Mammalian preimplantation embryo development is prone to high rates of early embryo demise. Two underlying causes for failed development include the effect of sub-optimal culture media and maternal lethal effect (MLE) genes. In line with the growing evidence, we hypothesize that embryo fate is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins under suboptimal culture conditions such as HTF medium and oxidative stress. Characterization of Nalp5, a MLE gene resulting in 2-cell embryo arrest, also found a significantly higher expression of pro-apoptotic proteins in knockout oocytes and embryos. With the use of two anti-apoptotic peptides, TAT-BH4 and Bax-inhibiting peptide (BIP), we attempted to improve embryo development. Our results found that neither peptide was able to improve embryo development in the Nalp5 model, or the HTF model. However, TAT-BH4 is capable of significantly improving developmental competence in embryos cultured under oxidative stress. Our findings suggest that supplementation of TAT-BH4 in embryo culture medium may offer a novel and cost-effective technique to improve embryogenesis of cultured embryos. However, further studies are still required.

Mammalian preimplantation embryo development is prone to high rates of early embryo demise. Two underlying causes for failed development include the effect of sub-optimal culture media and maternal lethal effect (MLE) genes. In line with the growing evidence, we hypothesize that embryo fate is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins under suboptimal culture conditions such as HTF medium and oxidative stress. Characterization of Nalp5, a MLE gene resulting in 2-cell embryo arrest, also found a significantly higher expression of pro-apoptotic proteins in knockout oocytes and embryos. With the use of two anti-apoptotic peptides, TAT-BH4 and Bax-inhibiting peptide (BIP), we attempted to improve embryo development. Our results found that neither peptide was able to improve embryo development in the Nalp5 model, or the HTF model. However, TAT-BH4 is capable of significantly improving developmental competence in embryos cultured under oxidative stress. Our findings suggest that supplementation of TAT-BH4 in embryo culture medium may offer a novel and cost-effective technique to improve embryogenesis of cultured embryos. However, further studies are still required.

Background Tetrahydrobiopterin (BH4)-sensitive phenylketonuria (PKU) can be treated with sapropterin dihydrochloride. We studied metabolic control and health-related quality of life (HRQoL) in PKU patients treated with BH4. Subjects and methods Based on the review of neonatal BH4 test results and mutation analysis in 41 PKU patients, 19 were identified as potentially BH4-sensitive (9 females, 10 males, age 4–18 years). We analyzed phenylalanine (phe) concentrations in dried blood samples, nutrition protocols, and HRQoL questionnaires (KINDL®) beginning from 1 year before, during the first 42 days, and after 3 months of BH4 therapy. Results Eight BH4-sensitive patients increased their phe tolerance (629±476 vs. 2131±1084 mg, p00.006) while maintaining good metabolic control (phe concentration in dried blood 283±145 vs. 304±136 μM, p01.0). Six of them were able to stop dietary protein restriction entirely. BH4- sensitive patients had average HRQoL scores that were comparable to age-matched healthy children. There was no improvement in HRQoL scores after replacing classic dietary treatment with BH4 supply, although personal reports given by the patients and their parents suggest that available questionnaires are inappropriate to detect aspects relevant to inborn metabolic disorders. Discussion BH4 can allow PKU patients to increase their phe consumption significantly or even stop dietary protein restrictions. Unexpectedly, this does not improve HRQoL as assessed with KINDL®, partly due to high scores even before BH4 therapy. Specific questionnaires should be developed for inborn metabolic disorders.

