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Assessment of protein aggregation using super-resolution microscopy
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Resumo(s)
O misfolding e a agregação de diversas proteínas estão frequentemente relacionados com o
aparecimento de determinadas doenças neurodegenerativas, como a doença de Parkinson e a doença de
Huntington. Todavia, ainda não é claro se a agregação das proteínas é uma causa ou consequência do
processo, nem o processo de agregação proteica no contexto celular é totalmente compreendido.
Técnicas inovadoras que permitam adquirir novos conhecimentos sobre como as proteínas
sofrem misfold e agregam são, portanto, de extrema importância para encontrar novas terapias e
tratamentos para estas doenças. A microscopia de super-resolução é uma destas técnicas, uma vez que
permite uma melhor análise da formação de agregados proteicos, em comparação com técnicas
convencionais.
Neste contexto, o objetivo principal deste projeto consistiu em implementar técnicas de
microscopia de super-resolução, nomeadamente microscopia de stimulated emission depletion (STED),
para analisar agregados intracelulares de proteínas relacionadas com doenças neurodegenerativas. Mais
concretamente, foram analisados agregados de alfa-sinucleína (aSyn), relacionada com a doença de
Parkinson, e de huntingtina (Htt), relacionada com a doença de Huntington. Durante o decorrer desta
investigação, foi ainda descoberta uma nova mutação para a-synucleína (K58N), a qual foi também
alvo de estudo.
Para realização deste projeto, células humanas de neuroglioma (células H4), foram transfetadas
com i) uma forma da proteína aSyn que possui uma proteína verde fluorescente melhorada (enhanced
green fluorescent protein – EGFP) truncada no seu C-terminal, a qual se designa SynT, para estudar
os agregados de aSyn; ii) uma forma da proteína huntigtina com 104 repetições de glutamina na sua
cauda de poliglutamina, para estudar a agregação de huntingtina (104QHtt-EGFP).
Durante esta investigação, foram realizadas técnicas de imunocitoquímica e os agregados
formados pelas proteínas foram estudados usando microscopia de fluorescência e microscopia de
superresolução. Foram ainda feitas análises de immunoblotting (western blot) para quantificar o nível
de expressão das proteínas em estudo.
Técnicas de imunocitoquímica foram usadas para estudar as inclusões que resultam do modelo
SynT + Sph-1-v5 e da expressão de 104QHtt-EGFP. As células transfetadas foram classificadas em
quatro categorias distintas quanto ao número de inclusões que apresentavam.
Para o estudo de aSyn, foi comparada a percentagem de células com inclusões e a percentagem
de inclusões por célula com um determinado tamanho, entre SynT WT e mutante K58N, e concluiu-se
que a mutação leva a um decréscimo significativo da percentagem de agregados por célula com mais
do que 3 µm de diâmetro. Também comparámos o nível de expressão proteica entre WT e mutantes
aSyn e SynT e observámos que não haviam diferenças estatisticamente significativas.
Para o estudo dos efeitos da Htt na formação de inclusões, observámos que apenas a forma
patológica da proteína com 104 repetições de glutamina levou à formação das mesmas. Estas inclusões
apresentavam uma semelhante distribuição em relação ao seu diâmetro.
Adicionalmente, foi feita microscopia STED nas condições onde tinham sido previamente
observadas inclusões, tanto para Htt como aSyn. Esta técnica permitiu obter imagens dos agregados
com melhor resolução do que aquela que tinha sido obtida com microscopia de fluorescência.
Em estudos futuros, será pertinente usar a microscopia de STED para obter imagens das
inclusões em diferentes orientações espaciais, de forma a obter um melhor conhecimento da sua
estrutura e composição. Para além disso, será ainda apropriado fazer estudos de co-localização com
proteínas que interagem com aSyn e Htt, uma vez que estas podem também podem estar presentes nos
agregados formados pelas respetivas proteínas. Também seria relevante estudar a localização
subcelular das inclusões, visto que tal não foi possível neste projeto.
