Determinació dels receptors de dopamina en mostres de cervell

Treball de recerca realitzat per alumnes d’ensenyament secundari i guardonat amb un Premi CIRIT per fomentar l'esperit científic del Jovent l’any 2008. Les alteracions del receptor de dopamina D2 són responsables de molts desordres neuronals que condueixen a malalties com el Parkinson, l’esquizofrènia i l’addicció a drogues. L’objectiu ha estat determinar quina espècie animal és més adient per substituir el teixit humà en l’estudi d’aquest receptor. Per dur a terme aquest estudi s’ha treballat amb nou espècies animals diferents en les quals s’ha relacionat, en funció de l’espècie, la concentració de receptor, la seva afinitat pels lligands agonistes i antagonistes, la seva relació evolutiva... El mètode més utilitzat per a la determinació i la quantificació de receptors hormonals als laboratoris d’investigació de les indústries farmacèutiques és la unió de radiolligands a membranes. Entre aquests experiments, els més emprats són els de desplaçament, en els quals el fàrmac no marcat competeix i desplaça el radiolligand dels centres d’unió del receptor i, tot seguit, es mesura la radioactivitat de la mostra amb un comptador de radioactivitat. Per fer aquest treball també ha calgut calcular la concentració de proteïnes per espectrofotometria i emprar tècniques d’homogeneïtzació i centrifugació. Després d’haver analitzat la concentració, l’afinitat i la relació filogenètica del receptor D2 de cada una de les espècies analitzades, es pot concloure que l’espècie ideal per estudiar aquest receptor, quan no es disposa de mostra humana, és un altre mamífer, i entre els estudiats es consideraria millor la vaca, ja que permet obtenir una gran quantitat de teixit, presenta un contingut apreciable de receptor i la seva afinitat per la dopamina és molt elevada.

Estudi de les alteracions en la capacitat neuroprotectora dels astròcits reactius en l'envelliment

En el periodo 2005-2008 hemos publicado tres artículos sobre las alteraciones de los astrocitos reactivos en el cerebro durante el envejecimiento. En el primer estudio, evaluamos la capacidad neuroprotectora de los astrocitos en un modelo experimental in vitro de envejecimiento. Los cambios en el estrés oxidativo, la captación del glutamato y la expresión proteica fueron evaluados en los astrocitos corticales de rata cultivados durante 10 y 90 días in vitro (DIV). Los astrocitos envejecidos tenían una capacidad reducida de mantener la supervivencia neuronal. Estos resultados indican que los astrocitos pueden perder parcialmente su capacidad neuroprotectora durante el envejecimiento. En el segundo estudio el factor neurotrófico derivado de la línea glial (GDNF) fue probado para observar sus efectos neurotróficos contra la atrofia neuronal que causa déficits cognitivos en la vejez. Las ratas envejecidas Fisher 344 con deficiencias en el laberinto de Morris recibieron inyecciones intrahippocampales de un vector lentiviral que codifica GDNF humano en los astrocitos o del mismo vector que codifica la proteína fluorescente verde humana como control. El GDNF secretado por los astrocitos mejoró la función de la neurona como se muestra por aumentos locales en la síntesis de los neurotransmisores acetilcolina, dopamina y serotonina. El aprendizaje espacial y la prueba de memoria demostraron un aumento significativo en las capacidades cognitivas debido a la exposición de GDNF, mientras que las ratas control mantuvieron sus resultados al nivel del azar. Estos resultados confirman el amplio espectro de la acción neurotrófica del GDNF y abre nuevas posibilidades de terapia génica para reducir la neurodegeneración asociada al envejecimiento. En el último estudio, examinamos cambios en la fosforilación de tau, el estrés oxidativo y la captación de glutamato en los cultivos primarios de astrocitos corticales de ratones neonatos de senescencia acelerada (SAMP8) y ratones resistentes a la senescencia (SAMR1). Nuestros resultados indican que las alteraciones en cultivos del astrocitos de los ratones SAMP8 son similares a las detectadas en cerebros enteros de los ratones SAMP8 de 1-5 meses de edad. Por otra parte, nuestros resultados sugieren que esta preparación in vitro es adecuada para estudiar en este modelo murino el envejecimiento temprano y sus procesos moleculares y celulares.

