Safety and efficacy investigations for new prenatal neuroprotective therapies. Applications in a model of intrauterine growth restriction (IUGR).

Abstract

[eng] Intrauterine growth restriction (IUGR) is defined as a significant reduction on fetal growth rate, resulting in a birth weight below the 10th percentile for the corresponding gestational age (Sharma et al., 2016). The prevalence accounts for 5-10% of all pregnancies, and amounts to approximately 600.000 cases in Europe, being a serious health problem worldwide (Kady and Gardosi, 2004). Placental insufficiency, the main cause of IUGR, chronically decreases the blood flow and nutrient supply to the developing fetus resulting in an adverse intrauterine environment with chronic hypoxia conditions and undernutrition. This situation results to a wide range of changes in brain development including grey (GM) and white matter (WM) injury (Esteban et al., 2010; Pla et al., 2020), which are associated with short- and long-term neurodevelopmental damage and cognitive dysfunctions (Mwaniki et al., 2012; Batalle et al., 2014; Eixarch et al., 2016). The most prevalent causes of brain damage of prenatal origin manifest as subtle neurological abnormalities. Indeed, IUGR has been proposed as the cause of one-quarter of special educational needs postnatally (Mackay et al., 2013). But currently, there is no efficient treatment which avoids deleterious consequences related to IUGR, especially in the neurodevelopmental field. To better understand, which basic cellular processes of brain development are altered due to IUGR, we established a novel in vitro model based on primary rabbit neuronal progenitor cells (NPCs) (Barenys et al., 2021). IUGR was surgically induced in one uterus horn in pregnant rabbits on gestational day (GD) 25. Neural progenitor cells (NPCs) growing as three-dimensional (3D) cell aggregates known as neurospheres were obtained from rabbit pups’ brains immediately after caesarean delivery at GD30. Neurospheres are able to mimic basic processes of brain development such as NPC proliferation, migration, differentiation into the brain effector cells neurons, oligodendrocytes and astrocytes, synaptogenesis, and network formation (Barenys et al., 2017; Breier et al., 2010; Gassmann et al., 2010a; Moors et al., 2009, 2007a; Schreiber et al., 2010). We revealed a significantly lower ability to form oligodendrocytes due to a slower differentiation rate in IUGR neurospheres. This result correlates very well with the clinical outcome of IUGR-generated white matter alterations. In addition, IUGR neurospheres presented an increased neurite length, which is consistent with previous in vivo studies (Pla et al., 2020). We have discovered for the first time that IUGR neurospheres respond differently than control to the exposure of the compound EGCG, which triggers migration alterations, and we have revealed the mechanism behind this difference: an overexpression of the adhesion molecule Integrin-β1 in IUGR. Because Integrin-β1 is implied in NPC migration but also in axonal growth and neuronal branching, this discovery gives new insights into the characterization of IUGR-induced neurodevelopmental alterations. The thesis addresses the medical necessity by assessing the safety and efficacy of the potential neuroprotective therapies

docosahexaenoic acid (DHA), melatonin (MEL), 3,3',5-triiodothyronine (T3), zinc, lactoferrin (LF) and its main metabolite sialic acid (SA), epigallocatechin gallate (EGCG) and derivatives in two different approaches: (1) exposure in vitro and (2) prenatal administration in vivo, both followed by the evaluation in vitro. DHA, and MEL, were identified as the best therapeutical agents for preventing/reverting impaired oligodendrogenesis caused by IUGR. LF and its main metabolite SA were revealed to reduce IUGR-induced neurite length extension. Finally, we integrated the discovered results about IUGR-induced changes in neurodevelopment into an “adverse outcome pathway” (AOP) approach and developed the putative AOP “Disrupted laminin-β1-integrin interaction leading to developmental neurotoxicity”. Overall, the novel neurosphere model is well suited for characterizing so far unknown neurodevelopmental effects on the cellular level induced by chemicals or IUGR. This new method opens the door to testing possible neuroprotective therapies for IUGR easily and cost- efficiently.

[spa] El retraso del crecimiento intrauterino (RCIU), un grave problema de salud en todo el mundo, se define como una reducción significativa de la tasa de crecimiento fetal que da lugar a un peso al nacer inferior al percentil 10 para la edad gestacional correspondiente. La insuficiencia placentaria, principal causa del RCIU, reduce el flujo sanguíneo y el aporte de nutrientes al bebé en desarrollo, lo que provoca daños cerebrales y deterioro cognitivo a corto y largo plazo. En este trabajo, desarrollamos por primera vez un modelo de neuroesfera de conejo in vitro basado en células progenitoras neurales (CPN) primarias que imita el desarrollo cerebral complicado por RCIU. Las neuroesferas son agregados celulares en 3D capaces de reproducir procesos básicos del neurodesarrollo como la proliferación, la migración, la diferenciación, la sinaptogénesis y la formación de redes de CPNs. Las neuroesferas del grupo RCIU mostraron una tasa significativamente reducida de diferenciación de oligodendrocitos, que se correlaciona muy bien con los resultados clínico que describen lesiones de la materia blanca. Además, las neuronas de las neuroesferas IUGR mostraron neuritas significativamente más largas. Encontramos que las neuroesferas IUGR responden de forma diferente a las neuroesferas control respecto al compuesto EGCG, que induce alteraciones en la migración. Esta diferencia se debe a la sobreexpresión de la molécula de adhesión integrina-β1, que está implicada en la migración de las CNP, así como en el crecimiento axonal y la ramificación neuronal. Mediante estudios toxicológicos y farmacológicos, evaluamos la seguridad y eficacia de potenciales terapias neuroprotectoras utilizando el novedoso modelo de neuroesfera de conejo in vitro. El DHA y la MEL se identificaron como terapias beneficiosas por sus efectos promotores de la diferenciación de los oligodendrocitos no sólo tras la exposición in vitro, sino también tras la administración prenatal durante la gestación en conejo. La LF y su principal metabolito SA redujeron la longitud de las neuritas debido a una posible interacción entre la integrina-β1 y la proteína de la matriz extracelular laminina. Finalmente, integramos los resultados obtenidos en una “adverse outcome pathway” (AOP) diseñando el AOP putativo "Disrupted laminin-β1-integrin interaction leading to developmental neurotoxicity". En general, el nuevo modelo de neuroesferas resulta muy adecuado para caracterizar de forma rápida y rentable las alteraciones desconocidas del neurodesarrollo causadas por sustancias químicas o por la RCI. En futuros estudios, también será importante medir los efectos y la seguridad de las terapias probadas no sólo en términos de neurodesarrollo, sino también en términos de parámetros generales de desarrollo.