The thesis describes the effects of various plant flavonoids (curcumin, epigallocatechin-3-gallate [EGCG], luteolin, apigenin, myricetin, quercetin, and cyanidin) on the physiological properties and viability of cultured human retinal pigment epithelial (RPE) cells. It is described that, with the exception of EGCG, all flavonoids tested decrease dose-dependently the RPE cell proliferation, migration, and secretion of VEGF. Luteolin, apigenin, myricetin, and quercetin decreased the viability of RPE cells at higher concentrations, by triggering cellular necrosis. Curcumin decreased the viability of RPE cells via induction of early necrosis and delayed apoptosis. The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, and increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein. Myricetin caused caspase-3 independent RPE cell necrosis mediated by free radical generation and activation of calpain and phospholipase A2. The myricetin- and quercetin-induced RPE cell necrosis was partially inhibited by necrostatin-1, a blocker of programmed necrosis. The author concludes that the intake of curcumin, luteolin, apigenin, myricetin, and quercetin as supplemental cancer therapy or in the treatment of retinal diseases should be accompanied by careful monitoring of the retinal function. Possible beneficial effects of EGCG and cyanidin in the treatment of retinal diseases should be examined in further investigations.

INTRODUCTION: Schizophrenia (SZ) and bipolar disorder (BD) are neurodegenerative psychotic disorders hallmarked by reductions in gray and white matter volume. Limitations in neuroimaging have led to the use of OCT to study retinal layer biomarkers and their relation to brain pathology. This thesis includes a meta-analysis of current literature and an exploratory analysis of retinal layer thickness in relation to SZ and BD. METHODS: For the meta-analysis, twelve articles were identified using PubMed, Web of Science, and Cochrane database. Diagnostic groups were proband (SZ and BD combined), SZ only, BD only, and healthy control (HC) eyes. Analyses utilized fixed and random effects models, in addition to assuring that bias was adjusted for and that results were cross-validated. Statistical analyses were performed using the “meta” package in R, with results reported as standard mean differences (SMD). The exploratory analysis included a total of 38 subjects (24 probands and 14 HC). Retinal measures were co-varied for age, sex, race, body mass index (BMI), and best-corrected visual acuity (BCVA). Correlations between retinal and clinical and cortical measures were also performed. Clinical data included illness duration, symptom severity, antipsychotic dosage, and smoking status. Neuroimaging data included gray matter (GM) thickness, gray matter volume, and intracranial volume (ICV). Linear effects and mixed effects models were used to study mean eye and right/left eye measures, respectively. Statistical analysis was done in R. RESULTS: A total of 820 patient eyes (541 SZ and 279 BD) and 904 HC eyes were used for the meta-analysis. Compared to HC eyes, probands, SZ, and BD eyes showed significant thinning the peripapillary retinal nerve fiber layer (RNFL), with atrophy greatest in the nasal, temporal, and superior regions. In addition, all diagnostic groups demonstrated significant reductions in the combined ganglion cell layer and inner plexiform layer (GCL-IPL) compared to HC. No significant differences were found for choroidal and macular measures. No significant relationships were seen from meta-regression analysis for clinical measures. For the exploratory analysis, retinal measures from a total of 24 probands (18 SZ and 6 BD) and 14 HC was studied. Compared to HC, probands showed reductions in overall RNFL in mean eye measures, while increases in the inner and outer RNFL were seen in left eye measures. No significant group differences were seen in the GCL, IPL, and inner nuclear layer (INL). The outer plexiform layer (OPL) showed significant thickening in probands and SZ compared to HC for all eye measures. Probands showed trending reductions in the outer nuclear layer (ONL) in the left eye compared to HC. No significant correlations were found between retinal layers and illness duration, overall PANSS (Positive and Negative Syndrome Scale) score, PANSS negative symptom subscore, and smoking status. PANSS positive symptom subscore showed significant and trending negative correlations to the RNFL and GCL, respectively. Antipsychotic medication dosage displayed a trending negative relationship with the IPL. GM thickness showed a significant and trending negative correlation to the RNFL and ONL, respectively. Furthermore, a trending inverse relationship was observed between GM volume and the OPL. Finally, ICV demonstrated a trending and significant negative relationship with GCL and OPL thickness, respectively. CONCLUSION: The meta-analysis showed that atrophy in RNFL and GCL-IPL measures are widely associated with psychosis. Furthermore, it supports previous findings of gray and white matter reductions in SZ and BD. The exploratory analysis showed psychosis-associated reductions in the RNFL and ONL layers, consistent with previous literature. Contradictory findings, the thickening of the ONL, can be attributed to the conflicting findings, but might also be explained by neuro-inflammatory pathways related to psychotic disorders.

The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 contributes to the timing of cell cycle withdrawal during development and, consequently, in organogenesis. Within the retina, this effector protein is up-regulated during the birth of neuronal and glial cells [Dev. Biol. (2000) 299]. However, its expression within the retinal pigment epithelium (RPE), a supporting cell layer that is essential for neural retina development and function, has not previously been reported. We show that p27Kip1 protein expression in the RPE occurs in two phases: an up-regulation during mid-to late embryonic stages and a down-regulation during the subsequent postnatal period. In the early phase of up-regulation, an inverse relationship is seen between expression of p27Kip1 and PCNA, an indicator of cycling cells. During both up-and down-regulation, the change in spatial pattern of expression proceeds in a central to peripheral manner, with p27Kip1 up-regulation paralleling retinal maturation. These data suggest that this cell cycle regulator may be an important factor controlling the timing of RPE cell cycle withdrawal.

