Please use this identifier to cite or link to this item: http://hdl.handle.net/10316/24392
Title: Loss of proteostasis in brain vascular endothelial cells in an in vitro model of alzheimer's disease
Authors: Fonseca, Ana Catarina Ribeiro da Graça 
Orientador: Pereira, Cláudia Maria Fragão
Alpoim, Maria Martins de Carvalho
Issue Date: 6-Mar-2014
Citation: FONSECA, Ana Catarina Ribeiro da Graça - Loss of proteostasis in brain vascular endothelial cells in an in vitro model of alzheimer's disease. Coimbra : [s.n.], 2014. Tese de doutoramento. Disponível na WWW: http://hdl.handle.net/10316/24392
Abstract: Abnormal accumulation of amyloid-â (Aâ) peptide in the brain is a pathological hallmark of Alzheimer's disease (AD). In addition to neurotoxic effects, Aâ also damages brain endothelial cells (ECs) and can thus contribute to the degeneration of cerebral vasculature, which has been proposed as an early pathogenic event in the course of AD able to trigger and/or potentiate the neurodegenerative process and cognitive decline. However, the mechanisms underlying Aâ-induced endothelial dysfunction are not completely understood. We hypothesized that Aâ impairs protein quality control mechanisms both in the secretory pathway and in the cytosol and also compromises Ca2+ and redox homeostasis leading to brain EC dysfunction and death by apoptosis.In the rat RBE4 cell line that was used as a model of brain ECs, exposure to toxic Aâ1-40 increased in a time-dependent manner the levels of several markers of ER stress-induced unfolded protein response (UPR) and impaired the ubiquitin-proteasome system increasing ubiquitinated proteins concomitantly with decreased overall proteasome activity and also the lysosome-dependent autophagic protein degradation pathway through inhibition of the autophagic flux. Under these conditions, Ca2+ homeostasis was significantly deregulated due to alterations of regulatory mechanisms at the plasma membrane, ER and mitochondria. Aâ1-40 inhibited the entry of Ca2+ to ER lumen and increase ER Ca2+ release and consequently decreased ER Ca2+ concentration that was accompanied by an increase in cytosolic and mitochondrial Ca2+ concentration. In order to compensate these alterations, cells increased SERCA2 levels to re-establish Ca2+ entrance into the ER and decreased IP3Rs and VDAC levels to counteract ER Ca2+ release and prevent excessive ER-to-mitochondria Ca2+ transfer. Additionally, STIM1 and Orai1 levels were decreased after prolonged exposure of brain ECs to Aβ1-40 and thus store-operated Ca2+ entry can not compensate ER Ca2+ depletion. In ECs treated with toxic Aβ1-40 a significant increase in reactive oxygen species (ROS) levels and depletion of antioxidant defences, namely glutathione and cytosolic Cu,Zn-superoxide dismutase (SOD1), was observed and ROS-induced responses to stress were triggered through nuclear translocation of the transcription factors Nrf2 and HIF-1α and expression of target genes. Finally, Aβ1-40 was shown to activate mitochondria- and ER stress-dependent apoptotic cell death pathways. Activation of the ER resident caspase-12 as well as increased levels of the ER stress-induced pro-apoptotic transcription factor CHOP were observed in Aβ1-40-treated cells. Furthermore, increased release of cytochrome c from mitochondria and activation of the downstream caspase- 9 were detected in cells treated with Aβ1-40, concomitantly with activation of the apoptosis’ effector caspase-3 and translocation of apoptosis-inducing factor to the nucleus demonstrating the involvement of caspase-dependent and -independent mechanisms during Aβ-induced EC death. In conclusion, Aâ impairs protein quality control mechanisms in brain ECs causing the deregulation of ER-mediated UPR, autophagic and proteasomal degradation pathways, concomitantly with impairment of mechanisms involved in Ca2+ and redox homeostasis. Despite ECs activate compensatory stress responses, these cells are not able to counteract the deleterious effects of Aâ and die by apoptosis. These evidences increase our knowledge about the mechanisms underlying brain endothelial dysfunction induced by Aâ and can contribute to the development of new therapeutic strategies for AD.
Description: Tese de doutoramento em Bioquímica, na especialidade de Biologia Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra
URI: http://hdl.handle.net/10316/24392
Rights: openAccess
Appears in Collections:FCTUC Ciências da Vida - Teses de Doutoramento

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