Os transtornos do espectro autista (TEA) são um grupo de doenças neuropsiquiátricas caracterizadas por um prejuízo na capacidade de comunicação e de interação social e por padrões comportamentais estereotipados. Os TEA são geneticamente heterogêneos o que dificulta a identificação das alterações genéticas que estão contribuindo para estes transtornos. No presente estudo, selecionamos como uma primeira abordagem o estudo de translocações cromossômicas, buscando encontrar genes candidatos para posteriores estudos funcionais. No primeiro caso, uma translocação de novo balanceada envolvendo os cromossomos 2q11 e Xq24, não identificamos nenhum candidato funcional rompido pelos pontos de quebra. Detectamos ainda a presença de uma isodissomia materna do cromossomo 5 nesta paciente. Este resultado sugere que, possivelmente, tanto a translocação cromossômica quanto a isodissomia devem estar contribuindo para a etiologia do TEA nesta paciente, caracterizando este como um caso de efeito poligênico. Já o estudo da translocação de novo balanceada (3,11)(p21,q22) revelou que o gene TRPC6, um canal de cálcio envolvido no desenvolvimento de dendritos e sinapses excitatórias, encontrava-se rompido no cromossomo 11 deste paciente. As análises dos neurônios e células progenitoras neurais deste paciente obtidas através da técnica de reprogramação celular e o estudo global de expressão gênica sugerem fortemente que o rompimento do gene TRPC6 é o fator etiológico do TEA neste caso. Por fim, nós também realizamos um estudo de expressão gênica global de pacientes autistas idiopáticos e verificamos que os genes diferencialmente expressos nestes pacientes estão principalmente envolvidos na regulação da dinâmica do citoesqueleto, indicando que este pode ser o processo biológico comumente afetado nos pacientes autistas. Nosso trabalho mostra que os estudos citogenéticos são importantes para a identificação de genes candidatos para os TEA e reforça a hipótese de que estes transtornos são causados por diferentes variantes genéticas mas que levam ao comprometimento de um processo biológico comum. Acreditamos que o modelo de reprogramação celular contribuirá para o entendimento da implicação de tais processos na etiologia dos TEA. / Autism spectrum disorders (ASD) are a group of neurodevelopmental diseases characterized by impairments in social and communicative skills and repetitive behaviors. The investigation of ASD causes is hampered by the genetic heterogeneity of these neurodevelopmental diseases. In the present study, we mapped the breakpoints associated to chromosomal translocations found in two autistic patients as a first screening approach, trying to identify single candidate genes that could be further investigated by functional analysis. In the first case, a de novo balanced translocation involving the chromosomes 2q11 and Xq24, we did not find any functionally known relevant gene disrupted by the breakpoints but, surprisingly, SNP-array data showed that the patient also presents a maternally inherited isodisomy on chromosome 5. In this case, is possible that ASD is caused by the combination of the molecular results caused by the translocation and the UPD on chromosome 5, which would characterize this case as an example of polygenic effects on ASD etiology. On the other hand, the study of a second case, a boy with a de novo balanced translocation (3;11)(p21;q22), revealed that TRPC6, a calcium channel involved in dendritic spine and excitatory synapse formation, was disrupted by the translocation on chromosome 11. Making use of cellular reprogramming to generate neurons and neuronal progenitor cells from this patient and expression analysis, we demonstrated that TRPC6 disruption can respond for the phenotype seen in this patient. Finally, we also performed a genome-wide expression analysis to investigate idiopathic autistic patients and we verified that ASD DEGs are mainly implicated in cytoskeleton dynamics, suggesting that the regulation of this cellular structure can be one of the common mechanisms of ASD etiology. Our work shows that cytogenetic studies are important for the identification of ASD candidate genes and reinforces the hypothesis that these disorders are caused by different genetic variants that are implicated in a common biological process. We believe that cellular reprogramming will contribute for the understanding of the implication of such biological processes in the etiology of ASD.

