Mitochondrial Fusion/Fission, Transport and Autophagy in Parkinson's Disease: When Mitochondria Get Nasty

Understanding the molecular basis of Parkinson's disease (PD) has proven to be a major challenge in the field of neurodegenerative diseases. Although several hypotheses have been proposed to explain the molecular mechanisms underlying the pathogenesis of PD, a growing body of evidence has highlighted the role of mitochondrial dysfunction and the disruption of the mechanisms of mitochondrial dynamics in PD and other parkinsonian disorders. In this paper, we comment on the recent advances in how changes in the mitochondrial function and mitochondrial dynamics (fusion/fission, transport, and clearance) contribute to neurodegeneration, specifically focusing on PD. We also evaluate the current controversies in those issues and discuss the role of fusion/fission dynamics in the mitochondrial lifecycle and maintenance. We propose that cellular demise and neurodegeneration in PD are due to the interplay between mitochondrial dysfunction, mitochondrial trafficking disruption, and impaired autophagic clearance

Microtubule Depolymerization Potentiates Alpha-Synuclein Oligomerization

Parkinson's disease (PD) is associated with perturbed mitochondria function and alpha-synuclein fibrillization. We evaluated potential mechanistic links between mitochondrial dysfunction and alpha-synuclein aggregation. We studied a PD cytoplasmic hybrid (cybrid) cell line in which platelet mitochondria from a PD subject were transferred to NT2 neuronal cells previously depleted of endogenous mitochondrial DNA. Compared to a control cybrid cell line, the PD line showed reduced ATP levels, an increased free/polymerized tubulin ratio, and alpha-synuclein oligomer accumulation. Taxol (which stabilizes microtubules) normalized the PD tubulin ratio and reduced alpha-synuclein oligomerization. A nexus exists between mitochondrial function, cytoskeleton homeostasis, and alpha-synuclein oligomerization. In our model, mitochondrial dysfunction triggers an increased free tubulin, which destabilizes the microtubular network and promotes alpha-synuclein oligomerization

Mitochondrial Dysfunction: The Road to Alpha-Synuclein Oligomerization in PD

While the etiology of Parkinson's disease remains largely elusive, there is accumulating evidence suggesting that mitochondrial dysfunction occurs prior to the onset of symptoms in Parkinson's disease. Mitochondria are remarkably primed to play a vital role in neuronal cell survival since they are key regulators of energy metabolism (as ATP producers), of intracellular calcium homeostasis, of NAD+/NADH ratio, and of endogenous reactive oxygen species production and programmed cell death. In this paper, we focus on mitochondrial dysfunction-mediated alpha-synuclein aggregation. We highlight some of the findings that provide proof of evidence for a mitochondrial metabolism control in Parkinson's disease, namely, mitochondrial regulation of microtubule-dependent cellular traffic and autophagic lysosomal pathway. The knowledge that microtubule alterations may lead to autophagic deficiency and may compromise the cellular degradation mechanisms that culminate in the progressive accumulation of aberrant protein aggregates shields new insights to the way we address Parkinson's disease. In line with this knowledge, an innovative window for new therapeutic strategies aimed to restore microtubule network may be unlocked

