京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20994号 / 医博第4340号 / 新制||医||1027(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 鈴木 茂彦, 教授 伊佐 正, 教授 大森 孝一 / 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21685号 / 医博第4491号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 辻川 明孝, 教授 高橋 淳, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Introduction: Inherited retinal diseases (IRDs) are one of the most common causes of incurable blindness in children and young adults. In the Czech Republic, prior to the start of our work, these disorders had not been the subject of a systematic research. The aim of the study was to identify, clinically characterize and molecular genetically analyse Czech patients with monogenic IRDs and based on the knowledge gained subsequently implement preventive and therapeutic measures to clinical practice. Material and methods: We have performed a comprehensive clinical examination, genealogical analysis and molecular genetic investigation in patients with IRDs and their family members. Detailed ocular examination included spectral domain optical coherence tomography, high-resolution fundus photography and autofluorescence imaging. DNA was isolated from venous blood samples or buccal cells. Causal variants were searched for using Sanger and massively parallel sequencing, and their pathogenicity was evaluated in the context of previously published data, bioinformatical analysis and segregation in available family members. Results: In total, 103 individuals from 76 Czech families diagnosed with IRDs were characterized and their data published. Specifically, we have described clinical and molecular genetic...

Inherited Retinal Degenerations (IRDs) are a heterogenous group of diseases which lead to vision impairment and can be found both in humans and in dogs. About 1 in 1,380 humans is estimated to suffer from an autosomal recessive IRD, which would be 5.5 million people worldwide, and many more are estimated to be unaffected carriers. This makes autosomal recessive IRDs likely the most common group of Mendelian diseases in humans. Today, about 300 genetic mutations have been connected to cause retinal diseases in humans. Whilst in dogs only 32 genes have been identified, numerous eye conditions have been described where the genetic cause has not yet been identified. This suggests that there are much more genetic causes to discover in the dog genome. Additionally, the dog serves well as a model organism to investigate IRDs as it is sharing morphological and genetic similarities with humans. For these reasons, proper software, a canine reference genome of high quality, and smart implementation of bioinformatic tools and methods are a big advantage to increase chances of finding new causative genetic variants and subsequently enable faster detection of possible preventions of the disease or at least alleviating its symptoms via early diagnosis. In this project, a pre-existing pipeline consisting of Bash scripts was stepwise improved with the goal to increase its efficiency. After controlling whether previous data could still be reproduced with the old pipeline in a first step, the software was exchanged to more updated versions in a second step. A main change was the replacement of the mapping tool Burrows-Wheeler Aligner (BWA) from bwa mem to bwa-mem2 mem, and the update of deprecated Genome Analysis Toolkit (GATK) 3.7 to version 4.3 or 4.4. Thirdly, the scripts were adapted from using the older canine reference genome CanFam3.1 to CanFam4. In a fourth step, for automatization and fastening the running time, the pipeline steps were implemented into the workflow management system Nextflow. Additionally, this step was partly aiming to make the pipeline in concordance with the FAIR-principles. All steps were tested on the same test data set, a Labrador retriever family trio, in which one genetic cause for a canine form of the IRD Stargardt disease in a previous study had been detected, namely an insertion in the ABCA4 gene. Lastly, the workflow was also tested on a second data set of a novel IRD of unknown genetic origin on two sibling pairs of Chinese Crested Dogs (CCR). The adjustment of the pipeline shows similar results regarding the change of mapping tool. Introducing the new reference genome revealed a drop of average coverage by one read average for when using CanFam4, while other results were similar. Using the new reference genome increased the number of unknown variants compared to findings with CanFam3.1. However, the known causative variant for the canine form of Stargardt disease, an insertion in ABCA4 gene, could be found in all cases. The run with Nextflow produced identical results to when the respective steps were run with Bash scripts, but it reduced the running time. Running the workflow on the new data set (CCR) and subsequent annotation and filtering indicate new candidates which could be further investigated as a potential cause for this currently unknown cause for an IRD.

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Vitamin E and selenium play key roles in preventing in vitro lipid peroxidation and free radical damage to retinal tissues. In this research, we studied the effects of hyperbaric oxygen on retinal structure in rats fed diets deficient in vitamin E and/or selenium. We also correlated any alterations in retinal structure with previously measured alterations in electroretinograms (ERGs). Age-matched rats were fed a basal diet deficient in both vitamin E and selenium (B diet), a basal diet supplemented with vitamin E alone (B+E diet), or selenium alone (B+Se diet), or with both micronutrients (B+E+Se). Half the rats in each group were treated (+ HBO) with hyperbaric oxygen (100% O2 at 3 ATA for 1·5 per hr day, 5 days per week) and half were not (-HBO). We previously found that the rats fed the B diet for 6 weeks and treated with HBO for 4 weeks (B+HBO group) had diminished a-wave ERG amplitudes. At this time point all rats in the B group and half of the rats in the B+E+Se group were killed for the structural studies reported here. Surprisingly, we found no evidence of photoreceptor cell necrosis [i.e. a decreased thickness of the outer nuclear layer (ONL)] in retinas from rats in the B+HBO group despite the diminished amplitude of the a-wave which arises from this retinal layer. Quantitative structural analyses of retinas from rats in the B+HBO, B-HBO, B+E+Se-HBO and B+E+Se+HBO groups also failed to reveal any significant differences in the cell height of the retinal pigmented epithelium (nasal, central or temporal regions) or the number of mitochondria, phagosomas or inclusion bodies in the central retinal pigment epithelium (RPE). The inner nuclear layer (INL) thickness was, however, consistently decreased in all retinal regions for the rats in the B+HBO group. Our previous work also showed that only rats fed the B+Se diet for 17 weeks and treated with HBO for 15 weeks (B+Se+HBO group) showed diminished a-wave and b-wave ERG amplitudes. At this time point rats in the B+E+Se, B+E, and B+Se groups were killed for structural studies reported here. Only rats in the B+Se+HBO group showed a significantly decreased (about 20%) thickness of the central ONL. This evidence of photoreceptor cell necrosis correlated very well with our previous observation of diminished a- and b-wave amplitudes only in the B+Se+HBO group (at week 17). Ultrastructural studies after 17 weeks of feeding the experimental diets revealed two different types of inclusion bodies in the central RPE. On the basis of morphological appearance we have termed these inclusion bodies 'electron-dense' and 'granulated'. The central RPE of rats in the B+Se+HBO and B+Se-HBO groups showed a larger number (P < 0·001) of 'granulated' inclusion bodies and a smaller number (P < 0·001) of 'electron-dense' inclusion bodies than rats in any other diet/treatment group at this time point. In marked contrast, there were no observable 'granulated' inclusion bodies and no significant differences in the number of electron-dense inclusion bodies found in the central RPE from rats in any diet/treatment group after 6 weeks of feeding the experimental diets. Our results are discussed with respect to the potential effects of lipid peroxidation on retinal morphology and on electroretinograms.