A síndrome de Prader Willi (PWS) é uma doença de neurodesenvolvimento; a principal hipótese de causa de PWS é a ausência da expressão de SNORD116. O SNORD116 fica na região 15q11-q13 que apresenta vários genes com imprinting genômico e é conhecida por ser controlada pela região de controle de imprinting PWS (PWS-IC) que se localiza sobreposta à região promotora e ao exon 1 do gene SNRPN. Em camundongos, uma proteína zinc finger (Zfp57) foi descrita como importante para o estabelecimento e manutenção do imprinting no Snrpn. Através de análise do ENCODE do Genome Browser, verificamos que outra proteína zinc finger (ZNF274) se liga ao SNORD116. ZNF274 é conhecida por formar um complexo com TRIM28 e SETDB1 que inibe a expressão através da trimetilação da lisina 9 na histona 3 (H3K9me3). No atual estudo mostramos que ZNF274 se liga ao SNORD116 preferencialmente ao alelo materno nas células-tronco pluripotente induzidas (iPSCs). Adicionalmente, as proteínas TRIM28 e SETDB1, que formam um complexo com a ZNF274, estão presentes na região do SNORD116, e a modificação H3K9me3 ocorre preferencialmente no alelo materno nas iPSCs. Na análise funcional, mostramos que o knockdown de SETDB1 isoladamente ou combinado com o knockdown de ZNF274 causa aumento na expressão de SNRPN e SNORD116 nas iPSCs. Além disso, ocorre redução do H3K9me3 e aumento da modificação relacionada à ativação da transcrição, H3K4me2 (dimetilação da lisina 4 na histona 3), na PWS-IC. Os knockdowns também afetam a metilação de DNA, ocasionando o aumento de 5-hidroximetliação de citosinas na PWS-IC. Em outros tipos celulares estudados, neurônios derivados de iPSCs e SHEDs, ZNF274 e a modificação H3K9me3 ocorrem em ambos os alelos dentro do SNORD116. É possível que, nas iPSCs, este complexo proteja a região imprintada da desmetilação do DNA de proteína(s) que atue(m) nessa região somente em células pluripotentes. Nossos achados possibilitam melhor compreensão dos mecanismos envolvidos no imprinting da região 15q11-q13, principalmente do SNORD116, e, consequentemente, disponibiliza novas ferramentas para o desenvolvimento de futuras terapias para PWS. / Prader-Willi syndrome (PWS) is a neurodevelopmental disorder. Loss of paternal copies of the cluster of SNORD116 C/D box snoRNAs and their host transcript, 116HG, on human chromosome 15q11-q13 imprinted region is considered to be the major responsible for PWS. PWS-imprinting center (PWS-IC) regulates 15q11-q13 imprinting. PWS-IC is located upstream and in the exon 1 of SNURF-SNRPN gene. In mice, Zfp57 plays an important role in establishment and maintenance of Snrpn imprinting. In human, ENCODE database indicates that ZNF274 binds to SNORD116. Moreover, ZNF274 are C2H2/KRAB zinc finger proteins as Zfp57. We have investigated the mechanism of repression of the maternal SNORD116. Here, we report that the ZNF274, in association with the histone H3 lysine 9 (H3K9) methyltransferase SETDB1, is part of a complex that binds to the silent maternal but not to the active paternal alleles in induced pluripotent stem cells (iPSCs). Knockdown of SETDB1 in PWS-specific iPSCs causes a decrease in the accumulation of H3K9 trimethylation (H3K9me3) at SNORD116. We also show that upon knockdown of SETDB1 in PWS-specific iPSCs, expression of maternally silenced 116HG RNA is partially restored. SETDB1 knockdown in PWS iPSCs also disrupts DNA methylation at the PWS-IC where a decrease in 5-methylcytosine is observed in association with a concomitant increase in 5-hydroxymethylcytosine. In iPSCs-derived neurons and stem cells from human exfoliated teeth (SHEDs) ZNF274/SETDB1 complex binding and H3K9me3 modification occur in both alleles. These observations suggest that the ZNF274/SETDB1 complex bound to the SNORD116 cluster may protect the PWS-IC from DNA demethylation during early development, as indicated by iPSCs. Our findings reveal novel epigenetic mechanisms that function to repress the maternal 15q11-q13 region. The better understanding of epigenetic mechanisms provides new tools for future therapy research.