Introdução: A Hiperfenilalaninemia por deficiência de fenilalanina hidroxilase (HPAPAH) é um erro inato do metabolismo no qual ocorre aumento dos níveis séricos de fenilalanina (Phe). Estudos recentes, realizados em várias populações, demonstraram que pacientes com HPA-PAH podem apresentar redução das concentrações plasmáticas de Phe mediante a administração oral de tetrahidrobiopterina (BH4). Objetivo: Identificar em uma amostra de pacientes brasileiros com HPA-PAH aqueles que são responsivos à administração de BH4 por via oral. Métodos: Para um paciente ser incluído no estudo, era necessário ter diagnóstico de HPA-PAH e idade igual ou superior a 7 anos, estar em tratamento dietético e apresentar nível de Phe igual ou superior a 6 mg/dL em todas as medidas realizadas no ano anterior à inclusão no estudo. No dia anterior à sobrecarga de BH4 (Dia 1), os pacientes foram submetidos a três coletas de sangue para mensuração dos níveis de Phe. No Dia 2, os pacientes receberam dose única de 20mg/Kg de BH4. As coletas de sangue foram, então, realizadas nos pontos de hora: 0, 4 e 8h (Dia 2) e 24h (Dias 3) após a ingestão do medicamento. Os níveis de Phe foram determinados através da espectrometria de massa in tandem. Foram utilizados dois critérios para definir a presença de responsividade ao BH4: Critério 1: redução 30% de Phe após 8h da administração do medicamento; Critério 2: redução 30% de Phe após 24h da administração do medicamento. Resultados: Dezoito pacientes foram incluídos no estudo, com mediana de idade de 14 anos, sendo 66,7% do sexo masculino. Onze apresentavam a forma clássica da doença e três a forma atípica. Três (forma clássica: 1, forma atípica: 2) e cinco (forma clássica: 2, forma atípica: 2 e forma não-definida: 1) pacientes foram considerados responsivos ao BH4 conforme critérios 1 e 2, respectivamente. Os níveis de Phe plasmáticos do dia anterior ao teste de sobrecarga não demonstraram variação nos pontos de hora (p=0,523). Entretanto, quando comparamos os níveis de Phe nos pontos de hora do dia pré e pós BH4, encontrou-se variação significativa entre eles (p=0,006). A análise da associação genótipo-fenótipo, para os pacientes com dados disponíveis (n=6) mostrou que a mesma é multifatorial. Conclusão: Nossos achados estão de acordo com a literatura, e indicaram que um número considerável de pacientes brasileiros com HPA-PAH poderá ser beneficiado com a administração oral de BH4. / Introduction: Hyperphenylalaninemia by phenylalanine hydroxylase deficiency (HPAPAH) is an inborn error of metabolism in which increased serum levels of phenylalanine (Phe) occur. Recent studies on several populations showed that patients with HPA-PAH can have their serum levels reduced when receiving oral tetrahydrobiopterin (BH4). Objective: to identify in a sample of Brazilian HPA-PAH the patients who are responsive to the oral administration of BH4. Methods: the following inclusion criteria were used: diagnosis of HPA-PAH, age 7 years, on dietary treatment and Phe levels 6 mg/dL in all tests performed one year prior to the inclusion in this study. On the day before the BH4 challenge (Day 1) 3 blood samples were obtained to measure Phe levels. Blood samples were also obtained at time points 0, 4, 8 hours (Day 2) and 24 h (Day 3) after the intake of the medication. Phe levels were determined by tandem mass spectrometry. Criteria used to define responsiveness to BH4 were: Criterion 1: Phe reduction 30% 8 hours after BH4 administration; Criterion 2: Phe reduction 30% 24 hours after BH4 administration. Results: a total of 18 patients with a mean age of 14 years were included in this study; of those, 66.7% were male. Eleven presented the classical form of the disease and 3, the atypical form. Three patients (classical form: 1, atypical form: 2) and 5 (classical form: 2; atypical form: 2; undefined form: 1) were considered responsive to BH4 according to criteria 1 and 2, respectively. Phe serum levels on the Day 1 did not show any change on the established time point schedule (p=0.523). However, when comparing levels of Phe between Days 1 and 2, significant variation was found (p=0.006). The phenotype – genotype association analysis of patients with available data (n=6) showed that the association is multifactorial. Conclusion: In accordance with the literature, our findings show that many Brazilian patients with HPA-PAH can benefit from the oral administration of BH4.

