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Introduction: Preterm birth is increasing worldwide and is a major cause of neonatal death. Survivors are at increased risk of neurodisability, cognitive, social and psychiatric disorders in later life. Alterations to the white matter can be assessed using diffusion tensor imaging (DTI) MRI and are associated with poor neurodevelopmental outcome. The pathogenesis of white matter injury is multifactorial and several clinical risk and resilience factors have been identified. DNA methylation (DNAm) is an epigenetic process which links stressful early life experience to later life disease and is associated with normal brain development, neuronal processes and neurological disease. Several studies have shown DNAm is altered by the perinatal environment, however its role in preterm white mater injury is yet to be investigated. Aims: 1. To examine the relationship between preterm birth and white matter integrity 2. To investigate the effect of neuroprotective treatments and deleterious clinical states on white matter integrity in preterm infants 3. To assess the best DTI method of quantifying white matter integrity in a neonatal population 4. To investigate the effect of preterm birth on DNA methylation and 5. To determine the clinical and imaging factors that contribute to the variance in DNA Methylation caused by preterm birth Methods: DTI data was acquired from preterm infants (< 32 weeks' gestation or < 1500 grams at birth) at term equivalent age (TEA) and term controls (> 37 weeks' gestation at birth). Region-of-interests (ROI) and tract-averaged methods of DTI analysis were performed to obtain measurements of fractional anisotropy (FA) and mean diffusivity (MD) in the genu of corpus callosum, posterior limb of internal capsule and centrum semiovale. Clinical data was collected for all infants and the effect of prematurity, neuroprotective agents and clinical risk factors on white matter integrity were analysed. 8 major white matter tracts were segmented using probabilistic neighbourhood tractography (PNT), a tract-averaged technique which also allowed the calculation of tract shape. The two DTI techniques were compared to evaluate agreement between results. DNA was collected from preterm infants and term controls at TEA, and a genome-wide analysis of DNAm was performed. DTI parameters from probabilistic neighborhood tractography (PNT) methodology and clinical risk and resilience factors were used to inform a principal components analysis to investigate the contribution of white matter integrity and clinical variables to variance in DNAm. Results: FA and MD were significantly affected by preterm birth on ROI analysis. In addition, DTI parameters were affected by clinical factors that included antenatal magnesium sulphate, histological chorioamnionitis and bronchopulmonary dysplasia. Evaluation of DTI methodology revealed good accuracy in repeated ROI measurements but limited agreement with tract-averaged values. Differential methylation was found within 25 gene bodies and 58 promoters of protein-coding genes in preterm infants, compared with controls. 10 of these genes have a documented association with neural function or neurological disease. Differences detected in the array were validated with pyrosequencing which captured additional differentially methylated CpGs. Ninety-five percent of the variance in DNAm in preterm infants was explained by 23 principal components (PC); corticospinal tract shape associated with 6th PC, and gender and early nutritional exposure associated with the 7th PC. Conclusions: Preterm birth is associated with alterations in white matter integrity which is modifiable by clinical risk factors and neuroprotective agents. ROI analysis may not provide sufficient representation of white matter tracts in their entirety. Prematurity is related to alterations in the methylome at sites that influence neural development and function. Differential methylation analysis has identified several promising candidate genes for future work and contributed to the understanding of the pathogenesis of preterm brain injury.

