocytic chloride channels is in its early stages. In recent years, we have moved from the identification of chloride channel activities present in astrocyte primary culture to the identification of the proteins involved in these activities, the determination of their 3D structure and attempts to gain insights about their physiological role. Here, we review the recent findings related to the main chloride channels identified in astrocytes: the voltage-dependent ClC-2, the calcium-activated bestrophin, the volume-activated VRAC (volume-regulated anion channel) and the stress-activated Maxi-Cl−. We discuss key aspects of channel biophysics and structure with a focus on their role in glial physiology and human disease.

La adrenoleucodistrofia ligada al cromosoma X (X-ALD, de sus siglas en inglés) es una enfermedad rara causada por mutaciones en el transportador peroxisomal ABCD1, presentando dos formas de manifestación clínica de neurodegeneración: demielinización aguda y letal en el cerebro de niños con la forma cerebral de X-ALD (CCALD), y degeneración crónica de los tractos de la médula espinal y neuropatía periférica en adultos con adrenomielopatía (AMN). Las alteraciones psiquiátricas son un signo temprano de CCALD y coexisten con la neurodegeneración periférica de la médula espinal en AMN, poniendo de manifiesto la patología cerebral. Teniendo en cuenta que la X-ALD es una condición genética quisimos determinar si las alteraciones psiquiátricas observadas pueden ser debidas, al menos en parte, a la formación aberrante de circuitos cerebrales durante el desarrollo y no a la neurodegeneración. Con este objetivo, usamos una aproximación de arriba hacia abajo, partiendo de datos de transcriptomas de pacientes, donde hemos buscado vías de neurodesarrollo desreguladas, hasta modelos murinos de X-ALD in vitro basados en el silenciamiento de los genes Abcd1 y Abcd2 para diseccionar la compartimentalización entre neuronas y astrocitos. Se presentan cuatro resultados principales. Primero, existen vías de neurodesarrollo desreguladas en CCALD, AMN y en los cultivos neuronales deficientes en ABCD, asociado con alteración en los procesos de neuritogénesis, espinogénesis, y axonogénesis. Segundo, un crecimiento aberrante de las espinas sinápticas es en parte debido a alteraciones en la vía canoníca de señalización Wnt puesto que la espinogénesis se recupera parcialmente mediante la activación de la vía de señalización Wnt tras la inhibición farmacológica de GSK-3. Tercero, la síntesis de colesterol y la localización del mismo se encuentran cambiadas en los astrocitos deficientes en ABCD. Cuarto, el silenciamiento de los transportadores ABCD causa alteraciones metabólicas en astrocitos incluyendo disminución de la oxidación de ácidos grasos, incremento del ratio NAD+/NADH, depleción de ATP, y disminución de la cantidad total de glutatión sugiriendo el deterioro conjunto de la defensa antioxidante y la bioenergética. Quinto, los astrocitos X-ALD presentan una señalización de calcio mediada por agonistas alterada y estrés del retículo endoplamático. Concluimos que i) la X-ALD presenta un componente de neurodesarrollo que puede explicar los síntomas psiquiátricos, y quizá contribuir a la progresión de la forma CCALD, y a la conversión de la AMN en una forma cerebral de la enfermedad, y ii) la deregulacion metabólica y de la excitabilidad en astrocitos apoya la disfunción global de los astrocitos que puede poner en peligro el papel computacional y homeostático de los astrocitos en los circuitos cerebrales. X-linked adrenoleukodystrophy (X-ALD) is a rare disease caused by mutations in the peroxisomal ABCD1 transporter, with two major clinical manifestations of neurodegeneration: acute and lethal brain demyelination in the child cerebral X-ALD (CCALD), and chronic degeneration of spinal-cord tracts and peripheral neuropathy in the adult adrenomyeloneuropathy (AMN). Psychiatric alterations are an early sign of CCALD and coexist with spinal-cord and peripheral neurodegeneration in AMN, revealing concomitant brain pathology. Since X-ALD is a genetic condition, we sought to determine whether psychiatric alterations could be due, at least in part, to abnormal formation of brain circuits during brain development and not to neurodegeneration. To this end, we used a top-down approach, moving from patient transcriptome data, where we searched for dysregulated neurodevelopmental pathways, to in vitro murine models of X-ALD based on Abcd1/Abcd2 silencing to dissect out astrocyte versus neurons compartmentalization. There are four major findings. First, developmental pathways are dysregulated in CCALD, CAMN and in ABCD-null neuronal cultures, associated with altered neuritogenesis, spinogenesis, and axonogenesis. Second, aberrant spine growth is in part due to alterations in canonical Wnt signalling alteration since the spinogenesis is partially rescued by activation of WNT pathways by pharmacological GSK-3 inhibition. Third, cholesterol synthesis and localization is changed in ABCD-null astrocytes. Four, silencing of ABCD transporters causes metabolic alterations in astrocytes including fatty acid oxidation impairment, increase of the ratio NAD+/NADH, ATP depletion, decreases in total glutathione, suggesting joint impairment of antioxidant defense and bioenergetics. Fifth, X-ALD astrocytes present altered agonist-induced calcium signaling and ER stress. We conclude that i) X-ALD has a neurodevelopmental component that may account for psychiatric symptoms, and perhaps contribute to the progression of CCALD, and to the conversion of AMN into a cerebral condition, and ii) metabolic and excitability dysregulation in astrocytes support global astrocytic dysfunction that may jeopardize computational and homeostatic role of astrocytes in neural circuits.

Significance Aging and neurodegenerative processes induce the formation of waste substances in the brain. Some of these substances accumulate in corpora amylacea (CA). We reveal that CA are released from the brain into the cerebrospinal fluid and are present in the cervical lymph nodes, into which cerebrospinal fluid drains through the meningeal lymphatic system. We also show that CA can be phagocytosed by macrophages. We conclude that CA can act as waste containers and hypothesize that CA are involved in a mechanism that cleans the brain. We also postulate that CA may contain clinical markers of brain disorders and may also play significant roles in some brain autoimmune diseases. These last points merit further study due to their possible clinical implications., Corpora amylacea (CA) in the human brain are granular bodies formed by polyglucosan aggregates that amass waste products of different origins. They are generated by astrocytes, mainly during aging and neurodegenerative conditions, and are located predominantly in periventricular and subpial regions. This study shows that CA are released from these regions to the cerebrospinal fluid and are present in the cervical lymph nodes, into which cerebrospinal fluid drains through the meningeal lymphatic system. We also show that CA can be phagocytosed by macrophages. We conclude that CA can act as containers that remove waste products from the brain and may be involved in a mechanism that cleans the brain. Moreover, we postulate that CA may contribute in some autoimmune brain diseases, exporting brain substances that interact with the immune system, and hypothesize that CA may contain brain markers that may aid in the diagnosis of certain brain diseases.

Peripheral nerve injuries (PNIs) may result in cellular and molecular changes in supraspinal structures possibly involved in neuropathic pain (NPP) maintenance. Activated glial cells in specific supraspinal subregions may affect the facilitatory role of descending pathways. Sterile chronic compression injury (sCCI) and complete sciatic nerve transection (CSNT) in rats were used as NPP models to study the activation of glial cells in the subregions of periaqueductal gray (PAG) and rostral ventromedial medulla (RVM). Molecular markers for activated astrocytes (glial fibrillary acidic protein, GFAP) and microglial cells (OX42) were assessed by quantitative immunohistochemistry and western blotting. The cellular distribution of CCL2/CCR2 was monitored using immunofluorescence. sCCI induced both mechanical and thermal hypersensitivity from day 1 up to 3 weeks post-injury. Unilateral sCCI or CSNT for 3 weeks induced significant activation of astrocytes bilaterally in both dorsolateral (dlPAG) and ventrolateral PAG (vlPAG) compared to naive or sham-operated rats. More extensive astrocyte activation by CSNT compared to sCCI was induced bilaterally in dlPAG and ipsilaterally in vlPAG. Significantly more extensive activation of astrocytes was also found in RVM after CSNT than sCCI. The CD11b immunopositive region, indicating activated microglial cells, was remarkably larger in dlPAG and vlPAG of both sides from sCCI- and CSNT-operated rats compared to naive or sham-operated controls. No significant differences in microglial activation were detected in dlPAG or vlPAG after CSNT compared to sCCI. Both nerve injury models induced no significant differences in microglial activation in the RVM. Neurons and activated GFAP+ astrocytes displayed CCL2-immunoreaction, while activated OX42+ microglial cells were CCR2-immunopositive in both PAG and RVM after sCCI and CSNT. Overall, while CSNT induced robust astrogliosis in both PAG and RVM, microglial cell activation was similar in the supraspinal structures in both injury nerve models. Activated astrocytes in PAG and RVM may sustain facilitation of the descending system maintaining NPP, while microglial activation may be associated with a reaction to long-lasting peripheral injury. Microglial activation via CCR2 may be due to neuronal and astrocytal release of CCL2 in PAG and RVM following injury.

