ATP13A2 and alpha-synuclein: a metal taste in autophagy

© The Korean Society for Brain and Neural Sciences. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Parkinson's Disease (PD) is a complex and multifactorial disorder of both idiopathic and genetic origin. Thus far, more than 20 genes have been linked to familial forms of PD. Two of these genes encode for ATP13A2 and alpha-synuclein (asyn), proteins that seem to be members of a common network in both physiological and disease conditions. Thus, two different hypotheses have emerged supporting a role of ATP13A2 and asyn in metal homeostasis or in autophagy. Interestingly, an appealing theory might combine these two cellular pathways. Here we review the novel findings in the interaction between these two proteins and debate the exciting roads still ahead.TLF is supported by Fundação para a Ciência e Tecnologia (SFRH/BD/74881/2010). TFO is supported by the DFG Center for Nanoscale Microscopy and Molecular Physiology of the Braininfo:eu-repo/semantics/publishedVersio

Zebrafish : a new model of Parkinson's disease

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2010Parkinson´s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease, affecting around 2% of the population with more than 65 years. The first gene implicated with PD was SNCA that encodes to alpha-synuclein. At the neuronal level, this protein was found in the Lewy Bodies, one of the pathologic hallmarks of the disease and it has been identified both in sporadic and familiar cases of PD. Its role in the disease is still unclear. Besides alpha-synuclein, other 15 genetic loci have been associated to PD. To clarify the role of alpha-synuclein in PD, several in vivo models have been developed, expressing wild-type and PD-associated mutant forms of the protein. However, none of the existing models fully recapitulates all of the hallmarks of the disease. To circumvent this limitation, new models must be developed. Zebrafish has been widely used as an animal model for studying several human disorders because, besides being a vertebrate, it has all the molecular background and bioimaging characteristics suitable for these studies. Here, we started to generate a Zebrafish transgenic model for human alpha-synuclein. In addition, through bioinformatic analysis, we found that four PD related genes encoding ATP13a2, Lrrk2, Synphilin-1, and Glucocerebrosidase, display high homology rates when compared to the human gene sequences. Moreover, despite a high homology in the entire sequence, specific conserved domains sequences are also highly homologous, suggesting a conserved functionality of the genes. In situ hybridization of ATP13a2 revealed that, although initially expressed in whole embryo’s body, it becomes more restricted to the brain area along the embryonic development. The generation of this novel PD transgenic model will allow further studies to better understand the mechanisms underlying the disease pathology. Taken together, these results will provide a novel and powerful system to explore more the molecular basis of PD and to open novel avenues for therapeutic intervention

The interplay between alpha-synuclein and ATP13A2 : towards the understanding of the molecular basis of Parkinson’s disease