Brain derived neurotrophic factor (BDNF) is a chemokine which levels are regulated by neuronal activity and could act as a sensor in front of distinct physiologic stimulus, activating the transcription of specific group of genes. In this work we show that BDNF induces the expression of BMP7 in neurons through TrkB receptor and MAPK/ERK pathways, an induction mechanism that is mediated in part by the release of the transcriptional repression exerted by p53 family proteins. BMP members in mammals are expressed in the growing nervous system where emerged as crucial regulators of dorsoventral patterning of the neural tube, neural cell fate determination, and cell death as well as terminal neural cell differentiation. In the earlier cerebral cortex development (at embryonic day 13, E13) BMPs predominantly induce cell death and inhibit the proliferation, as a mechanism for the regulation of cell number and phenotype within the developing cortex. Subsequently they exert sequential actions promoting neuronal differentiation at E16 and increasingly with time, they promote astrocytic differentiation and inhibit oligodendrocytes generation. This thesis demonstrates that BMP7 injection at midgestation alters the laminar distribution of pyramidal neurons in the cerebral cortex while GABAergic neurons distribution was not affected. We observed that abnormal high levels of BMP7 during cerebral cortex development induce the premature radial glia maturation into astrocytes impairing the radial migration of upper layers pyramidal neurons that remained accumulated in lower cortical regions. We also observed that altered BMP7 levels during midgestation lead to corpus callosum malformation. Although corpus callosum agenesis can be due to multiple causes, our analysis show that the correct pattern of BMP7 expression is necessary for the proper maturation of intermediate structures such as the glial wedge, the induseum griseum and the subcallosal sling, that provide essential guidepost signals for the proper corpus callosum development. Based on these results, it is proposed a physiologic model where the expression of BDNF induced by the initial electrical activity in the perinatal period would induce in turn, an increase in BMP7 expression. Both chemokines may act co-ordinately maturating neurons and glial cells at the end of neurogenic period. The alteration of BDNF and BMP7 spatio-temporal expression patterns could dramatically affect the proper cerebral cytoarchitecture and consequently the cerebral functioning. Indeed, different traumas occurred during embryonic and perinatal development are associated with an imbalance in BDNF and BMP7 levels. To check this hypothesis we reproduced an embryonic sublethal hypoxia, a pathological condition that can be associated to altered BDNF and BMP7 expression. Moreover, perinatal reduction of oxygen input can dramatically affect the cerebral cortex developmental program. As a result, many behavioural and learning disorders in infants have been associated to this pathological condition. We observed that this condition reduces BMP7 expression and signalling in the cerebral cortex promoting the differentiation of cortical progenitors into the oligodendrocytes in detrimental to the astroglial fate in vitro and in vivo. So, our findings indicate that changes on BMP7 expression in the tightly regulated developmental program of the central nervous system might importantly modify the cellular fate choice of cortical progenitors. When this change occurs during the critic perinatal developmental period, it could compromise the normal brain functionality in the affected individual. / “FUNCIÓN Y REGULACIÓN DE LA PROTEINA MORFOGENÉTICA DE HUESO (BMP7) EN EL DESARROLLO DE LA CORTEZA” TEXTO: "Brain derived neurotrophic factor" (BDNF) es una citoquina regulada por la actividad neuronal y puede actuar como sensor en respuesta a distintos estímulos fisiológicos, activando grupos específicos de genes. En este trabajo demuestro que BDNF induce la expresión de BMP7 en neuronas a través del receptor TrkB y la vía de señalización MAPK/ERK. Un mecanismo de inducción mediado en parte por la liberación de la represión transcripcional ejercida por la familia de proteínas p53. La inyección intraventricular de BMP7 durante la corticogenesis altera la distribución de las neuronas piramidales en la corteza cerebral. BMP7 induce la maduración prematura de la glia radial hacia astrocito alterando la migración radial de las neuronas piramidales de capas altas, que quedan anormalmente acumuladas en capas corticales inferiores. Niveles anormales de BMP7 durante fases gestacionales intermedias provocan malformación del cuerpo calloso (CC). Aunque la agénesis del CC puede ser debida a múltiples causas, nuestros análisis muestran que BMP7 es necesario para la formación de poblaciones de la línea media (glial wedge, induseum griseum y subcallosal sling) que participan en mecanismos de guía axonal necesarios para el desarrollo del CC. Proponemos un modelo fisiológico donde la expresión de BDNF inducida por el aumento de actividad eléctrica perinatal induciría a su vez un aumento de los niveles de BMP7. Ambas citoquinas actuarían conjuntamente madurando de una manera sincrónica las poblaciones neuronales y gliales de la corteza cerebral. La modificación del patrón de expresión espacio-temporal de ambas citoquinas podría afectar la composición celular y por tanto la correcta funcionalidad de la corteza cerebral. De hecho, diferentes traumas producidos durante el desarrollo embrionario y perinatal, donde se observa alteración de los niveles de BDNF y BMP7, están asociados a distintos desordenes neurológicos. En este trabajo reproducimos una hipoxia embrionaria sub-letal y observamos que los niveles de expresión y señalización de BMP7 están reducidos en animales hipóxicos. Esta reducción en los niveles de BMP7 promueve la diferenciación de los progenitores corticales hacia un fenotipo oligodendroglial en detrimento del fenotipo astroglial. Por tanto, BMP7 es vital para la correcta determinación de diferentes progenitores neurales durante el desarrollo cortical.

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