Increased amyloid-β (Aβ) load in the brain, neurite degeneration, neuronal loss, and decreased levels of several neurotrophins are among the characteristics of Alzheimer’s disease (AD). Generation of Aβ occurs when the amyloid precursor protein (APP) is proteolytically processed by β- and γ-secretases in the amyloidogenic pathway. However, Aβ formation is prevented if APP is cleaved by α- and γ- secretases in the non-amyloidogenic pathway. The normal function of APP is still not fully known. It seems clear that the different fragments that are produced during proteolytic processing have different bioactive properties. APP and its metabolites have been implicated in neurite outgrowth, synaptogenesis, cell adhesion, neuroprotection and apoptosis. The aim of this thesis was to investigate how neurotrophic factors affect the synthesis and processing of APP and its two mammalian paralogues the APP-like protein-1 and-2 (APLP1 and APLP2). We also wanted to determine how the expression levels of α- and β- secretases were affected in response to these factors. In addition, we wanted to analyze if the levels and function of the most well characterized APP adaptor protein, Fe65, was regulated during neuronal differentiation. Our results show that retinoic acid (RA), insulin-like growth factor-1 (IGF-1), and brain derived neurotrophic factor (BDNF) all regulate expression levels and processing of the APP protein family. Interestingly, the increased processing of the APP family involves different signaling pathways. The PI3-K/Akt pathway is involved in IGF-1-induced APP and APLP1, but not APLP2, processing. In addition, RA-induced expression of the α-secretase, a disintegrin and metalloproteinase (ADAM) 10 is dependent on PI3-K, whereas PKC is involved in RA-induced expression of another α-secretase, ADAM17/TACE. Furthermore, we present evidence that maturation of the adaptor protein Fe65, as well as its docking to APP, increases concomitant with neuronal differentiation. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript.

Made available in DSpace on 2016-06-02T20:21:29Z (GMT). No. of bitstreams: 1 DissHCSS.pdf: 1174664 bytes, checksum: 6d1af63d33e89cf1251e02e9083170e2 (MD5) Previous issue date: 2003-01-24 / The genes DCRA and DSCR1 are located in the Down Syndrome Critical Region , a specific portion of human chromosome 21 responsible for the main traits of the disease. In the present work we have used the two-hybrid system as an approach to find proteins that interact with DCRA and DSCR1. This method is based on the properties of the yeast GAL4 protein, which consists of separable domains responsible for DNA-binding and transcriptional activation. The open reading frames of DCRA and DSCR1 were cloned in frame with the GAL4 DNA-binding domain. These constructions were used to analyse protein interactions using a human foetal brain cDNA library, cloned in frame with the GAL4 transcriptional activation domain. No DCRA protein partners were found in any of the screenings performed; nevertheless, the analysis allowed the detection of several false positive clones. In the analysis of DSCR1 we identified two proteins, UXT (ubiquitously expressed transcript) and APLP1 (amyloid precursor-like protein 1). These results may help elucidate a new function for DSCR1 in the nucleus, probably related gene expresion. / Os genes DCRA e DSCR1 estão localizados no cromossomo 21 mais especificamente na Região Crítica da Síndrome de Down que, em triplicata é responsável por alguns fenótipos da Síndrome. Neste projeto, visando encontrar proteínas que interagem com as proteínas DCRA e DSCR1, foi empregado o método do Duplo- Híbrido que utiliza as propriedades da proteína GAL 4 de levedura Saccharomyces Cerevisiae. Com esta finalidade fez-se a sub-clonagem das ORFs DCRA e DSCR1 em plasmídeos que contém um domínio de ligação ao DNA da proteína GAL 4. Estas construções foram utilizadas para analisar possíveis interações proteícas utilizando uma biblioteca de cérebro fetal construída em um plasmídeo em fase com o domínio de ativação da transcrição da proteína GAL 4. Nas análises com a proteína DCRA não foi possível confirmar nenhuma interação, porém possibilitou o estabelecimento de vários falso positivos dentro da técnica. Por outro lado, identificação de novos ligantes a proteína DSCR1, que são as proteínas UXT e APLP1, revela um novo papel da DSCR1 dentro da célula, provavelmente relacionado à regulação gênica.

Alzheimer’s disease (AD) is a neurodegenerative disorder which is histopathologically characterised by amyloid plaques and neurofibrillary tangles. Amyloid plaques consist of the amyloid β-peptide (Aβ) that can form aggregates in the brain. Aβ is generated from the amyloid precursor protein (APP) through proteolytic cleavage. APP belongs to a conserved protein family that also includes the two paralogues, APP-like proteins 1 and 2 (APLP1 and APLP2). Despite the immense amount of research on APP, motivated by its implication in AD, the function of this protein family has not yet been determined. In this thesis, we have studied the expression and proteolytic processing of the APP protein family. Our results are consistent with previous findings that suggest a role for APP during neuronal development. Treatment of cells with retinoic acid (RA) resulted in increased synthesis. In addition, we observed that RA treatment shifted the processing of APP from the amyloidogenic to the non-amyloidogenic pathway. The proteins in the APP family have been hard to distinguish both with respect to function and proteolytic processing. However, for development of new drugs with APP processing enzymes as targets this is of great importance. Our studies suggest similarities, but also differences in the mechanism regulating the processing of the different paralogues. We found that brain-derived neurotrophic factor (BDNF) had different impact on the members of the APP family. Most interestingly, we also found that the mechanism behind the increased processing in response to IGF-1 was not identical between the homologous proteins. In summary, our results indicate that in terms of regulation APLP1 and APLP2 differ more from each other than from APP. Our studies open up the possibility of finding means to selectively block Aβ production without interfering with the processing and function of the paralogous proteins.