Thèses sur le sujet « Autophagy dysfunction »

Long lifespan of evolutionary higher organisms including humans is associated with the challenge to maintain viability of post mitotic cells, such as neurons, for decades. Autophagy is increasingly recognized as an important prosurvival pathway in oxidative and proteotoxic stress conditions. Autophagy degrades cytosolic macromolecules in response to starvation and is involved in the selective degradation of damaged/toxic organelles, such as mitochondria. With age autophagy function declines, and is also compromised in several neurodegenerative diseases. We identified a novel role for autophagy in the maintenance of mitochondrial health, specifically respiratory complex I. Intriguingly, galactose-induced cell death of autophagy deficient cells was rescued by preventing ROS production at complex I or bypassing complex I-linked respiration. We propose that aberrant ROS production via complex I in response to autophagy deficiency could be pathogenic and result in neurodegeneration and preventing this could be an interesting therapeutic target. Furthermore, we found that vertebrates have evolved mechanisms to induce autophagy in response to oxidative stress. This involves the oxidation of the autophagy receptor p62, which promotes autophagy flux and the clearance of autophagy cargo, resulting in increased stress resistance in mammalian cells and survival under stress in flies. In addition, we obtained data revealing an important role for redox-regulated cysteines in NDP52 for the degradation of mitochondria via mitophagy and tools were created to study the role of other autophagy receptors in autophagy initiation and selective autophagy.

The single greatest risk factor for the development of idiopathic Parkinson’s Disease is advancing age. The differences at the cellular level that cause some individuals to develop this highly debilitating disease over healthy ageing are not fully understood. Mitochondrial dysfunction has been implicated in the pathogenesis of Parkinson’s disease (PD) since the drug MPTP, known to cause Parkinson’s like symptoms, was shown to invoke its deleterious effect through inhibition of Complex I (CI) of the mitochondrial electron transport chain. Since this discovery in the 1980s, several causative genes in the much rarer familial forms of PD have been shown to encode proteins which function within, or in association with mitochondria. Through inherited cases of the disorder the process through which mitochondria are removed, mitophagy, a specialized form of autophagy has also been associated with the pathogenesis that leads to en masse cell death in this disorder. This work explores the interplay between mitochondrial deficiencies, through complex I dysfunction, and changes to autophagic processes. The methodologies to enable these observations are also described in detail with the development of novel and specialized techniques necessary to answer many of the specific research questions. The mechanisms behind complex I deficiency’s impact upon cellular processes is also explored as part of this thesis. Mitochondria and autophagy are irrevocably linked through mitochondrial dynamics, to this end an exploration of the greater impact complex I dysfunction has upon mitochondrial motility, fission and fusion was investigated. As the most prevalent neurodegenerative movement disorder of old age, understanding the molecular changes that result in Parkinson's Disease is vital to increase knowledge and offer novel therapeutic targets. Parallel studies in human upper midbrain tissue and cybrid cell lines within this work have revealed significant changes to both autophagy and mitochondrial dynamics in response to complex I deficiency. Given that mitochondrial ‘health’ and autophagic regulation directly impact upon one another identifying how exactly these may contribute to neuronal loss will hopefully allow therapeutic modulation at a point of PD pathogenesis where cells can still be retained.

Type 2 diabetes is a global disease which affects an increasing number of peopleevery year. At the heart of the disease lies insulin resistance in the target tissues,primarily fat and muscle. The insulin resistance is caused by the failure of a complexsignalling network, and several mechanistic hypotheses for this failure havebeen proposed. Herein, we evaluate a hypothesis that revolves around the proteinmammalian target of rapamycin (mTOR) and its feedback signals to insulin receptorsubstrate-1 (IRS1). In particular, we have re-examined this hypothesis andrelevant biological data using a mathematical modelling approach. During the course of modelling we gained several important insights. For instance,the model was unable to reproduce the relation between the EC50-valuesin the dose-response curves for IRS1 and its serine residue 312 (Ser-312). Thisimplies that the presented hypothesis, where the phosphorylation of Ser-312 liesdownstream of the tyrosine phosphorylation of IRS1, is inconsistent with the provideddata, and that the hypothesis or the data might be incorrect. Similarly, wealso realized that in order to fully account for the information in the dose-responsedata, time curves needed to be incorporated into the model. A preliminary model is presented, which explains most of the data-sets, butstill is unable to describe all the details in the data. The originally proposed hypothesisas an explanation to the given data has been revised, and our analysisserves to exemplify that an evaluation of a mechanistic hypothesis by mere biochemicalreasoning often misses out on important details, and/or leads to incorrectconclusions. A model-based approach, on the other hand, can efficiently pin-pointsuch weaknesses, and if combined with a comprehensive understanding of biologicalvariation and generation of experimental data, mathematical modelling canprove to be a method of great potential in the search for mechanistic explanationsto the cause of insulin resistance in type 2 diabetics.

Head and neck cancer is one of the most common cancers worldwide. The current standard of care for head and neck cancer includes surgical resection of the tumor followed by chemoradiation. This targeted head and neck radiation causes dysfunction of the salivary glands, which leads to xerostomia, mucositis, dysphagia, dental caries, and malnutrition. These side effects greatly decrease patient quality of life and increase their financial responsibility. Current therapies available to ameliorate these negative side effects are expensive, only provide short-term relief, and many of them have negative side effects of their own. Therefore, another therapy is needed to prevent salivary gland dysfunction or restore its function following targeted head and neck radiation. Autophagy is a homeostatic cellular mechanism that could be targeted as a therapeutic mechanism in the salivary glands following targeted head and neck radiation. Autophagy is a catabolic process necessary to maintain cellular homeostasis. It has been shown to play a beneficial role in a variety of disease states including diabetes mellitus, obesity, and cancer. The role of autophagy in the response of cancerous tissue to radiation has been vastly studied. However, the role autophagy plays in normal tissue response to radiation remains poorly understood and much more research in this area is needed.Atg5^(f/f);Aqp5-Cre mice have a conditional knockout of Atg5, a gene necessary for autophagy, in the salivary glands. These mice have unchanged baseline levels of apoptosis, proliferation, and stimulated salivary flow rates when compared to wild-type mice. Therefore, they are a useful model to investigate the role of autophagy in the response of the salivary glands to targeted head and neck radiation. These Atg5^(f/f);Aqp5-Cre autophagy-deficient mice display increased radiosensitivity following targeted head and neck radiation. Furthermore, post-therapy use of CCI-779, a rapalogue and inducer of autophagy, allowed for restoration of salivary gland function following targeted head and neck radiation. Taken together, these results implicate autophagy as playing a beneficial role in normal salivary function following radiation. Therefore, autophagy could be utilized by normal salivary gland tissue following targeted head and neck radiation to maintain salivary gland function.