Paper de RIP140 en la fisiopatogenia de la adrenoleucodistròfia lligada a l'X.

La adrenoleucodistrofia ligada al X (X-ALD) es un enfermedad neurometabólica fatal caracterizada por una desmielinización cerebral progresiva infantil (CCALD) o por una neurodegeneración de la médula espinal (adrenomieloneuropatía, AMN), insuficiencia adrenal y acumulación de ácidos grasos de cadena muy larga (AGCML) como el ácido hexacosanoico (C26:0) en tejidos. La enfermedad está causada por mutaciones en el gen ABCD1 el cual codifica para un transportador peroxisomoal que importa AGCML. El ratón knockout para Abcd1 (Abcd1-) desarrolla alteraciones en la médula espinal que mimetizan el modelo de enfermedad AMN con inicio de los síntomas a los 20 meses. Previamente, nuestro grupo evidenció mediante análisis de transcriptómica, una desregulación mitocondrial en el modelo murino Abcd1- . En este trabajo demostramos que tanto en el ratón Abcd1- como en la sustancia blanca afectada de pacientes X-ALD hay una depleción mitocondrial. Para poder explicar esta depleción, estudiamos los niveles de un repressor de la biogenesis mitocondrial, RIP140. En cultivo organotípico de cortes de médula espinal observamos un aumento de los niveles proteicos de RIP140 en el ratón Abcd1- y también un aumento mediado por C26:0. Estos resultados indican que la sobreexpresión de RIP140 puede ser la responsable de la depleción mitocondrial presente en el ratón Abcd1- y una posible nueva diana terapèutica para la X-ALD.

Ultrasonografía transcraneal: utilidad en los Trastornos del Movimiento.

El diagnóstico de enfermedad de Parkinson Idiopática (EPI) y otros trastornos del movimiento (TM) se basa en criterios clínicos, aunque la ultrasonografía transcraneal ha demostrado cierta utilidad en el diagnóstico. En este trabajo, describimos los hallazgos ultrasonográficos en 291 pacientes con TM y evaluamos su utilidad clínica. Encontramos que la hiperecogenicidad de la sustancia nigra (SN) es más frecuente y extensa en la EPI y que el área de hiperecogenicidad de la SN y el tamaño del tercer ventrículo son útiles para predecir EPI, parálisis supranuclear progresiva y atrofia multisistémica. Según nuestros resultados, la ultrasonografía transcraneal es una técnica complementaria válida y segura para el diagnóstico de EPI.

Agonistes de neurotrofines com a teràpia neuroprotectora per la Esclerosi Múltiple

