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Assessment of neurometabolic dysfunction and editing mitochondrial genome in the context of leigh syndrome by using induced pluripotent stem cell models
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Abstract(s)
A Síndrome de Leigh (LS) é uma doença mitocondrial rara e complexa que inicia normalmente durante a infância. É causada por danos na produção de energia devido a mutações no ADN nuclear e/ou mitocondrial. Os doentes com LS apresentam uma vasta gama de sintomas, tais como hipotonia e atraso no desenvolvimento neurológico. A LS tem sido associada ao metabolismo lipídico desde que foi demonstrado que níveis mais elevados de certos ácidos gordos no plasma aumentam o risco de desenvolvimento da doença. Este trabalho teve como objetivos: 1) validar um modelo
de células estaminais pluripotentes induzidas (iPSC) com base na edição do genoma nuclear para estudar a LS; 2) investigar possíveis alterações nestas células por exposição a diferentes tipos de lípidos; e 3) gerar um novo modelo da LS através da edição do genoma mitocondrial. Para os dois primeiros objetivos, foram geradas células progenitoras neurais (NPCs) humanas com uma mutação nuclear específica da LS a partir de iPSCs humanas, assim como NPCs isogénicas de controlo. Ensaios de imunocitoquímica e qPCR ajudaram-nos a confirmar a eficácia da geração de NPCs. Curiosamente, a análise de Western blot indicou várias alterações em proteínas relacionadas com o metabolismo mitocondrial em NPCs com uma mutação nuclear específica da LS (nLS-like NPCs) quando comparados com NPCs de controlo, apoiando a validação deste modelo para estudar a disfunção mitocondrial em LS. Além disso, a análise metabólica do secretoma derivado de nLS-like NPCs revelou diferenças em vários metabolitos importantes, com destaque para o lactato e o piruvato. Surpreendentemente, ambos estão diminuídos em nLS-like NPCs, estando talvez relacionados com um aumento da glicólise e consumo de piruvato no interior das células. Relativamente ao segundo
objetivo, descobrimos que concentrações crescentes de um ácido gordo de cadeia longa (LCFA), ácido oleico, e dois ácidos gordos de cadeia curta (SCFAs), propionato de sódio e butirato de sódio, podem aumentar a viabilidade tanto das NPCs de murganho como de humanos. Contudo, doses mais elevadas de SCFAs promoveram o efeito inverso em
nLS-like NPCs, indicando que as NPCs dos doentes com LS poderão ser mais sensíveis a elevados níveis destes tipos de lípidos de cadeia curta saturados. Em relação ao último objetivo, em primeiro lugar foi realizada a identificação dos
locais-alvo no ADN mitocondrial (mtDNA) associados a LS. Depois procedemos à sua confirmação, através da extração do mtDNA de células renais embrionárias humanas (HEKCs) e iPSCs, as primeiras para otimização e as últimas como a linha de células-alvo adequada. De seguida, procedemos à amplificação dos locais-alvo através de PCR e, seguidamente, por Sanger sequencing. Entretanto, utilizando a ferramenta de edição do genoma mitocondrial mediada por DdCBE (CRISPR/Cas), foi possível realizar a clonagem por Golden Gate cloning, o que tornou possível a inserção das mutações pretendidas. No conjunto, os resultados mostraram que as iPSCs poderão ser muito promissoras para o estudo da patogénese e possíveis terapias da LS. Além disso, os resultados revelaram não apenas que lípidos saturados de cadeia curta parecem impactar de forma negativa as NPCs dos doentes com LS, mas também que a edição do genoma mitocondrial em iPSCs parece ser um método exequível para gerar modelos celulares da LS associados a mutações mitocondriais.