Amyotrophic Lateral Sclerosis (ALS) is a rare and fatal neurodegenerative disorder resulting in the loss of motor neurons from the spinal cord and frontal cortex. The patterns of neurodegeneration, affected regions, age of onset, and time course of disease progression are all highly variable between and within variants of the disease. Familial ALS (fALS), inherited versions of ALS due to genetic changes, accounts for between 5-20% of all ALS cases, while the rest are sporadic, with either no causative mutation identified or no familial history of ALS. Recently, the discovery of C9orf72 hexanucleotide repeat expansions have been identified as one of the most common causes of familial ALS, with some patients presenting with dual phenotypes of ALS and frontotemporal dementia, leading to new hypotheses about the nature of neurodegenerative diseases. Despite the continued discovery of new ALS causative genes, little is known about the pathogenesis of the disease. While almost all variants include the presence of intracellular protein inclusions, the site of the protein plaques and involved proteins varies between genetic and phenotypic variants of this disease. Due to the lack of clear pathogenic mechanisms, several hypotheses have been developed to explain the process of neurodegeneration. Autophagy, the process of self-eating, leading to destruction of damaged or excess proteins and organelles, has been implicated as being altered in ALS. Multiple variants have demonstrated altered mitochondrial morphology and cellular energetic dynamics, which could explain previous observations that implicate the process of apoptosis in cellular death. Many of the involved proteins in ALS have functional roles for intracellular, nucleocytoplasmic, and axonal transport of various proteins or RNA. These three competing hypotheses are currently the most prominent hypotheses in the pathogenesis of ALS, and have largely been considered as separate and competing in past research. Is there a chance that the true pathogenesis leading to neuronal destruction via apoptosis involve all three hypotheses? Altered protein and RNA transport dynamics could lead to changes in cellular stress responses or overload autophagy pathways, leading to exacerbated cellular stress responses, leading to alterations in mitochondrial morphology and eventually cell death via apoptosis.

Castration resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer with still limited therapeutic outcomes due to the development of resistance to standard treatments. Unraveling the molecular mechanisms at the basis of the pro-death activity of novel anticancer compounds is necessary to increase the treatment strategies for this pathology. Here, we demonstrated that δ-tocotrienol (δ-TT, a vitamin E derivative) can induce apoptosis in human CRPC cell lines (PC3 and DU145) through modulation of the endoplasmic reticulum (ER) stress-autophagy axis. In these cells, δ-TT also triggers paraptosis, a non-canonical cell death mechanism characterized by cytoplasmic vacuolation resulting from mitochondrial/ER swelling and requiring protein synthesis. Mechanistically, we observed that δ-TT downregulates OXPHOS protein levels and inhibits mitochondrial respiration in PC3 and DU145 cells, leading to reduced oxygen consumption, ATP depletion and AMPK activation. Moreover, δ-TT treatment resulted in Ca2+ homeostasis alteration and ROS production, followed not only by apoptosis/paraptosis but also by mitochondrial fission and mitophagy. Taken together, these data demonstrate that in CRPC δ-TT can trigger both apoptosis and paraptosis, involving ER stress and autophagy. In addition, they suggest that δ-TT specifically alters mitochondrial morphology and function, inducing Ca2+ overload- and oxidative stress-mediated cell death.

Parkinson's disease (PD) is an incurable neurodegenerative motor disorder caused by the inexorable loss of dopamine neurones from the substantia nigra pars compacta. Cell loss is characterised by the perturbation of multiple physiological processes (including mitochondrial function, autophagy and dopamine homeostasis) and much of this pathophysiology can be reproduced in vitro using the mitochondrial toxin MPP+ (1-methyl-4-phenylpyridinium). It was hypothesised that MPP+ toxicity could be modelled using protein-protein interaction networks (PPIN) in order to better understand the interplay of systems-level processes that result in eventual cell death in MPP+ models and PD. Initially, MPP+ toxicity was characterised in the human, dopamine-producing cell line BE(2)-M17 and it was confirmed that the neurotoxin resulted in time and dose dependent apoptosis. A radio-label pulse-chase assay was developed and demonstrated that MPP+ induced decreased autophagic flux preceded cell death. Autophagic dysfunction was consistent with lysosome deacidification due to cellular ATP depletion. Pertinent PPINs were sampled from publically available data using a seedlist of proteins with validated roles in MPP+ toxicity. These PPINs were subjected to a series of analyses to identify potential therapeutic targets. Two topological methods based on betweenness centrality were used to identify target proteins predicted to be critical for the crosstalk between mitochondrial dysfunction and autophagy in the context of MPP+ toxicity. Combined knockdown of a subset of target proteins potentiated MPP+ toxicity and the combined resulted in cellular rescue. Neither of these effects was observed following single knockdown/overexpression confirming the need for multiple interventions. Cellular rescue occurred via an autophagic mechanism; prominent autophagosomes were formed and it was hypothesised that these structures allowed for the sequestration of damaged proteins. This thesis demonstrates the value of PPINs as a model for Parkinson's disease, from network creation through target identification to phenotypic benefit.

Autophagy serves as the sole catabolic mechanism for degrading organelles and protein aggregates. Increasing evidence implicates autophagic dysfunction in Alzheimer's disease (AD) and other neurodegenerative diseases associated with protein misprocessing and accumulation. Under physiologic conditions, the autophagic/lysosomal system efficiently recycles organelles and substrate proteins. However, reduced autophagy function leads to the accumulation of proteins and autophagic and lysosomal vesicles. These vesicles contain toxic lysosomal hydrolases as well as the proper cellular machinery to generate amyloid-beta, the major component of AD plaques. Here, we provide an overview of current research focused on the relevance of autophagic/lysosomal dysfunction in AD pathogenesis as well as potential therapeutic targets aimed at restoring autophagic/lysosomal pathway function.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
A Doença de Crohn é uma doença inflamatória crónica que pode afetar qualquer parte do trato gastrointestinal, embora comprometa preferencialmente o íleo. Apesar da incessante investigação a sua etiologia e patogénese permanecem desconhecidas. Até ao momento várias hipóteses têm sido avançadas na compreensão desta doença. Contudo, a teoria atual considera que se trata de uma doença complexa multifatorial que ocorre em indivíduos com predisposição genética, e que determinados fatores ambientais e microbianos são responsáveis pelo desenvolvimento de uma resposta inume inadequada. O possível envolvimento de um organismo infecioso, em particular a Escherichia coli Aderente-Invasiva (AIEC), tem estado sob investigação. A análise da flora bacteriana associada à mucosa ileal revelou uma anormal colonização da AIEC nos pacientes com Doença de Crohn. Estas bactérias são capazes de aderir e invadir as células epiteliais intestinais, assim como, penetrar e replicar extensivamente no interior dos macrófagos, sem induzir a morte da célula hospedeira. Por outro lado, a permeabilidade intestinal está significativamente aumentada nos indivíduos com Doença de Crohn. A AIEC diminui a resistência elétrica transepitelial e altera a estrutura morfológica das junções celulares, o que pode contribuir para esse aumento de permeabilidade. Pensa-se que as células M poderão constituir um potencial alvo de entrada que permite a interação bacteriana com os macrófagos da lâmina própria. Estudos in vitro têm demonstrado que os macrófagos infetados produzem grandes quantidades de fator de necrose tumoral α, e induzem a formação de agregados de células muitos semelhantes aos granulomas epitelioides. Estas estruturas representam uma das marcas histológicas características da Doença de Crohn. Crohn's disease is a chronic inflammatory disease that can affect any part of the gastrointestinal tract, although preferably compromise the ileum. Despite ongoing research its etiology and pathogenesis remain unknown. So far several hypotheses have been advanced in the understanding of this disease. However, the current theory considers that it is a complex multifactorial disease that occurs in individuals with a genetic predisposition and certain environmental and microbial factors are responsible for developing a response immune inadequate. The possible involvement of an infectious organism, in particular adherent-invasive Escherichia coli (AIEC) has been under investigation. The analysis of the bacterial flora associated with ileal mucosa revealed an abnormal AIEC colonization in patients with Crohn's disease. These bacteria are able to adhere to and invade intestinal epithelial cells, as well as penetrate and replicate extensively within macrophages without inducing the death of the host cell. On the other hand, is significantly increased intestinal permeability in patients with Crohn's disease. The AIEC decreases the transepithelial electrical resistance changes and the morphological structure of cell junctions, which may contribute to this increased permeability. It is believed that M cells might constitute a potential target input which allows the bacterial interaction with macrophages in the lamina propria. In vitro studies have demonstrated that infected macrophages produce large quantities of tumor necrosis factor α, and induce the formation of cell aggregates similar to many epithelioid granulomas. These structures represent one of the marks histological features of Crohn's disease.