Les neurotrofines son factors tròfics que poden induir la supervivencia, la diferenciació i el creixement de les neurones i aquesta és la principal raó per la qual les estudiem en el context de les malalties neurológiques. Les propietats nombrades son crucials per a la cerca d’efectes funcionals pel que fa a tractaments de malalties neurológiques. Avui en dia. donada la seva activitat neuroprotectora, s’ha intentat l’administració extena de neurotrofines com una terapia per diverses enfermetats cerebrals, pero fins ara han tingut poc o cap resultat donada la inhabilitat d’aquestes molècules per creuar la barrera hematoencefàlica i pels seus efectes secondaris, com ara el dolor neuroilogic. Per això, l’aplicació de petites molècules similars a les neurotrofines es considerada com unapossible sol•lució com un possible tractament neuroprotector amb el cervell com a diana. L’objectiu principal d’aquest projecte és testar l’eficacia d’un compost replicant de la neurotrofina pel tractament de algunes enfermetats neurològiques i per a estudiar el mecanisme d’acció d’aquesta molècula. Els resultats obtinguts fins al moment mostren que hem desenvolupat e identificat un compost replicant de la neurotrofina (G79) que manté la seva capacitat com a factor de creixement nerviós (NGF) en un assaig funcional d’NGF (diferenciació i tests de supervivència). A més a més, hem obtingut una proba de concepte per a la eficacia d’aquest compost com un agent terapèutic en diversos models in vivo e in vitro d’Esclerosi Múltiple, glaucoma, enfermetat de Parkinson y Esclerosi Lateral Amiotrófica. En conclusió, els resultats obtinguts durant aquests dos anys suggereixen que la molècula replicant d’NGF G79 és un bon candidat per a ser desenvolutat com a part d’una estratégica terapeutica la diferenciació neuronal, promou la supervivència, activa la fosforilització de TrkA i TrkB, vies de senyalització específiques de la neurotrofina. Aquesta molècula ademés pot creuar la barrera hematoencefàlica per vies de transport actiu, millora el score clínic en animals infectats per Encefalomielitis Autoinmune Experimental, protegeix les cèlules gangliars retinals en el model in vivo de Glaucoma, promou la supervivència de les cèlules en els models in vitro de l’enfermetat de Parkinson i en ELA. En resum, els nostres resultats sugereixen que les molècules replicants de neurotrofina poden desenvoluparse com part d’una estratègia terapéutica neuroprotectora.

Oral administration of the KATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis

Background Multiple Sclerosis (MS) is an acquired inflammatory demyelinating disorder of the central nervous system (CNS) and is the leading cause of nontraumatic disability among young adults. Activated microglial cells are important effectors of demyelination and neurodegeneration, by secreting cytokines and others neurotoxic agents. Previous studies have demonstrated that microglia expresses ATP-sensitive potassium (KATP) channels and its pharmacological activation can provide neuroprotective and anti-inflammatory effects. In this study, we have examined the effect of oral administration of KATP channel opener diazoxide on induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Methods Anti-inflammatory effects of diazoxide were studied on lipopolysaccharide (LPS) and interferon gamma (IFNy)-activated microglial cells. EAE was induced in C57BL/6J mice by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Mice were orally treated daily with diazoxide or vehicle for 15 days from the day of EAE symptom onset. Treatment starting at the same time as immunization was also assayed. Clinical signs of EAE were monitored and histological studies were performed to analyze tissue damage, demyelination, glial reactivity, axonal loss, neuronal preservation and lymphocyte infiltration. Results Diazoxide inhibited in vitro nitric oxide (NO), tumor necrosis factor alpha (TNF-¿) and interleukin-6 (IL-6) production and inducible nitric oxide synthase (iNOS) expression by activated microglia without affecting cyclooxygenase-2 (COX-2) expression and phagocytosis. Oral treatment of mice with diazoxide ameliorated EAE clinical signs but did not prevent disease. Histological analysis demonstrated that diazoxide elicited a significant reduction in myelin and axonal loss accompanied by a decrease in glial activation and neuronal damage. Diazoxide did not affect the number of infiltrating lymphocytes positive for CD3 and CD20 in the spinal cord. Conclusion Taken together, these results demonstrate novel actions of diazoxide as an anti-inflammatory agent, which might contribute to its beneficial effects on EAE through neuroprotection. Treatment with this widely used and well-tolerated drug may be a useful therapeutic intervention in ameliorating MS disease.