Leigh Syndrome (LS) is a rare and complex mitochondrial disorder that usually starts during childhood. It is caused by damage in energy production due to mutations in the nuclear and/or mitochondrial DNA. LS patients present a wide range of symptoms, such as hypotonia and neurodevelopmental delay. LS has been associated with lipid metabolism since it was showed that higher levels of certain fatty acids in the plasma resulted in increased risk of developing the disease. This thesis is focused on: 1) validating a nuclear genome edited induced pluripotent stem cell (iPSC)-based model to study LS; 2) investigating possible alterations in these cells by exposure to different lipids; and 3) generating a novel iPSC-based LS model through mitochondrial genome editing. For the first two aims, neural progenitor cells (NPCs) with a LS-specific nuclear mutation were generated from human iPSCs and isogenic control iPSC-derived NPCs were used as control cells. Immunocytochemistry and qPCR techniques helped us to confirm the efficacy of the NPC generation. More interestingly, Western blot analysis indicated several alterations in metabolic- and mitochondrial-related proteins in NPCs with a LS-specific nuclear mutation (nLS-like NPCs) when compared with isogenic control cells, supporting the validation of this nuclear genome edited iPSC-based model to study mitochondrial dysfunction in LS. Moreover, metabolic analysis of the secretome derived from nLS-like NPCs also showed differences in several important metabolites, with highlights to lactate and pyruvate. Curiously, both metabolites were decreased in nLS-like NPCs, perhaps being related to the high glycolysis levels and pyruvate consumption inside the cells. Regarding the second objective, we found that increasing concentrations of one long-chain fatty acid (LCFA), oleic acid, and two short-chain fatty acids (SCFAs), sodium propionate and sodium butyrate, can increase the viability of both mouse and human NPCs. However, higher doses of SCFAs promoted the reverse effect in nLS-like NPCs, indicating that NPCs in LS patients are more sensitive in dealing with large amounts of these types of short and saturated lipids. Regarding the last aim, firstly the identification of LS mitochondrial DNA (mtDNA) target sites was performed. Their confirmation was assessed by extracting mtDNA from human embryonic kidney cells (HEKCs) and iPSCs, the first for optimization and the last as the appropriate target cell line. Then, the amplification of the target sites was performed by PCR and then Sanger sequencing. Meanwhile, using the DdCBE-mediated mitochondrial base editing tool (CRISPR/Cas), it was possible to perform the Golden Gate cloning, which made possible the insertion of the desired mutations. Altogether, the results showed that the iPSC-based model might be very promising to study the pathogenesis and potential therapies of LS. In addition, the results revealed not only a significant impact of short saturated lipids in the NPCs from LS patients, but also that mitochondrial genome editing appears to be a feasible method to generate iPSC-based LS models associated with mitochondrial mutations.
Leigh Syndrome (LS) is a rare and complex mitochondrial disorder that usually starts during childhood. It is caused by damage in energy production due to mutations in the nuclear and/or mitochondrial DNA. LS patients present a wide range of symptoms, such as hypotonia and neurodevelopmental delay. LS has been associated with lipid metabolism since it was showed that higher levels of certain fatty acids in the plasma resulted in increased risk of developing the disease. This thesis is focused on: 1) validating a nuclear genome edited induced pluripotent stem cell (iPSC)-based model to study LS; 2) investigating possible alterations in these cells by exposure to different lipids; and 3) generating a novel iPSC-based LS model through mitochondrial genome editing. For the first two aims, neural progenitor cells (NPCs) with a LS-specific nuclear mutation were generated from human iPSCs and isogenic control iPSC-derived NPCs were used as control cells. Immunocytochemistry and qPCR techniques helped us to confirm the efficacy of the NPC generation. More interestingly, Western blot analysis indicated several alterations in metabolic- and mitochondrial-related proteins in NPCs with a LS-specific nuclear mutation (nLS-like NPCs) when compared with isogenic control cells, supporting the validation of this nuclear genome edited iPSC-based model to study mitochondrial dysfunction in LS. Moreover, metabolic analysis of the secretome derived from nLS-like NPCs also showed differences in several important metabolites, with highlights to lactate and pyruvate. Curiously, both metabolites were decreased in nLS-like NPCs, perhaps being related to the high glycolysis levels and pyruvate consumption inside the cells. Regarding the second objective, we found that increasing concentrations of one long-chain fatty acid (LCFA), oleic acid, and two short-chain fatty acids (SCFAs), sodium propionate and sodium butyrate, can increase the viability of both mouse and human NPCs. However, higher doses of SCFAs promoted the reverse effect in nLS-like NPCs, indicating that NPCs in LS patients are more sensitive in dealing with large amounts of these types of short and saturated lipids. Regarding the last aim, firstly the identification of LS mitochondrial DNA (mtDNA) target sites was performed. Their confirmation was assessed by extracting mtDNA from human embryonic kidney cells (HEKCs) and iPSCs, the first for optimization and the last as the appropriate target cell line. Then, the amplification of the target sites was performed by PCR and then Sanger sequencing. Meanwhile, using the DdCBE-mediated mitochondrial base editing tool (CRISPR/Cas), it was possible to perform the Golden Gate cloning, which made possible the insertion of the desired mutations. Altogether, the results showed that the iPSC-based model might be very promising to study the pathogenesis and potential therapies of LS. In addition, the results revealed not only a significant impact of short saturated lipids in the NPCs from LS patients, but also that mitochondrial genome editing appears to be a feasible method to generate iPSC-based LS models associated with mitochondrial mutations.
Description
Tese de mestrado, Ciências Biofarmacêuticas, 2022, Universidade de Lisboa, Faculdade de Farmácia.
Keywords
Fatty acids Genome editing Leigh syndrome Mitochondria Stem cells Teses de mestrado - 2022