Alterações micrometeorológicas em vinhedos pelo uso de coberturas de plástico

The objective of this work was to evaluate the effects of plastic covering on the microclimate of vineyards (Vitis vinifera L., cultivar Moscato Giallo), in Serra Gaúcha region in Rio Grande do Sul State, Brazil. The experiment was performed in Flores da Cunha, RS, during the 2005/2006 growing season. It comprised uncovered and covered rows of vines, using a 160 µm thick plastic film. Photossinthetically active radiation (PAR), air temperature and humidity, and wind velocity were monitored: over the plastic covering; between the film and the canopy; over the uncovered canopy; and close to grapes of both treatments. Reference evapotranspiration was estimated for both treatments. From the incoming PAR, 67.5% reached the covered canopy, 16% reached the covered grapes, and 36% reached the uncovered grapes. The plastic covering increased by 3.4ºC the maximum air temperatures close to plants. Diurnal air relative humidity was reduced, while water vapor pressure and vapor pressure deficit were increased because of the plastic covering, which also reduced in 88% the wind velocity in comparison to open air. The reference evapotranspiration on the covered canopy was 35% lower than in open air. Although increasing diurnal air temperatures, the plastic covering may reduce the evaporative demand on vineyards, by reducing the incoming solar radiation and the wind velocity.O objetivo deste trabalho foi avaliar os efeitos da cobertura de plástico sobre alguns elementos meteorológicos que formam o microclima de vinhedos de Vitis vinifera L., cultivar Moscato Giallo, em Flores da Cunha, na Serra Gaúcha. O experimento foi conduzido na safra 2005/2006, com os tratamentos: fileiras de plantas descobertas; e fileiras de plantas cobertas com plástico transparente tipo ráfia, com 160 µm de espessura. Medições contínuas de radiação fotossinteticamente ativa, temperatura e umidade do ar, e velocidade do vento foram realizadas: acima da cobertura; entre a cobertura e o dossel; sobre o dossel descoberto; e junto aos cachos de ambos tratamentos. Estimou-se a evapotranspiração de referência nos dois tratamentos. Da radiação fotossinteticamente ativa incidente, 67,5% chegou ao dossel coberto, 16% atingiu os cachos cobertos e 36% chegou aos cachos descobertos. A cobertura aumentou em 3,4°C as temperaturas máximas do ar junto às plantas. A umidade relativa do ar diurna foi menor, enquanto a pressão de vapor e o deficit de saturação foram superiores debaixo da cobertura. A velocidade do vento foi reduzida pela cobertura em 88%. No ambiente coberto, a evapotranspiração de referência foi 65% daquela do ambiente externo. Embora aumente as temperaturas diurnas, a cobertura de plástico promoveu redução na demanda evaporativa do vinhedo

Mitochondria and protein homeostasis in Parkinson's disease : from quality control pathways to an integrated network