Liberación de mercurio por parte de las obturaciones de amalgama dental: tipo, cantidad, método de determinación y posibles efectos adversos

Las obturaciones dentales de amalgama constituyen la fuente principal de exposición permanente de bajo nivel al vapor de mercurio (Hg°) y al mercurio inorgánico (Hg(II)) para la población general. La dosis de mercurio absorbido procedente de la amalgama es de 2.7 ¿g/día/persona para una cantidad promedio de 7.4 obturaciones. Si esta cantidad consistiera enteramente en mercurio inorgánico (Hg(II)), estaría muy por debajo de la cifra de 15 ¿g/día para una persona de 65 kg que la OMS considera como ingesta tolerable de mercurio inorgánico. En el caso de una exposición permanente a la misma cantidad, pero de vapor de mercurio (Hg0), se obtendría una concentración de 0.18 mg/m3 que puede compararse con la concentración de referencia de la EPA de 0.3 mg/m3 o con el nivel de riesgo mínimo de la ATSDR de 0.2 mg/m3. Varios estudios clínicos longitudinales y aleatorizados han evaluado la relación entre la concentración urinaria de mercurio y la exposición al mercurio procedente de las obturaciones dentales de amalgama en niños, particularmente vulnerables al Hg0, así como los posibles efectos neurológicos de tal exposición. La concentración promedio de mercurio en orina en los niños tratados con amalgama, con un promedio de 18,7 superficies obturadas, aumentó hasta un pico de 3.2 ¿g/L a los 2 años de iniciado el tratamiento y a los 7 años de seguimiento había descendido hasta los niveles basales y no se detectó ninguna alteración en las distintas exploraciones de monitorización neuropsicológica. De la misma manera, diversas investigaciones epidemiológicas no han aportado ninguna evidencia del papel de la amalgama en la posible causa o exacerbación de trastornos degenerativos como la esclerosis lateral amiotrófica, la enfermedad de Alzheimer, la esclerosis múltiple o el Parkinson. La extracción de las obturaciones de amalgama produce un aumento transitorio de los niveles de mercurio en sangre inmediatamente después de extraer las obturaciones de amalgama, pero de pequeña magnitud y que se normaliza a los 100 días, por lo que el efecto del dique de goma tiene una relevancia toxicológica menor. La conclusión de esta revisión es que la amalgama dental continúa siendo un excelente material de obturación.

Emerging functions of myelin associated proteins during development neuronal plasticity and and neurpdegeneration.

Adult mammalian central nervous system (CNS) axons have a limited regrowth capacity following injury. Myelin-associated inhibitors (MAIs) limit axonal outgrowth and their blockage improves the regeneration of damaged fiber tracts. Three of these proteins, Nogo-A, MAG and OMgp, share two common neuronal receptors: NgR1, together with its co-receptors (p75(NTR), TROY and Lingo-1), and the recently described paired immunoglobulin-like receptor B (PirB). These proteins impair neuronal regeneration by limiting axonal sprouting. Some of the elements involved in the myelin inhibitory pathways may still be unknown, but the discovery that blocking both PirB and NgR1 activities leads to near-complete release from myelin inhibition, sheds light on one of the most competitive and intense fields of neuroregeneration study during in recent decades. In parallel with the identification and characterization of the roles and functions of these inhibitory molecules in axonal regeneration, data gathered in the field strongly suggest that most of these proteins have roles other than axonal growth inhibition. The discovery of a new group of interacting partners for myelin-associated receptors and ligands, as well as functional studies within or outside the CNS environment, highlights the potential new physiological roles for these proteins in processes such as development, neuronal homeostasis, plasticity and neurodegeneration.

The psychosocial difficulties in brain disorders that explain short term changes in health outcomes.

BACKGROUND: This study identifies a set of psychosocial difficulties that are associated with short term changes in health outcomes across a heterogeneous set of brain disorders, neurological and psychiatric. METHODS: Longitudinal observational study over approximately 12 weeks with three time points of assessment and 741 patients with depression, bipolar disorders, multiple sclerosis, parkinson's disease, migraine, traumatic brain injury and stroke. The data on disability was collected with the checklist of the International Classification of Functioning, Disability and Health. The selected health outcomes were the Short Form 36 and the World Health Organization Disability Assessment Schedule. Multilevel models for change were applied controlling for age, gender and disease severity. RESULTS: The psychosocial difficulties that explain the variability and change over time of the selected health outcomes were energy and drive, sleep, and emotional functions, and a broad range of activities and participation domains, such as solving problems, conversation, areas of mobility and self-care, relationships, community life and recreation and leisure. CONCLUSIONS: Our findings are of interest to researchers and clinicians for interventions and health systems planning as they show that in addition to difficulties that are diagnostic criteria of these disorders, there are other difficulties that explain small changes in health outcomes over short periods of time.

Microglia, Calcification and Neurodegenerative Diseases

Neurodegeneration is a complex process involving different cell types andneurotransmitters. A common characteristic of neurodegenerative disorders such asAlzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis, Huntington’s disease (HD) and Amyotrophic Lateral Sclerosis (ALS) is the occurrence of a neuroinflammatoryreaction in which cellular processes involving glial cells (mainly microglia and astrocytes) and T cells are activated in response to neuronal death. This inflammatory reaction has recently received attention as an unexpected potential target for the treatment of these diseases.Microglial cells have a mesenchymal origin, invade the central nervous system (CNS)prenatally (Chan et al., 2007b) and are the resident macrophages in the CNS (Ransohoff &Perry, 2009). They comprise approximately 10-20% of adult glia and serve as the CNS innateimmune system. In neurodegenerative diseases, microglia is activated by misfoldedproteins. In the case of AD, amyloid- (A ) peptides accumulate extracellularly and activate the microglia locally. In the case of PD, ALS and HD, the misfolded proteins accumulate intracellularly but are still associated with activation of the microglia (Perry et al., 2010). Reactive microglia in the substantia nigra and striatum of PD brains have been described, and increased levels of proinflammatory cytokines and inducible nitric oxide synthase havebeen detected in these brain regions, providing evidence of a local inflammatory reaction (Hirsch & Hunot, 2009). The injection of lipopolysaccharide (a potent microglia activator) into the substantia nigra produces microglial activation and the death of dopaminergic cells. These findings support the hypothesis that microglial activation and neuroinflammationcontribute to PD pathogenesis (Herrera et al., 2000)...

Dietary resveratrol prevents alzheimer's markers and increases life span in SAMP8

Resveratrol is a polyphenol that is mainly found in grapes and red wine and has been reported to be a caloric restriction (CR) mimetic driven by Sirtuin 1 (SIRT1) activation. Resveratrol increases metabolic rate, insulin sensitivity, mitochondrial biogenesis and physical endurance, and reduces fat accumulation in mice. In addition, resveratrol may be a powerful agent to prevent age-associated neurodegeneration and to improve cognitive deficits in Alzheimer's disease (AD). Moreover, different findings support the view that longevity in mice could be promoted by CR. In this study, we examined the role of dietary resveratrol in SAMP8 mice, a model of age-related AD. We found that resveratrol supplements increased mean life expectancy and maximal life span in SAMP8 and in their control, the related strain SAMR1. In addition, we examined the resveratrol-mediated neuroprotective effects on several specific hallmarks of AD. We found that long-term dietary resveratrol activates AMPK pathways and pro-survival routes such as SIRT1 in vivo. It also reduces cognitive impairment and has a neuroprotective role, decreasing the amyloid burden and reducing tau hyperphosphorylation.

Dietary resveratrol prevents alzheimer's markers and increases life span in SAMP8

Resveratrol is a polyphenol that is mainly found in grapes and red wine and has been reported to be a caloric restriction (CR) mimetic driven by Sirtuin 1 (SIRT1) activation. Resveratrol increases metabolic rate, insulin sensitivity, mitochondrial biogenesis and physical endurance, and reduces fat accumulation in mice. In addition, resveratrol may be a powerful agent to prevent age-associated neurodegeneration and to improve cognitive deficits in Alzheimer's disease (AD). Moreover, different findings support the view that longevity in mice could be promoted by CR. In this study, we examined the role of dietary resveratrol in SAMP8 mice, a model of age-related AD. We found that resveratrol supplements increased mean life expectancy and maximal life span in SAMP8 and in their control, the related strain SAMR1. In addition, we examined the resveratrol-mediated neuroprotective effects on several specific hallmarks of AD. We found that long-term dietary resveratrol activates AMPK pathways and pro-survival routes such as SIRT1 in vivo. It also reduces cognitive impairment and has a neuroprotective role, decreasing the amyloid burden and reducing tau hyperphosphorylation.