Macroautophagy, also referred to as autophagy, is an intracellular process aimed to degrade and recycle cytoplasmic components, including long-lived proteins and damaged organelles. Due to the pivotal role of autophagy in maintaining cellular proteostasis, its dysfunction is associated with a wide number of human diseases such as cancer, cardiomyopathies and neurodegenerative disorders. In these pathological conditions, autophagy is initially activated as a survival mechanism but subsequently becomes defective, leading to cell damage. Many recent studies aim to better understand why autophagy is compromised in pathological conditions. Consequently restoration of defective autophagy now appears as an important therapeutic strategy in disease contexts. Several small molecules acting as autophagy modulators, such as plant polyphenols, or natural compounds present in fruit and vegetables, have been proposed as potential therapeutic applications. Plant polyphenols are able to regulate autophagy through different pathways. In particular, resveratrol and epigallocatechin-3 gallate (EGCG) stimulate the autophagic pathway via CamKK-AMPK-mTOR signalling. Polyphenols can also activate the sirtuins (SIRT), family of class III histone deacetylases, which are also involved in autophagy modulation. SIRT-induction results in many cellular outcomes and is considered responsible for the epigenetic effects of polyphenols. Oleuropein is the main polyphenol found in the olive tree and its main product, olive oil. Our previous studies have highlighted the beneficial effects of oleuropein aglycone (OLE), both in neuroblastoma cell lines (N2a) and in TgCRND8 mice, a model of Aβ deposition. In the latter, food supplementation with OLE resulted in remarkable plaque reduction and in the reduction of cognitive impairment when compared to non-OLE fed littermates. These protective effects were strongly correlated to an increased activation of autophagy in OLE fed mice. In light of the benefits associated with the upregulation in autophagy, the aim of this thesis was to investigate the cellular and molecular effectors of OLE-induced autophagy in vitro, by use of cultured human neuroblastoma cells (SH-SY5Y) and in vivo, using our TgCRND8 mice. Our in vitro results showed that OLE supplementation induces a rapid release of Ca2+ from the endoplasmic reticulum stores which, in turn, activates CAMKKβ with subsequent phosphorylation and activation of AMPK. The interplay between AMPK activation and mTOR inhibition shown in the OLE-fed animal model supports the idea that autophagy activation by OLE proceeds through mTOR inhibition. SIRT1 activation, another mechanism that synergizes with OLE-induced Ca2+-CaMKKβ-AMPK-mTOR signalling was also found in N2a cells. Given our findings for OLE dependent promotion of autophagy in our in vitro and in vivo neurodegeneration models, we aimed to determine whether OLE promotion of autophagy is ubiquitous and could protect against other pathological conditions displaying autophagy dysfunction. We selected an in vitro model of cardiomyopathy characterized by overexpression of monoamine oxidase-A (MAO-A). It is well established that catecholamine and serotonin degradation by MAO-A produces H2O2, which then disrupts nuclear translocation of TFEB, a master regulator of autophagy, causing autophagosome accumulation and ultimately cell death. Using this model we have shown that OLE treatment counteracts the effects of the MAO-A/H2O2 axis by improving mitochondrial function and decreasing cell necrosis. We demonstrate that these protective outcomes are, at least in part, related to the activation in autophagy. Indeed, increased autophagy observed in cardiac cells treated with OLE was a measure of the increase in autophagic vacuoles and autophagy-specific marker (LC3II) expression. Double immunofluorescence imaging of RFP-GFP-LC3 after 6 h of OLE treatment showed an increase of the autophagic flux; in addition, nuclear translocation of TFEB in OLE-treated cells was also observed. Together these data suggest that OLE treatment evokes transcriptional regulation of autophagy. In conclusion, our findings demonstrate that the underlying molecular mechanism of OLE stimulated autophagy includes the activation of the Ca2+-CaMKK-AMPK-mTOR signalling pathway. The identified molecular underpinnings of OLE treatment are indeed similar to other plant polyphenols such as resveratrol and EGCG. We show further that SIRT1-activation could synergize to maintain OLE-induced autophagy. TFEB translocation to the nucleus supports the importance of the transcriptional regulation of autophagy, findings that warrent further investigation. The results of this thesis add to the growing knowledge base of the molecular mechanisms of OLE-induced autophagy and provide strong evidence that similar to other plant polyphenols OLE can be a potential therapeutic against age-related diseases associated with autophagy dysfunction, including neurodegeneration and cardiovascular diseases.