Tese de doutoramento em Ciências da Saúde (Ciências Biomédicas), apresentada à Faculdade de Medicina da Universidade de CoimbraParkinson’s disease (PD) is the second most prevalent neurodegenerative disorder affecting almost 2% of people over 65 years of age. The primary neuropathological hallmark of PD is the degeneration of the nigrostriatal dopaminergic pathway which, by depleting dopamine in the brain, initiates abnormal motor symptoms including resting tremor, bradykinesia, postural instability, gait difficulty and rigidity. A second relevant hallmark is the presence of intracellular inclusion bodies, known as Lewy bodies that mainly contain aggregated α-synuclein. While the etiology involved in the development of PD is unknown, 90-95% of PD cases occurs sporadically and correlates, in part, with mitochondrial dysfunction and oxidative stress. Furthermore, the identification of single genes associated to the familial forms of PD has revolutionized this field of research, providing unique opportunities to pursue novel mechanisms and clues to the pathogenesis of PD. The sustained study of the cellular functions of each PD-related gene indicates that protein misfolding and aggregation, as well as, dysfunction of their quality control systems may play a crucial role in the cascade of deleterious events implicated in the neurodegenerative process of PD. Thus, clear insights into how mitochondrial dysfunction, oxidative stress and protein homeostasis systems interconnect, overlap or converge to produce nigral neuronal degeneration is essential to understand the pathogenesis of sporadic PD (sPD). In this thesis, we initially addressed the potential implications of an altered structural and functional crosstalk between mitochondria and the endoplasmic reticulum (ER), two important metabolic organelles for the maintenance of cellular protein homeostasis. We reported that mitochondrial dysfunction induced by an acute stimulus of the neurotoxin MPP+ renders cells more susceptible to develop an ER stress response. We found that MPP+ was able to evoke a sustained flux of Ca2+ from the ER to mitochondria which subsequently triggered ER- and mitochondria-dependent apoptotic pathways. Our findings highlight the inevitable role of ER to mitochondria Ca2+ fluxes and their requirement for a mitochondria-dependent cell death induction, enclosing a feedback loop whereas mitochondria signals ER and ER induces further mitochondrial alterations, leading to the activation of cascade of signals that culminates in apoptotic cell death. Furthermore, we also found that ER stress response strengthens mitochondrial stress-induced abnormalities. We demonstrated that sustained ER stress caused by accumulation of unfolded or misfolded proteins potentiated Ca2+ overload and impairment of mitochondrial function mainly characterized by dissipation of mitochondrial membrane potential and substantial decline in the mitochondrial respiratory chain complex I activity. These cumulative events led to induction of apoptotic cell death. In addition, we assessed the role of mitochondrial metabolism in the regulation of the autophagy-lysosomal pathway, a major cellular homeostatic process that mediates the degradation of long-lived proteins and dysfunctional or superfluous organelles in eukaryotic cells. We demonstrated that prolonged metabolic failure due to mitochondrial dysfunction, either in cellular models harboring sPD subjects mtDNA (sPD cybrids), cells depleted of all mtDNA (Rho0 cells), or in MPP+-treated rat cortical neurons causes a functional decline in the activity of the autophagic system. This defect is a consequence of alterations in microtubules (MT) assembly that hamper mitochondria and autophagosome transport along the MT network toward the lysosomal compartment. Consequently, deficient autophagic turnover potentiates the accumulation of α-synuclein oligomers and, ultimately, prompts apoptosis. Our data identify for the first time the PDassociated defects in mitochondrial dysfunction as the basis for the selective transport abnormalities and highly characteristic pattern of neuritic dystrophy associated to the autophagic pathology in PD. Finally, we dissected the molecular mechanisms by which mitochondrial metabolism in sPD can affect MT-directed autophagic turnover that, in turn, regulates intracellular protein homeostasis. We demonstrated that sirtuin 2 (SIRT2), a NAD+ dependent protein deacetylase, controls the functional ability of the autophagic system by modulating the acetylation status of the MT cytoskeleton. The studies presented here provide novel insights into the multiple mechanisms that dictate the association between mitochondria, intracellular metabolism and proteotoxicity in sPD, and contribute with new findings that could have important therapeutic implications to halt or retard the progression of sPD pathology.A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum, afectando cerca de 2% dos indivíduos com idade superior a 65 anos. A marca neuropatológica primária desta doença é a disfunção da via dopaminérgica nigroestriatal, da qual resulta a depleção de dopamina no cérebro, conduzindo aos principais sintomas motores que caracterizam a clínica da doença e que incluem tremor de repouso, bradicinesia, instabilidade postural e rigidez muscular. Outra característica neuropatológica da DP é a presença de corpos de inclusão intracelulares, designados de Corpos de Lewy, que são maioritariamente constituídos por α-synucleína na forma agregada. Embora a etiologia da doença permaneça por esclarecer, sabe-se que 90-95% dos casos de DP ocorrem de uma forma esporádica e pensa-se que, em parte, se correlacionam com a disfunção da mitocôndria e com o stresse oxidativo. A identificação de genes associados às formas familiares da DP revolucionou a forma de pensar dos investigadores