Implications of glial nitric oxide in neurodegenerative diseases

Nitric oxide (NO) is a pleiotropic janus-faced molecule synthesized by nitric oxide synthases (NOS) which plays a critical role in a number of physiological and pathological processes in humans. The physiological roles of NO depend on its local concentrations, as well as its availability and the nature of downstream target molecules. Its double-edged sword action has been linked to neurodegenerative disorders. Excessive NO production, as the evoked by inflammatory signals, has been identified as one of the major causative reasons for the pathogenesis of several neurodegenerative diseases. Moreover, excessive NO synthesis under neuroinflammation leads to the formation of reactive nitrogen species and neuronal cell death. There is an intimate relation between microglial activation, NO and neuroinflammation in the human brain. The role of NO in neuroinflammation has been defined in animal models where this neurotransmitter can modulate the inflammatory process acting on key regulatory pathways, such as those associated with excitotoxicity processes induced by glutamate accumulation and microglial activation. Activated glia express inducible NOS and produce NO that triggers calcium mobilization from the endoplasmic reticulum, activating the release of vesicular glutamate from astroglial cells resulting in neuronal death. This change in microglia potentially contributes to the increased age-associated susceptibility and neurodegeneration. In the current review, information is provided about the role of NO, glial activation and age-related processes in the central nervous system (CNS) that may be helpful in the isolation of new therapeutic targets for aging and neurodegenerative diseases

Critical evaluation of the anatomical location of the Barrington nucleus: relevance for deep brain stimulation surgery of pedunculopontine tegmental nucleus

Deep brain stimulation (DBS) has become the standard surgical procedure for advanced Parkinson’s disease (PD). Recently, the pedunculopontine tegmental nucleus (PPN) has emerged as a potential target for DBS in patients whose quality of life is compromised by freezing of gait and falls. To date, only a few groups have published their long-term clinical experience with PPN stimulation. Bearing in mind that the Barrington (Bar) nucleus and some adjacent nuclei (also known as the micturition centre) are close to the PPN and may be affected by DBS, the aim of the present study was to review the anatomical location of this structure in human and other species. To this end, the Bar nucleus area was analysed in mouse, monkey and human tissues, paying particular attention to the anatomical position in humans, where it has been largely overlooked. Results confirm that anatomical location renders the Bar nucleus susceptible to influence by the PPN DBS lead or to diffusion of electrical current. This may have an undesirable impact on the quality of life of patients

7-Nitroindazole down-regulates dopamine/DARPP-32 signaling in neostriatal neurons in a rat model of Parkinson's disease

Neuronal nitric oxide synthase (nNOS) is involved in the regulation of diverse intracellular messenger systems in the brain. Nitric Oxide (NO) contributes to inducing signaling cascades that involve a complex pattern of phosphorylation of DARPP-32 (in Thr-34), which controls the phosphoproteins involved in neuronal activation. However, the role of NO in the pathophysiology of Parkinson's disease (PD) and its effect in striatal neurons have been scarcely explored. In the present work, we investigate the effects of a nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI) in the nigrostriatal pathway of striatal 6-hydroxydopamine (6-OHDA) lesioned rats. Our quantitative histological findings show that treatment with 7-NI significantly reduced 6-OHDA-induced dopaminergic damage in the dorsolateral striatum and Substantia Nigra pars compacta (SNpc). Moreover, 6-OHDA lesioned rats show a significant increase of nNOS+ and Phospho-Thr34-DARPP-32+ cells, accompanied by a consequent decrease of total DARPP-32+ cells, which suggests an imbalance of NO activity in the DA-depleted striatum, which is also reflected in behavioral studies. Importantly, these effects are reverted in the group treated with 7-NI. These results show a clear link between the state of phosphorylation of DARPP-32 and parkinsonism, which is regulated by nNOS. This new evidence suggests a prominent role for nitric oxide in the neurotransmitter balance within the basal ganglia in the pathophysiology of experimental parkinsonism.This work was supported by grants from the Spanish Ministry of Science (SAF 2007-62262), FIS (PI/2010/02827) and CIBERNED (Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas)

Persistent phagocytic characteristics of microglia in the substantia nigra of long-term Parkinsonian macaques

Patients with Parkinson's disease show persistent microglial activation in the areas of the brain where the degeneration of dopaminergic neurons takes place. The reason for maintaining this activated state is still unknown, but it is thought that this persistent microglial activation may contribute to the degeneration of dopaminergic neurons. In this study, we report the microanatomical details of microglia and the relationship between microglia and neurons in the substantia nigra pars compacta of Parkinsonian monkeys years after insult with MPTP. We observed that microglial cells appear polarized toward dopaminergic neurons in MPTP-treated macaques compared to untreated animals and present clear phagocytic characteristics, such as engulfing gliaptic contacts, an increase in Golgi apparatus protein machinery and ball-and-chain phagocytic buds. These results demonstrate that activated microglia maintain phagocytic characteristics years after neurotoxin insult, and phagocytosis may be a key contributor to the neurodegenerative process