Introdução: A Hiperfenilalaninemia por deficiência de fenilalanina hidroxilase (HPAPAH) é um erro inato do metabolismo no qual ocorre aumento dos níveis séricos de fenilalanina (Phe). Estudos recentes, realizados em várias populações, demonstraram que pacientes com HPA-PAH podem apresentar redução das concentrações plasmáticas de Phe mediante a administração oral de tetrahidrobiopterina (BH4). Objetivo: Identificar em uma amostra de pacientes brasileiros com HPA-PAH aqueles que são responsivos à administração de BH4 por via oral. Métodos: Para um paciente ser incluído no estudo, era necessário ter diagnóstico de HPA-PAH e idade igual ou superior a 7 anos, estar em tratamento dietético e apresentar nível de Phe igual ou superior a 6 mg/dL em todas as medidas realizadas no ano anterior à inclusão no estudo. No dia anterior à sobrecarga de BH4 (Dia 1), os pacientes foram submetidos a três coletas de sangue para mensuração dos níveis de Phe. No Dia 2, os pacientes receberam dose única de 20mg/Kg de BH4. As coletas de sangue foram, então, realizadas nos pontos de hora: 0, 4 e 8h (Dia 2) e 24h (Dias 3) após a ingestão do medicamento. Os níveis de Phe foram determinados através da espectrometria de massa in tandem. Foram utilizados dois critérios para definir a presença de responsividade ao BH4: Critério 1: redução 30% de Phe após 8h da administração do medicamento; Critério 2: redução 30% de Phe após 24h da administração do medicamento. Resultados: Dezoito pacientes foram incluídos no estudo, com mediana de idade de 14 anos, sendo 66,7% do sexo masculino. Onze apresentavam a forma clássica da doença e três a forma atípica. Três (forma clássica: 1, forma atípica: 2) e cinco (forma clássica: 2, forma atípica: 2 e forma não-definida: 1) pacientes foram considerados responsivos ao BH4 conforme critérios 1 e 2, respectivamente. Os níveis de Phe plasmáticos do dia anterior ao teste de sobrecarga não demonstraram variação nos pontos de hora (p=0,523). Entretanto, quando comparamos os níveis de Phe nos pontos de hora do dia pré e pós BH4, encontrou-se variação significativa entre eles (p=0,006). A análise da associação genótipo-fenótipo, para os pacientes com dados disponíveis (n=6) mostrou que a mesma é multifatorial. Conclusão: Nossos achados estão de acordo com a literatura, e indicaram que um número considerável de pacientes brasileiros com HPA-PAH poderá ser beneficiado com a administração oral de BH4. / Introduction: Hyperphenylalaninemia by phenylalanine hydroxylase deficiency (HPAPAH) is an inborn error of metabolism in which increased serum levels of phenylalanine (Phe) occur. Recent studies on several populations showed that patients with HPA-PAH can have their serum levels reduced when receiving oral tetrahydrobiopterin (BH4). Objective: to identify in a sample of Brazilian HPA-PAH the patients who are responsive to the oral administration of BH4. Methods: the following inclusion criteria were used: diagnosis of HPA-PAH, age 7 years, on dietary treatment and Phe levels 6 mg/dL in all tests performed one year prior to the inclusion in this study. On the day before the BH4 challenge (Day 1) 3 blood samples were obtained to measure Phe levels. Blood samples were also obtained at time points 0, 4, 8 hours (Day 2) and 24 h (Day 3) after the intake of the medication. Phe levels were determined by tandem mass spectrometry. Criteria used to define responsiveness to BH4 were: Criterion 1: Phe reduction 30% 8 hours after BH4 administration; Criterion 2: Phe reduction 30% 24 hours after BH4 administration. Results: a total of 18 patients with a mean age of 14 years were included in this study; of those, 66.7% were male. Eleven presented the classical form of the disease and 3, the atypical form. Three patients (classical form: 1, atypical form: 2) and 5 (classical form: 2; atypical form: 2; undefined form: 1) were considered responsive to BH4 according to criteria 1 and 2, respectively. Phe serum levels on the Day 1 did not show any change on the established time point schedule (p=0.523). However, when comparing levels of Phe between Days 1 and 2, significant variation was found (p=0.006). The phenotype – genotype association analysis of patients with available data (n=6) showed that the association is multifactorial. Conclusion: In accordance with the literature, our findings show that many Brazilian patients with HPA-PAH can benefit from the oral administration of BH4.