Hereditary transthyretin amyloidosis (ATTR amyloidosis) is a multi-system protein folding disorder that results from >100 described mutations in the transthyretin (TTR) gene. In the disease, non-natively folded TTR, originally produced by the liver, travels throughout circulation and deposits extracellularly at downstream target organs. The multi-tissue etiology of the disease makes it difficult to study in vitro, while no mouse model accurately recapitulates disease pathology. Therefore, we utilized patient-specific induced pluripotent stem cells (iPSCs) to test the hypothesis that production of and exposure to destabilized TTRs results in distinct cellular and molecular changes. The liver’s contribution to the deposition of TTR at distal tissues is understudied. As a result, in Aim 1 we sought to assess the effects of destabilized TTR production on effector hepatic cells. To this end, we utilized gene editing to generate isogenic, patient iPSCs expressing either mutant or wild-type TTR. Combining this tool with single cell RNAseq, we identified hepatic proteostasis factors, including unfolded protein response (UPR) pathways, whose expression coincided with the production of destabilized TTR. Enhancing endoplasmic reticulum (ER) proteostasis within patient hepatic cells via exogenous activation of adaptive UPR signaling, we demonstrated preferential reduction in the secretion of pathogenic TTR. In turn, we demonstrated that production of disease-associated TTR correlates with expression of proteostasis factors capable of regulating TTR secretion and in turn downstream pathogenesis. ATTR amyloidosis patients exhibit extreme phenotypic variation (e.g. TTR fibril deposits at cardiac tissue and/or peripheral nerves). In Aim 2, we sought to define responses of target cell types to pathologically-diverse TTRs. To accomplish this, we profiled transcriptomic changes resulting from exposure to a variety of destabilized TTRs to determine 1) target cell response to TTR exposure and 2) how this response changes across diverse variants and cell types. In doing so, we found that TTR exposure elicits distinct variant- and cell type-specific transcriptional responses. Herein, we addressed our central hypothesis by profiling destabilized TTR production within hepatic cells and TTR exposure at target cell types. Collectively, these data may result in the discovery of unidentified and potentially druggable pathologically-associated pathways for ATTR amyloidosis and other systemic amyloid diseases.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22700号 / 医科博第115号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 近藤 玄, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The liver is a crucial human organ with a complex architecture. Although the liver has great regeneration potential, deadly liver diseases are associated with irreversible hepatocytes damage. Currently, a liver transplant is the only treatment for liver failure. A shortage of donors forced extensive research for alternative treatments. The most promising hepatocyte source could be obtained from the differentiation of induced pluripotent stem cells (iPSCs). This technology can give us great amounts of pluripotent cells without ethical restrictions, which could be available in a variety of haplotypes to minimize the possibility of rejection. There are many reprogramming protocols available. However, there is still no standardised method to obtain clinical grade iPSCs. From those stem cells, it is possible to obtain hepatic-like cells (HLCs) by direct differentiation in vitro. HLCs express multiple hepatocyte-specific features, but their names signal that they still show fetal liver identity. A variety of hepatic differentiation protocols were described, although the process of hepatic differentiation must be improved in order to be translated into the clinic. Along with genes, microRNA (miRNA) is the well-known controller of cell fate. MiRNA is a type of non-coding RNA (ncRNA) which can influence gene transcription by inhibiting gene expression. In contrast to genes, many of the miRNA can affect up to thousands of genes simultaneously. Another group of ncRNA, which is a subject of potential differences are small nucleolar RNA (snoRNA). SnoRNA are involved in RNA chemical modifications by acting as a guide, mostly for ribosomal RNA (rRNA), but some of them have additional functions. In this study, a new iPSCs line was generated from skin fibroblasts using lipotransfection