ROCK/Cdc42-mediated microglial motility and gliapse formation lead to phagocytosis of degenerating dopaminergic neurons in vivo

The role of microglial motility in the context of adult neurodegeneration is poorly understood. In the present work, we investigated the microanatomical details of microglia-neuron interactions in an experimental mouse model of Parkinson’s disease following the intraperitoneal injection of MPTP. The specific intoxication of dopaminergic neurons induces the cellular polarization of microglia, leading to the formation of body-to-body neuron-glia contacts, called gliapses, which precede neuron elimination. Inhibiting ROCK/Cdc42-mediated microglial motility in vivo blocks the activating features of microglia, such as increased cell size and number of filopodia and diminishes their phagocyting/secreting domains, as the reduction of the Golgi apparatus and the number of microglia-neuron contacts has shown. High-resolution confocal images and three-dimensional rendering demonstrate that microglia engulf entire neurons at one-to-one ratio, and the microglial cell body participates in the formation of the phagocytic cup, engulfing and eliminating neurons in areas of dopaminergic degeneration in adult mammals

Pasados y presente. Estudios para el profesor Ricardo García Cárcel

Ricardo García Cárcel (Requena, 1948) estudió Historia en Valencia bajo el magisterio de Joan Reglà, con quien formó parte del primer profesorado de historia moderna en la Universidad Autónoma de Barcelona. En esta universidad, desde hace prácticamente cincuenta años, ha desarrollado una extraordinaria labor docente y de investigación marcada por un sagaz instinto histórico, que le ha convertido en pionero de casi todo lo que ha estudiado: las Germanías, la historia de la Cataluña moderna, la Inquisición, las culturas del Siglo de Oro, la Leyenda Negra, Felipe II, Felipe V, Austrias y Borbones, la guerra de la Independencia, la historia cultural, los mitos de la historia de España... Muy pocos tienen su capacidad para reflexionar, ordenar, analizar, conceptualizar y proponer una visión amplia y llena de matices sobre el pasado y las interpretaciones historiográficas. A su laboriosidad inimitable se añade una dedicación sin límites en el asesoramiento de alumnos e investigadores e impulsando revistas, dosieres, seminarios o publicaciones colectivas. Una mínima correspondencia a su generosidad lo constituye este volumen a manera de ineludible agradecimiento

Papel del óxido nítrico en la evolución del parkinsonismo y en las discinesias causadas por L-Dopa en ratas y en primates no humanos.

El objetivo general de esta tesis es investigar la interacción entre el sistema nitrérgico y dopaminérgico en modelos experimentales de parkinsonismo en ratas y en primates no huamnos (M. fascicularis). Los objetivos específicos son: 1) Observar si el tratamiento sub-crónico con un inhibidor específico de la nNOS (7-NI) influye en la lesión de la vía nigroestriatal causada por la microinyección de la neurotoxina 6-OHDA en el estriado dorsal en ratas, 2) Verificar si existe algún efecto de la inhibición específica de nNOS en los monos que serán intoxicados con MPTP por vía intravenosa (i.v.) y en los cuales se inducirán discinesias mediante el tratamiento crónico con L-DOPA, 3) Estudiar si existe algún efecto del tratamiento dopaminérgico con L-DOPA sobre la inervación simpática cardíaca en los monos parkinsonizados con MPTP y 4) Estudiar el post-mortem de los monos para ver los mecanismos moleculares que puedan mejorar las discinesias a nivel intraestriatal. La metodología que se ha utilizado en esta tesis incluye una serie de diferentes técnicas experimentales. El tipo de técnicas utilizadas son múltiples y bien ejecutadas y comprenden: a) Una lesión estereotáxica en ratas por una inyección vía intraestriatal de 6-OHDA, con y sin tratamiento del 7-NI; b) inducción del parkinsonismo crónico experimental en macacos por inyecciones de MPTP, con o sin tratamiento terapéutico de levodopa y 7-NI; c) test de comportamiento apropiados con el fin de evaluar el número de rotaciones después del tratamiento de apomorfina en ratas lesionadas y el grado de parkinsonismo y discinesias en monos intoxicados con MPTP y d) Western Blot en tejido de corazón de los monos para cuantificar los niveles de las diferentes proteínas examinadas en el estudio. Los principales resultados obtenidos en esta tesis demuestran que: En ratas lesionadas con 6-OHDA: a) la denervación dopaminérgica unilateral induce un aumento en los niveles de nNOS; b) la administración de un inhibidor específico, el 7-NI, protege la ruta nigroestriatal y revierte el comportamiento parkinsoniano y c) esto se consigue mediante la reducción de los niveles de DARPP-32 fosforilada y el aumento de DARPP-32 total, que son modulados de manera inversa por el tratamiento con 6-OHDA. En los monos intoxicados con MPTP y tratados con Levodopa: i) El 7-NI disminuye la gravedad (intensidad y duración) de las discinesias en monos tratados crónicamente con levodopa, apenas alterando la actividad anti-parkinsoniana de la levodopa; ii) en el tejido cardíaco, el tratamiento con MPTP disminuye los niveles de catecolaminas y aumenta la expresión de la fosfo-TH-40, que puede resultar tóxico para las células cardíacas y c) el tratamiento crónico con levodopa revierte el resultado del parkinsonismo, posiblemente induciendo un efecto cardioprotector mediante el aumento de la forma fosforilada de la proteína Hsp-27. Palabras clave: 6-OHDA, MPTP, Óxido Nítrico, 7-NI, discinesias, L-DOPA y denervación simpática cardíaca. SUMMARY OF PhD THESIS The general aim of the Doctoral thesis is to investigate the interaction between the dopaminergic and nitrergic system models induced experimental parkinsonism in rats lesioned with 6-OHDA and MPTP intoxicated monkeys (M. fascicularis). The specific objectives in this PhD thesis are: 1) Observe sub-chronic treatment with a specific inhibitor of nNOS (7-NI) influences in the nigrostriatal pathway injury caused by microinjection of the neurotoxin 6-OHDA in the dorsal striatum in rats, 2 ) check if there is any effect of specific inhibition of nNOS in the monkeys to be subjected to intoxication with MPTP intravenously (i.v.) and which induce dyskinesias by chronic treatment with L-DOPA, 3) Study whether there is any effect of treatment with L-DOPA on sympathetic heart innervation in MPTP-treated monkeys and 4) post-mortem study in monkeys to see the molecular mechanisms that may improve dyskinesias at intrastriatal level. The methodology used in this thesis includes a number of different experimental techniques. The type of techniques used are multiple and well executed. They included: a) stereotactic lesion in the rat by intrastriatal injection of 6-OHDA, with and without treatment of 7-NI; b) induction of experimental Parkinsonism in macaques by MPTP injections, with or without therapeutic treatment of levodopa and 7-NI; c) appropriate behavioral tests in order to evaluate the number of rotations after apomorfine treatment in lesioned rats and the degree of parkinsonism and dyskinesia in monkeys and d) Western blots on heart tissue extracts to quantify levels of the different proteins examined in the study. Main results obtained demonstrate that: in 6-ODHA lesioned rats: a) unilateral dopaninergic denervation induces an increase in the levels of nNOS; b) the administration of a specific inhibitor, the 7-NI, protects nigrostriatal pathway and reverses the parkinsonian behavior and c) this is achieved by reducing the levels of phosphorylated DARPP-32 and increasing those of total DARPP-32, which are inversely modulated by the 6-OHDA treatment. In MPTP-intoxicated and levodopa-treated-monkeys: i) 7-NI decreases the severity (both intensity and duration) of dyskinesias in chronic levodopa treated-monkeys, leaving unaffected the anti-parkinsonian activity of levodopa; ii) in the cardiac tissue, MPTP treatment decreases catecholamine levels and increases the expression of phospho-TH-40, which may result toxic for cardiac cells and iii) chronic levodopa treatment reverts parkinsonian outcome, possibly inducing a cardio-protective effect by increasing the phosphorylated form of Hsp-27

Nigral degeneration correlates with persistent activation of cerebellar Purkinje cells in MPTP-treated monkeys

In the present work we analyze the cerebellum of chronic parkinsonian monkeys in order to clarify whether chronic mesencephalic depletion is associated with long term activation of the cerebellar neurons in chronic Parkinsonism. In our study, we observed a persistent activation of Purkinje cells in the cerebellum of chronic parkinsonian macaques, characterized by the expression of c-Fos, which correlated with dopaminergic degeneration. These results are compatible with the results observed in fMRI in Parkinson’s disease patients, and may contribute to the understanding of additional alterations in the brain circuitry in Parkinsonis

Moving ahead in the application of the Service-Learning methodology to teaching in the Faculty of Philology.

Se ha seguido formando al profesorado de la Facultad de Filología en la metodología Aprendizaje-Servicio, desarrollando propuestas didácticas y reflexionando sobre las experiencias llevadas a cabo.Universidad Complutense de MadridDecanatoFac. de FilologíaFALSEsubmitte

Philology teacher training in Service Learning.

Formación del profesorado de la Facultad de Filología en la metodología Aprendizaje-Servicio para posteriormente desarrollar propuestas didácticas en las que se implemente dicha metodología.Depto. de Estudios Ingleses: Lingüística y LiteraturaFac. de FilologíaFALSEsubmitte