京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24045号 / 生博第471号 / 新制||生||63(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 井垣 達吏, 教授 上村 匡, 教授 鈴木 淳 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Une des pathologies les plus répandues dans les pays développés est la dégénérescence maculaire liée à l’âge (DMLA). Elle se présente sous différentes formes dont la plus sévère est caractérisée par la présence de formation de néo-vascularisation provenant de la choroïde (CNV) migrant vers la rétine. Malheureusement, le diagnostic peut être seulement posé une fois que les symptômes apparaissent et que la vision est déjà affectée. Récemment, un vif engouement s’est porté sur les microARN (miARN), devenus des molécules d’intérêt pour le développement de biomarqueurs et représentant également un important potentiel thérapeutique. Nous avons donc émis l’hypothèse que l’identification de miRNA dans un contexte de DMLA avec CNV permettrait de caractériser leurs rôles, tant au niveau de l’identification de biomarqueurs que de nouvelles cibles thérapeutiques. Le premier objectif de notre projet était d’identifier chez l’humain une signature de miARN spécifique à la forme humide de la DMLA. Pour ce faire, nous avons réalisé un criblage des miARN dans l’humeur vitrée de patients. Nous avons observé une augmentation des niveaux de miR-146a et une diminution de miR-106b et miR-152 spécifique à la DMLA. Cette signature fut confirmée dans le plasma de cette même cohorte de patients. D’autre part, l’exploration de bases de données AMD Gene Consortium (AGC) et Ingenuity Pathway Analysis (IPA) a démontré une relation entre les miARN détectés et des mutations génétiques associées à la forme DMLA avec CNV. En effet, nous avons identifié un SNP (single-nucleotide polymorphism ou SNP) (rs1063320) dans un site de liaison de miR-152 du gène HLA-G. Le second objectif était d’explorer la possibilité d’utiliser, du point de vue thérapeutique, un des miARN identifiés au 1er objectif. Nous avons, tout d’abord, quantifié l’expression de miR-146a, miR-106b et miR152 dans un des modèles classiques de la DMLA avec CNV chez la souris (brûlures par laser). Le candidat retenu fut miR-106b, puisqu’en plus d’être impliqué dans l’angiogenèse, son expression dans le modèle animal reflétait de plus près celle obtenue chez les patients. Suite à ces résultats, nous avons: exploré A) le mécanisme influençant la diminution de l’expression de miR-106b et B) l’effet de sa surexpression sur l’angiogenèse. D’abord en A), nous avons observé une activation de la voie de PERK dans notre modèle animal de CNV induite par laser menant à une diminution de l’expression de MCM7 et du polycistron miR-106b~25. . Ensuite en B), nous avons pu observer l’effet anti-angiogénique de miR-106b sur la migration des cellules endothéliales in vitro et sur la formation de NV in vivo. L’action anti-angiogénique de miR-106b pourrait ainsi avoir un potentiel thérapeutique important sur la formation de la CNV dans la DMLA. / Age-related macular degeneration (AMD) is a leading cause of blindness worldwide affecting individuals over the age of 60. The neovascular form (NV AMD) is characterized by choroidal neovascularization (CNV) and is responsible for the majority of central vision impairment. Unfortunately, diagnostic of AMD can only be done after symptoms have appeared and loss of visual field occurred. Recently, there is a growing interest in microRNAs (miRNAs) for their eventual use in the development of new biomarkers or new therapeutic strategies. Our hypothesis is that wet AMD is associated with specific signature of several miRNAs that can be used as biomarkers for the disease. These miRNAs can be harnessed for therapeutic interventions. Our first objective was to identify a specific signature of miRNAs for wet AMD by using non-biased microRNA arrays and individual TaqMan qPCRs. We profiled miRNAs in the vitreous humour and plasma of patients with