of episomal vectors. This method is free from exogene integration and shows low cytotoxicity. A pluripotency of generated cells was confirmed by morphological assessment, immunocytochemical staining, and spontaneous differentiation assay. To be sure that the genome of the cells was not changed, karyotype analysis was performed. Next, HLCs were derived from those iPSCs using a four-stage hepatic differentiation protocol. The obtained HLCs were characterised using, among others, a hepatic gene expression analysis. Cells after differentiation express mature and fetal hepatic markers, which is consistent with previous results. The attempt to improve differentiation using transient overexpression of master hepatic transcription factor – HNF4α, was not sufficient, as shown by gene expression analysis and whole slide scanning. Previous studies failed to point out the genetic inhibitors of hepatic maturation and non-coding RNA (ncRNA) profiles of iPSCs – derived HLCs were not investigated. In this study, the sequencing of ncRNA was performed in order to compare the expression profiles of HLCs on two stages of differentiation (Day 20 and 24) with mature hepatocytes. The obtained results indicate that HLCs express miRNA, which control hepatic differentiation and maintain their fetal liver character. In comparison to mature hepatocytes, differentially expressed miRNAs in HLCs control the pathways of fatty acid metabolism and synthesis, proteoglycan in cancer, the Hippo signaling pathway, ECM-receptor interaction and adherens junction. Some of those highly expressed miRNAs can potentially block maturation by inhibiting epithelial-mesenchymal transition (EMT) which has an impact that is essential during hepatic differentiation. However, this should be resolved in future research. In this work, differentially expressed snoRNA were also identified. A total of 68% of differentially expressed snoRNAs was C/D box class. This is interesting because this snoRNa class was previously indicated as capable to be processed by an miRNA processing pathway. Many of the differentially expressed snoRNAs belong to the imprinted loci, in which a different expression in a human were analysed before. In obteined dataset, copies of SNORD115 were upregulated in a liver, but not in HLCs, which is consistent with an earlier comparison of a liver and other endoderm organs. Additionally, an analysis of obtained sequencing data allowed for a discovery of 19 novel snoRNA genes. In summary, this work shows a new approach to the reprogramming of a fibroblast and investigates the involvement of miRNAs and snoRNAs in the dynamics of hepatic differentiation. This study has shed a light on the molecular and regulatory mechanisms that underlie the complex process of liver differentiation and will hopefully allow existing problems with the use of in vitro derived hepatocytes to be overcome. A dataset generated here can be the foundation for a hepatic-specialised rybosomes theory and enabled to discover novel snoRNA genes.:1. INTRODUCTION 11 1.1. PLURIPOTENT STEM CELLS 11 1.1.1. Pluripotency 11 1.1.2. IPSCs 13 1.1.3. Reprogramming methods 14 1.1.4. IPSCs as an alternative cell source for disease modelling and regenerative medicine 16 1.2. LIVER 18 1.2.1. Liver anatomy and function 18 1.2.2. Liver embryonal development 20 1.3. HEPATIC DIFFERENTIATION OF IPSCS IN VITRO 22 1.3.1. HLCs 22 1.3.2. Differentiation protocols into hepatocytes 24 1.4. NCRNA 25 1.4.1. MiRNA 26 1.4.2. SnoRNA 28 2. AIMS 31 3. MATERIALS 32 3.1. EQUIPMENT 32 3.2. SOFTWARE 32 3.3. ENZYMES, KITS AND TRANSFECTION REAGENTS 33 3.4. SOLUTIONS AND REAGENTS 33 3.5. CELL LINES 34 3.6. CELL CULTURE MEDIA AND CYTOKINES 34 3.7. PLASMIDS 35 3.8. PCR REAGENTS AND PRIMERS 35 3.8.1. PCR reagents 35 3.8.2. PCR primers 35 3.9. ANTIBODIES 36 4. METHODS 37 4.1. CELL BIOLOGY 37 4.1.1. Derivation and culture of primary human foreskin fibroblasts 37 4.1.2. Counting cells 37 4.1.3. Cryo-preservation of cells 37 4.1.4. Thawing of cryo-preserved cells 38 4.1.5. Cell reprogramming 38 4.1.6. Cultivation and expansion of iPSCs 39 4.2. IMMUNOCYTOCHEMISTRY 39 4.3. IN VITRO SPONTANEOUS DIFFERENTIATION 39 4.4. KARYOTYPE ANALYSIS 40 4.5. RNA ISOLATION 40 4.6. QUANTITATIVE PCR 40 4.7. PERIODIC ACID-SCHIFF (PAS) STAINING 41 4.8. INDOCYANINE GREEN UPTAKE AND RELEASE 41 4.9. PLASMID TRANSFECTION 42 4.10. HEPATIC DIFFERENTIATION 42 4.11. WHEAT GERM AGGLUTININ STAINING 42 4.12. VALIDATION OF HEPATIC DIFFERENTIATION EFFICIENCY 43 4.13. RNA