Introdução: A Hiperfenilalaninemia por deficiência de fenilalanina hidroxilase (HPAPAH) é um erro inato do metabolismo no qual ocorre aumento dos níveis séricos de fenilalanina (Phe). Estudos recentes, realizados em várias populações, demonstraram que pacientes com HPA-PAH podem apresentar redução das concentrações plasmáticas de Phe mediante a administração oral de tetrahidrobiopterina (BH4). Objetivo: Identificar em uma amostra de pacientes brasileiros com HPA-PAH aqueles que são responsivos à administração de BH4 por via oral. Métodos: Para um paciente ser incluído no estudo, era necessário ter diagnóstico de HPA-PAH e idade igual ou superior a 7 anos, estar em tratamento dietético e apresentar nível de Phe igual ou superior a 6 mg/dL em todas as medidas realizadas no ano anterior à inclusão no estudo. No dia anterior à sobrecarga de BH4 (Dia 1), os pacientes foram submetidos a três coletas de sangue para mensuração dos níveis de Phe. No Dia 2, os pacientes receberam dose única de 20mg/Kg de BH4. As coletas de sangue foram, então, realizadas nos pontos de hora: 0, 4 e 8h (Dia 2) e 24h (Dias 3) após a ingestão do medicamento. Os níveis de Phe foram determinados através da espectrometria de massa in tandem. Foram utilizados dois critérios para definir a presença de responsividade ao BH4: Critério 1: redução 30% de Phe após 8h da administração do medicamento; Critério 2: redução 30% de Phe após 24h da administração do medicamento. Resultados: Dezoito pacientes foram incluídos no estudo, com mediana de idade de 14 anos, sendo 66,7% do sexo masculino. Onze apresentavam a forma clássica da doença e três a forma atípica. Três (forma clássica: 1, forma atípica: 2) e cinco (forma clássica: 2, forma atípica: 2 e forma não-definida: 1) pacientes foram considerados responsivos ao BH4 conforme critérios 1 e 2, respectivamente. Os níveis de Phe plasmáticos do dia anterior ao teste de sobrecarga não demonstraram variação nos pontos de hora (p=0,523). Entretanto, quando comparamos os níveis de Phe nos pontos de hora do dia pré e pós BH4, encontrou-se variação significativa entre eles (p=0,006). A análise da associação genótipo-fenótipo, para os pacientes com dados disponíveis (n=6) mostrou que a mesma é multifatorial. Conclusão: Nossos achados estão de acordo com a literatura, e indicaram que um número considerável de pacientes brasileiros com HPA-PAH poderá ser beneficiado com a administração oral de BH4. / Introduction: Hyperphenylalaninemia by phenylalanine hydroxylase deficiency (HPAPAH) is an inborn error of metabolism in which increased serum levels of phenylalanine (Phe) occur. Recent studies on several populations showed that patients with HPA-PAH can have their serum levels reduced when receiving oral tetrahydrobiopterin (BH4). Objective: to identify in a sample of Brazilian HPA-PAH the patients who are responsive to the oral administration of BH4. Methods: the following inclusion criteria were used: diagnosis of HPA-PAH, age 7 years, on dietary treatment and Phe levels 6 mg/dL in all tests performed one year prior to the inclusion in this study. On the day before the BH4 challenge (Day 1) 3 blood samples were obtained to measure Phe levels. Blood samples were also obtained at time points 0, 4, 8 hours (Day 2) and 24 h (Day 3) after the intake of the medication. Phe levels were determined by tandem mass spectrometry. Criteria used to define responsiveness to BH4 were: Criterion 1: Phe reduction 30% 8 hours after BH4 administration; Criterion 2: Phe reduction 30% 24 hours after BH4 administration. Results: a total of 18 patients with a mean age of 14 years were included in this study; of those, 66.7% were male. Eleven presented the classical form of the disease and 3, the atypical form. Three patients (classical form: 1, atypical form: 2) and 5 (classical form: 2; atypical form: 2; undefined form: 1) were considered responsive to BH4 according to criteria 1 and 2, respectively. Phe serum levels on the Day 1 did not show any change on the established time point schedule (p=0.523). However, when comparing levels of Phe between Days 1 and 2, significant variation was found (p=0.006). The phenotype – genotype association analysis of patients with available data (n=6) showed that the association is multifactorial. Conclusion: In accordance with the literature, our findings show that many Brazilian patients with HPA-PAH can benefit from the oral administration of BH4.

