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Estudio de los mecanismos moleculares y celulares de la función protectora de la Apolipoproteína D

AutorBajo-Grañeras, Raquel
DirectorGanfornina, M. D. ; Sánchez, Diego
Fecha de publicación2012
EditorUniversidad de Valladolid
Resumen[EN]: ApoD is a secreted Lipocalin that has been functionally associated with aging, degeneration and nervous system damage, and with many cancer types as well. Recent work in model organisms like plants, flies and mice1-5 has shown that ApoD participates as a survival mechanism, conserved across species, against diverse oxidative stress situations. In this thesis we propose four objectives aiming to understand the ApoD protective role in various physiological and pathological situations. To carry out these objectives we have used biological models with high sensitivity to and/or high levels of oxidative stress. The experimental approach includes analyses at the following levels: 1) gene expression assays (microarrays or qPCR arrays), 2) biochemical assays (enzyme activity, lipid peroxidation, or dopamine levels), 3) cell cultures (cell lines or primary cultures), 4) tissue analyses (mouse nervous system tissue or human colorectal adenocarcinoma), and 5) locomotor behavior analyses in the whole organism (in the mouse experimental model). We further ask whether ApoD function changes in different tissues or whether it works through a general mechanism of action everywhere it is expressed. The first three objectives of this work have been performed in the nervous system, an essentially post-mitotic tissue highly sensitive to stress. We have focused on glial cells (astrocytes), because this cell type predominantly responds against pro-oxidative situations; and on dopaminergic neurons because they are particularly sensitive to this type of stress. The last thesis objective aims at studying ApoD function in a proliferating tissue that is able to support high levels and tolerance to the oxidative stress caused by its high rate of ROS production (human colorectal cancer). Thus, we have been able to contrast the similarities and differences between both physiological situations to contextualize the relevance and impact of ApoD.
In summary, the presence of ApoD in the neuronal environment is necessary for an adequate protection against oxidative damage in the nervous system since it affects the transcriptional profile of the early response to this kind of stress. We have shown that ApoD preferentially alters the neuronal and oligodendroglial transcriptome with changes in expression of genes involved in neuronal excitability, synaptic transmission, management of myelin and the response to oxidative stress (Objective 1). After demonstrating the influence of ApoD in a proper glial response that could cushion the neurodegeneration associated with oxidative stress, we directed our study to the role of ApoD in the important glia-glia and glia-neuron interactions within the nervous system. We show that ApoD is secreted by astrocytes in response to the ROS-generator paraquat, and that it has a beneficial effect on the functionality of the locomotor system in the mouse, particularly on the dopaminergic system. Our data demonstrate that ApoD expression is induced by the activation of the JNK signaling pathway and that it functions as an autocrine mechanism to protect astrocytes against oxidative stress. In addition, ApoD modulates astroglial reactivity and alters the astrocytes transcriptional response upon oxidative stress. The addition of human ApoD to mouse astrocytes promotes their survival, further indicating the existence of mechanisms conserved across species. ApoD contributes to the endurance of astrocytes and reduces their reactivity both in vitro and in vivo. These two effects are sufficient to improve the functionality of the nigrostriatal dopaminergic system (Objective 2). The observed decrease in the impact of damage in neurons of the substantia nigra could be due to a combination of the benefits of a healthy surrounding glia and the direct effects of ApoD on neurons. Among other glial factors released to extracellular medium, ApoD could perform direct effects on the viability of neurons. We tested this hypothesis and found that ApoD is effective even in PINK1 deficient dopaminergic neurons (a Parkinson's disease model) and that these beneficial effects are mediated by ERK signaling pathway activation which promotes cell survival (Objective 3).
After observing what happens in the nervous system, where ApoD plays a protective role both for glia and for damaged neurons, we wanted to study a very different model to confirm if ApoD protective effects are applicable. For this purpose we have used human colorectal cancer tissues and a cell line of colon cancer. Both strategies have allowed us to observe a negative correlation between the ApoD expression and cancer progression. This represents a paradox because oxidative stress increases along cancer progression. Our study shows that cancer cells are able to respond to prooxidant stimuli. Even though ApoD expression is low in the stromal cells, it increases in the dysplastic epithelium. Finally, ApoD modifies neither the proliferation rate nor apoptosis levels in control conditions, but it promotes apoptosis under oxidative stress conditions. Therefore ApoD might become a therapeutic resource to promote cancer cell death when they are under stress (Objective 4). The general conclusion extracted from these results is that ApoD is a protein able to perform protective effects in different systems upon oxidative stress, promoting cell survival in glial and neurons (essentially post-mitotic cells), but promoting cell death in neoplastic cells under oxidative stress. Our work also uncovers some of the mechanisms by which this apparently pleiotropic protein is able to control the survival/death balance in both physiological and pathological conditions of diverse etiology.
DescripciónTesis Doctoral presentada por Raquel Bajo Grañeras para optar al grado de doctora por la Universidad de Valladolid, Facultad de Medicina (Departamento de Bioquímica y Biología y Fisiología).-- Sujeta a Licencia Creative Commons.
Versión del editorhttp://uvadoc.uva.es/handle/10324/1714
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