da área, proporcionando oportunidades únicas para estudar novos mecanismos envolvidos na etiopatogenia da DP. O estudo continuado das funções celulares de cada um dos genes relacionados com a DP indica que o misfolding e a agregação proteica, bem como a disfunção dos sistemas de controlo de qualidade proteica, desempenham um papel crucial na cascata de eventos implicada no processo de neurodegeneração da DP. Assim, uma visão clara de como e quando a disfunção mitocondrial, o stresse oxidativo e os mecanismos de homeostase proteica se entrecruzam, sobrepõem ou convergem para produzir degeneração nigorestriatal é essencial para a compreensão da fisiopatologia da DP do tipo esporádico (sDP). Nesta tese, abordámos, inicialmente, as potenciais implicações das alterações na interação estrutural e funcional entre a mitocôndria e o retículo endoplasmático (RE), dois organelos importantes envolvidos na manutenção da homeostase proteica celular. Reportamos que a disfunção mitocondrial induzida por um estímulo agudo da neurotoxina MPP+ aumenta a susceptibilidade das células para o desenvolvimento de uma resposta ao stresse do RE. Verificámos que o MPP+ foi capaz de evocar um fluxo contínuo de Ca2+ entre o RE e a mitocôndria, desencadeando, subsequentemente, a activação de vias apoptóticas dependentes do RE e da mitocôndria. Os resultados obtidos destacam o papel inevitável dos fluxos de Ca2+ entre o RE e a mitocôndria, e a sua relevância para a indução de morte celular dependente da mitocôndria, estabelecendo-se um ciclo de retrocontrolo em que a mitocôndria sinaliza o RE e o RE induz alterações posteriores na mitocôndria, conduzindo à activação de uma cascata de sinalização que culmina na morte celular por apoptose. Do mesmo modo, observámos que o stresse do RE potencia alterações ao nível da função mitocôndrial. Demonstramos que stresse do RE prolongado devido à acumulação de proteínas mal processadas no seu interior conduz a um fluxo excessivo de Ca2+ para o interior mitocôndria e a um comprometimento da função mitocondrial, caracterizada principalmente pela dissipação do potencial de membrana e por um decréscimo substancial da actividade da cadeia respiratória mitocondrial. Cumulativamente, estes eventos desencadeam a indução de morte celular por apoptose. Por outro lado, avaliámos o papel do metabolismo mitocondrial na regulação da via autofágica-lisossomal, um processo homeostático celular que medeia a degradação de proteínas de longa duração e organelos disfuncionais em células eucarióticas. Demonstramos que uma falência metabólica prolongada devido a disfunção mitocondrial quer em células híbridas portadoras de ADN mitocondrial de doentes de sDP (cíbridos sDP), em células depletadas do seu ADN mitocondrial (células Rho0) ou em culturas primárias de neurónios corticais de rato tratados com MPP+, provoca um declínio funcional na actividade do sistema autofágico. Este defeito é uma consequência de alterações estruturais na rede microtubular (MT), comprometendo o transporte de mitocôndrias e autofagossomas para o compartimento lisossomal, onde ocorre degradação. Consequentemente, um ineficaz turnover autofágico potencia a acumulação de oligómeros de α-sinucleína e, em última análise, desencadeia apoptose. Assim, os dados obtidos identificam pela primeira vez os defeitos na função mitocondrial associados à DP como a base para alterações específicas no transporte intracelular e no padrão característico da distrofia neurítica associado à patologia autofágica na DP. Por fim, analisámos os mecanismos moleculares através dos quais o metabolismo mitocondrial em sDP pode afectar o turnover autofágico dependente da rede MT, o qual, por sua vez, regula a homeostase proteica intracelular. Demonstramos que a sirtuina 2 (SIRT2), uma desacetilase citoplasmática dependente do NAD+, controla a capacidade funcional do sistema autofágico, modulando o estado de acetilação do MTs. Os estudos apresentados fornecem novas pistas relativamente aos múltiplos mecanismos que determinam a associação entre mitocôndria, metabolismo intracelular e proteotoxicidade e contribuem com novas perspectivas que poderão ter importantes implicações terapêuticas no sentido impedir ou retardar a progressão da sPD.O trabalho apresentado nesta tese foi financiado pelo projecto PTDC/SAUNEU/102710/2008 concedido à Professora Doutora Sandra Morais Cardoso pela Fundação para a Ciência e Tecnologia (FCT-MEC, Portugal). A autora usufruiu de uma bolsa de Doutoramento (SFRHD/BD/38743/2007) concedida pela FCT-MEC, Portuga

Manutenção do campo de golfe. A importância da sua gestão ambiental

Mestrado em Arquitectura Paisagista - Instituto Superior de AgronomiaThe evolution of the golf courses design has neglected the initial concept of sustainability, due to the emergence of a style that ignored the natural shape and the existing forms of the land. Such style has been considered responsible for the increase of the environmental impacts associated with maintenance activities. The current challenge is to make golf more sustainable by applying a proper environmental management, which plays a vital role and is essential to its success, contributing to the achievement of environmental and economic goals. In this thesis one aims to demonstrate the importance of environmental management in maintaining a golf course. ISO 14001 concepts and GEO requirements will be applied in the management of the Oeiras golf course. The use of GEO OnCourseTM standards, specific for golf courses, will allow the certification of the implemented procedures. Collectively, the use of these strategies will allow the reduction of environmental impacts, in addition to enhancing the preservation of natural areas, protection of soil and water resources, environmental awareness of golfers and the general public, and the minimization of maintenance costs