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
A doença de Alzheimer (DA) é uma patologia neurodegenerativa crónica e progressiva que afecta o bem-estar físico, psicológico e social de quase 50 milhões de pessoas em todo o mundo, incluindo 6% da população portuguesa acima de 60 anos. A DA é caracterizada pela acumulação de placas neuríticas, contendo o peptídeo beta-amilóide (Aβ), e de tranças neurofibrilares da proteína Tau. A doença é também acompanhada por perda sináptica, degeneração axonal e morte neuronal, resultando em défices cognitivos e de memória. A acumulação patológica de proteínas é pronunciada na DA, o que sugere que a macroautofagia (doravante denominada autofagia) desempenha um papel fundamental na patogénese da doença. A autofagia é um sistema de degradação e reciclagem de componentes celulares em células eucarióticas. Esses componentes são inicialmente rodeados por vesículas com dupla membrana denominadas de autofagossomas, que depois se fundem com lisossomas iniciando a degradação do seu conteúdo. Este é um processo fortemente regulado por uma série de proteínas envolvidas na autofagia (ATG), que por sua vez são recrutadas por outra proteína (i.e. WIPI), caracterizada pela presença da repetição do domínio triptofano-ácido aspártico (WD), que interage com fosfoinositois e dessa forma controla a formação do autofagossoma. A autofagia ocorre constitutivamente nos neurónios, inclusivamente no cone de crescimento axonal e nos locais sinápticos, existindo diferenças notáveis entre a autofagia dendrítica e axonal. Contudo, as alterações na autofagia intra-axonal no contexto da DA são ainda pouco compreendidas.Estudos recentes indicam que os autofagossomas sofrem modificações com a idade e que o decaímento na sua produção pode ser restaurado, se manipulado extrinsecamente. Com base nestas evidências e em alguns resultados preliminares prévios, colocámos a hipótese de que oligómeros de Aβ (AβO) podem afetar a biogénese dos autofagossomas a nível intra-axonal nas fases iniciais da DA, que este poderá ser um dos primeiros efeitos dos AβOs, e que este mecanismo poderá exacerbar o efeito do envelhecimento na autofagia a nível dos axónios. Isto poderá explicar a disfunção sináptica induzida por AβO observada antes que qualquer dano celular seja detetável, suportando a ideia de que um aumento modesto da autofagia talvez possa ser suficiente para neutralizar a agregação de proteínas ao longo do tempo, sem efeitos colaterais consideráveis.Assim, os principais objetivos desta tese foram estudar: 1) o efeito dos AβO na biogénese dos autofagossomas; 2) as vias de sinalização envolvidas na disfunção intra-axonal da autofagia induzida pelos AβO; 3) o efeito dos AβO no transporte axonal dos autophagosomas. Resumidamente, os resultados obtidos mostram que os níveis de autofagia intra-axonal são alterados em resposta à estimulação de neurónios do hipocampo com AβO. A análise quantitativa de proteínas ligadas às etapas iniciais da biogénese do autofagossoma revelou alterações significativas durante as fases iniciais da DA, tanto na porção distal do axónio como nas regiões pré-sinápticas. Em particular, descobrimos que os AβOs aumentam a fosforilação da proteína WIPI2 e os seus níveis totais, o que se espera que tenha um impacto no alongamento e na selagem/ fecho do autofagossoma. Os resultados também indicaram que a exposição aos AβOs induz, em primeiro lugar, uma reorganização de proteínas relacionadas à autofagia, que, juntamente com as mudanças observadas no estado de fosforilação de WIPI2, podem indicar a formação de estruturas autofagossómicas aberrantes, conforme evidenciado em estudos anteriores. Dada a importância da fosforilação da WIPI2 como regulador molecular da formação de autofagossomas, este trabalho também se focou na identificação e caracterização de participantes envolvidos neste mecanismo molecular. O nosso estudo mostrou que a toxicidade induzida por AβO afeta esta via de sinalização e que a resposta é mediada não só pelo receptor N-metil-D-aspartato (NMDA) mas também pela proteína quinase II dependente de cálcio / calmodulina (CaMKII). Diante dos resultados obtidos, a última parte deste trabalho focou-se no transporte axonal de autofagossomas, para entender de que forma essas alterações na biogénese do autofagossoma podem também regular e influenciar a motilidade dessas vesículas na porção distal do axónio. Nesta parte do trabalho, descobrimos que os AβOs aumentam o número de autofagossomas imaturos na porção distal do axónio, influênciam o seu transporte e induzem movimentos aleatórios do autofagossoma. Isto indica que a incubação de neurónios do hipocampo com AβOs, não regula apenas as proteínas ATG, mas também afeta o correcto transporte dos autofagossomas ao longo dos axónios.De uma forma geral, mostrámos que a proteína WIPI2 é um alvo muito promissor para estudos futuros sobre a DA e que a sua fosforilação pode ser um alvo terapêutico potencialmente importante para esta doença neurodegenerativa.
Alzheimer’s disease (AD) is a chronic and progressive neurodegenerative disorder that affects the physical, psychologic and social well-being of nearly 50 million people worldwide, including 6% of the Portuguese population over 60 years old. The most common hallmarks of AD, besides extracellular amyloid-beta (Aβ) accumulation and neurofibrillary tangles, are synaptic loss, axonal degeneration and neuronal death, resulting in memory and cognitive deficits. Since the pathological accumulation of proteins is pronounced in AD, it has been suggested that macroautophagy (henceforward termed autophagy) plays a role in the pathogenesis of the disease. In fact, autophagy dysfunction has been associated to several neurodegenerative disorders. Autophagy is a system for degradation and recycling of cellular components in eukaryotic cells. These cellular wastes are initially engulfed by double-membraned vesicles called autophagosomes, which then fuse with lysosomes initiating the degradation of their content. This is a highly regulated process by a series of proteins defined as autophagy-related (ATG) proteins, which can be recruited by a tryptophan-aspartic acid (WD) repeat protein interacting with phosphoinositides (WIPI) that controls autophagosome formation. Bulk autophagy occurs constitutively in neurons throughout development, in a spatially localized manner in the growth cone and at synaptic sites and, interestingly, there are remarkable differences between dendritic and axonal autophagy. Noteworthy, the mechanisms involved in intra-axonal autophagy in the context of AD, which has implications in axonal viability, are still poorly understood.Recent evidence demonstrated that autophagosomes suffer modifications with age and that the decay in autophagosome production can be restored, if extrinsically manipulated. Based on this evidence and in some previous preliminary results from our laboratory, we hypothesized that Aβ Oligomers (AβO) may impair intra-axonal autophagosome biogenesis in early phases of AD and this may be one of the first effects of AβO, exacerbating the effect of aging with a consequent impairment of the axonal function. This might explain how AβO can induce synaptic failure before any cell damage is detectable and raises the idea that perhaps a modest increase in autophagy could be sufficient to counteract protein aggregation over time, without considerable deleterious side effects.Therefore, the main goals of this thesis were to study: 1) the effects of AβO on autophagosome biogenesis; 2) the pathways involved in AβO-induced dysfunction of intra-axonal autophagy; 3) the effect of AβO on the axonal transport of autophagosomes along the axon.Briefly, the results obtained demonstrate that the presence of AβO changes the levels of intra-axonal autophagy. Quantitative analysis of proteins linked to the initial steps of the autophagosome biogenesis revealed significant alterations during the early stages of AD, both in the distal portion of the axon and at presynaptic regions. In particular, we found that AβO increases WIPI2 protein phosphorylation and total levels, which is expected to have an impact on autophagosome elongation and sealing. The results also indicated that exposure to AβO induces, first, a reorganization of autophagy-related proteins, which, together with the observed changes in the phosphorylation state of WIPI2, might indicate the formation of aberrant autophagosomal structures, as evidenced in previous studies. Given the importance of WIPI2 phosphorylation as a molecular switch for autophagosome formation, this work also focused on the characterization of the players involved in this molecular mechanism. Our study showed that not only AβO-induced toxicity affects this signaling pathway but also that the response is mediated by both N-methyl-D-aspartate (NMDA) receptor and calcium / calmodulin-dependent protein kinase II (CaMKII). In light of the results obtained, the last part of this our work focused on the axonal transport of autophagosomes, to understand how these alterations in autophagosome biogenesis can also regulate and influence the motility of these vesicles in the distal portion of the axon. In this part of the work we found that AβO increases the number of immature autophagosomes in the axonal distal portion, influences their transport and induces autophagosome random movements. This indicates that incubation of hippocampal neurons with AβOs, not only regulates ATG proteins but also affects autophagic transport at the early stages of its toxicity.Together, we show that WIPI2 is a very promising protein for future studies in AD and that its phosphorylation might be an interesting potential therapeutical target for this neurodegenerative disorder.
FCT
Outro - This work was financed by the Erasmus+ program, the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme under project CENTRO-01-0145-FEDER-000008.