NV AMD. We identified a disease-associated increase in miR-146a and a decrease in miR-106b and miR-152 in the vitreous humour, which was reproducible in plasma. Moreover, miR-146a/miR-106b ratios discriminated patients with NV AMD with an area under the Receiver Operating Characteristic curve (ROC AUC) of 0,977 in vitreous humour and 0,915 in plasma, suggesting potential for a blood-based diagnostic. Furthermore, using the AGC and IPA database, we mapped a NV AMD-associated single nucleotide polymorphism (SNP) (rs1063320) in a binding site for miR-152-3p in the HLA-G gene. Our second objective was to explore the therapeutic potential of a specific miRNA (identified in objective 1). First, we explored the expression levels of miR-146a, miR-106b and miR-152 in the laser burn mouse model. We demonstrated that levels of miR-106b were significantly decreased in this mouse model of CNV. We divided this objective in two parts: Part A, explore mechanisms causing miR-106b downregulation and part B, study the therapeutic potential of miR-106b on the inhibition of angiogenesis and CNV formation. First, we showed that expression of the miR-106b-25 cluster is negatively regulated by the ER stress pathway of protein kinase RNA-like ER kinase (PERK) and a reduction in levels of MCM7, the host gene of miR-106b. �����Second, we demonstrated that therapeutic delivery of miR-106b to the retina with lentiviral vectors protects against aberrant retinal neovascularization in two distinct mouse models of pathological neo-vascularization. Results from this study suggest that miRNAs, such as miR-106b, have the potential to be used as multitarget therapeutics for conditions characterized by aberrant retinal neovascularization.

Indiana University-Purdue University Indianapolis (IUPUI) / Eukaryotic cells rapidly modulate protein synthesis in response to environmental cues through the reversible phosphorylation of eukaryotic initiation factor 2 (eIF2α~P) by a family of eIF2α kinases. The eIF2 delivers initiator Met-tRNAiMet to the translational apparatus, and eIF2α~P transforms its function from a translation initiation factor into a competitive inhibitor of the guanine nucleotide exchange factor (GEF) eIF2B, which is responsible for the recycling of eIF2-GDP to the translationally-competent eIF2-GTP state. Reduced eIF2-GTP levels lower general protein synthesis, which allows for the conservation of energy and nutrients, and a restructuring of gene expression. Coincident with global translational control, eIF2α~P directs the preferential translation of mRNA encoding ATF4, a transcriptional activator of genes important for stress remediation. The term Integrated Stress Response (ISR) describes this pathway in which multiple stresses converge to phosphorylate eIF2α and enhance synthesis of ATF4 and its downstream effectors. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary across a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2α~P, while a notable cohort of key regulators are subject to preferential translation. From this latter group, we identify IBTKα as being subject to both translational and transcriptional induction during eIF2α~P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKα mRNA involves the stress-induced relief of two inhibitory uORFs in the 5’-leader of the transcript. Also identified as being subject to preferential translation is mRNA encoding the bifunctional aminoacyl tRNA synthetase EPRS. During eIF2α~P, translational regulation of EPRS is suggested to occur through the bypass of a non-canonical upstream ORF encoded by a CUG start codon, highlighting the diversity by which upstream translation initiation events can regulate expression of a downstream coding sequence. This body of work provides for a better understanding of how translational control during stress is modulated genome-wide and for the processes by which this mode of gene regulation in the ISR contributes to cell fate.