The retina is one of the most metabolically active tissues in the body and the high energetic demand is met by a well-organized vascular network. Aberrant vasculature is a prominent feature of many vision-threatening diseases, and although angiogenic pathways have been extensively studied the limited efficacy of therapies currently available for the treatment of these diseases suggests that there is more to be elucidated. The caspase family of proteases is best known for their roles in programmed cell death and inflammation, however members of this family have been found to have essential functions independent of cell death. Caspase-8, in particular, has been previously shown to be essential for embryonic vascular development, however, a requirement for caspase-8 in postnatal vascular development has not been established and it is unclear how caspase-8 exerts its function. In this study, we investigate the cell specific roles of caspase-8 in the development of the retinal vasculature using the postnatal mouse retina as our model and identified endothelial caspase-8 as a mediator of canonical Wnt signaling. Inducible endothelial cell-specific caspase-8 knockout (Casp8 iECKO) resulted in a delay in early angiogenesis and barrier establishment, and an increase in inflammation and premature vascular remodeling compared to littermate controls. Assessment of Lef1, a downstream effector of the Wnt pathway, confirmed that this phenotype was a result of inhibited Wnt signaling. We additionally show that caspase-8 mediates this pathway through degradation of its substrate HDAC7. HDAC7 has been shown previously to bind to β-catenin blocking its nuclear translocation. Caspase-8 mediated HDAC7 degradation restores β-catenin translocation and downstream Wnt signaling. We also explore the function of caspase-8 in myeloid cells – microglia and macrophages – during angiogenesis. We used an inducible myeloid-specific caspase-8 knockout (Casp8 imGKO) mouse and found that loss of caspase-8 in these cells did not affect angiogenesis. However, Casp8 imGKO resulted in a reduction in microglia number and a change in their morphology specifying a role for caspase-8 in mediating cell survival and activation in microglia. Altogether we show that caspase-8 exerts cell specific functions during retinal angiogenesis that are independent of cell death. We elucidate a novel role of caspase-8 in mediating Wnt/β-catenin signaling, and implicate caspase-8 as a potential therapeutic target in pathological angiogenesis.

<p>Down syndrome (DS) is a genetic condition caused by the triplication of human chromosome  21 and presents with many phenotypes including decreased brain size, hypocellularity in the brain,  and assorted ocular phenotypes. Some of the ocular phenotypes seen are increased risk of cataracts,  accommodation difficulties, increased risk of refractive errors, and increased retinal thickness. The  Ts65Dn mouse model of DS is a classically used mouse model as it presents a number of  phenotypes also seen in those with DS. Some of these phenotypes include decreased brain volume,  abnormal synaptic plasticity, and ocular phenotypes. These ocular phenotypes include decreased  visual acuity, cataracts, and increased retinal thickness. The Ts65Dn mouse model is trisomic for <em>Dyrk1a</em>, a gene of interest in DS research. We hypothesize that there will be a genotypic and sex effect of retinal thickness and retinal ganglion cell (RGC) count at postnatal day 15 in the Ts65Dn  mouse model of DS. Retinal slices were taken from male and female trisomic and euploid Ts65Dn  mice at P15 and fluorescently labeled for RGCs and bipolar cells via immunohistochemistry. The  retinas were measured for total retinal thickness and RNA-binding protein (RBPMS) positive cells in the RGC layer were counted. There was no genotypic or sex effect when comparing retinal  thickness in trisomic mice as compared to euploid mice. There was a genotypic effect of RBPMS  positive cell count in which the trisomic mice had a higher number of RBPMS positive cells than  euploid mice. Increased retinal thickness along with increased RGC number have both been  implicated with decreased apoptosis in the retina. In the Ts65Dn mouse model along with in  individuals with DS, this could be due to an increase in DYRK1A protein levels reducing apoptosis.  In future studies, determining DYRK1A’s influence in retinal thickness and RGC number could  result in a treatment for overactive <em>DYRK1A</em> that could normalize retinal thickness and RGC  number in those with DS.</p>