La tétrahydrobioptérine (BH4) est le cofacteur requis pour l’activité des enzymes responsables de la synthèse de la dopamine, la sérotonine et l’oxyde nitrique. Par conséquent, la BH4 est nécessaire au bon déroulement de nombreux processus physiologiques centraux et périphériques dont la neurotransmission, la réponse inflammatoire, la régulation du stress oxydatif, la fonction vasculaire et endothéliale et le métabolisme. Or, de par sa structure chimique, la BH4 est une molécule facilement oxydable et dégradable, et des diminutions des niveaux centraux de BH4 ont été observées dans de nombreuses maladies neuropsychiatriques à composante inflammatoire dont la maladie d’Alzheimer et la dépression majeure. Le déficit de BH4 apparait donc comme un mécanisme pouvant participer à l’étiologie de ces pathologies et en aggraver les symptômes. Malgré ces observations, les effets d’une administration de BH4 sur la neurophysiologie et les comportements en s’y rapportant restent largement inexplorés. L’objectif de cette thèse a donc été de caractériser les effets d’une administration périphérique de BH4 sur la fonction cérébrale et le comportement chez la souris. Nous nous sommes intéressés aux effets de BH4 sur le système dopaminergique mésolimbique et la motivation en condition physiologique puis en situation d’inflammation aigue. Dans un second temps, nous avons exploré le potentiel thérapeutique de la BH4 dans le traitement de la maladie d’Alzheimer. Nous avons tout d’abord démontré que la BH4 traversait la barrière hémato-encéphalique et qu'une injection périphérique permettait d’augmenter les niveaux centraux de BH4. Nos travaux ont montré qu’en condition physiologique, l’administration de BH4 potentialise la libération de dopamine dans le nucleus accumbens et les comportements motivés. En condition de neuroinflammation aiguë induite par le LPS, l’administration de BH4 permet d’atténuer la neuroinflammation. Ces données suggèrent un potentiel bénéfique de la BH4 sur les troubles motivationnels induits par l’inflammation. Dans notre deuxième étude, nous avons démontré que l’administration chronique de BH4 permet de corriger les déficits mnésiques observés dans le modèle murin triple transgénique de la maladie d’Alzheimer. La supplémentation en BH4 induit également une diminution de la neuroinflammation ainsi qu’une amélioration de la tolérance au glucose. Cependant, ces améliorations mnésiques, métaboliques et inflammatoires ne s’accompagnent pas d’une diminution des pathologies amyloïde et tau.Ainsi, l’ensemble de ces travaux a permis une meilleure caractérisation des effets neurobiologiques et comportementaux de la BH4 et renforce son potentiel thérapeutique. / Tetrahydrobiopterin (BH4) is the required cofactor for the activity of the enzymes involved in the synthesis of dopamine, serotonin and nitric oxide. BH4 is therefore necessary for many central and peripheral physiological processes including neurotransmission, inflammatory response, oxidative stress regulation, vascular and endothelial function, and metabolism. However, BH4 can be easily oxidized and degraded and decreased BH4 brain levels has been observed in many neuropsychiatric diseases including Alzheimer's disease and major depression. Consequently, the decrease in BH4 levels in these pathologies could contribute to the onset and aggravation of symptoms. Despite these observations, the effects of BH4 administration on brain function and related behavior remain largely unexplored. The aim of this thesis was to characterize the effects of peripheral BH4 administration on brain function and behavior in mice. We investigated the effects of BH4 on the mesolimbic dopaminergic system and motivation in physiological condition and during acute inflammation. Then, we explored the therapeutic potential of BH4 in the treatment of Alzheimer's disease. We first demonstrated that BH4 crossed the blood-brain barrier and that a peripheral injection of BH4 increased its cerebral levels. Our results also showed that under physiological condition, BH4 administration potentiates dopamine release into the nucleus accumbens and motivated behaviors. In condition of acute LPS-induced neuroinflammation, BH4 administration helps to reduce neuroinflammation. Therefore, BH4 may have beneficial effects on dopaminergic and motivational disturbances induced by inflammation. In our second study, we demonstrated that chronic administration of BH4 reversed memory deficits observed in the transgenic triple murine model of Alzheimer's disease. We also observed a decrease in neuroinflammation and an improvement in glucose tolerance. However, these memory, metabolic and inflammatory improvements are not accompanied by a decrease in amyloid and tau pathologies. This work had contributed to a better characterization of the neurobiological and behavioral effects of the BH4 and reinforces its therapeutic potential.