Although a rare disease, light chain (LC) amyloidosis (AL) is the most common systemic amyloidosis in developed countries. It is caused by an overproduction of immunoglobulin LC proteins in bone marrow plasma cells. In AL amyloidosis, LCs that are prone to misfolding and insolubility will aggregate, form fibrils, and deposit themselves in various tissues, thereby causing organ dysfunction. The most fatal manifestation of AL amyloidosis is associated with cardiac involvement, defined by the presence of extracellular AL amyloid deposits within the heart. Cardiac amyloid infiltration typically leads to diastolic dysfunction followed by heart failure and has a median survival of approximately 6 months from the time of diagnosis if untreated. Clinical observation suggests that a reduction in circulating LCs results in an improvement in heart failure symptoms despite minimal changes in amyloid deposition. This has led to the concept that LCs themselves are cytotoxic to cardiomyocytes. Recent studies indicate that AL LCs induce oxidative stress, cellular dysfunction, and apoptosis (programmed cell death) in cardiomyocytes via a p38α mitogen-activated protein kinase (MAPK) mechanism. They may therefore be a target for amyloidosis therapy. By understanding how LCs cause cardiac dysfunction, we can target this process with therapies and utilize downstream measures of LC activity as diagnostic and prognostic tools. The objective of this study was to determine the role of autophagy in AL amyloidosis. Autophagy is the intracellular process of degrading aging or dysfunctional cellular components. Autophagy can be beneficial by preventing proteotoxicity and providing nutrients, amino acids, and other necessities during times of cellular stress. On the other hand, increased autophagy, like apoptosis, may mediate cellular death depending on the type of stimulus and its duration. Autophagy is induced by a variety of stimuli, including oxidative stress. AL has been demonstrated to increase reactive oxygen species (ROS), and it is unknown if autophagy mediates cardiomyocyte dysfunction in AL cardiac amyloidosis. We thus sought to determine if it is a factor in amyloid cardiotoxicity. We explored the ERK1/2, p38, and JNK MAPK pathways in particular, since MAPK signaling cascades regulate several transcription factors involved in the cell cycle and p38α has been implicated in ROS-induced cardiac AL amyloidosis. Adult rat ventricular myocytes (ARVM) were harvested from healthy adult male rats and exposed to a variety of experimental conditions in vitro. ARVM were treated with vehicle control, human LC obtained from a patient without cardiac involvement, a positive control (aldosterone), and human AL light chains obtained from a patient with AL cardiac amyloidosis in the presence or absence of UO126, SB203580, or SP600125 (specific inhibitors of ERK1/2, p38, and JNK, respectively). The resulting protein expression levels of autophagy indicators LC3II and ATG4B in cardiomyocytes were analyzed by Western blotting. The ratio of phosphorylated to total ERK1/2 protein expression was also explored. We found that AL light chains did not contribute to autophagy via the ERK1/2, p38, or JNK pathways. In contrast to our previous unpublished findings, the protein levels of autophagy indicators in AL-treated ARVM did not differ from vehicle control levels, suggesting that AL did not activate autophagy. However non-cardiomyopathic light chains (LC) did increase LC3II expression in ARVM, despite their human source exhibiting no clinical indications of cardiac involvement. This implies that autophagy induced by non-cardiomyopathic LCs may be beneficial and protect against the development of the cardiotoxicity seen in AL cardiac amyloidosis. Further studies are necessary to understand the effect of autophagy in the heart and its role in cardiac amyloidosis. Continuing to explore the underlying mechanisms of AL light chain toxicity will contribute to the development of diagnostic, prognostic, and treatment strategies for AL amyloidosis.

博士
國立陽明大學
分子醫學博士學位學程
103
Progressive impairment of mitochondrial function and increased oxidative damage contribute to the pathogenesis of neurodegenerative disorders such as Huntington’s disease (HD). Exposure to 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, results in mitochondrial dysfunction and oxidative stress in neurons. Sulfiredoxin and sestrin2 are sulfinic acid reductase capable of reducing hyperoxidized peroxiredoxin back to the catalytically active thiol form in an ATP-dependent manner that, under severe oxidative stress, catalyze the formation of a sulfinic acid phosphoric ester on peroxiredoxins, which can subsequently be reduced by thiol equivalents such as thioredoxin. Thus, these proteins may have critical effects in cellular defense against oxidative stress upon mitochondrial inhibition. We have previously demonstrated that preconditioning of cortical neurons with brain-derived neurotrophic factor (BDNF) attenuates neurotoxicity induced by 3-NP. However, whether sulfiredoxin and sestrin2 mediate BDNF-dependent neuroprotection remains unknown. In this thesis, we tested the hypothesis that BDNF may enhance the expression of sulfiredoxin and sestrin2 that contribute to neuroprotection against 3-NP toxicity in primary cortical neurons and characterized the upstream regulatory mechanisms underlying BDNF induction of these two proteins. We found that BDNF transiently induced the expression of sulfiredoxin at both mRNA and protein levels. BDNF also enhanced expression of c-Jun that required prior phosphorylation of extracellular signal-regulated kinase (ERK)1/2. Further, ERK1/2 inhibitor PD98059, c-Jun siRNA, and sulfiredoxin siRNA all abrogated both BDNF-induced sulfiredoxin and BDNF-mediated 3-NP resistance. Together, these results established the first signaling cascade of “BDNF → ERK1/2 phosphorylation → c-Jun → sulfiredoxin → 3-NP resistance”. For sestrin2 induction by BDNF, however, the regulatory mechanisms appear to be different. We found that BDNF increased formation of nitric oxide (NO) with subsequent production of 3',5'-cyclic guanosine monophosphate (cGMP) and also transiently induced expression of cGMP-dependent protein kinase-1 (PKG-1). Interestingly, BDNF triggered physical interaction of nuclear factor-kappaB (NF-κB) subunits p65/p50 heterodimer with PKG-1 in nuclei and enhanced binding of this p65/p50/PKG-1 complex to the sestrin2 promoter. Consistently, BDNF induction of sestrin2 was abolished by L-NG-Nitroarginine methyl ester (L-NAME), 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), KT5823, and SN50, the respective inhibitor of nitric oxide synthase (NOS), soluble guanylate cyclase (sGC), PKG, and NF-κB. The siRNAs targeting at p65 and p50 exerted similar effects in attenuating BDNF-dependent sestrin2 induction. Finally, 3-NP-induced production of reactive oxygen species (ROS) was suppressed by BDNF preconditioning; further, this BDNF effect was reversed by KT5823, SN50, and sestrin2 siRNA. Taken together, these results established the second signaling cascade of “BDNF → NO/cGMP/PKG → NF-κB → sestrin2 → 3-NP resistance”. Apart from oxidative stress, 3-NP-induced cytotoxicity also involves autophagy activation. However, whether BDNF-dependent neuroprotection against 3-NP involves autophagy and, if so, its underlying mechanisms remain to be fully delineated. We therefore further characterized the crucial roles of autophagy regulation in this experimental paradigm. We found that 3-NP increased the ratio of LC3-II/LC3-I, an index of autophagy. The autophagy inhibitor bafilomycin A1 (Baf-A1) attenuated cell death induced by 3-NP. Further, we found BDNF downregulated the LC3-II/LC3-I ratio, indicating its capability of autophagy inhibition, either with or without 3-NP exposure. The p62/sequestosome 1, a scaffold protein interacting with polyubiquitinated protein aggregates for subsequent transportation into autophagosomes, plays a vital role in autophagy regulation. In our model system, BDNF time-dependently induced expression of p62. Interestingly, inhibition of p62 expression by its siRNA increased the basal levels of autophagy as evidenced by higher ratio of LC3-II/LC3-I; further, p62 siRNA also abrogated the BDNF effects in attenuating autophagy with or without 3-NP exposure as well as the neuroprotective action of BDNF against 3-NP. Further, BDNF triggered phosphorylation of mTOR and, more importantly, the mTOR inhibitor rapamycin attenuated BDNF-dependent p62 expression and its neuroprotective effects against 3-NP. Thus, in addition to antioxidant effects, we proposed the third neuroprotective signaling cascade of “BDNF → mTOR phosphorylation → p62 → autophagy inhibition → 3-NP resistance” in cortical neurons. Overall, this thesis demonstrated that BDNF may elicit a multitude of signal transduction pathways involving both antioxidation and autophagy regulation to confer neuronal resistance against mitochondrial dysfunction, at least in vitro. Because mitochondria dysfunction and oxidative stress play critical roles in the pathogenic mechanisms underlying various neurodegeneration disorders such as Huntington’s disease and Alzheimer’s disease, our results provide novel information regarding the neuroprotective mechanisms of BDNF against mitochondrial dysfunction. These in vitro results pave the foundation for future investigation of these signaling cascades in the relevant animal models in vivo.

