Hipertrofia del cuerpo carotídeo en hipoxia crónicamecanismos de activación, proliferación y diferenciación de los progenitores neurales en el sistema nervioso periférico

Resumo

The goal of this doctoral thesis is to achieve a better understanding of the biology and behavior of the recently discovered adult carotid body neural stem cells. The work focuses on two main objectives. First, to study from a physiological and cellular standpoint, the mechanisms involved in the activation of carotid body neural stem cells; and secondly, to unravel the genes and molecular pathways involved in stemness, maintenance and differentiation of these cells. An O2-sensitive glomus cell-stem cell synapse induces carotid body growth in chronic hypoxia Adult stem cells reside within specific �niches� which provide the appropriate environment to maintain their ability for self-renewal and multipotency. These cells are normally in a dormant state that protects them from stressors; the manner by which they are selectively activated to progress from quiescence to differentiated mature cells is still to be resolved (Suda et al., 2011; Chell and Frisen, 2012). Neural stem cells (NSC), which resemble embryonic radial glia-like cells, and are able to generate new neurons and glial cells, persist in two niches in the adult mammalian central nervous system: the subventricular zone (SVZ) and the subgranular layer of the hippocampus (SGZ) (Alvarez-Buylla and Lim, 2004; Zhao et al., 2008). Central neurogenesis is crucial for numerous brain functions and its impairment could be involved in some neuropsychiatric disorders (Kriegstein and Alvarez-Buylla, 2009; Ming and Song). NSC can sense neuronal activity, as a result of which adult neurogenesis is modulated by experience and environmental stimuli. However, the coupling of lineage progression to physiological demand remains poorly understood (Hoglinger et al., 2004; Liu et al., 2005; Ge et al., 2006; Song et al., 2012). Multipotent NSC of glial lineage also exist in the adult carotid body (CB), a neural crest-derived paired organ located in the carotid bifurcation (Pardal et al., 2007). The CB is composed of clusters (glomeruli) of neuron-like glomus (type I) cells that are electrically excitable and have numerous secretory vesicles containing neurotransmitters and neuropeptides. Glomus cells are surrounded by processes of glia-like sustentacular (type II) cells. This organ is the main arterial chemoreceptor that mediates reflex hyperventilation during hypoxemia. Glomus cells, the primary O2-sensing elements in the CB, depolarize in response to hypoxia, thereby releasing neurotransmitters that activate sensory nerve fibers terminating in the brainstem respiratory center (Lopez-Barneo et al., 2001). In addition to this fundamental role in acute oxygen sensing, the CB exhibits a remarkable structural plasticity that is uncommon for a neural tissue, which is manifested upon chronic exposure to hypoxia. The CB grows to several times its normal size during acclimatization in high altitude dwellers (Arias-Stella and Valcarcel, 1976) or in hypoxemic patients suffering cardiopulmonary disorders (Heath et al., 1982). We have shown that the glia-like type II cells, selectively expressing glial fibrillary acidic protein (GFAP), are NSC and contribute to CB growth in hypoxia. These cells form clonal colonies in vitro that are enriched in proliferating Nestin positive(+) progenitors that give rise to mature glomus cells and other neural crest cell lineages. Similarly, cell fate experiments in vivo have demonstrated that NSC contribute to the generation of new glomus cells in animals exposed to sustained hypoxia (Pardal et al., 2007). Stem cells in the CB neurogenic center are quiescent under normoxic conditions. Nonetheless, they become activated upon lowering blood O2 tension (hypoxia), a well-defined and controllable variable. Therefore, the CB niche provides an ideal model in which to study activity-dependent neurogenesis and to explore the mechanisms whereby stem cells switch from dormancy to cycling. Herein we show that, unexpectedly, CB NSC proliferation in vitro is insensitive to hypoxia over a broad range of O2 tensions. We provide compelling structural and functional evidence supporting the existence of abundant direct �synaptic� contacts between mature neuron-like, O2-sensitive, glomus cells and glia-like progenitors, thus optimizing the activity-dependent stimulation of stem cells. The release of stored neurotransmitters from glomus cells during hypoxia induces the proliferation of progenitor cells and growth of the CB. Among the substances released by glomus cells we have identified endothelin-1 (ET-1), an agent involved in neural crest progenitor specification and migration (Shin et al., 1999; Bonano et al., 2008), as a powerful activator of CB stem cell proliferation in vitro and in vivo. In this way, O2- sensing glomus cells mediate both the acute activation of the respiratory center and the chronic induction of CB growth upon exposure to hypoxia. Gene expression profile in adult carotid body stem cells The molecular mechanisms underlying CB stem cell proliferation and differentiation are poorly known. We have set up conditions in vitro to enrich neurosphere cultures in undifferentiated (NSundiff) or differentiated (NSdiff) cells. We performed gene expression studies by microarray techniques to identify molecules and pathways involved in the biology of CB progenitor cells. Microarray results were validated by PCR analyses of individual molecules, thus confirming that proliferation genes and markers of stemness and undifferentiated state are mainly expressed in the NSundiff sample, whereas neuronal genes are highly expressed in differentiated cultures. The analysis in silico performed by IPA software shows that CB stem cells conserve the ability to give rise to several neural crest derivatives. The results confirm that multipotent CB progenitors are neural crest-derived stem cells that persist in the adult organ. CB stem cells cannot be prospectively isolated due to the lack of selective membrane markers suitable for cell sorting. We have identified CD10, a membrane metalloendopeptidase, as the most highly expressed surface marker in NSundiff. CD10 has allowed the sorting of a purified CB progenitor population, which is being currently studied and characterized in our laboratory. In summary, this study increases our knowledge on the molecular and cellular properties of CB stem cells, the only neurogenic niche known so far in the adult peripheral nervous system. Our results could be relevant for the understanding of hypoxia-associated pathologies, and for the use of CB stem cells in cell therapy against neurodegenerative disorders.