Publicação
The role of LRRK2 in Parkinson’s disease : from function to dysfunction
| Resumo: | Parkinson’s disease (PD) belongs to the group of neurodegenerative disorders and it is currently considered the most common progressive movement disorder. Neurodegenerative disorders, such as Alzheimer’s, Huntington’s, frontotemporal dementia and amyotrophic lateral sclerosis, share several dysfunctional molecular pathways and impairments in basic cell mechanisms. Despite intense efforts to understand to decipherthe triggers underlying these disorders, to date, there is no effective cure. This results in a growing number of cases and, consequently, in a complex social and economic problem. Therefore, it is of extreme importance to understand the common biological mechanisms involved in the pathogenesis of this devastating group of diseases, in order to develop effective therapies. The majority of the PD cases are sporadic, however, in the last decades, it has been recognized that rare genetic mutations are patholgical for PD in a number of inherited cases. Futhermore, these mutations can be as well a risk factor for sporadic PD, supporting the idea that familial and sporadic PD can share common pathlogical mechanisms. This study focused on a key player protein in PD, Leucine-rich repeat kinase 2 (LRRK2). Mutations in LRRK2 gene are the most frequent cause of autosomal dominant forms of PD and they are also consider a risck factor for sporadic cases. A central catalytic GTPase and kinase core, flanked by protein interaction domains, composes this large and complex multi-domain protein. The most frequent LRRK2 PD-related mutation occurs at the animoacid 2019, a glycine subtitution for a serine (G2019S), precisely on the kinase domain of the protein resulting in its toxic gain of function. LRRK2 is known to play a role in distinct cellular mechanisms such as vesicular trafficking, microtubule network regulation and mitochondrial morphology. However, the function of LRRK2 in these important mechanisms and their related pathways is not fully understood, which is crucial for developing new therapeutic targets. Here, we investigated LRRK2 function by characterizing/identifying its protein interactors and, in particular, by exploring its relationship with two central proteins in neurodegenerative disorders, α-synuclein and Tau. In PD brain samples, we show that levels of LRRK2 are positively correlated to an increase in α-synuclein phosphorylation and aggregation in affected brain regions, where both proteins co-localize in neurons and Lewy body inclusions. In a cell line model, this co-localization also occurs in α-synuclein inclusions and knocking down LRRK2 promotes formation of smaller inclusions. Moreover, we show an interaction between α-synuclein and LRRK2 under endogenous and over-expression conditions. These results shed light on the complex interaction of these two central PD proteins and, in particular, on underlying molecular mechanisms involved in a disease scenario. Furthermore, we demonstrate that LRRK2 also interacts with Tau protein in a cell line model, in which co-expression of both proteins promotes accumulation of Tau protein. This accumulation occurs independently of LRRK2 kinase activity and it gives rise to formation of high molecular weight Tau species and increased levels of Tau secretion. Moreover, we suggest that these effects are a consequence of an impairment of proteasomal Tau degradation and that this impairment is promoted by LRRK2. Consistently, a LRRK2-knockout mouse displayed lower levels of Tau in the brain, when compared with transgenic animals expressing human wild-type LRRK2. Our results highlight the compromised status of cellular and molecular neurodegenerative mechanisms. The identification of LRRK2 interactors is crucial to placing the protein in known biochemical pathways. To that end, we performed a screen to identify LRRK2-interacting proteins. The results obtained confirmed that this is a multifaceted protein, involved in a variety of molecular functions and biochemical pathways. α-synuclein and Tau are two proteins present in the list of interactors, which validates previously reported results. The role of LRRK2 on the cytoskeleton is also highlighted by the presence of several protein interactors linked to microtubule dynamics, which lead us to explore the effect of LRRK2 on mechanical properties of the cell. Applying a combined microscopy tecniques in cell indentation experiments, we confirmed that different distribution patterns of LRRK2 result in differential states of cell stiffness. We found that the stiffest cells exhibit a diffuse pattern of LRRK2 distribution, such that LRRK2 is dispersed throughout the entire cell, interacting with microtubule-related proteins and compromising cytoskeletal dynamics. The identification of novel interactos resulted in a better understanding of LRRK2 patho-physiological role. Taken together, our results presented in this thesis provide novel insight into the function of LRRK2 and its particular role in neurodegenerative diseases. Ultimately, this knowledge is essential for the understanding of the molecular underpinnings of PD and for the development of novel therapeutics. |
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| Autores principais: | Guerreiro, Patricia I. da Silva |