碩士
國立陽明大學
生化暨分子生物研究所
101
Autophagy is a highly conserved degradation process, in which aggregations of damaged proteins are eliminated in affected cells to assure that energy is available under starvation or other stressful conditions. Many studies have demonstrated that an increase in cellular oxidative stress has a strong association with the induction of autophagy. Indeed, intracellular reactive oxygen species (ROS) play an essential role in the regulation of autophagy through multiple signaling pathways to protect mammalian cells from further oxidative damage. Most importantly, it has been reported that autophagy is involved in the pathophysiology of various human diseases, including aging, neurodegenerative diseases, spinal muscular atrophy and mitochondrial diseases. Myoclonic epilepsy with ragged-red fibers (MERRF) syndrome is one of the rare mitochondrial diseases, and the patients usually carry an A to G transition at nucleotide position 8344 of mitochondrial DNA (mtDNA) in the affected tissue cells. Previous studies showed that an increase of the intracellular ROS level is accompanied by increased secondary lysosomes in the primary culture of skin fibroblasts from MERRF patients as compared with those of normal skin fibroblasts. This indicates that MERRF skin fibroblasts may have an increased autophagy activity. In the present study, I first demonstrated that skin fibroblasts from MERRF patients (M1-M3) had higher autophagy activities as compared with those of normal skin fibroblasts (N1-N3). In addition, I treated normal skin fibroblasts with 5 M rotenone, a mitochondrial respiratory enzyme Complex Ⅰ inhibitor, to induce oxidative stress. The results showed an increase of intracellular ROS levels accompanied by pronounced autophagy activity in rotenone-treated normal skin fibroblasts. By pretreatment of the cells with antioxidants including 2 M N-acetylcysteine and vitamin C, both the autophagy activity and the ROS levels were decreased concurrently in MERRF skin fibroblasts and rotenone-treated normal skin fibroblasts, respectively. Finally, I found that treatment of cells with bafilomycin A1, an inhibitor of autophagy, would affect the cell viability of MERRF skin fibroblasts. Taken together, I suggest that the upregulation of autophagy in human cells with mitochondrial dysfunction is a response to the elevated oxidative stress and plays a role in the protection of the cells from oxidative damage. Furthermore, the higher levels of autophagy activity in MERRF skin fibroblasts may be an adaptive response for the cells to survive under mild oxidative stress, and would be involved in the pathophysiology of the mitochondrial diseases caused by pathogenic mtDNA mutations.

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Hipertensão pulmonar (HP) é um estado hemodinâmico definido por uma pressão arterial pulmonar média (mPAP)> 20 mmHg em repouso, medida através de cateterismo do coração direito. Hipertensão arterial pulmonar (HAP), subgrupo de HP, descrita pela Organização Mundial de Saúde como Grupo I, é uma doença crónica e progressiva caracterizada por um aumento persistente da resistência vascular pulmonar e pela sobrecarga do ventrículo direito, que resulta em insuficiência cardíaca e morte. O início e a progressão da HAP dependem da interação entre cardiomiócitos e células da vasculatura pulmonar, especialmente células endoteliais (ECs), que regulam a homeostasia vascular. No entanto, a interação entre esses tipos de células ainda é pouco conhecida, a nível molecular.O presente estudo tem como objetivo avaliar a comunicação intercelular entre ECs e cardiomiócitos. Pelo facto de a inflamação estar associada à HAP, decidimos expor as células endoteliais da veia umbilical humana (HUVEC) ao lipopolissacarídeo (LPS), usado como um estímulo inflamatório. O meio condicionado recolhido das HUVEC foi adicionado a células H9c2, um modelo de cardiomiócitos. Posteriomente, foram avaliados os níveis de Cx43 e de marcadores de autofagia (i.e., p62 e a razão LC3II/LC3I) nas células. Os resultados mostraram que alterações do secretoma das HUVEC, induzidas pelo LPS modula os níveis de Cx43 e dos marcadores de autofagia em células H9c2. Resultados semelhantes foram observados in vivo utilizando um modelo animal de rato de PAH induzido por monocrotalina (MCT).Estes resultados sugerem que, na HAP, as ECs secretam fatores para o sangue, nomeadamente mediadores inflamatórios, que afetam a função dos cardiomiócitos.
Pulmonary hypertension (PH) is a haemodynamic state defined as a mean pulmonary arterial pressure (mPAP) > 20 mmHg at rest, measured by right heart catheterisation. Pulmonary arterial hypertension (PAH), described as a subgroup of PH by World Health Organization Group I PH, is a chronic and progressive disorder characterised by a persistent increase in pulmonary vascular resistance and overload of the right ventricle, leading to heart failure and death. The onset and progression of PAH depends on the crosstalk between cardiomyocytes and cells of the pulmonary vasculature, especially endothelial cells (ECs), which regulate vascular homeostasis. However, the interaction between these cell types is still poorly understood at a molecular level.The present study aimed to evaluate the crosstalk between ECs and cardiomyocytes. We mimicked PAH-associated inflammation by exposing human umbilical vein endothelial cells (HUVEC) to the inflammatory stimulus lipopolysaccharide (LPS). The HUVEC-conditioned medium was then added to cardiac cell line (H9c2 cells).The levels of Cx43 and autophagy markers (i.e. p62 and LC3I/LC3II) in rat ventricular H9c2 cells exposed to a conditioned medium were evaluated. The results showed that conditioned medium from HUVECs modulated the levels of Cx43 and autophagy markers in H9c2 cells. Similar results were observed in vivo using a monocrotaline (MCT)-induced PAH rat model.Together, these results suggest that in PAH, ECs secrete factors to the blood which are similar to inflammatory mediators, with these factors affecting cardiomyocyte function.