Programmed cell death by apoptosis is required for normal development and tissue homeostasis. Perturbations of the critical signaling pathways that regulate apoptosis drive a number of pathologic diseases; therefore, a deep understanding of the apoptotic regulatory networks and methods for therapeutically modulating them is highly warranted. As constituents of the intrinsic pathway of apoptosis, pro-apoptotic BCL-2 family proteins respond to internal signals of cell stress to activate cell death through permeabilization of the outer mitochondrial membrane to release cytochrome c and other apoptogenic factors. Anti-apoptotic BCL-2 proteins block apoptosis by forming stable heterodimers with pro-apoptotic BAX and BAK. Specifically, the C-terminal binding groove of BCL-2 sequesters the BCL-2 homology 3 (BH3) death domain of BAX to prevent BAX oligomerization. The N-terminal BH4 domain of BCL-2 is also believed to confer anti-apoptotic activity but the mechanism by which this occurs remains unknown. A direct, inhibitory interaction between the BCL-2 BH4 domain and BAX was established through application of a peptide stapling technology to preserve the α-helical character of the BH4 domain outside the context of full-length BCL-2. Photoaffinity labeling identified a new mode of BH4 domain interaction at the C-terminal face of BAX, revealing an additional layer of apoptotic regulation. Examination of the conformational activation of full-length BAX in a lipid membrane by hydrogen-deuterium exchange mass spectrometry revealed that the BCL-2 BH4 helix blocks the BAX conformational changes triggered by an activating BH3 peptide to preserve the inactive BAX fold. Thus, the mechanistic paradigm for BCL-2 inhibition of BAX has been expanded to allow future opportunities for modulation of apoptosis by mimicking or inhibiting the BH4 motif.

Background Tetrahydrobiopterin (BH4)-sensitive phenylketonuria (PKU) can be treated with sapropterin dihydrochloride. We studied metabolic control and health-related quality of life (HRQoL) in PKU patients treated with BH4. Subjects and methods Based on the review of neonatal BH4 test results and mutation analysis in 41 PKU patients, 19 were identified as potentially BH4-sensitive (9 females, 10 males, age 4–18 years). We analyzed phenylalanine (phe) concentrations in dried blood samples, nutrition protocols, and HRQoL questionnaires (KINDL®) beginning from 1 year before, during the first 42 days, and after 3 months of BH4 therapy. Results Eight BH4-sensitive patients increased their phe tolerance (629±476 vs. 2131±1084 mg, p00.006) while maintaining good metabolic control (phe concentration in dried blood 283±145 vs. 304±136 μM, p01.0). Six of them were able to stop dietary protein restriction entirely. BH4- sensitive patients had average HRQoL scores that were comparable to age-matched healthy children. There was no improvement in HRQoL scores after replacing classic dietary treatment with BH4 supply, although personal reports given by the patients and their parents suggest that available questionnaires are inappropriate to detect aspects relevant to inborn metabolic disorders. Discussion BH4 can allow PKU patients to increase their phe consumption significantly or even stop dietary protein restrictions. Unexpectedly, this does not improve HRQoL as assessed with KINDL®, partly due to high scores even before BH4 therapy. Specific questionnaires should be developed for inborn metabolic disorders.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13111号 / 論医博第2129号 / 新制||医||1022(附属図書館) / (主査)教授 齊藤 博英, 教授 松原 和夫, 教授 林 康紀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM