Diferencias sexuales y regulación de la ruta de Notch durante la gliosis reactiva

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-03-2015Glial cells are responsible of homeostasis maintenance in the central nervous system (CNS). When facing a harmful stimulus in the CNS, glial cells generate a spatiotemporally organized response, known as reactive gliosis, which main objective is to protect endangered or stressed cells. However, and given that reactive gliosis also implies inflammatory processes, its fine regulation is essential to prevent the chronification of neuroinflammation. Chronic inflammation in the CNS underlies as the main characteristic of several neurodegenerative diseases, in which sex differences in their incidence or progression have been detected. In the first part of the present work sex differences in reactive gliosis were studied, given their possible role in the generation of sex differences in neuroinflammatory or neurodegenerative diseases. In the cortical stab wound injury model no sex differences were found in the number or reactive status of astroglia, but astrocytes from males expressed CCL2 (involved in immune cells recruitment and glial reactivity) in a higher frequency than astrocytes from females. In the closest area to the wound edge males presented more microglial cells than females and those cells from males displayed a more neuroprotective-biased profile than microglia from females. These differences in the glial response to the injury were accompanied by a higher neuronal survival in males than in females. In order to deeply analyze the molecular causes of sex differences in glial response, an in vitro model of lipopolysaccharide (LPS)-induced astrogliosis was used. In that model, we studied the Notch signaling pathway regulation by LPS and by different neuroprotective hormones, such as estrogens, progesterone and the insulinlike growth factor 1 (IGF-1). LPS downregulated the Notch signaling pathway, acting through two different molecular mechanisms. These effects were completely reverted by IGF-1, whereas estrogenic compounds were only able to revert part of them. Notch pathway is not involved in proinflammatory cytokines production, nor in the morphological changes, neither in the proliferative effects of astrocytes exposed to LPSLas células de glía son las encargadas del mantenimiento de la homeostasis en el sistema nervioso central (SNC). Ante un estímulo lesivo, estas células emiten una respuesta organizada espaciotemporalmente, conocida como gliosis reactiva, cuya función principal es la protección de las células en riesgo. Sin embargo, la gliosis reactiva también incluye procesos inflamatorios, por lo que su regulación fina es imprescindible para evitar la cronificación de la neuroinflamación. La inflamación crónica del SNC subyace como principal característica de muchas enfermedades neurodegenerativas, en las que se han detectado diferencias sexuales en incidencia y progresión. En la primera parte de este trabajo estudiamos las diferencias sexuales en la gliosis reactiva, como posible factor relevante en la génesis de las diferencias sexuales en las enfermedades neuroinflamatorias y/o neurodegenerativas. En un modelo de lesión cortical penetrante no se encontraron diferencias en el número o reactividad de la astroglía; pero los astrocitos de machos expresaban en mayor proporción CCL2, proteína que regula el reclutamiento de células del sistema inmunológico y la reactividad glial. En la zona más próxima a la lesión los machos mostraban una mayor cantidad de células de microglía, y con características propias de un perfil más neuroprotector (menos proinflamatorio) que la microglía de hembras. Estas diferencias en la zona próxima a la lesión fueron acompañadas de una mayor supervivencia neuronal en machos que en hembras. Con el objetivo de profundizar en las causas moleculares de las diferencias sexuales en la respuesta glial se empleó un modelo de astrogliosis in vitro inducida por el lipopolisacárido bacteriano (LPS). En dicho modelo se estudió la regulación de la ruta de Notch por parte del LPS, y de diversas hormonas con funciones neuroprotectoras como los compuestos estrogénicos, la progesterona y el factor de crecimiento insulínico tipo 1 (IGF-1). El LPS redujo la actividad de la ruta de Notch mediante dos mecanismos moleculares distintos; el IGF-1 revirtió totalmente, y los compuestos estrogénicos parcialmente, los efectos del LPS sobre la ruta. Dicha ruta no media la producción de citoquinas proinflamatorias, ni los cambios morfológicos, ni la proliferación de los astrocitos expuestos al estímulo inflamatori

Sex differences in the inflammatory response of primary astrocytes to lipopolysaccharide