Dissertação de Mestrado em Química Farmacêutica Industrial apresentada à Faculdade de Farmácia
A hipertensão arterial pulmonar (HAP) é uma doença rara, caraterizada por uma pressão arterial pulmonar elevada, causada por uma obliteração progressiva das pequenas artérias pulmonares que podem levar à insuficiência cardíaca direita. Esta oclusão arterial é principalmente devida à disfunção das células endoteliais e à proliferação anormal do tecido muscular liso circundante. Doentes com HAP têm uma qualidade de vida reduzida e uma baixa esperança média de vida devido à falta de tratamentos eficazes. Na verdade, as terapias existentes, além de caras, apenas proporcionam pequenas melhorias a curto prazo, justificando a procura de novos agentes terapêuticos. Terpenos de baixo peso molecular têm sido descritos como agentes terapêuticos eficazes em patologias vasculares. Portanto, é plausível que os óleos essenciais (OEs) ricos nesses compostos também exerçam os mesmos efeitos. Além disso, é provável que sinergismos entre os terpenos presentes nesses extratos possam culminar em abordagens ainda mais eficientes. De facto, os óleos essenciais são reconhecidos como uma fonte de compostos bioativos, com muitas evidências científicas validando o seu potencial preventivo/terapêutico.Neste contexto, o presente estudo tem como objetivo avaliar o efeito de dois OEs, Lavandula viridis e Thymus zygis subps. sylvestris, e dos seus principais compostos, 1,8-cineol e timol respetivamente, em várias características associadas à HAP, tais como autofagia comprometida, comunicação intercelular ineficiente e angiogénese desregulada. O presente trabalho assenta na hipótese que células endoteliais submetidas a hipóxia (de forma a mimetizar a patologia) desenvolvem características de HAP que poderão ser prevenidas ou revertidas pelo tratamento com OEs e/ou terpenos. Utilizando diferentes abordagens complementares, o presente trabalho mostra que os OEs/terpenos estimulam a angiogénese em vários modelos in vitro e ex vivo. Demostra-se pela primeira vez que os OEs/terpenos promovem a migração e modulam a formação de tubos, e aumentam o número de sprouts angiogénicos em anéis de aorta de rato. Por último, o efeito desses compostos foi avaliado na autofagia das células endoteliais e os resultados mostram que a presença dos OEs e do 1,8-cineol levam a um aumento dos níveis de LC3-II, sugerindo um aumento da autofagia. Além disso, em células endoteliais da artéria pulmonar humana, HPAEC, o OE de Lavandula viridis promove o fluxo autofágico. Acreditamos que estes resultados abrem novos caminhos para o desenvolvimento de abordagens preventivas ou terapêuticas eficazes para a disfunção endotelial associada à HAP.
Pulmonary arterial hypertension (PAH) is a rare disorder characterized by an elevated pulmonary arterial pressure, caused by a progressive obliteration of small pulmonary arteries, that can ultimately lead to right heart failure. This artery occlusion is primarily due to endothelial cell dysfunction and abnormal proliferation of the surrounding smooth muscle tissue. PAH patients have reduced quality of life as well as a low average life expectancy due to the lack of effective treatments. Indeed, current therapies provide small improvements with short-term benefits and are very expensive. Therefore, efficient and less costly treatments are required.Terpenes of low molecular weight have been described as effective therapeutic agents in vascular-related disorders. Therefore, it is conceivable that essential oils (EOs) rich in these compounds may also have these beneficial effects. Moreover, it is likely that synergisms between terpenes present in these extracts, may result in more efficient approaches. Indeed, EOs are recognized as a source of bioactive compounds, with many scientific evidences validating their preventive/therapeutic potential.In this context, the present study aims to evaluate the effect of selected EOs, namely Lavandula viridis and Thymus zygis subps. sylvestris, and their major compounds, 1,8-cineole (for Lavandula viridis) and thymol (for Thymus zygis subps. sylvestris), on several features associated with PAH, like compromised autophagy, inefficient intercellular communication and dysregulated angiogenesis. We hypothesize that endothelial cells (ECs) subjected to hypoxia (to mimic the disease) will develop PAH features that can be prevented and/or reverted by the treatment with essential oils and/or isolated terpenes.Using different complementary approaches, we demonstrate that EOs/terpenes stimulate angiogenesis in different in vitro and ex vivo models. Indeed, we demonstrate that the treatments promote migration and modulate tube formation in ECs and increase the number of angiogenic sprouts in rat aortic rings. Furthermore, we evaluated the effect of these compounds in the autophagic response and showed that the presence of EOs and 1,8-cineole led to an accumulation of LC3-II, suggesting an increase of autophagy. Moreover, in human pulmonary arterial endothelial cells, HPAEC, Lavandula viridis EO enhanced the autophagic flux.We believe that these results will open new avenues for the development of effective preventive/therapeutic approaches for endothelial dysfunction associated with PAH.

Autophagy is a critical cellular maintenance machinery in cells, and prevents the accumulation of toxic protein aggregates, organelles or lipid droplets through degradation via the lysosome. In macro-autophagy, autophagosome first engulfs around aggregates or cellular debris and subsequently fuses with a lysosome that is sufficiently acidic (pH 4.5–5.5), where the contents are then degraded via lysosomal enzymes. Autophagy inhibition as a result of lysosomal acidification dysfunction (pH > 5.5) have been reported to play a major role in various diseases pathogenesis. Hence, there is a pressing need to target lysosomal pH to rescue autophagy. Nanoparticles are attractive materials which has been shown to be efficiently uptaken into cellular organelles and can serve as an agent to specifically localize into lysosomes and modulate its pH. Lipotoxicity, induced by chronic exposure to free fatty acids, and exposure to neurotoxins (e.g. MPP+), elevates lysosomal pH in pancreatic beta cells (Type II Diabetes, T2D) and hepatocytes (Non-alcoholic fatty liver disease, NAFLD), and PC-12 cells (Parkinson’s Disease), respectively. We first tested the lysosome acidification capability of photo-activable nanoparticles (paNPs) and poly (lactic-co-glycolic) acid nanoparticles (PLGA NPs) in a T2D model. Both NPs lowered lysosomal pH in pancreatic beta cells under lipotoxicity and improved insulin secretion function. However, paNPs only release acids upon UV trigger, limiting its applicability in vivo, while PLGA NPs degrade upon lysosome localization. We further showed that PLGA NPs are able to rescue MPP+ induced cell death in a PD model, though it has a slow degradation rate. To attain the most efficacious nanoparticle with a fast degradation and acidification rate, we synthesized acidic nanoparticles (acNPs) based on tetrafluorosuccinic and succinic acids to form optimized nanoparticles. The acNPs showed faster rescue of cellular function compared to PLGA NPs in the PD model. Finally, we tested the acNPs in NAFLD model, and where lysosomal pH reduction by acNPs restored autophagy, reduced lipid accumulation, and improved mitochondria function in high-fat diet mice. In sum, nanoparticles are of potential therapeutic interest for pathologies associated with lysosomal acidity impairment. Future studies include testing the acNPs in NASH disease model and clinical studies.
2022-05-18T00:00:00Z