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2016Parkinson’s disease (PD) is the most common neurodegenerative disorder with motor impairment. While PD is clinically well characterized, the molecular and cellular basis underlying both the onset and progression of the disorder are still unknown. Gaining deeper knowledge on PD pathophysiology has been hindered by the fact that only a minority of PD patients have a defined genetic cause, with the remaining 90% of the cases being classified as sporadic. Thus far, mutations in more than 20 genes are considered risk factors for developing PD. These PDrelated genes are linked to several distinct intracellular pathways, hardening the quest to pinpoint the exact molecular imbalance responsible for PD onset. One of these pathways, the Endolysosomal, has recently gained notoriety due to its importance in alpha-Synuclein homeostasis (α-Syn). α-Syn is a small protein with unknown function and, perhaps, the most extensively studied in PD context. Several point mutations and gene multiplications in α-Syn gene, SNCA, have been linked to PD and the protein is found in Lewy Bodies (LB), the pathological hallmark of the disease. In the Endolysosomal machinery another PD-associated protein has been under the spotlight: ATP13A2. This protein is a transmembrane ATPase located at the late endosomes and lysosomes, with yet unknown function, that is also present in LB. In the last years important steps have been taken towards understanding the interplay between ATP13A2 and α-Syn, with contradictory results reported. Inarguable though is that ATP13A2 can, at least partially, affect the intracellular fate of α-Syn. Our work confirms that the Endolysosomal pathway plays an important role in α-Syn homeostasis. We start by showing that two proteins members of this pathway, Raba8a and ATP13A2, could alter α-Syn aggregation in a well-established cellular model. Nevertheless, while we found that Raba8a exerts its effect by direct binding to α-Syn, the PD-associated protein ATP13A2 may affect specific cellular mechanisms. We describe that, in human cells, a mutation in ATP13A2, a duplication of 22 base pairs (Dup22), enhances α-Syn aggregation and increases its resistance to proteinase K digestion. The mutated protein could also promote the formation of oligomers and higher α-Syn molecular weight species. In addition, the dynamics between α-Syn and ATP13A2 Dup22 can severely impact cellular homeostasis. Here we report that α-Syn and ATP13A2 Dup22 can be found in a reticular, membranar structure, which we found to be composed by endoplasmic reticulum (ER). This alteration in the ER morphology was correlated with an unmitigated increase in ER stress that culminates with the activation of apoptotic pathways and cell death. Besides ER changes we also identified significant alterations in mitochondria morphology, along with increased susceptibility to oxidative stress. Altogether our work provides novel insights into the effect of Endolysosomal proteins on α-Syn. Additionally we describe that the interaction of two PDassociated proteins, ATP13A2 and α-Syn, may trigger a cascade of deleterious intracellular events that involve ER stress and mitochondria alterations, and ultimately lead to cell death. Most importantly, our findings shed new light on the cellular dyshomeostasis that may underlie the development of PD.A doença de Parkinson (PD) é a condição neurodegenerativa com sintomas motores mais comum. Embora o quadro clínico da doença esteja bem caracterizado, os mecanismos moleculares e celulares responsáveis pelo seu aparecimento e progressão são ainda desconhecidos. O estudo aprofundado sobre a patofisiologia de PD tem sido dificultado pelo escasso número de pacientes que apresentam uma causa genética definida, sendo que aproximadamente 90% dos casos são classificados como esporádicos. Atualmente, um total de 20 genes são considerados factores de risco para o aparecimento de PD. O envolvimento desses genes em diferentes mecanismos intracelulares confere complexidade adicional à investigação da causa da doença. Um desses mecanismos é a via endo-lisossomal, a qual tem recentemente ganho relevo devido à sua importância na homeostasia da proteína alpha-sinucleina (α-Syn). A α-Syn é uma proteína com função desconhecida e, provavelmente, a mais estudada no contexto de PD. Várias mutações e multiplicações no gene da α-Syn, SNCA, foram descritas em doentes com PD e a proteína está presente nos Corpos de Lewy (LBs), considerados um dos principais marcos histopatológicos da doença. A via endo-lisossomal é regulada por várias proteínas, sendo que a ATP13A2 tem merecido destaque por estar geneticamente associada a PD. Esta proteína é uma ATPase transmembranar com localização nos endossomas e lisossomas. À semelhança da α-Syn, a ATP13A2 é também encontrada nos LBs e a sua função é ainda desconhecida. Nos últimos anos têm sido dados passos importantes para melhor compreender a interação entre estas duas proteínas, embora resultados contraditórios tenham sido descritos. Apesar de algumas discrepâncias, é indiscutível que a ATP13A2 afecta, pelo menos parcialmente, o destino intracelular da α-Syn. O nosso estudo confirma que a via endo-lisossomal desempenha um papel crucial na homeostasia da α-Syn. Inicialmente, mostramos que duas proteínas que intervêm nesta via, ATP13A2 e Raba8a, têm a capacidade de alterar a formação de inclusões intracelulares de α-Syn. No entanto, embora a Raba8a exerça o seu efeito através de uma interação direta com a α-Syn os nossos resultados indicam que a ATP13A2 afecta mecanismos intracelulares específicos. Em células humanas, uma mutação familiar na ATP13A2, que consiste na duplicação de 22 pares bases no gene (Dup22), aumenta a formação de inclusões de α-Syn e a sua resistência à digestão com proteinase K. Esta proteína mutada é também capaz de promover a formação de oligómeros e espécies de peso molecular superior de α-Syn. Paralelamente, a dinâmica de interação entre α-Syn e ATP13A2 Dup22 tem um efeito severo na homeostasia celular. Neste trabalho reportamos que a α-Syn e a ATP13A2 Dup22 co-localizam em estruturas reticulares membranosas, as quais descobrirmos serem compostas por reticulo endoplasmático (ER). Esta alteração da morfologia do ER está associada a um aumento do stress do ER, que culmina na ativação de mecanismos apoptóticos e morte celular. Para além do ER, também observámos alterações significativas na morfologia da mitocôndria acompanhada por um aumento de susceptibilidade a stress oxidativo. Em resumo, estes resultados oferecem novos conhecimentos sobre o efeito de proteínas associadas à via endo-lisossomal na homeostasis da α-Syn. Adicionalmente descrevemos que a interação de duas proteínas associadas a PD, ATP13A2 e α-Syn, ativa uma cascata perniciosa de eventos intracelulares que envolvem stress do ER e alterações na mitocôndria, e culminam em morte celular. Mais importante, estas descobertas fornecem uma melhor compreensão dos mecanismos celulares que poderão influenciar o aparecimento da doença de PD