La enfermedad de Alzheimer es la forma mas común de demencia en edades avanzadas, la cual afecta a 40 millones de personas alrededor del mundo. Es una enfermedad compleja que afecta no solo a neuronas, sino también a astrocitos. La Apolipoproteína E4 (ApoE4) ha sido descrita como el factor de riesgo genético más importante de la forma esporádica de la enfermedad y además, los astrocitos son los principales secretores de esta. La investigación sobre los mecanismos patogénicos causados por esta apolipoproteína está centrada en su rol extracelular. Por el contrario, nosotros analizamos las desregulaciones intracelulares que causa la ApoE4. En nuestro estudio, nos centramos en 2 principales procesos fisiológicos alterados en la enfermedad de Alzheimer: la señalización de calcio y las funciones mitocondriales. Utilizamos como modelo celular, astrocitos inmortalizados que expresan la forma humana ApoE3 (no asociada a ninguna patología) o la ApoE4. Utilizando indicadores de calcio fluorescentes y la técnica de Imagen de Calcio, determinamos que los astrocitos ApoE4 tienen alteraciones de la homeostasis de calcio, ya que presentan menores niveles de calcio intracelular basal pero mayores señales de calcio inducidas por estímulos purinérgicos en comparación con los astrocitos ApoE3. Una mayor actividad de la V-ATPasa y por lo tanto, una mayor entrada de calcio al lisosoma en los astrocitos ApoE4 explica estas alteraciones. Además, la salida de calcio lisosomal después de la activación de receptores purinérgicos es seguida por una mayor movilización de calcio del retículo endoplásmico en los astrocitos ApoE4 comparados con los ApoE3. La entrada de calcio extracelular es similar en ambos tipos celulares. Nuestros estudios también demuestran que la falta de lipoproteínas en el medio extracelular aumenta la magnitud de las respuestas de calcio inducidas por receptores purinérgicos en los astrocitos ApoE3, ya que la entrada de calcio extracelular amplifica la liberación del calcio lisosomal en estas células. Esta característica se pierde en las células ApoE4, en las cuales la magnitud de las respuestas de calcio no se ve afectada por las lipoproteínas extracelulares. Por otro lado, describimos alteraciones en las dinámicas mitocondriales determinadas por microscopia a tiempo real y el marcaje fluorescente de mitocondrias. En particular, las mitocondrias de células ApoE4 no realizan fisión después de la inhibición de la fosforilación oxidativa mitocondrial, mientras que las mitocondrias de los astrocitos ApoE3 si la realizan. Asimismo, los astrocitos ApoE4 tienen un incremento en la movilidad mitocondrial, una reducción de Parkina, proteína involucrada en la mitofagia y una reducción del ADN mitocondrial en comparación con los astrocitos ApoE3. En resumen, demostramos por primera vez que la ApoE4 endógena altera la señalización del calcio y las funciones mitocondriales en astrocitos. Teniendo en cuenta que la ApoE4 se expresa a lo largo de toda la vida de los individuos, estas alteraciones astrocíticas podrían aparecer en las primeras etapas de la enfermedad de Alzheimer. Para avanzar en la detección de estas primeras etapas de la patología, y ya que las proteínas del líquido cefalorraquídeo reflejan las funcionalidad de las células cerebrales, nosotros hemos identificado un grupo de proteínas astrocíticas presentes en este líquido relacionadas con la enfermedad de Alzheimer y que proponemos como firma funcional astrocítica. Esta firma esta compuesta de las proteínas S100B, ApoE, prostaglandina D2 sintetasa, cistatina 3, proteína integral de membrana 2C y clusterina. En general, la ApoE4 endógena altera las funciones de los astrocitos, un fenómeno que puede contribuir a la enfermedad de Alzheimer, además de a su detección a través de biomarcadores del líquido cefalorraquídeo. Alzheimer’s disease is the most common form of dementia in advanced ages affecting more than 40 million people around the world. It is a complex disease that affects not only neurons but also astrocytes. Apolipoprotein E4 (ApoE4) has been described as the most important genetic risk factor for the sporadic form of the disease and interestingly, astrocytes are its main secretors. The research about its pathogenic mechanisms has mainly focused on its extracellular role. On the contrary, we analysed the dysregulations that endogenous intracellular ApoE4 causes on astrocytes. We focused on 2 principal physiological processes altered in Alzheimer’s disease: calcium signalling and mitochondrial functions. As cellular model, we used immortalized astrocytes that express human ApoE3 (non-associated with any pathology) or ApoE4. Using fluorescent calcium indicators and the technique of Calcium Imaging, we determined that ApoE4 astrocytes have altered calcium homeostasis as they have lower basal intracellular calcium levels but higher purinergic-induced calcium signals compared to ApoE3 astrocytes. A high V-ATPase activity, and hence, higher lysosomal calcium uptake in ApoE4 astrocytes explains these alterations. Moreover, lysosomal calcium release after purinergic receptor activation is followed by higher endoplasmic reticulum (ER) calcium mobilization in ApoE4 than in ApoE3 astrocytes. Extracellular calcium entry is similar in both cell types. Our studies also demonstrated that the lack of lipoproteins in the extracellular medium upregulates the magnitude of purinergic-elicited calcium responses in ApoE3 astrocytes, as extracellular calcium entry amplifies lysosomal calcium release. This feature is missing in ApoE4 astrocytes being the magnitude of calcium responses unaffected by extracellular lipoproteins. On the other hand, we described alterations in mitochondrial dynamics determined by real-time microscopy and fluorescent mitochondria labelling. In particular, ApoE4 cell mitochondria do not perform fission after inhibition of mitochondrial oxidative phosphorylation whereas ApoE3 astrocyte mitochondria perform it. In addition, ApoE4 astrocytes have increased mitochondrial motility, reduction of Parkin, a protein involved in mitophagy, and reduction in mitochondrial DNA content compared to ApoE3 astrocytes. In summary, we demonstrated, for the first time, that endogenous ApoE4 alters calcium signalling and mitochondrial functions in astrocytes. Taking into account that ApoE is expressed throughout the life of individuals, these astrocytic alterations might appear in the early stages of the Alzheimer’s disease. In order to advance in the detection of such early phases of the pathology, and since cerebrospinal fluid proteins reflect the cellular function of brain cells, we next identified a group of astrocytic proteins present in the cerebrospinal fluid related to Alzheimer’s disease that we propose as a functional astrocytic signature for early stages of the disease. This signature is composed of S100B, ApoE, prostaglandin D2 synthetase, cystatin 3, integral membrane protein 2C and clusterin. Overall, endogenous ApoE4 alters astrocyte functions, a phenomenon that can contribute to Alzheimer’s disease, but also, to its detection through cerebrospinal fluid biomarkers.