Dietary resveratrol prevents alzheimer's markers and increases life span in SAMP8

Resveratrol is a polyphenol that is mainly found in grapes and red wine and has been reported to be a caloric restriction (CR) mimetic driven by Sirtuin 1 (SIRT1) activation. Resveratrol increases metabolic rate, insulin sensitivity, mitochondrial biogenesis and physical endurance, and reduces fat accumulation in mice. In addition, resveratrol may be a powerful agent to prevent age-associated neurodegeneration and to improve cognitive deficits in Alzheimer's disease (AD). Moreover, different findings support the view that longevity in mice could be promoted by CR. In this study, we examined the role of dietary resveratrol in SAMP8 mice, a model of age-related AD. We found that resveratrol supplements increased mean life expectancy and maximal life span in SAMP8 and in their control, the related strain SAMR1. In addition, we examined the resveratrol-mediated neuroprotective effects on several specific hallmarks of AD. We found that long-term dietary resveratrol activates AMPK pathways and pro-survival routes such as SIRT1 in vivo. It also reduces cognitive impairment and has a neuroprotective role, decreasing the amyloid burden and reducing tau hyperphosphorylation.

Targeting microglial KATP channels to treat neurodegenerative diseases: a mitochondrial issue

Neurodegeneration is a complex process involving different cell types and neurotransmitters. A common characteristic of neurodegenerative disorders is the occurrence of a neuroinflammatory reaction in which cellular processes involving glial cells, mainly microglia and astrocytes, are activated in response to neuronal death. Microglia do not constitute a unique cell population but rather present a range of phenotypes closely related to the evolution of neurodegeneration. In a dynamic equilibrium with the lesion microenvironment, microglia phenotypes cover from a proinflammatory activation state to a neurotrophic one directly involved in cell repair and extracellular matrix remodeling. At each moment, the microglial phenotype is likely to depend on the diversity of signals from the environment and of its response capacity. As a consequence, microglia present a high energy demand, for which the mitochondria activity determines the microglia participation in the neurodegenerative process. As such, modulation of microglia activity by controlling microglia mitochondrial activity constitutes an innovative approach to interfere in the neurodegenerative process. In this review, we discuss the mitochondrial KATP channel as a new target to control microglia activity, avoid its toxic phenotype, and facilitate a positive disease outcome.

Targeting microglial KATP channels to treat neurodegenerative diseases: a mitochondrial issue

Neurodegeneration is a complex process involving different cell types and neurotransmitters. A common characteristic of neurodegenerative disorders is the occurrence of a neuroinflammatory reaction in which cellular processes involving glial cells, mainly microglia and astrocytes, are activated in response to neuronal death. Microglia do not constitute a unique cell population but rather present a range of phenotypes closely related to the evolution of neurodegeneration. In a dynamic equilibrium with the lesion microenvironment, microglia phenotypes cover from a proinflammatory activation state to a neurotrophic one directly involved in cell repair and extracellular matrix remodeling. At each moment, the microglial phenotype is likely to depend on the diversity of signals from the environment and of its response capacity. As a consequence, microglia present a high energy demand, for which the mitochondria activity determines the microglia participation in the neurodegenerative process. As such, modulation of microglia activity by controlling microglia mitochondrial activity constitutes an innovative approach to interfere in the neurodegenerative process. In this review, we discuss the mitochondrial KATP channel as a new target to control microglia activity, avoid its toxic phenotype, and facilitate a positive disease outcome.