ISOLATION AND SEQUENCING 43 4.14. BIOINFORMATIC ANALYSIS 44 4.14.1. Sequencing quality and mapping 44 4.14.2. Analysis of differential expressed ncRNAs 44 4.14.3. Target pathways prediction of differentially expressed miRNAs 44 4.14.4. Identification of novel ncRNAs candidates 45 5. RESULTS 46 5.1. GENERATION OF IPSCS USING EPISOMAL VECTORS 46 5.1.1. Cell transfection 46 5.1.2. Establishment of iPSCs line 48 5.2. PLURIPOTENCY CHARACTERISATION OF THE IPSCS 49 5.2.1. Pluripotency markers 49 5.2.2. Spontaneous differentiation assay 50 5.2.3. Karyotype 52 5.3. HEPATIC DIFFERENTIATION OF IPSCS AND HLCS CHARACTERISATION 53 5.3.1. iPSCs hepatic differentiation 53 5.3.2. Expression of hepatic markers 54 5.3.3. Hepatic gene expression in HLCs 56 5.3.4. Hepatic functions in HLCs 58 5.4. HNF4A OVEREXPRESSION DURING DIFFERENTIATION 59 5.4.1. Cell transfection during differentiation 59 5.4.2. Comparison of hepatic differentiation efficiency 60 5.4.3. Whole slide scanning 62 5.5. NON-CODING RNA ANALYSIS 64 5.5.1. Non-coding RNA sequencing quality 64 5.5.2. MicroRNA analysis 68 5.5.3. SnoRNA analysis 79 5.5.4. Short reads snoRNA analysis 84 5.5.5. New gene candidates 85 6. DISCUSSION 88 6.1. METHODICAL STRATEGY 88 6.2. CHARACTERISATION OF GENERATED IPSCS 89 6.3. HEPATIC DIFFERENTIATION OF IPSCS 89 6.3.1. Characterisation of HLCs 89 6.3.2. Protocol with HNF4a overexpression 90 6.3.3. Differentially expressed miRNA 90 6.3.4. Differentially expressed snoRNA 93 6.4. NOVEL SNORNA GENES 95 7. SUMMARY 96 8. REFERENCES 99 9. APPENDIX 118 ERKLÄRUNG ÜBER DIE EIGENSTÄNDIGE ABFASSUNG DER ARBEIT 122. ACKNOWLEDGEMENTS 123

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13464号 / 論医博第2251号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 金子 新, 教授 江藤 浩之, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12788号 / 論医博第2068号 / 新制||医||1000(附属図書館) / 30807 / 京都大学大学院医学研究科医学専攻 / (主査)教授 瀬原 淳子, 教授 髙橋 淳, 教授 山下 潤 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18850号 / 医博第3961号 / 新制||医||1007(附属図書館) / 31801 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 吉村 長久, 教授 中畑 龍俊 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19561号 / 医博第4068号 / 新制||医||1013(附属図書館) / 32597 / 京都大学大学院医学研究科医学専攻 / (主査)教授 吉村 長久, 教授 戸口田 淳也, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The discovery of resetting human somatic cells via introduction of four transcription factors into an embryonic stem cell-like state that enables the generation of any cell type of the human body has revolutionized the field of medical science. The generation of patient-derived iPSCs and the subsequent differentiation into the cells of interest has been, nowadays, widely used as model system for various inherited diseases. The aim of this thesis was to generate iPSCs and to subsequently derive NPCs which can be differentiated into neurons in order to model the two most common forms of the NCLs: LINCL which is caused by mutations within the TPP1 gene, encoding a lysosomal enzyme, and JNCL which is caused by mutations within the CLN3 gene, affecting a lysosomal transmembrane protein. It was shown that patient-derived fibroblasts can be successfully reprogrammed into iPSCs by using retroviral vectors that introduced the four transcription factors POU5F1, SOX2, KLF4 and MYC. The generated iPSCs were subsequently differentiated into expandable NPCs and finally into mature neurons. Phenotype analysis during the different stages, namely pluripotent iPSCs, multipotent NPCs and finally differentiated neurons, revealed a genotype-specific progression of the disease. The earliest events were observed in organelle disruption such as mitochondria, Golgi and ER which preceded the accumulation of subunit c of the mitochondrial ATPase complex that was only apparent in neurons. However, none of these events led to neurodegeneration in vitro. The established disease models recapitulate phenotypes reported in other NCL disease models such as mouse, dog and sheep model systems. More importantly, the hallmark of the NCLs, accumulation of subunit c in neurons, could be reproduced during the course of disease modeling which demonstrates the suitability of the established system. Moreover, the derived expandable NPC populations can be used for further applications in drug screenings. Their robust phenotypes such as low levels of TPP1 activity in LINCL patient-derived NPCs or cytoplasmic vacuoles, containing storage material, observed in CLN3 mutant NPCs, should serve as possible phenotypic read-outs.