<p>Abstract</p> <p>Background</p> <p>Numerous neurological and psychiatric disorders show sex differences in incidence, age of onset, symptomatology or outcome. Astrocytes, one of the glial cell types of the brain, show sex differences in number, differentiation and function. Since astrocytes are involved in the response of neural tissue to injury and inflammation, these cells may participate in the generation of sex differences in the response of the brain to pathological insults. To explore this hypothesis, we have examined whether male and female astrocytes show a different response to an inflammatory challenge and whether perinatal testosterone influences this response.</p> <p>Methods</p> <p>Cortical astrocyte cultures were prepared from postnatal day 1 (one day after birth) male or female CD1 mice pups. In addition, cortical astrocyte cultures were also prepared from female pups that were injected at birth with 100 μg of testosterone propionate or vehicle. Cultures were treated for 5 hours with medium containing lipopolysaccharide (LPS) or with control medium. The mRNA levels of IL6, interferon-inducible protein 10 (IP10), TNFα, IL1β, Toll-like receptor 4 (TLR4), steroidogenic acute regulatory protein and translocator protein were assessed by quantitative real-time polymerase chain reaction. Statistical significance was assessed by unpaired <it>t</it>-test or by one-way analysis of variance followed by the Tukey <it>post hoc </it>test.</p> <p>Results</p> <p>The mRNA levels of IL6, TNFα and IL1β after LPS treatment were significantly higher in astrocytes derived from male or androgenized females compared to astrocytes derived from control or vehicle-injected females. In contrast, IP10 mRNA levels after LPS treatment were higher in astrocytes derived from control or vehicle-injected females than in those obtained from males or androgenized females. The different response of male and female astrocytes to LPS was due neither to differences in the basal expression of the inflammatory molecules nor to differences in the expression of the LPS receptor TLR4. In contrast, the different inflammatory response was associated with increased mRNA levels of translocator protein, a key steroidogenic regulator, in female astrocytes that were treated with LPS.</p> <p>Conclusions</p> <p>Male and female cortical astrocytes respond differentially to an inflammatory challenge and this may be predetermined by perinatal testosterone exposure.</p

Developmental Sex Differences in the Metabolism of Cardiolipin in Mouse Cerebral Cortex Mitochondria

Cardiolipin (CL) is a mitochondrial-specific phospholipid. CL content and acyl chain composition are crucial for energy production. Given that estradiol induces CL synthesis in neurons, we aimed to assess CL metabolism in the cerebral cortex (CC) of male and female mice during early postnatal life, when sex steroids induce sex-dimorphic maturation of the brain. Despite the fact that total amount of CL was similar, its fatty acid composition differed between males and females at birth. In males, CL was more mature (lower saturation ratio) and the expression of the enzymes involved in synthetic and remodeling pathways was higher, compared to females. Importantly, the sex differences found in CL metabolism were due to the testosterone peak that male mice experience perinatally. These changes were associated with a higher expression of UCP-2 and its activators in the CC of males. Overall, our results suggest that the perinatal testosterone surge in male mice regulates CL biosynthesis and remodeling in the CC, inducing a sex-dimorphic fatty acid composition. In male's CC, CL is more susceptible to peroxidation, likely explaining the testosterone-dependent induction of neuroprotective molecules such as UCP-2. These differences may account for the sex-dependent mitochondrial susceptibility after perinatal hypoxia/ischemia.Instituto de Investigaciones Bioquímicas de La Plat

Developmental Sex Differences in the Metabolism of Cardiolipin in Mouse Cerebral Cortex Mitochondria

Cardiolipin (CL) is a mitochondrial-specific phospholipid. CL content and acyl chain composition are crucial for energy production. Given that estradiol induces CL synthesis in neurons, we aimed to assess CL metabolism in the cerebral cortex (CC) of male and female mice during early postnatal life, when sex steroids induce sex-dimorphic maturation of the brain. Despite the fact that total amount of CL was similar, its fatty acid composition differed between males and females at birth. In males, CL was more mature (lower saturation ratio) and the expression of the enzymes involved in synthetic and remodeling pathways was higher, compared to females. Importantly, the sex differences found in CL metabolism were due to the testosterone peak that male mice experience perinatally. These changes were associated with a higher expression of UCP-2 and its activators in the CC of males. Overall, our results suggest that the perinatal testosterone surge in male mice regulates CL biosynthesis and remodeling in the CC, inducing a sex-dimorphic fatty acid composition. In male’s CC, CL is more susceptible to peroxidation, likely explaining the testosterone-dependent induction of neuroprotective molecules such as UCP-2. These differences may account for the sex-dependent mitochondrial susceptibility after perinatal hypoxia/ischemia

Developmental Sex Differences in the Metabolism of Cardiolipin in Mouse Cerebral Cortex Mitochondria

Cardiolipin (CL) is a mitochondrial-specific phospholipid. CL content and acyl chain composition are crucial for energy production. Given that estradiol induces CL synthesis in neurons, we aimed to assess CL metabolism in the cerebral cortex (CC) of male and female mice during early postnatal life, when sex steroids induce sex-dimorphic maturation of the brain. Despite the fact that total amount of CL was similar, its fatty acid composition differed between males and females at birth. In males, CL was more mature (lower saturation ratio) and the expression of the enzymes involved in synthetic and remodeling pathways was higher, compared to females. Importantly, the sex differences found in CL metabolism were due to the testosterone peak that male mice experience perinatally. These changes were associated with a higher expression of UCP-2 and its activators in the CC of males. Overall, our results suggest that the perinatal testosterone surge in male mice regulates CL biosynthesis and remodeling in the CC, inducing a sex-dimorphic fatty acid composition. In male's CC, CL is more susceptible to peroxidation, likely explaining the testosterone-dependent induction of neuroprotective molecules such as UCP-2. These differences may account for the sex-dependent mitochondrial susceptibility after perinatal hypoxia/ischemia.Instituto de Investigaciones Bioquímicas de La Plat

Estradiol Meets Notch Signaling in Developing Neurons

The transmembrane receptor Notch, a master developmental regulator, controls gliogenesis, neurogenesis, and neurite development in the nervous system. Estradiol, acting as a hormonal signal or as a neurosteroid, also regulates these developmental processes. Here we review recent evidence indicating that estradiol and Notch signaling interact in developing hippocampal neurons by a mechanism involving the putative membrane receptor G protein-coupled receptor 30. This interaction is relevant for the control of neuronal differentiation, since the downregulation of Notch signaling by estradiol results in the upregulation of neurogenin 3, which in turn promotes dendritogenesis

Notch signaling in astrocytes mediates their morphological response to an inflammatory challenge

In the nervous system, Notch pathway has a prominent role in the control of neuronal morphology and in the determination of the astrocyte fate. However, the role of Notch in morphological astrocyte plasticity is unknown. Here, we have explored the role of Notch activity on the morphological reactivity of primary astrocytes in response to LPS, an inflammatory stimulus. We found that LPS induces reactive astrocyte morphology by the inhibition of Notch signaling via NFκB activation and Jagged upregulation. In contrast, IGF-1, an anti-inflammatory molecule, inhibits LPS-induced reactive astrocyte morphological phenotype by enhancing Notch signaling through the inhibition of NFκB and the activation of MAPK. Therefore, Notch signaling pathway emerges as a mediator of the regulation of astrocyte morphology by inflammatory and anti-inflammatory stimuli

Gonadal hormones and the control of reactive gliosis

Astrocytes and microglia respond to central nervous system (CNS) injury with changes in morphology, proliferation, migration and expression of inflammatory regulators. This phenomenon is known as reactive gliosis. Activation of astrocytes and microglia after acute neural insults, such as stroke or traumatic CNS injury, is considered to be an adaptive response that contributes to minimize neuronal damage. However, reactive gliosis may amplify CNS damage under chronic neurodegenerative conditions. Progesterone, estradiol and testosterone have been shown to control reactive gliosis in different models of CNS injury, modifying the number of reactive astrocytes and reactive microglia and the expression of anti-inflammatory and proinflammatory mediators. The actions of gonadal hormones on reactive gliosis involve different mechanisms, including the modulation of the activity of steroid receptors, such as estrogen receptors a and 13, the regulation of nuclear factor-kappa B mediated transcription of inflammatory molecules and the recruitment of the transcriptional corepressor c-terminal binding protein to proinflammatory promoters. In addition, the Parkinson's disease related gene parkin and the endocannabinoid system also participate in the regulation of reactive gliosis by estradiol. The control exerted by gonadal hormones on reactive gliosis may affect the response of neural tissue to trauma and neurodegeneration and may contribute to sex differences in the manifestation of neurodegenerative diseases. However, the precise functional consequences of the regulation of reactive gliosis by gonadal hormones under acute and chronic neurodegenerative conditions are still not fully clarified. (C) 2012 Elsevier Inc. All rights reserved

Developmental Sex Differences in the Metabolism of Cardiolipin in Mouse Cerebral Cortex Mitochondria

Cardiolipin (CL) is a mitochondrial-specific phospholipid. CL content and acyl chain composition are crucial for energy production. Given that estradiol induces CL synthesis in neurons, we aimed to assess CL metabolism in the cerebral cortex (CC) of male and female mice during early postnatal life, when sex steroids induce sex-dimorphic maturation of the brain. Despite the fact that total amount of CL was similar, its fatty acid composition differed between males and females at birth. In males, CL was more mature (lower saturation ratio) and the expression of the enzymes involved in synthetic and remodeling pathways was higher, compared to females. Importantly, the sex differences found in CL metabolism were due to the testosterone peak that male mice experience perinatally. These changes were associated with a higher expression of UCP-2 and its activators in the CC of males. Overall, our results suggest that the perinatal testosterone surge in male mice regulates CL biosynthesis and remodeling in the CC, inducing a sex-dimorphic fatty acid composition. In male's CC, CL is more susceptible to peroxidation, likely explaining the testosterone-dependent induction of neuroprotective molecules such as UCP-2. These differences may account for the sex-dependent mitochondrial susceptibility after perinatal hypoxia/ischemia.Instituto de Investigaciones Bioquímicas de La Plat