Publicação
The role of alpha-synuclein phosphorylation in synucleinopathies
| Resumo: | Alpha-synuclein (aSyn) is a pre-synaptic protein linked to Parkinson’s disease (PD) both by genetic and pathological evidence. The gene encoding for aSyn was the first to be associated with familial forms of PD. In concert, aSyn was identified as the main component of Lewy Bodies (LBs), one of the pathological hallmarks of PD along with the loss of dopaminergic neurons from the substantia nigra. The discovery that the same protein –aSyn- and its aggregation propensity were involved in the pathogenesis of both sporadic and genetic PD cases propelled the aSyn research field. Remarkably, further findings characterize a range of diseases with parkinsonian features and presenting aSyn aggregates, the so-called synucleinopathies. These include amongst others PD, Parkinsons’s disease with dementia, dementia with Lewy Bodies (LBs), and Multiple System Athrophy (MSA). Nonetheless, the exact mechanisms responsible for aSyn aggregation and toxicity still remain unknown. Try to understand the molecular pathways behind aSyn misfolding properties is crucial to shed light into the neurodegeneration process, and in the search for therapeutic treatments that may alleviate the social burden of PD and related diseases. This thesis focuses on the study of the role of aSyn phosphorylation, a post-translational modification (PTM) that was shown to be essential in modulating aSyn function, aggregation and toxicity. aSyn is indeed phosphorylated on serine 129 (Ser-129) in physiological conditions, phosphorylation that goes awry during the pathogenesis of the disease resulting in 90% of aSyn phosphorylated in the LBs of brain from patients and transgenic animals models of PD. The importance of this modification became soon specific since antibodies used against this residue were adopted as common staining procedure for LBs structures. Recently, other phosphorylation sites have received intense attention; in particular tyrosine 125 (Tyr-125) phosphorylation levels were shown to be significantly reduced in diseased brains. Despite the great effort to discover the kinases mediating aSyn phosphorylation; the contribution of this modification to aSyn toxicity and aggregation remains elusive. This is primarily due to the usage of several different methodological approaches, the difficulty to study in vivo such a rapid and reversible modification and the absence of phosphorylation mutants’ forms that can properly mimic the effect of this PTM. For these reasons, the purpose of this thesis was to investigate the pathways involved in aSyn phosphorylation using a simple, effective and well-established model for neurodegenerative diseases: the yeast Saccharomyces cerevisiae. This model has been extensively used to identify several pathways involved in PD and to generate a consistent model for aSyn aggregation that mimics the disease one. Therefore, we took advantage of the power of yeast to investigate the effects of aSyn phosphorylation on Ser-129 co-expressing human aSyn and known kinases able to phosphorylate it; namely the Polo-like kinases family (PLKs). We studied aSyn inclusions formation and toxicity and we then proceeded to validate the results in established mammalian cell model of PD. Our approach demonstrated a unique role for one of the member of the kinases family -PLK2- in aSyn inclusion formation. We showed that PLK2 expression and aSyn phosphorylation on Ser-129 are both required for aSyn inclusion formation. The role of aSyn Tyr-125 phosphorylation was then studied in an established oligodendroglial model of MSA. We demonstrated that this modification can prevent aSyn aggregation propensity, but only when phosphorylation on Ser-129 occurred. We then identified novel tyrosine kinases inhibitors that may be involved in the disease, but further experimental data will elucidate these potential targets. Furthermore, using yeast we implemented a functional screening and characterized novel kinases, but most importantly, new pathways involved in aSyn pathobiology. ATG1, a kinase involved in autophagy, represented one of the major modulator of aSyn aggregation and toxicity in yeast; however this novel player requires further validation in mammalian systems to establish its potential for therapeutic purposes. Altogether, these results provide novel insights and implications for the function of aSyn phosphorylation in PD: a co-operative role for serine and tyrosine residue embodies a novel clue to unravel aSyn misfolding behavior. Our data represent a unique opportunity to tackle one side of aSyn phosphorylation function ad further validate the importance of this PTM in aSyn pathobiology. |
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| Autores principais: | Basso, Elisa, 1983- |