We have identified a novel Cyclin, called Cyclin O, which is able to bind and activate Cdk2 in response to intrinsic apoptotic stimuli. We have focused on the study of Cyclin Oα and Cyclin Oβ, alternatively spliced products of the gene. Upon treatment with different stress stimuli, transfected Cyclin Oα accumulates in dense aggregations in the cytoplasm compatible with being Stress Granules (SGs). Furthermore, we have seen that Cyclin Oβ and a point mutant of the N-terminal part of the protein constitutively localize to the SGs. Although both alpha and beta isoforms are proapoptotic, only Cyclin Oα can bind and activate Cdk2. On the other hand, we have demonstrated that Cyclin O is upregulated by Endoplasmic Reticulum (ER) stress and is necessary for ER stress-induced apoptosis. Cyclin O activates specifically the PERK pathway and interacts with the PERK inhibitor protein p58IPK. Moreover, Cyclin O participates in the activation of other eIF2α kinases. We have also observed that a pool of Cyclin O is located in active mitochondria, suggesting a function of the protein linked to oxidative metabolism.Hemos identificado una nueva Ciclina, llamada Ciclina O, que es capaz de unirse y activar Cdk2 en respuesta a estímulos apoptóticos intrínsecos. Nos hemos centrado en el estudio de la Ciclina Oα y la Ciclina Oβ, productos de splicing alternativo del gen. En respuesta a diferentes tipos de estrés, la Ciclina Oα se acumula en agregaciones citoplásmicas densas que podrían corresponder a Gránulos de Estrés (SGs). Además, hemos visto que la Ciclina Oβ y un mutante puntual de la parte N-terminal de la proteína se localizan constitutivamente en los SGs. Aunque las dos isoformas alfa y beta son proapoptóticas, solo la Ciclina Oα es capaz de unirse y activar Cdk2. Por otro lado, hemos demostrado que los niveles de Ciclina O se incrementan en respuesta al estrés de Retículo Endoplásmico (RE) y que esta proteína es necesaria para la inducción de apoptosis dependiente de estrés de RE. La Ciclina O activa específicamente la vía de PERK e interacciona con la proteína inhibidora de PERK p58IPK. Además, la Ciclina O participa en la activación de otras quinasas de eIF2α. La Ciclina O se localiza en mitocondrias activas, lo que sugiere una función de la proteína ligada al metabolismo oxidativo.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Autonomic innervation of the heart begins in utero and continues during the neonatal phase of life. A balance between the sympathetic and parasympathetic arms of the autonomic nervous system is required to regulate heart rate as well as the force of each contraction. Our lab studies the development of sympathetic innervation of the early postnatal heart in a conditional knockout (cKO) of Src homology protein tyrosine phosphatase 2 (Shp2). Shp2 is a ubiquitously expressed non-receptor phosphatase involved in a variety of cellular functions including survival, proliferation, and differentiation. We targeted Shp2 in post-migratory neural crest (NC) lineages using our novel Periostin-Cre. This resulted in a fully penetrant mouse model of diminished cardiac sympathetic innervation and concomitant bradycardia that progressively worsen. Shp2 is thought to mediate its basic cellular functions through a plethora of signaling cascades including extracellular signal-regulated kinases (ERK) 1 and 2. We hypothesize that abrogation of downstream ERK1/2 signaling in NC lineages is primarily responsible for the failed sympathetic innervation phenotype observed in our mouse model. Shp2 cKOs are indistinguishable from control littermates at birth and exhibit no gross structural cardiac anomalies; however, in vivo electrocardiogram (ECG) characterization revealed sinus bradycardia that develops as the Shp2 cKO ages. Significantly, 100% of Shp2 cKOs die within 3 weeks after birth. Characterization of the expression pattern of the sympathetic nerve marker tyrosine hydroxylase (TH) revealed a loss of functional sympathetic ganglionic neurons and reduction of cardiac sympathetic axon density in Shp2 cKOs. Shp2 cKOs exhibit lineage-specific suppression of activated pERK1/2 signaling, but not of other downstream targets of Shp2 such as pAKT (phosphorylated-Protein kinase B). Interestingly, restoration of pERK signaling via lineage-specific expression of constitutively active MEK1 (Mitogen-activated protein kinase kinase1) rescued TH-positive cardiac innervation as well as heart rate. These data suggest that the diminished sympathetic cardiac innervation and the resulting ECG abnormalities are a result of decreased pERK signaling in post-migratory NC lineages.