Cada cop més evidencies suggereixen que els astròcits participen en les altes funcions cerebrals, controlant des de la transmissió sinàptica fins a les ones cerebrals globals i els processos d’aprenentatge i memòria. Diferents mecanismes han sigut proposats com a responsables d’aquests processos mediats per astròcits, entre ells, l’alliberació de gliotransmissors a partir de les senyals de calci així com la de lactat semblen els principals efectors. L’existència d’aquest control de les funcions cerebrals per part dels astròcits suggereix que aquestes cèl·lules poden regular les funcions cerebrals en resposta a experiència tan com les neurones, constituint el fenomen de plasticitat astrocitària. En neurones s’ha demostrat que el conegut factor de transcripció CREB, coordina les plasticitats sinàptica i intrínseca. El fet que, en astròcits, l’activació de CREB també està regulada per activitat cerebral, situa aquest factor de transcripció com a la diana ideal per promoure canvis dependents d’activitat en astròcits. En aquesta tesi hem analitzat l’efecte de l’activació de la transcripció depenent de CREB en astròcits, centrant-nos en l’excitabilitat del calci i en el metabolisme d’aquestes cèl·lules. Hem demostrat que l’activació de la transcripció depenent de CREB redueix les senyals citosòliques de calci a través del mitocondri a la vegada que augmenta l’alliberació de lactat, dos canvis que poden tenir impacte en la transmissió sinàptica. Una altra contribució important d’aquest estudi es l’anàlisi molecular dels mitocondris dels astròcits, que ha revelat que aquestes cèl·lules poden utilitzar metabòlits que no són glucosa, com ara àcids grassos, per respondre a les necessitats metabòliques energètiques. Els nostres resultats estableixen el CREB en astròcits con un eix de la plasticitat astrocitària i revelen la interacció entre la plasticitat i el metabolisme energètic en astròcits. Aquests descobriments constitueixen un avenç mecanístic i conceptual en el coneixement de la biologia dels astròcits i com aquestes cèl·lules poden controlar l’aprenentatge i la memòria. An increasing body of evidence suggests that astrocytes participate in higher-brain functions, controlling from synaptic transmission to global brain waves and learning and memory processes. Different mechanisms have been proposed to mediate these astrocyte-dependent processes, astrocytic lactate release and calcium-dependent gliotransmission being the main known effectors. The existence of control of brain functions by astrocytes suggests that astrocytes may shape brain functions in response to experience as much as neurons, thus constituting the phenomenon of astrocyte plasticity. In neurons, the transcription factor CREB is the best known coordinator of synaptic and intrinsic plasticity. The fact that, in astrocytes, CREB activation is also activity-dependent, positions CREB as an ideal target to promote plasticity-related changes in astrocytes, too. In this thesis, we have analyzed the effect of the activation of CREB-dependent transcription in astrocytes, specifically regarding calcium signals and metabolism. We have demonstrated that activation of CREB-dependent transcription reduces cytosolic calcium events via mitochondria and increases in lactate release, which may have impact on synaptic transmission. An important contribution of the study is the molecular analysis of astrocytic mitochondria, which has revealed that astrocytes may use fuels other than glucose such as fatty acids to meet basic energy metabolic demands. Taken together, our results establish astrocytic CREB as a hub in astrocyte-plasticity and shed light on the interplay between plasticity and energy metabolism in astrocytes; these findings constitute a conceptual and mechanistic advance in the knowledge of astrocytic biology and how these cells may control learning and memory.

Targeted expression of genes coding for proteins specific to astrocytes, oligodendrocytes and myelin was performed in frontal cortex area 8 of Creutzfeldt-Jakob disease methionine/methionine and valine/valine (CJD MM1 and VV2, respectively) compared with controls. GFAP (glial fibrillary acidic protein) mRNA was up-regulated whereas SLC1A2 (solute carrier family 1 member 2, coding for glutamate transporter 1: GLT1), AQ4 (aquaporin 4), MPC1 (mitochondrial pyruvate carrier 1) and UCP5 (mitochondrial uncoupled protein 5) mRNAs were significantly down-regulated in CJD MM1 and CJD VV2, and GJA1 (connexin 43) in CJD VV2. OLIG1 and OLIG2 (oligodendocyte transcription factor 1 and 2, respectively), SOX10 (SRY-Box10) and oligodendroglial precursor cell (OPC) marker NG2 (neuronal/glial antigen) 2 were preserved, but GALC (coding for galactosylceramidase), SLC2A1 (solute carrier family 2 member 1: glucose transporter member 1: GLUT1) and MCT1 (monocarboxylic acid transporter 1) mRNA expression levels were significantly reduced in CJD MM1 and CJD VV2. Expression levels of most genes linked to myelin were not altered in the cerebral cortex in CJD. Immunohistochemistry to selected proteins disclosed individual variations but GFAP, Olig-2, AQ4 and GLUT1 correlated with mRNA levels, whereas GLT1 was subjected to individual variations. However, MPC1, UCP5 and MCT1 decrease was more closely related to the respective reduced neuronal immunostaining. These observations support the idea that molecular deficits linked to energy metabolism and solute transport in astrocytes and oligodendrocytes, in addition to neurons, are relevant in the pathogenesis of cortical lesions in CJD.

Aging-related tau astrogliopathy (ARTAG) is defined by the presence of two types of tau-bearing astrocytes: thorn-shaped astrocytes (TSAs) and granular/fuzzy astrocytes in the brain of old-aged individuals. The present study is focused on TSAs in rare forms of ARTAG with no neuronal tau pathology or restricted to entorhinal and transentorhinal cortices, to avoid bias from associated tauopathies. TSAs show 4Rtau phosphorylation at several specific sites and abnormal tau conformation, but they lack ubiquitin and they are not immunostained with tau-C3 antibodies which recognize truncated tau at Asp421. Astrocytes in ARTAG have atrophic processes, reduced glial fibrillary acidic protein (GFAP) and increased superoxide dismutase 2 (SOD2) immunoreactivity. Gel electrophoresis and western blotting of sarkosyl-insoluble fractions reveal a pattern of phospho-tau in ARTAG characterized by two bands of 68 and 64 kDa, and several middle bands between 35 and 50 kDa which differ from what is seen in AD. Phosphoproteomics of dissected vulnerable regions identifies an increase of phosphorylation marks in a large number of proteins in ARTAG compared with controls. GFAP, aquaporin 4, several serine-threonine kinases, microtubule associated proteins and other neuronal proteins are among the differentially phosphorylated proteins in ARTAG thus suggesting a hyper-phosphorylation background that affects several molecules, including many kinases and proteins from several cell compartments and various cell types. Finally, present results show for the first time that tau seeding is produced in neurons of the hippocampal complex, astrocytes, oligodendroglia and along fibers of the corpus callosum, fimbria and fornix following inoculation into the hippocampus of wild type mice of sarkosyl-insoluble fractions enriched in hyper-phosphorylated tau from selected ARTAG cases. These findings show astrocytes as crucial players of tau seeding in tauopathies.

Lafora disease (LD), the most devastating adolescence-onset epilepsy, is caused by mutations in the EPM2A or EPM2B genes, which encode the proteins laforin and malin, respectively. Loss of function of one of these proteins, which are involved in the regulation of glycogen synthesis, induces the accumulation of polyglucosan bodies (PGBs)—known as Lafora bodies (LBs) and associated with neurons—in the brain. Ageing and some neurodegenerative conditions lead to the appearance of another type of PGB called corpora amylacea, which are associated with astrocytes and contain neo-epitopes that can be recognized by natural antibodies. Here we studied the PGBs in the cerebral cortex and hippocampus of malin knockout mice, a mouse model of LD. These animals presented not only LBs associated with neurons but also a significant number of PGBs associated with astrocytes. These astrocytic PGBs were also increased in mice from senescence-accelerated mouse-prone 8 (SAMP8) strain and mice with overexpression of Protein Targeting to Glycogen (PTG(OE)), indicating that they are not exclusive of LD. The astrocytic PGBs, but not neuronal LBs, contained neo-epitopes that are recognized by natural antibodies. The astrocytic PGBs appeared predominantly in the hippocampus but were also present in some cortical brain regions, while neuronal LBs were found mainly in the brain cortex and the pyramidal layer of hippocampal regions CA2 and CA3. Our results indicate that astrocytes, contrary to current belief, are involved in the etiopathogenesis of LD.

BACKGROUND: cAMP signaling produces dramatic changes in astrocyte morphology and physiology. However, its involvement in phenotype acquisition and the transcriptionally mediated mechanisms of action are largely unknown. RESULTS: Here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of cAMP pathways. A bulk of astroglial transcripts, 6221 annotated genes, were differentially regulated by cAMP signaling. cAMP analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. Moreover, numerous genes typically activated in reactive cells, such as scar components and immunological mediators, were repressed by cAMP. GSEA analysis contrasting gene expression profiles with transcriptome signatures of acutely isolated astrocytes and in situ evaluation of protein levels in these cells showed that cAMP signaling conferred mature and in vivo-like transcriptional features to cultured astrocytes. CONCLUSIONS: These results indicate that cAMP signaling is a key pathway promoting astrocyte maturation and restricting their developmental and activation features. Therefore, a positive modulation of cAMP signaling may promote the normal state of differentiated astrocytes and favor the protection and function of neuronal networks. This work was supported by grants from Spanish Ministry of Economy and Competitiveness (BFU2010-22132 and BFU2013-48822-R) and from Catalonian Government (2014SGR-01178) to F.A

[cat] La Leucoencefalopatia Megalencefàlica amb Quists subcorticals (MLC) és un tipus rar de leucodistròfia vacuolitzant de progressió lenta, que presenta com a principals característiques clíniques macrocefàlia acusada durant els primers anys de vida, deteriorament de les funcions motores, epilèpsia i retard mental de grau mig. Actualment encara es desconeix el mecanisme fisiopatològic de la malaltia, i per tant ni hi ha cap tractament possible per als pacients. S’han descrit dos gens implicats en la malaltia MLC. El primer gen descobert s’anomena MLC1 i codifica per una proteïna de membrana que porta el mateix nom. El segon gen s’anomena GLIALCAM i codifica per una proteïna transmembrana de tipus I que també porta el mateix nom. S’ha decrit que la proteïna GlialCAM actua com a subunitat ß de MLC1 ja que es capaç de dirigir-la i concentrar-la a les unions cel·lulars. Per altra banda, GlialCAM també s’ha descrit com a subunitat auxiliar del canal de Cl- ClC-2 ja que és capaç de modificar les propietats d’activació i rectificació del canal. Recentment s’ha descrit mutacions en ClC-2 associades a un tipus de leucodistròfia vacuolitzant. L’objectiu general d’aquesta Tesi és avançar en la comprensió del possible mecanisme d’acció i la funció de la proteïna GlialCAM i ClC-2 i així aprofundir en el coneixement de en el seu paper en les cèl·lules glials així com en la fisiopatologia de les leucoencefalopaties vacuolitzants. Per a realizar aquest objectiu es van realitzar estudis d’estructura-funció de GlialCAM mitjançant la caracterització bioquímica i funcional de noves mutacions en GLIALCAM associades a MLC. S’ha obtingut una classificació de les mutacions en GLIALCAM en funció del defecte que presentaven. S’ha descrit mutacions defectives en la expressió proteica, defectives en la homooligomerització i en el tràfic a les unions cel·lulars, defectives únicament en el tràfic cel·lular, mutacions sensibles a la manca de MLC1 i mutacions defectives en la internalització de la proteïna. Paral·lelament es va aprofundir en la relació bioquímica entre GlialCAM i ClC-2 a partir de l’estudi bioquímic i funcional de mutacions en CLCN2 associades a leucoencefalopaties vacuolitzants. S’ha observat que GlialCAM augmenta la funció del canal ClC-2 a través de la modificació del gating del canal i de la estabilització de ClC-2 a la membrana plasmàtica però no sembla que millori la sortida de ClC-2 del reticle endoplasmàtic. A més, aquesta estabilització requereix de la formació d’homocomplexes de GlialCAM. Per últim, es va generar i caracteritzar un model knock-down de la proteïna ClC-2 per aprofundir el paper de ClC-2 en els astròcits. Així com també es va avançar en la relació bioquímica i funcional entre GlialCAM i ClC-2 en la fisiologia astrocitària. S’ha descrit que en astròcits en cultiu en condicions d’alta concentració de K+, similar al que succeiria en situacions d’alta activitat neuronal, ClC-2 es transloca de l’aparell de Golgi a les membranes cel·lulars, modificant les seves propietats funcionals per l’efecte de GlialCAM. En canvi, en astròcits deficients de MLC1, ClC-2 es troba retinguda citoplasmàticament. Aquest fet, indicaria que aquestes proteïnes podrien tenir un paper en el procés de sifoneig del K+, i per tant, la deslocalització de ClC-2 podria donar lloc a un desordre en l’homeòstasi d’aigua i ions., [eng] Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a rare type of vacuolating leukodystrophy. Currently still unknown pathophysiological mechanism of the disease, and therefore there is no effective treatment possible for patients. There are two genes involved in the MLC disease. Gene was first discovered was MLC1 and this encodes for a membrane protein with the same name. The second gene is called GLIALCAM and encodes for a transmembrane protein type I that also carries the same name. In our group is has been described that GlialCAM acts as a protein ß subunit of MLC1 because it is able to direct and concentrate in the cellular junctions. Moreover, GlialCAM also act as auxiliary subunit of CLC-2 Cl channel as it is capable of modifying the activation and rectification properties of the channel. Recently, mutations in ClC-2 have been associated with a rare type of vacuolating leukodystrophy. The principal aim of this study is to advance in the knowledge of GlialCAM and ClC-2 in the glial cells and into the pathogenesis of vacuolating leukodistrophies. To accomplish the study, the group performed a biochemical and funcional characterization of new mutations in GLIALCAM associated with MLC. We suggest that the HEPACAM mutations described up to now can be classified in several groups. Some mutations can affect GlialCAM protein expression, affect its ability to cis-homooligomerize and consequently reduce their localization in cell–cell junctions. Some mutations can affect specifically only transinteractions between GlialCAM molecules or may be unstable without MLC1 and finally some mutations affect the protein internalization. Parallel progress was made in the biochemical relationship between GlialCAM and ClC-2 from biochemical and functional studies of mutations in CLCN2 associated with vacuolating leukodystrophies. GlialCAM has been observed to increase ClC-2 function by the modification of its gating and the stabilization of ClC-2 in the plasma membrane. In addition, the stabilization requires a previous formation of GlialCAM’s homocomplexes. Finally, a knock-down model of the CLC-2 protein was generated and characterized to deepen the role of CLC -2 astrocytes. As well as progress was made in biochemistry and functional relationship between CLC -2 and GlialCAM in the astrocitic physiology. It has been reported that astrocytes cultured in conditions of high concentrations of K + , similar to what happens in situations of high neuronal activity , CLC -2 translocates from the Golgi apparatus to the cell membrane , changing the its functional properties for the purpose of GlialCAM . However, in MLC1 deficient astrocytes, CLC -2 is retained intracellularly. This would indicate that these proteins could play a role in the potassium siphoning, and therefore the relocation of CLC -2 could lead to disorder in the homeostasis of water and ions.

El estudio de los astrocitos se ha vuelto muy relevante en el ámbito de las neurociencias desde el descubrimiento, a principios de los años 90, de que aquellas “células de soporte” podían responder a la actividad sináptica. Actualmente, está aceptado que los astrocitos poseen la capacidad de modular la transmisión sináptica a través de la liberación de moléculas que actúan tanto en el terminal pre-sináptico como en el post-sináptico, los llamados gliotransmisores. Sin embargo, todavía quedan muchas cuestiones por resolver sobre el papel que juega esta población de células gliales en la fisiología cerebral. En el presente trabajo, hemos estudiado un campo ampliamente explorado en neuronas pero poco conocido en astrocitos: la transcripción dependiente de CREB. Este factor de transcripción expresado en todos los tipos celulares integra respuestas de todo tipo de estímulos y, por tanto, regula numerosos procesos tanto fisiológicos como patológicos. En neuronas, CREB es un factor clave en los mecanismos de plasticidad sináptica, que son la base para la adquisición de nuevas memorias, y también participa en los procesos neuroprotectores desencadenados por numerosas patologías cerebrales. Dado que los astrocitos participan tanto en procesos de regulación de la actividad sináptica como en neuroprotección, nuestro estudio pretende determinar en qué medida estas funciones están reguladas por CREB. Para ello hemos caracterizado, en cultivos primarios de astrocitos, los mecanismos moleculares de la activación de CREB tras la estimulación con los transmisores ATP y NE, y hemos estudiado los programas genéticos inducidos por la activación de CREB a través de diferentes vías de señalización. Finalmente, hemos determinado el papel protector del CREB astrocitario en un modelo murino de daño cerebral agudo. Los principales hallazgos de esta tesis son: (i) una nueva vía de señalización que activa CREB en astrocitos, (ii) la asociación de los programas transcripcionales de CREB con el metabolismo oxidativo, la homeostasis y la comunicación celular, (iii) el papel neuroprotector del CREB astrocitario en un modelo de daño cerebral a través de un incremento de la supervivencia neuronal y regeneración axonal, un descenso de la inflamación y un rescate energético a través de la estimulación de vías metabólicas mitocondriales., The study of astrocytes has become highly relevant in the realm of neurosciences since the discovery in the early 90’s that those “supportive cells” can respond to synaptic activity. Nowadays, it is accepted that astrocytes have the ability to modulate synaptic transmission through the release of molecules that act at both pre-synaptic and post-synaptic sites, the so-called gliotransmitters. However, there are still many questions to be solved about the role of this glial cell population in brain physiology. In the present work, we studied a field that has been largely explored in neurons, but that is poorly known in astrocytes: the CREB-dependent transcription. This widely-expressed transcription factor integrates the cellular responses of multiple stimuli, thus regulating numerous physiological and pathological functions. In neurons, CREB is a key player in the mechanisms that underlie synaptic plasticity, which is the molecular basis of memory acquisition, and it is involved in neuroprotective processes triggered by several brain disorders. Given the fact that astrocytes also play a role in synaptic activity and neuroprotection, we ought to know whether those functions are regulated by astrocytic CREB. Thus, we studied the molecular mechanisms of CREB activation in cell cultures upon stimulation with the transmitters ATP and NE and characterized the transcriptional programs triggered by CREB activation through different signalling pathways. Finally, we determine the protective roles of astrocytic CREB in a mouse model of traumatic brain injury. The main findings of this thesis are: (i) a novel signalling pathway for CREB activation in astrocytes, (ii) the functional association of CREB-dependent transcriptional programs with oxidative metabolism, homeostasis and intercellular communication, and (iii) the neuroprotective roles of astrocytic CREB in a model of traumatic brain injury, through an increase in neuronal survival and axonal regeneration, a decrease in inflammation and a rescue of bioenergetic failure by increasing the mitochondrial metabolic pathways.