| Assunto: | LRRK2 Doença de Parkinson a-sinucleína Tau Interação proteica Teses de doutoramento - 2015 |
| Ano: | 2015 |
| 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: | Parkinson’s disease (PD) belongs to the group of neurodegenerative disorders and it is currently considered the most common progressive movement disorder. Neurodegenerative disorders, such as Alzheimer’s, Huntington’s, frontotemporal dementia and amyotrophic lateral sclerosis, share several dysfunctional molecular pathways and impairments in basic cell mechanisms. Despite intense efforts to understand to decipherthe triggers underlying these disorders, to date, there is no effective cure. This results in a growing number of cases and, consequently, in a complex social and economic problem. Therefore, it is of extreme importance to understand the common biological mechanisms involved in the pathogenesis of this devastating group of diseases, in order to develop effective therapies. The majority of the PD cases are sporadic, however, in the last decades, it has been recognized that rare genetic mutations are patholgical for PD in a number of inherited cases. Futhermore, these mutations can be as well a risk factor for sporadic PD, supporting the idea that familial and sporadic PD can share common pathlogical mechanisms. This study focused on a key player protein in PD, Leucine-rich repeat kinase 2 (LRRK2). Mutations in LRRK2 gene are the most frequent cause of autosomal dominant forms of PD and they are also consider a risck factor for sporadic cases. A central catalytic GTPase and kinase core, flanked by protein interaction domains, composes this large and complex multi-domain protein. The most frequent LRRK2 PD-related mutation occurs at the animoacid 2019, a glycine subtitution for a serine (G2019S), precisely on the kinase domain of the protein resulting in its toxic gain of function. LRRK2 is known to play a role in distinct cellular mechanisms such as vesicular trafficking, microtubule network regulation and mitochondrial morphology. However, the function of LRRK2 in these important mechanisms and their related pathways is not fully understood, which is crucial for developing new therapeutic targets. Here, we investigated LRRK2 function by characterizing/identifying its protein interactors and, in particular, by exploring its relationship with two central proteins in neurodegenerative disorders, α-synuclein and Tau. In PD brain samples, we show that levels of LRRK2 are positively correlated to an increase in α-synuclein phosphorylation and aggregation in affected brain regions, where both proteins co-localize in neurons and Lewy body inclusions. In a cell line model, this co-localization also occurs in α-synuclein inclusions and knocking down LRRK2 promotes formation of smaller inclusions. Moreover, we show an interaction between α-synuclein and LRRK2 under endogenous and over-expression conditions. These results shed light on the complex interaction of these two central PD proteins and, in particular, on underlying molecular mechanisms involved in a disease scenario. Furthermore, we demonstrate that LRRK2 also interacts with Tau protein in a cell line model, in which co-expression of both proteins promotes accumulation of Tau protein. This accumulation occurs independently of LRRK2 kinase activity and it gives rise to formation of high molecular weight Tau species and increased levels of Tau secretion. Moreover, we suggest that these effects are a consequence of an impairment of proteasomal Tau degradation and that this impairment is promoted by LRRK2. Consistently, a LRRK2-knockout mouse displayed lower levels of Tau in the brain, when compared with transgenic animals expressing human wild-type LRRK2. Our results highlight the compromised status of cellular and molecular neurodegenerative mechanisms. The identification of LRRK2 interactors is crucial to placing the protein in known biochemical pathways. To that end, we performed a screen to identify LRRK2-interacting proteins. The results obtained confirmed that this is a multifaceted protein, involved in a variety of molecular functions and biochemical pathways. α-synuclein and Tau are two proteins present in the list of interactors, which validates previously reported results. The role of LRRK2 on the cytoskeleton is also highlighted by the presence of several protein interactors linked to microtubule dynamics, which lead us to explore the effect of LRRK2 on mechanical properties of the cell. Applying a combined microscopy tecniques in cell indentation experiments, we confirmed that different distribution patterns of LRRK2 result in differential states of cell stiffness. We found that the stiffest cells exhibit a diffuse pattern of LRRK2 distribution, such that LRRK2 is dispersed throughout the entire cell, interacting with microtubule-related proteins and compromising cytoskeletal dynamics. The identification of novel interactos resulted in a better understanding of LRRK2 patho-physiological role. Taken together, our results presented in this thesis provide novel insight into the function of LRRK2 and its particular role in neurodegenerative diseases. Ultimately, this knowledge is essential for the understanding of the molecular underpinnings of PD and for the development of novel therapeutics. |
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