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2018
Alzheimer’s disease (AD) is a devastating neurodegenerative disorder, characterized by neuronal loss and gradual cognitive impairment, a serious public health problem, affecting more than 30 million people worldwide. The presence of two well-known abnormal protein aggregates in cerebral cortex and hippocampus characterize AD pathologically: senile plaques in specific areas of the brain, extracellular, and composed of insoluble A peptides; and neurofibrillary tangles, intracellular aggregates, mostly consisted by hyperphosphorylated Tau, a microtubule-associated protein localized in axons. Several authors have described for decades that protein aggregation process can induce toxicity for neurons causing synaptic dysfunction, neuroinflammation and oxidative stress. One major aspect of AD pathology, that is observed in both humans and mouse models of the disease is the accumulation of senile plaques, containing A peptides, leading to a neuronal dysfunction and cell death. Neuronal cell survival depends on a health and effective mitochondrial quality control, but also a balance between autophagic and lysosomal pathways. Data has demonstrated the crucial role of both macroautophagy (referred to here as autophagy) and lysosomal pathways in maintaining cellular homeostasis, as well in neuronal survival, degrading and decreasing the amount of misfolded proteins and impaired organelles, like that preventing the accumulation of toxic protein aggregates. Beclin-1 is a protein involved in several biological functions so relevant in several human diseases, such as heart disease, pathogen infection, development and neurodegenerative disorders. However, as one of the main proteins responsible of autophagy regulation, it has been shown that Beclin-1 levels are reduced in AD patient’s brain. For several years, a lot of research has been focused on a family of protein deacetylases, Sirtuins (SIRTs), and its crucial role in a variety of cellular biological systems, including neuroinflammation, melanocortin system, energy balance, the ubiquitin-proteasome system; and central nervous system regulation. SIRT1’s activity can influence autophagic pathway, acting on components of the autophagic machinery. Despite of this, it has not been described in neuronal cells the effect of SIRT1 on deacetylation of Beclin-1, which can result in deregulation of the autophagic pathway. Autophagy impairment plays a key role in sporadic Alzheimer’s disease (sAD) neurodegenerative process. Nevertheless, the mechanism(s) that lead to a deficiency in autophagy in AD remains elusive. In this work we identify, for the first time, that Beclin-1 acetylation status is responsible for autophagosomes maturation and is implicated in the alterations in autophagy observed in AD neurodegeneration. We observed that Beclin-1 is deacetylated by SIRT1 and acetylated by p300. In addition, Beclin-1 acetylation inhibits autophagosomes maturation, leading to impairment in autophagic flux. We also analyzed some proteins, known to be involved in the maturation of autophagosomes, such as Rab 7 that ABSTRACT vi participates in the fusion step with lysosomes. We observed that an overexpression of Rab 7 and the formation of large perinuclear lysosome clusters are in accordance with an increase in lysosomal biogenesis determined by an increase in LAMP-2A and Cathepsin D expression in sAD cells. Thus, our data provide strong evidence that Beclin-1 acetylation impairs the autophagic flux and despite lysosomal biogenesis is triggered as a compensatory response, autophagosome fusion with lysosomes is compromised contributing to AD neurodegeneration.
A doença de Alzheimer (DA) é um distúrbio neurodegenerativo devastador, caracterizado por uma perda de neurónios e por um comprometimento gradual cognitivo, um grave problema de saúde pública, afetando mais de 30 milhões de pessoas em todo o mundo. A acumulação não normal de duas proteínas específicas no córtex cerebral e no hipocampo, caracteriza a DA patologicamente: as placas senis em áreas específicas do cérebro, depósitos extracelulares, e constituídos por peptídeos de -amilóide insolúveis; e as tranças neurofibrilares, agregados intracelulares, constituídos principalmente pela Tau hiperfosforilada, uma proteína associada aos microtúbulos, localizada nos axónios. Por várias décadas, diversos autores têm descrito que o processo de acumulação proteica, pode induzir toxicidade aos neurónios, levando a uma disfunção sináptica, a uma neuroinflamação e a um estresse oxidativo. Uma das características mais importantes da patologia DA observada quer em humanos, quer em modelos de ratinho que apresentam a doença, é a acumulação das placas senis, as quais contêm peptídeos ricos em proteína -amilóide, levando a uma disfunção neuronal e morte celular. A sobrevivência neuronal, depende tanto de um ótimo bem-estar e de um controlo efetivo de qualidade a nível mitocondrial, mas também de um equilíbrio entre as vias autofágica e lisossomal. Dados científicos têm demonstrado o papel fundamental de ambas as vias, a via da macroautofagia (designada aqui como autofagia) e a via lisossomal, na manutenção da homeostasia celular, bem como na sobrevivência neuronal, na degradação e diminuição da quantidade de proteínas disfuncionais e organelos deficientes, prevenindo assim a acumulação de agregados proteicos tóxicos. A Beclin-1 é uma proteína envolvida em várias funções biológicas e importante em diferentes patologias, como por exemplo, nas doenças cardíacas, na infeção por patógenos, no desenvolvimento e na neurodegeneração. No entanto, como uma das principais proteínas responsáveis pela regulação da via autofágica, foi demonstrado que os níveis de Beclin-1 estão reduzidos em cérebros de doentes com DA. Durante vários anos, a ciência focou-se numa família de proteínas de deacetilase de histonas, as Sirtuínas (SIRTs), e no seu papel de muito importância em diversos processos biológicos e celulares, incluindo na neuroinflamação, no sistema de melanocortina e no balanço energético, e no sistema proteossómico; e na regulação do sistema nervoso central. A atividade da SIRT1 pode influenciar o processo autofágico, atuando sobre os seus componentes presentes na maquinaria autofágica. No entanto, ainda não foi descrito o seu efeito de deacetilação na Beclin-1, em neurónios, podendo levar a um comprometimento do próprio processo autofágico. Um comprometimento por parte da autofagia vai desempenhar um papel crucial no processo neurodegenerativo da doença de Alzheimer do tipo esporádico (DAs). Contudo, o (s) mecanismo (s) responsável (eis) pela incapacidade da via autofágica na DA permanece inconclusivo. Neste trabalho identificámos, pela viii RESUMO primeira vez que o estado de acetilação da Beclin-1 é responsável pela maturação dos autofagossomas, e que está implícito nas alterações da via autofágica observada na neurodegeneração da DA. Verificámos que a Beclin-1 é deacetilada pela SIRT1 e acetilada pela p300. Para além disso, a acetilação da Beclin-1 inibe a maturação dos autofagossomas, levando a um comprometimento do fluxo autofágico. Também analisámos algumas proteínas, bastante conhecidas por estarem envolvidas na maturação dos autofagossomas, tais como a Rab7, a qual participa na etapa de fusão com os lisossomas. Observámos que uma sobre-expressão da Rab7 e a formação de grandes aglomerados lisossomais perinucleares estão de acordo com um aumento da biogénese lisossomal, determinada por um aumento na expressão de LAMP-2A e da Cathepsin D em células com DAs. Assim, os nossos resultados mostram fortes evidências de que a acetilação da Beclin-1 compromete o fluxo autofágico e, apesar da biogénese lisossomal ser desencadeada como uma resposta compensatória, a fusão dos autofagossomas com os lisossomas é prejudicada, contribuindo para a neurodegeneração da DA.