La Leucoencefalopatia Megalencefàlica amb quists subcorticals, també anomenada MLC, és un tipus rar de leucodistròfia vacuolitzant. Actualment encara es desconeix el mecanisme fisiopatològic de la malaltia, i per tant ni hi ha cap tractament possible per als pacients. S’han descrit dos gens implicats en la malaltia MLC. El primer gen descobert s’anomena MLC1 i codifica per una proteïna de membrana que porta el mateix nom. El segon gen s’anomena GLIALCAM i codifica per una proteïna transmembrana de tipus I que també porta el mateix nom. S’ha decrit que la proteïna GlialCAM actua com a subunitat ß de MLC1 ja que es capaç de dirigir-la i concentrar-la a les unions cel•lulars. Per altra banda, GlialCAM també s’ha descrit com a subunitat auxiliar del canal de Cl- ClC-2 ja que és capaç de modificar les propietats d’activació i rectificació del canal. En la present tesi s’han generat i estudiat diferents models animals i cel•lulars per a l’estudi de la malaltia. En primer lloc, s’ha generat i s’ha caracteritzat un model de ratolí knock-out per a Mlc1. Gràcies a aquest model s’ha observat que la proteïna MLC1 és únicament astrocitària i que la proteïna GlialCAM no es independent de MLC1, ja que en absència d’aquesta es troba deslocalitzada en el cerebel. També s’ha pogut descriure per primer cop la implicació del canal de Cl- ClC-2 en la fisiopatologia, ja que els seus nivells de proteïna disminuixen en el cerebel i el canal es troba gairebé inactiu en els oligodendròcits de l’animal knock-out. Les característiques fenotípiques que presenta el model de ratolí equivalen a les característiques observades en els pacients en fases inicials de la malaltia, ja que l’animal tot i que mostra presència de vacuoles no presenta deteriorament motor i macrocefàlia aparent. També s’ha generat un model de peix zebra knock-out per a zmlc1. Aquest model presenta avantatges respecte el ratolí, com per exemple el baix cost o l’aplicació de tècniques genètiques a gran escala. Aquest model ha permés observar de nou que realment GlialCAM necessita a MLC1 per a la seva correcta localització. També s’ha observat que l’ortòleg zGlialCAMa conserva la seva funció de entre espécies ja que també es capaç de modificar les corrents de ClC-2. Aquests resultats obtinguts amb els models s’han pogut comparar amb el cervell d’una pacient. Aquest cervell demostra que MLC1 és necessària per a la correcta localització de GlialCAM en la regió del cerebel. Per altra banda, s’han desenvolupat diferents models cel•lulars. Primerament s’han estudiat els astròcits del ratolí knock-out. Aquestes cel•lules mancades de MLC1 també presenten vacuoles per tot el citoplasma, però no mostren canvis en la localització ni en els nivells de proteïna de GlialCAM i ClC-2. Aquest fet juntament amb altres estudis del grup van fer pensar si la condició necessària per a que es veguessin afectades aquestes proteïnes estaria relacionada amb el procés del sifoneig de K+. Estudis realitzats en astròcits de rata demostren que en condicions d’un alt contingut de K+, com per exemple durant una alta activitat neuronal, GlialCAM i ClC-2 és localitzen juntament a les membranes cel•lular i ClC-2 canvia les seves propietat de canal. Paral•lelament, estudis realitzats en oligodendròcits de rata també demostren que aquest fet també succeix en aquest tipus cel•lular., Megalencefalic leukoencephalopathy with subcortical cysts, also known as MLC, is a rare type of leukodystrophy. Currently still unknown pathophysiological mechanism of the disease, and therefore there is no effective treatment possible for patients. There are two genes involved in the MLC disease. Gene was first discovered was MLC1 and this encodes for a membrane protein with the same name. The second gene is called GLIALCAM and encodes for a transmembrane protein type I that also carries the same name. In our group is has been described that GlialCAM acts as a protein ß subunit of MLC1 because it is able to direct and concentrate in the cellular junctions. Moreover, GlialCAM also act as auxiliary subunit of CLC-2 Cl channel as it is capable of modifying the activation and rectification properties of the channel. In this work we have developed two different models to study the physiopathology. The results show that GlialCAM affected by the absence of MLC1. It has been also demonstrated that ClC-2 is implicated in the disease.These results were compared with a patient brian and has been shown that MLC1 is important for the correct location of GlialCAM in the cerbellum. Have also been developed a different cellular models. The results with this models show that GlialCAM and ClC-2 could have a functional role in the process of potassium siphoning.

In X-ALD, mutation/deletion of ALD gene (ABCD1) and the resultant very long chain fatty acid (VLCFA) derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD). The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal beta-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs) (1 and 3) in both cell types. However, higher induction of ELOVL's in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of antiapoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL) and cell survival (phospho-Erk1/2) proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid) leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD.

[spa] La Leucoencefalopatía Megalencefálica con Quistes Subcorticales (MLC) es un tipo raro de leucodistrofia vacuolizante, que presenta como principales características clínicas macrocefalia, deterioro de las funciones motoras, epilepsia y retraso mental medio. Sin embargo, el diagnóstico de MLC se confirma mediante imágenes de resonancia magnética, donde el encéfalo se presenta atrofiado e hinchado, muestra una sustancia blanca anormalmente difusa y hay presencia de quistes subcorticales. Desde el punto de vista fisiopatológico, una biopsia obtenida de un paciente de MLC muestra la presencia de numerosas vacuolas situadas en las láminas más externas de la mielina. Se ha encontrado un primer gen responsable de la enfermedad en el 75% de los pacientes afectados, denominado MLC1. Se han descrito alrededor de 60 mutaciones, aunque existen pacientes que manifiestan las características clínicas de la enfermedad pero no presentan mutaciones en MLC1 ni presentan ligamiento con su locus, sugiriendo que existe al menos otro gen involucrado en la enfermedad. En el 25% de pacientes restantes, la enfermedad se manifiesta de dos maneras diferentes: en un caso, los enfermos presentan las mismas características clínicas que los pacientes con mutaciones en MLC1; y en el otro, presentan síntomas transitorios y los pacientes mejoran, llegando incluso a que la enfermedad remitiera. El gen MLC1 codifica para una proteína transmembrana que lleva el mismo nombre. Su función es todavía desconocida. Aunque muestra un bajo grado de homología con el canal de potasio Kv1.1 no se ha podido detectar actividad de canal iónico en diferentes sistemas heterólogos. No obstante, dicha homología, su confinamiento en la membrana plasmática y el fenotipo característico vacuolizante de los pacientes sugieren que la proteína podría estar mediando la translocación de iones a través de la superficie celular. El total desconocimiento del rol preciso de la proteína MLC1 ha imposibilitado el entendimiento del mecanismo patofisiológico de la enfermedad, y por ello, no se ha podido desarrollar ningún tratamiento efectivo para los pacientes afectados. Es por ello que nuestro grupo quiso apostar por estrategias innovadoras (combinación de bioquímica y genética) para poder encontrar otros genes responsables de la enfermedad. Usando técnicas de purificación por afinidad combinada con métodos de proteómica cuantitativa encontramos a GlialCAM como una proteína que estaba asociada con MLC1. Es por eso que decidimos estudiar (en colaboración) si los pacientes que no tenían mutaciones en MLC1 podían presentar mutaciones en GLIALCAM. Tras el análisis de 40 de estos pacientes encontramos que cuando los enfermos tenían las características clínicas típicas de MLC presentaban dos mutaciones en GLIALCAM (herencia recesiva); mientras que en el caso de aquellos que mejoraban a lo largo del tiempo, éstos solo presentaban una mutación (herencia dominante), demostrando que GLIALCAM es el segundo gen de MLC. En este estudio también se ha podido determinar que mutaciones dominantes en GLIALCAM podían también causar otras enfermedades como la macrocefalia familiar benigna y la macrocefalia con retraso mental, con o sin autismo. Estudios bioquímicos posteriores han permitido avanzar en el entendimiento de la relación que existe entre MLC1 y GlialCAM. Así se ha demostrado que GlialCAM actúa como una molécula escolta, necesaria para localizar específicamente a MLC1 en uniones celulares. De esta forma pudimos descubrir que las mutaciones en GLIALCAM provocaban un defecto en el tráfico de la proteína debido a una deficiente oligomerización. Como consecuencia, estas mutaciones provocaban la deslocalización de los complejos de MLC1-GlialCAM en las uniones astrocitarias. De forma interesante, GlialCAM permite estabilizar la proteína MLC1, sugiriendo nuevas aproximaciones terapéuticas para los pacientes afectos con MLC. Tras el descubrimiento de GlialCAM como segundo gen de MLC gracias a la aproximación proteómica, y tras comprobar que no todo GlialCAM estaba asociado a MLC1, nos planteamos volver a realizar estudios de proteómica para intentar encontrar posibles proteínas que pudiesen estar interaccionando con GlialCAM. De esta manera encontramos que el canal de cloruro ClC-2, estaba asociado con GlialCAM, y pudimos comprobar que GlialCAM también actuaba como molécula escolta para localizar específicamente a ClC-2 en las uniones entre células. Además, también era capaz de modificar sus propiedades de canal, así como aumentar su función, demostrándose interacción directa entre ambas proteínas. Igualmente que para el caso de MLC1, las mutaciones encontradas en GLIALCAM fallaban en la capacidad de concentrar a ClC-2 en las uniones astrocitarias. Por tanto, la función de GlialCAM podría ser necesaria para agrupar tanto a MLC1 como a ClC-2 en tales uniones, particularmente en los pies terminales astrocitarios, donde podrían estar llevando a cabo su función. ClC-2 podría ser necesario para desarrollar un flujo de Cl- transcelular o para compensar gradientes electroquímicos iónicos que pueden estar ocurriendo en dichas uniones durante cambios en la osmolaridad. El descubrimiento de GlialCAM como una subunidad auxiliar de ClC-2 incrementa la compleja regulación de este canal y proporciona nuevas ideas acerca del papel que ClC-2 puede estar desempeñando en las células gliales así como se sugiere que pueda estar involucrado en la fisiopatología de MLC., [eng] Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by early-onset macrocephaly and delayed-onset neurological deterioration. Recessive MLC1 mutations are observed in 75% of patients with MLC. Genetic-linkage studies failed to identify another gene. We have showed that some patients without MLC1 mutations display the classical phenotype; others improve or become normal but retain macrocephaly. To find another MLC-related gene, we used quantitative proteomic analysis of affinity-purified MLC1 as an alternative approach and found that GlialCAM, an IgG-like cell adhesion molecule, is a direct MLC1-binding partner. Analysis of 40 MLC patients without MLC1 mutations revealed multiple different GLIALCAM mutations. Patients with the classical phenotype had two mutations, and patients with the improving phenotype had one mutation. In addition, patients with dominant GLIALCAM mutations, could also had macrocephaly and mental retardation with or without autism. Therefore, we found that GLIALCAM is the second gene found to be mutated in MLC. Furthermore, we demonstrated that GlialCAM functions as an MLC1 beta-subunit, needed for proper localization of MLC1 in cell-cell junctions. We also demonstrated that MLC1 and GlialCAM form homo- and hetero-complexes and that MLC-causing mutations in GLIALCAM mainly reduce the formation of GlialCAM homo-complexes, leading to a defect in the trafficking of GlialCAM alone to cell junctions. GLIALCAM mutations also affect the trafficking of its associated molecule MLC1, explaining why GLIALCAM and MLC1 mutations lead to the same disease: MLC. In this thesis, we also identify GlialCAM as a chloride channel ClC-2 binding partner. GlialCAM and ClC-2 colocalize in Bergmann glia, in astrocyteastrocyte junctions at astrocytic end-feet around blood vessels, and in myelinated fiber tracts. GlialCAM targets ClC-2 to cell junctions, increases ClC- 2 mediated currents, and changes its functional properties. Disease-causing GLIALCAM mutations abolish the targeting of the channel to cell junctions. Hence, we describes the first auxiliary subunit of ClC-2 and suggests that ClC-2 may play a role in the pathology of MLC disease.

This research was originally published in Journal of Biological Chemistry 287(9): 6454-6468 (2012) © the American Society for Biochemistry and Molecular Biology".-- El pdf es el manuscrito revisado de autor., Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E 2(PGE 2) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE 2 and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE 2 production after LPS. The results show that astrocytes respond to LPS by a COX- 2-dependent production of prostanoids, mainly vasoactive PGE 2, and suggest that the coordinated down-regulation of COX-1 facilitates PGE 2 production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported in part by Spanish Ministry of Science and Innovation Grant SAF2008-04515 and by European Community FP7/2007-2013 Project, Agreement 201024.

[cat] En els últims anys, diversos treballs han demostrat que els astròcits participen activament en el desenvolupament i la plasticitat del sistema nerviós central, així com en la modulació de la neurotransmissió. Característicament, la majoria de les accions descrites dels astròcits sobre la fisiologia i la patologia neuronal són mitjançades per secreció vesicular. En el aquest treball s’han identificat les molècules implicades en l’exocitosi de cèl•lules astroglials. Malgrat que alguns components són comuns amb les neurones, altres com sintaxina 4, VAMP3 i SNAP23 s’expressen de forma específica en les cèl•lules astroglials. Tractaments activadors o de maduració diferencialment regulen l’expressió de diferents isoformes de proteïnes exocítiques en cèl•lules glials in vitro. Així, l’activació amb citocines proinflamatòries augmenta l’expressió d’algunes SNAREs i els seus reguladors en glia, com sintaxina4 i munc18b. La correlació entre els nivells d’expressió d’aquestes proteïnes exocítiques i l’augment de la secreció de mediadors d’activació i inflamació suggereix un important paper d’aquestes molècules en la secreció de cèl•lules activades. Amb l’objectiu d’estudiar la secreció regulada per calci en astròcits madurs s’ha obtingut un fenotip madur glial in vitro mitjançant l’activació de la via del AMPc. L’anàlisi global amb "gene set enrichment analysis" del transcriptoma astrocitari ha demostrat que l’increment en els nivells intracel•lulars d’AMPc reprimeix la immaduresa i activació dels astròcits i promou la seva maduració. Aquesta maduració dependent de la via del AMPc augmenta l’expressió de proteïnes exocítiques, com per exemple VAMP2, així com la via de secreció regulada per calci dels pèptids ANP i SgII. Finalment, mitjançant assajos de pèrdua de funció, es demostra un paper d’aquestes proteïnes en la secreció de pèptids glials. Aquests resultats suggereixen que diferents molècules d’exocitosi mitjancen diferents processos de secreció glials. Per altra banda, en aquesta tesi s’ha identificat un nou component de la via de secreció astroglial, tant in vitro com in vivo, la SgIII. En cèl•lules neuroendocrines SgIII actua com un receptor de direccionament a grànuls secretors. En cèl•lules astroglials SgIII presenta una forma molecular i una dinàmica de secreció diferencial a cèl•lules neuroendocrines. A més, hem demostrat una notable sobreexpressió d’aquesta proteïna en astròcits reactius en lesions traumàtiques, la qual cosa suggereix una participació de SgIII en els mecanismes de protecció o dany cerebral en lesions del sistema nerviós central., [eng] In recent years, several studies have demonstrated that astrocytes influences neuronal development, function and plasticity through vesicular transmitter release. However, secretory pathways and the involved molecular mechanisms in astroglial cells are poorly known. In this study, we showed that a variety of SNARE and Munc18 isoforms were expressed by cultured astrocytes, with syntaxin-4, Munc18c, SNAP-23 and VAMP-3 being the most abundant variants. Exocytotic protein expression was differentially regulated by activating and differentiating agents. Specifically, proteins controlling Ca2+-dependent secretion in neuroendocrine cells were up-regulated after long-term 8Br-cAMP administration in astrocytes, but not by proinflammatory cytokines. We also analyzed the global transcriptome of cultured astroglial cells incubated with activators of cAMP pathways. cAMP analogs strongly upregulated genes involved in typical functions of mature astrocytes, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. Gene Set Enrichment Analysis and evaluation in situ of gene expression in astrocytes in different states showed that cAMP signaling conferred a mature and in vivo–like transcriptional profile to cultured astrocytes. Moreover, 8Br-cAMP treatment greatly increased the cellular content of exocytotic proteins such as VAMP-2 and stimulated Ca2+-dependent secretion of secretogranin-2 and ANP. Regulation of both exocytotic protein expression and Ca2+-dependent peptide secretion in astrocytes by differentiating and activating agents suggested that glial secretory pathways were adjusted in different physiological states. In this thesis, we showed the expression, transcriptional regulation, trafficking and release of the secretory pathway component SgIII in astroglial cells. In endocrine cells, SgIII is a key sorting receptor for peptide hormones while astrocytes produced and released a non-processed form. Moreover, SgIII expression was specifically upregulated in reactive astrocytes after perforating brain injury. These results showed that SgIII is a reliable component of the astrocyte secretory pathway and suggest important roles for glial SgIII in the glia–neuron communication.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, in the majority of cases caused by mutations in the MLC1 gene. MRI from MLC patients shows diffuse cerebral white matter signal abnormality and swelling, with evidence of increased water content. Histopathology in a MLC patient shows vacuolation of myelin, which causes the cerebral white matter swelling. MLC1 protein is expressed in astrocytic processes that are part of blood- and cerebrospinal fluid-brain barriers. We aimed to create an astrocyte cell model of MLC disease. The characterization of rat astrocyte cultures revealed MLC1 localization in cell-cell contacts, which contains other proteins described typically in tight and adherent junctions. MLC1 localization in these contacts was demonstrated to depend on the actin cytoskeleton: it was not altered when disrupting the microtubule or the GFAP networks. In human tissues, MLC1 and the protein Zonula Occludens 1 (ZO-1), which is linked to the actin cytoskeleton, co-localized by EM immunostaining and were specifically co-immunoprecipitated. To create an MLC cell model, knockdown of MLC1 in primary astrocytes was performed. Reduction of MLC1 expression resulted in the appearance of intracellular vacuoles. This vacuolation was reversed by the co-expression of human MLC1. Re-examination of a human brain biopsy from an MLC patient revealed that vacuoles were also consistently present in astrocytic processes. Thus, vacuolation of astrocytes is also a hallmark of MLC disease. (C) 2011 Elsevier Inc. All rights reserved.

The use of non-steroidal anti-inflammatory drugs (NSAIDs) in Alzheimer's disease (AD) is controversial because conclusions from numerous epidemiological studies reporting delayed onset of AD in NSAID users have not been corroborated in clinical trials. The purpose of this personal view is to revise the case for NSAIDs in AD therapeutics in light of: (i) the last report from the only primary prevention trial in AD, ADAPT, which, although incomplete, points to significant protection in long-term naproxen users, and (ii) the recently proposed dynamic model of AD evolution. The model contends that there is a clinical silent phase in AD that can last up to 20 years, the duration depending on life style habits, genetic factors, or cognitive reserve. The failure of many purported disease-modifying drugs in AD clinical trials is forcing the view that treatments will only be efficacious if administered pre-clinically. Here we will argue that NSAIDs failed in clinical trials because they are disease-modifying drugs, and they should be administered in early stages of the disease. A complete prevention trial in cognitively normal individuals is thus called for. Further, the shift of anti-inflammatory treatment to early stages uncovers a knowledge void about the targets of NSAIDs in asymptomatic individuals. AD researchers have mostly relied on post-mortem analysis of A beta plaque-laden brains from demented patients or animal models, thus drawing conclusions about AD pathogenesis based on late symptoms. We will discuss evidence in support that defective, not excessive, inflammation underlies AD pathogenesis, that NSAIDs are multifunctional drugs acting on inflammatory and non-inflammatory targets, and that astrocytes and microglia may play differing roles in disease progression, with an emphasis of ApoE epsilon 4 as a key, undervalued target of NSAIDs. According to a meta-analysis of epidemiological data, NSAIDs afford an average protection of 58%. If this figure is true, and translated into patient numbers, NSAID treatment may revive as a worth pursuing strategy to significantly reduce the socio-economical burden imposed by AD.

La fosfatidilcolina (PtdCho) és el fosfolípid majoritari de les membranes cel·lulars. La inhibició de la seva síntesi ha estat relacionada amb la mort cel·lular apoptòtica, que ha estat estudiada principalment mitjançant inhibidors farmacòlogics. La manca d'especificitat dels inhibidors utilitzats ha posat de relleu la necessitat d'utilitzar un model cel·lular on només la via de síntesi de PtdCho estigui afectada. La línia cel·lular CHO-MT58 presenta una mutació termosensible en l'enzim CTP:fosfocolina citi¬dililtransferasa (CCT), enzim regulador de la síntesi de PtdCho.Quan es cultiven les cèl·lules CHO-MT58 a 40ºC, observem una ràpida inhibició de la síntesi de PtdCho, un descens del contingut cel·lular de PtdCho i la pèrdua de viabilitat cel·lular en una seqüència temporal definida. La mort cel·lular produïda per la inhibició de la síntesi de PtdCho en cèl·lules CHO-MT58 genera poca condensació de la cromatina i fragmentació del DNA i no hi ha activació de caspasa-3 si es compara amb la mort apoptòtica induïda pel tractament amb Actinomicina D (ActD). A més, en la inhibició de la síntesi de PtdCho en cèl·lules CHO-MT58 es produeix en paral·lel un increment de la catepsina D i l'aparició de vesícules autofàgiques identificades en el microscopi electrònic de transmissió. Quan seguim el procés de l'autofàgia amb el marcador LC3 podem observar que l'aparició del fragment LC3-II, indicatiu de la presència d'autofagosomes, no disminueix al llarg dels temps a 40ºC suggerint un bloqueig de l'autofàgia causat per la inhibició de la síntesi de PtdCho en cèl·lules CHO-MT58.Per tant, la inhibició de la síntesi de PtdCho en cèl·lules CHO-MT58 a 40ºC provoca mort cel·lular no apoptòtica tot aturant l'autofàgia que actuaria com a mecanisme constitutiu de supervivència. Addicionalment, les catepsines actuarien com a possible mecanisme alternatiu de mort cel·lular.D'altra banda, actualment es sap que els astròcits tenen un paper importantíssim en la regulació de la transmissió sinàptica i en la plasticitat neuronal. Aquestes noves funcions dels astròcits són possibles perquè són cèl·lules excitables, tot i que la seva excitabilitat no es basa en potencials d'acció sinó en ones de Ca2+ intra- i intercel·lulars. Els mecanismes que controlen la senyalització via Ca2+ en astròcits, però, no es coneixen del tot. El nostre projecte ha demostrat que les vesícules acídiques de tipus lisosomals (VAL) són reservoris intracel·lulars de Ca2+ i el NAADP és un segon missatger capaç d'alliberar-ne el Ca2+ en astròcits corticals. Així hem determinat nivells endògens de NAADP i que aquests s'incrementen després d'estimulació amb diferents agonistes. Per altra banda, el NAADP-AM, un anàleg del NAADP permeable a la membrana plasmàtica, és capaç d'induir respostes de Ca2+ a diferents concentracions a través de l'alliberament de Ca2+ de les VAL en forma de campana (inhibició a altes concentracions de NAADP-AM). A més, hem demostrat l'expressió del receptor de NAADP en vesícules acídiques de tipus lisosomal. Per altra banda, la inhibició dels receptors de NAADP amb Ned-19 i la destrucció de les VAL amb GPN inhibeixen en part les respostes de calci induïdes per ATP, Endotelina-1 (ET-1) i Acetilcolina (Ach) però no per Bradiquinina. Finalment, el NAADP i les VAL també tenen un paper en l'excitabilitat astrocitària en la modulació de la magnitud de les ones de Ca2+ entre astròcits induïdes per estrès mecànic. Per tant, podem concloure com l'excitabilitat i senyalització via Ca2+ dels astròcits és un fenomen complex en el qual participen rutes de senyalització clàssiques (IP3 i ER; cADPR i RyR) però també nous segons missatgers i reservoris intracel·lulars de calci com ara el NAADP i les VAL., Phosphatidylcholine (PtdCho) is the major phospholipid of the cell membrane. Inhibition of their synthesis has been linked to apoptotic cell death, when it has been studied primarily using pharmacological inhibitors. The lack of specificity of these inhibitors has highlighted the need to use a cell line where the only PtdCho synthesis is affected. The cell line CHO-MT58 has a termosensitive mutation in the enzyme Thermal CTP:phosphocholine cytidyltransferase (CCT), the regulatory enzyme of PtdCho synthesis.When CHO MT58 cells are grown at 40°C, we observed a rapid inhibition of PtdCho synthesis, a decrease in cellular mass of PtdCho and loss of cell viability in a defined temporal sequence. The cell death caused by inhibition of PtdCho synthesis in CHO-MT58 cells generates little chromatin condensation and late DNA fragmentation without activation of caspase-3 when compared with the apoptotic death induced by treatment with Actinomycin D (ActD). In addition, CHO-MT58 cells at 40ºC induce an increase of cathepsin D and the emergence of autophagic vesicles identified in transmission electron microscopy. When we follow the autophagic process using LC3 as marker, we show the appearance of LC3-II fragment, indicative of the presence of autofagosomes and LC3-II form do not decrease over time at 40ºC, suggesting a blockade of autophagy caused by inhibition of PtdCho synthesis in CHO-MT58 cells. Therefore, inhibition of PtdCho synthesis in CHO MT58 cells at 40ºC causes non apoptotic cell death stopping the autophagy that acts as a constitutive survival mechanism. In addition, cathepsin acts as a possible alternative mechanism of cell death.Moreover, it is currently known that astrocytes play a huge role in the regulation of synaptic transmission and neuronal plasticity. These new features are possible because astrocytes are excitable cells, although their excitability is not based on action potentials, but in Ca2+ waves intra- and intercellularly. The mechanisms that control Ca2+ signalling in astrocytes are not completely known. Our project has shown that the lysosome-related vesicles are intracellular Ca2+ stores and NAADP is a second messenger able to release their Ca2+ in cortical astrocytes. Thus we determined endogenous NAADP levels and how they increase after stimulation with different agonists. Furthermore, the NAADP-AM, a cell permeable analog of NAADP, is able to induce Ca2+ responses at different concentrations by releasing Ca2+ from lysosome-related vesicles in a bell-shape manner (inhibition at high concentrations of NAADP-AM). In addition, we have demonstrated the expression of the NAADP receptor in lysosome-related vesicles. Furthermore, inhibition of NAADP receptors by Ned-19 and the destruction of lysosome-related vesicles with GPN partly inhibit Ca2+ responses induced by ATP, Endothelin-1 (ET-1) and Acetylcholine (Ach) but not Bradykinin. Finally, the Ca2+ signalling by NAADP and lysosome-related vesicles also play a role in the astrocitic excitability with a modulation of the magnitude of the Ca2+ waves between astrocytes induced by mechanical stress. Therefore, we concluded that the excitability and Ca2+ signalling of astroyctes is a complex phenomenon in which classical signalling pathways (ER and IP3, cADPR and RyR) as well as new Ca2+ stores and intracellular second Ca2+ messengers such as NAADP and lysosome-related vesicles.