The misfolding and aggregation of several proteins is one of the central features of various neurodegenerative diseases, such as Parkinson’s and Huntington’s disease. However, whether protein aggregation is a cause or a consequence of the disease process is still unclear. Also, the process of protein aggregation in the cellular context is not fully understood. Novel techniques that allow further insight into how these proteins misfold and form inclusions are of major importance to find new therapies for these diseases. Super resolution microscopy is one of these techniques since it allows us to better assess the aggregates formed by misfolded proteins and the mechanisms leading to their assemble, thus putting us in a better position to uncover solutions to address neurodegeneration. Taking this into account, the aim of this project was to use super-resolution microscopy techniques to image and study the aggregation of different proteins (a-synuclein and huntingtin) in human cell cultures that model two of these neurodegenerative diseases (Parkinson’s and Huntington’s disease). We hoped to gain insight into the mechanisms that lead to the misfolding and aggregation of these proteins, to find new therapeutic targets and strategies. While the project was ongoing, a novel mutation of a-synuclein protein (K58N) was discovered, and its study was added to the project. The project involved several cell biology and biochemical techniques, such as the expression of variants of wild type and mutant proteins in human cells, the performance of immunostainings with different antibodies against the proteins studied and the assessment of the aggregation of two aggregation-prone proteins, a-synuclein and huntingtin, using fluorescence microscopy and superresolution microscopy. We used immunocytochemistry assays to study the inclusions form by the model used for aSyn, which consisted of a aSyn truncated form fused with a non-fluorescent GFP fragment (SynT). We compared the percentage of cells with inclusions and the percentage of inclusions with a certain size per cell between WT and mutant K58N SynT, and concluded that the mutation leads to the decrease of the percentage of aggregates with more than 3 µm, compared to WT. We also compared the level of protein expression between WT and mutant aSyn and SynT and no significant differences were found. For the study of the effects of Htt on inclusion formation, we observed that only the pathological form of Htt with a extended number of glutamines led to the formation of inclusions. These inclusions had a similar size distribution. Additionally, we conducted STED microscopy on the conditions that led to the formation of aggregates, both for aSyn and Htt. This technique allowed us to better image the inclusions formed, with a much higher resolution than the one obtained with fluorescence microscopy. In future studies, STED microscopy should be used to image inclusions in different display orientations, allowing a better insight of their structure and composition. Furthermore, the colocalization of proteins that interact with aSyn and Htt should be studied, to better assess the protein composition of the inclusions formed. Finally, it would also be pertinent to study the subcellular localization of the inclusions.
The misfolding and aggregation of several proteins is one of the central features of various neurodegenerative diseases, such as Parkinson’s and Huntington’s disease. However, whether protein aggregation is a cause or a consequence of the disease process is still unclear. Also, the process of protein aggregation in the cellular context is not fully understood. Novel techniques that allow further insight into how these proteins misfold and form inclusions are of major importance to find new therapies for these diseases. Super resolution microscopy is one of these techniques since it allows us to better assess the aggregates formed by misfolded proteins and the mechanisms leading to their assemble, thus putting us in a better position to uncover solutions to address neurodegeneration. Taking this into account, the aim of this project was to use super-resolution microscopy techniques to image and study the aggregation of different proteins (a-synuclein and huntingtin) in human cell cultures that model two of these neurodegenerative diseases (Parkinson’s and Huntington’s disease). We hoped to gain insight into the mechanisms that lead to the misfolding and aggregation of these proteins, to find new therapeutic targets and strategies. While the project was ongoing, a novel mutation of a-synuclein protein (K58N) was discovered, and its study was added to the project. The project involved several cell biology and biochemical techniques, such as the expression of variants of wild type and mutant proteins in human cells, the performance of immunostainings with different antibodies against the proteins studied and the assessment of the aggregation of two aggregation-prone proteins, a-synuclein and huntingtin, using fluorescence microscopy and superresolution microscopy. We used immunocytochemistry assays to study the inclusions form by the model used for aSyn, which consisted of a aSyn truncated form fused with a non-fluorescent GFP fragment (SynT). We compared the percentage of cells with inclusions and the percentage of inclusions with a certain size per cell between WT and mutant K58N SynT, and concluded that the mutation leads to the decrease of the percentage of aggregates with more than 3 µm, compared to WT. We also compared the level of protein expression between WT and mutant aSyn and SynT and no significant differences were found. For the study of the effects of Htt on inclusion formation, we observed that only the pathological form of Htt with a extended number of glutamines led to the formation of inclusions. These inclusions had a similar size distribution. Additionally, we conducted STED microscopy on the conditions that led to the formation of aggregates, both for aSyn and Htt. This technique allowed us to better image the inclusions formed, with a much higher resolution than the one obtained with fluorescence microscopy. In future studies, STED microscopy should be used to image inclusions in different display orientations, allowing a better insight of their structure and composition. Furthermore, the colocalization of proteins that interact with aSyn and Htt should be studied, to better assess the protein composition of the inclusions formed. Finally, it would also be pertinent to study the subcellular localization of the inclusions.
Descrição
Tese de Mestrado, Biologia Molecular e Genética, 2022, Universidade de Lisboa, Faculdade de Ciências
Palavras-chave
alfa-sinucleína doença de Parkinson huntingtina doença de Huntington microscopia de super-resolução Teses de mestrado - 2022