Fasudil attenuates aggregation of α-synuclein in models of Parkinson’s disease

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, yet disease-modifying treatments do not currently exist. Rho-associated protein kinase (ROCK) was recently described as a novel neuroprotective target in PD. Since alpha-synuclein (alpha-Syn) aggregation is a major hallmark in the pathogenesis of PD, we aimed to evaluate the anti-aggregative potential of pharmacological ROCK inhibition using the isoquinoline derivative Fasudil, a small molecule inhibitor already approved for clinical use in humans. Fasudil treatment significantly reduced alpha-Syn aggregation in vitro in a H4 cell culture model as well as in a cell-free assay. Nuclear magnetic resonance spectroscopy analysis revealed a direct binding of Fasudil to tyrosine residues Y133 and Y136 in the C-terminal region of alpha-Syn. Importantly, this binding was shown to be biologically relevant using site-directed mutagenesis of these residues in the cell culture model. Furthermore, we evaluated the impact of long-term Fasudil treatment on alpha-Syn pathology in vivo in a transgenic mouse model overexpressing human alpha-Syn bearing the A53T mutation (alpha-Syn(A53T) mice). Fasudil treatment improved motor and cognitive functions in alpha-Syn(A53T) mice as determined by Catwalk (TM) gait analysis and novel object recognition (NOR), without apparent side effects. Finally, immunohistochemical analysis revealed a significant reduction of alpha-Syn pathology in the midbrain of alpha-Syn(A53T) mice after Fasudil treatment. Our results demonstrate that Fasudil, next to its effects mediated by ROCK-inhibition, directly interacts with alpha-Syn and attenuates alpha-Syn pathology. This underscores the translational potential of Fasudil as a disease-modifying drug for the treatment of PD and other synucleinopathies

Compilation of parameterized seismogenic sources in Iberia for the SHARE European-scale seismic source model.

Abstract: SHARE (Seismic Hazard Harmonization in Europe) is an EC-funded project (FP7) that aims to evaluate European seismic hazards using an integrated, standardized approach. In the context of SHARE, we are compiling a fully-parameterized active fault database for Iberia and the nearby offshore region. The principal goal of this initiative is for fault sources in the Iberian region to be represented in SHARE and incorporated into the source model that will be used to produce seismic hazard maps at the European scale. The SHARE project relies heavily on input from many regional experts throughout the Euro-Mediterranean region. At the SHARE regional meeting for Iberia, the 2010 Working Group on Iberian Seismogenic Sources (WGISS) was established; these researchers are contributing to this large effort by providing their data to the Iberian regional integrators in a standardized format. The development of the SHARE Iberian active fault database is occurring in parallel with IBERFAULT, another ongoing effort to compile a database of active faults in the Iberian region. The SHARE Iberian active fault database synthesizes a wide range of geological and geophysical observations on active seismogenic sources, and incorporates existing compilations (e.g., Cabral, 1995; Silva et al., 2008), original data contributed directly from researchers, data compiled from the literature, parameters estimated using empirical and analytical relationships, and, where necessary, parameters derived using expert judgment. The Iberian seismogenic source model derived for SHARE will be the first regional-scale source model for Iberia that includes fault data and follows an internationally standardized approach (Basili et al., 2008; 2009). This model can be used in both seismic hazard and risk analyses and will be appropriate for use in Iberian- and European-scale assessments

The Interplay between Alpha-Synuclein Clearance and Spreading

Parkinson’s Disease (PD) is a complex neurodegenerative disorder classically characterized by movement impairment. Pathologically, the most striking features of PD are the loss of dopaminergic neurons and the presence of intraneuronal protein inclusions primarily composed of alpha-synuclein (α-syn) that are known as Lewy bodies and Lewy neurites in surviving neurons. Though the mechanisms underlying the progression of PD pathology are unclear, accumulating evidence suggests a prion-like spreading of α-syn pathology. The intracellular homeostasis of α-syn requires the proper degradation of the protein by three mechanisms: chaperone-mediated autophagy, macroautophagy and ubiquitin-proteasome. Impairment of these pathways might drive the system towards an alternative clearance mechanism that could involve its release from the cell. This increased release to the extracellular space could be the basis for α-syn propagation to different brain areas and, ultimately, for the spreading of pathology and disease progression. Here, we review the interplay between α-syn degradation pathways and its intercellular spreading. The understanding of this interplay is indispensable for obtaining a better knowledge of the molecular basis of PD and, consequently, for the design of novel avenues for therapeutic intervention

The interplay between alpha-Synuclein clearance and spreading

Parkinson's Disease (PD) is a complex neurodegenerative disorder classically characterized by movement impairment. Pathologically, the most striking features of PD are the loss of dopaminergic neurons and the presence of intraneuronal protein inclusions primarily composed of alpha-synuclein (alpha-syn) that are known as Lewy bodies and Lewy neurites in surviving neurons. Though the mechanisms underlying the progression of PD pathology are unclear, accumulating evidence suggests a prion-like spreading of alpha-syn pathology. The intracellular homeostasis of alpha-syn requires the proper degradation of the protein by three mechanisms: chaperone-mediated autophagy, macroautophagy and ubiquitin-proteasome. Impairment of these pathways might drive the system towards an alternative clearance mechanism that could involve its release from the cell. This increased release to the extracellular space could be the basis for alpha-syn propagation to different brain areas and, ultimately, for the spreading of pathology and disease progression. Here, we review the interplay between alpha-syn degradation pathways and its intercellular spreading. The understanding of this interplay is indispensable for obtaining a better knowledge of the molecular basis of PD and, consequently, for the design of novel avenues for therapeutic intervention.publishersversionpublishe