El desenvolupament de noves teràpies per als trastorns bipolars, caracteritzats per l'alternança d'episodis de depressió i d'eufòria exacerbada, es veu dificultat pel desconeixement de l'etiologia de la malaltia i del mecanisme d'acció dels fàrmacs que estabilitzen l'estat d'ànim del pacient, com el valproat, la carbamazepina, la lamotrigina, i el liti. En aquest sentit, una possibilitat que ha estat poc explorada és que les cèl·lules diana d'aquests fàrmacs siguin els astròcits, un tipus de glia que darrerament està emergent com a element actiu en els processos de transmissió sinàptica, fet que ens ha portat a estudiar els efectes del liti en cultius d'astròcits.Hem caracteritzat l'increment que produeix el liti en la velocitat de síntesi de fosfatidilcolina (PC) en astròcits, que ja apareix amb el tractament agut amb liti, però és especialment significatiu a partir de les 24 hores de tractament. El liti potencia la ruta de Kennedy per a la síntesi de PC, un efecte que es veu parcialment revertit per l'addició d'inositol, indicant que la inhibició de la IMPasa per part del liti hi estaria implicada. Els efectes del liti no es limiten a la PC sinó que es produeix una alteració general del metabolisme lipídic dels astròcits. El liti inhibeix la síntesi de fosfatidilinositol i de fosfatidiletanolamina, un efecte que es dóna de manera similar amb valproat o carbamazepina que, de manera oposada al liti, també provoquen una forta inhibició en la síntesi de PC. Paral·lelament, el tractament crònic amb liti sembla reduïr la síntesi de novo d'àcids grassos i de colesterol. Globalment, els efectes observats ens fan pensar que en incrementar la síntesi de PC el liti podria produir una reducció en els nivells de diacilglicerol cel·lulars, fet que podria estar implicat amb el mecanisme terapèutic d'aquest ió.Si la diana terapèutica del liti es troba en els astròcits, és probable que actuï a nivell de les molècules que aquests alliberen per tal de modular la transmissió sinàptica. En aquest sentit hem comprovat que el tractament amb liti, a dosis lleugerament superiors a les terapèutiques, incrementa l'alliberació de TNF-α, òxid nítric, i prostaglandina E2 (PGE2) en cultius d'astròcits estimulats amb lipopolisacàrid bacterià (LPS). Sorprenentment en emprar una dosi de liti inferior, dins del rang terapèutic del fàrmac, s'observa una reducció en l'alliberació de PGE2, un efecte reproduït pel valproat, la carbamazepina i la lamotrigina. L'efecte dual del liti en funció de la concentració aplicada, es deu a una reducció en la producció d'àcid araquidònic en resposta a senyals de Ca2+ citosòlic, paral·lela a un increment en la inducció de la ciclooxigenasa-2 (COX-2) en astròcits estimulats amb LPS. Aquest fet podria estar relacionat amb l'estreta finestra terapèutica d'aquest fàmac, i contribueix a relacionar els astròcits amb el mecanisme d'acció dels estabilitzadors de l'estat d'ànim., Bipolar disorder is a common disease characterized by an alternating pattern of depression and mania episodes. Bipolar patients are treated with the mood stabilizing drugs, like lithium, valproate and carbamazepine, however the action mechanism of these drugs remains unknown. As most of the studies on this issue have been focused in neurons, we decided to analyze the effects of lithium in cultured astrocytes, a type of glial cells that are emerging as active elements in the regulation of synaptic transmission.We have characterized the increase on phosphatidylcholine (PC) synthesis induced by lithium in cultured astrocytes, an effect that appears with the acute treatment and is enhanced after 24 hours of treatment with lithium. Lithium potentiates the Kennedy pathway for the synthesis of PC, in an IMPase (inositol monophosphatase) inhibition dependent manner. Lithium effects are not limited to PC but it produces a broad alteration on astrocytic lipid metabolism. Lithium inhibits phosphatidylinositol and phosfatidylethanolamine synthesis, an effect also induced by the treatment with valproate or carbamazepine, which don't stimulate PC synthesis as lithium but inhibit it. On the other hand, chronic lithium treatment reduces fatty acids and cholesterol "de novo" synthesis. Overall, we hypothesized that lithium, increasing PC synthesis, induces a reduction on cellular diacylglycerol levels, which in turn could be related with the therapeutic mechanism of this ion.If astrocytes are the lithium's cellular target, it might actuate modifying the release of signaling molecules by these cells. Accordingly to this hypothesis, we have found that lithium treatment, at concentrations higher than its therapeutic window, increases TNF-alpha, nitric oxide and prostaglandin E2 (PGE2) release in cultured astrocytes stimulated with bacterial lipopolisaccharide (LPS). Surprisingly, using a lower lithium concentration, corresponding to its therapeutic range, astrocytes shown a reduction on PGE2 release, an effect replicated by the treatment with valproate, carbamazepine or lamotrigine. The dual effect of lithium on the PGE2 release is due to an inhibition of arachydonic acid release in response to cytosolic Ca2+ signals that courses in parallel to an increase on cyclooxygenase expression in astrocytes stimulated with LPS. This dual role could be related with the thin therapeutic window of lithium, and contributes to link astrocytes with the mechanism of action of mood stabilizers.