| Assunto: | Alfa-sinucleína Fosforilação Doença de Parkinson Doenças neurodegenerativas Neurociências Teses de doutoramento - 2014 |
| Ano: | 2014 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Alpha-synuclein (aSyn) is a pre-synaptic protein linked to Parkinson’s disease (PD) both by genetic and pathological evidence. The gene encoding for aSyn was the first to be associated with familial forms of PD. In concert, aSyn was identified as the main component of Lewy Bodies (LBs), one of the pathological hallmarks of PD along with the loss of dopaminergic neurons from the substantia nigra. The discovery that the same protein –aSyn- and its aggregation propensity were involved in the pathogenesis of both sporadic and genetic PD cases propelled the aSyn research field. Remarkably, further findings characterize a range of diseases with parkinsonian features and presenting aSyn aggregates, the so-called synucleinopathies. These include amongst others PD, Parkinsons’s disease with dementia, dementia with Lewy Bodies (LBs), and Multiple System Athrophy (MSA). Nonetheless, the exact mechanisms responsible for aSyn aggregation and toxicity still remain unknown. Try to understand the molecular pathways behind aSyn misfolding properties is crucial to shed light into the neurodegeneration process, and in the search for therapeutic treatments that may alleviate the social burden of PD and related diseases. This thesis focuses on the study of the role of aSyn phosphorylation, a post-translational modification (PTM) that was shown to be essential in modulating aSyn function, aggregation and toxicity. aSyn is indeed phosphorylated on serine 129 (Ser-129) in physiological conditions, phosphorylation that goes awry during the pathogenesis of the disease resulting in 90% of aSyn phosphorylated in the LBs of brain from patients and transgenic animals models of PD. The importance of this modification became soon specific since antibodies used against this residue were adopted as common staining procedure for LBs structures. Recently, other phosphorylation sites have received intense attention; in particular tyrosine 125 (Tyr-125) phosphorylation levels were shown to be significantly reduced in diseased brains. Despite the great effort to discover the kinases mediating aSyn phosphorylation; the contribution of this modification to aSyn toxicity and aggregation remains elusive. This is primarily due to the usage of several different methodological approaches, the difficulty to study in vivo such a rapid and reversible modification and the absence of phosphorylation mutants’ forms that can properly mimic the effect of this PTM. For these reasons, the purpose of this thesis was to investigate the pathways involved in aSyn phosphorylation using a simple, effective and well-established model for neurodegenerative diseases: the yeast Saccharomyces cerevisiae. This model has been extensively used to identify several pathways involved in PD and to generate a consistent model for aSyn aggregation that mimics the disease one. Therefore, we took advantage of the power of yeast to investigate the effects of aSyn phosphorylation on Ser-129 co-expressing human aSyn and known kinases able to phosphorylate it; namely the Polo-like kinases family (PLKs). We studied aSyn inclusions formation and toxicity and we then proceeded to validate the results in established mammalian cell model of PD. Our approach demonstrated a unique role for one of the member of the kinases family -PLK2- in aSyn inclusion formation. We showed that PLK2 expression and aSyn phosphorylation on Ser-129 are both required for aSyn inclusion formation. The role of aSyn Tyr-125 phosphorylation was then studied in an established oligodendroglial model of MSA. We demonstrated that this modification can prevent aSyn aggregation propensity, but only when phosphorylation on Ser-129 occurred. We then identified novel tyrosine kinases inhibitors that may be involved in the disease, but further experimental data will elucidate these potential targets. Furthermore, using yeast we implemented a functional screening and characterized novel kinases, but most importantly, new pathways involved in aSyn pathobiology. ATG1, a kinase involved in autophagy, represented one of the major modulator of aSyn aggregation and toxicity in yeast; however this novel player requires further validation in mammalian systems to establish its potential for therapeutic purposes. Altogether, these results provide novel insights and implications for the function of aSyn phosphorylation in PD: a co-operative role for serine and tyrosine residue embodies a novel clue to unravel aSyn misfolding behavior. Our data represent a unique opportunity to tackle one side of aSyn phosphorylation function ad further validate the importance of this PTM in aSyn pathobiology. |
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