The World Health Organization estimates that there are 285 million people suffering from visual impairment worldwide. The top two causes of uncorrectable vision loss are glaucoma and age-related macular degeneration (AMD), with 112 million people anticipated to be impacted by glaucoma by 2040 and nearly 15% of U.S. adults aged 43-86 predicted to be diagnosed with AMD over the next 15 years. To slow the progression of these ophthalmic diseases, the most valuable preventive action is timely detection and treatment by an ophthalmologist. However, over 50% of glaucoma cases go undetected due to lack of timely assessment by a medical expert. This thesis seeks to transform artificial intelligence (AI) into a trustworthy partner to clinicians, aiding in expediting diagnostic screening for obvious cases and serving as corroboration/a ‘second opinion’ in ambiguous cases. In order to develop AI algorithms that can be trusted as team-mates in the clinic, the AI must be robust to data collected at various sites/from various patient populations, its decision-making mechanisms must be explainable, and to benefit the broadest population (for whom expensive imaging equipment and/or specialist time may not be available), it must be portable. This thesis addresses these three challenges (1) by developing and evaluating robust deep learning (DL) algorithms for detection of glaucoma and AMD from data collected at multiple sites or using multiple imaging modalities, (2) by making AI interpretable, through: (a) comparison of image concepts used by DL systems for decision-making with image regions fixated upon by human experts during glaucoma diagnosis, and (b) through odds ratio ranking of clinical biomarkers most indicative of AMD risk used by both experts and AI, and (3) by enhancing theimage quality of data collected via a portable OCT device using deep-learning based super-resolution generative adversarial network (GAN) approaches. The resulting robust deep learning algorithms achieve accuracy as high as 95% at detection of glaucoma and AMD from optical coherence tomography (OCT) and OCT angiography images/volumes. The interpretable AI-concept/expert-eye-movement comparison showed the importance of three OCT-report sub-regions used by both AI and human experts for glaucoma detection. The pipeline described here for evaluating AI robustness and validating interpretable image concepts used by deep learning systems in conjunction with expert eye movements has the potential to help standardize the acceptance of new AI tools for use in the clinic. Furthermore, the eye movement collection protocols introduced in this thesis may also help to train current medical residents and fellows regarding key features employed by expert specialists for accurate and efficient eye disease diagnosis. The odds ratio ranking of AMD biomarkers distinguished the top two clinical features (choroidal neovascularization and geographic atrophy) most indicative of AMD risk that are agreed upon by both AI and experts. Lastly, GAN-based super-resolution of portable OCT images boosted performance of downstream deep learning systems for AMD detection, facilitating future work toward embedding AI algorithms within portable OCT systems, in order for a larger population to gain access to potentially sight-saving technology. By enhancing AI robustness, interpretability, and portability, this work paves the way for ophthalmologist-AI teams to achieve augmented performance compared to human experts or AI alone, leading to expedited eye disease detection, treatment, and thus better patient outcomes.

Indiana University-Purdue University Indianapolis (IUPUI) / Fetal alcohol spectrum disorder (FASD) is a result of prenatal alcohol exposure, producing a wide range of defects including craniofacial, sensory, motor and cognitive deficits. Many ocular abnormalities are frequently associated with FASD including microphthalmia, optic nerve hypoplasia, and cataracts. FASD is highly prevalent in low socioeconomic populations, where it is also accompanied by higher rates of malnutrition and alcoholism. Using zebrafish as a model to study FASD retinal defects has been extremely insightful in understanding the ethanol-induced retinal defects at the cellular level. Zebrafish embryos treated with ethanol from mid-blastula transition through somitogenesis (2-24 hours post fertilization; hpf) showed defects similar to human ocular deficits including microphthalmia, optic nerve hypoplasia, and photoreceptor differentiation defects. Ethanol exposure altered critical transcription factor expression involved in retinal cell differentiation. Retinoic acid (RA) and folic acid (FA) nutrient co-supplementation rescued optic nerve and photoreceptor differentiation defects. Ethanol exposure during retinal morphogenesis stages (16-24 hpf), produced retinal defects like those seen with ethanol exposure between 2-24 hpf. Significantly, during ethanol-sensitive time window (16-24 hpf), RA co-supplementation moderately rescued these defects, whereas, FA cosupplementation showed significant rescue of optic nerve and photoreceptor differentiation. RA, but not FA, supplementation after ethanol exposure could restore ethanol-induced optic nerve and photoreceptor differentiation defects. Ethanol exposure did not affect timing of retinal cell differentiation induction, but later increased retinal cell death and proliferation. Ethanol-treated embryos showed increased retinal proliferation in the outer nuclear layer (ONL), inner nuclear layer (INL), and ciliary marginal zone (CMZ) at 48 hpf and 72 hpf. In order to identify the genesis of ethanol-induced persistent retinal defects, ethanol effects on retinal stem cell populations in the CMZ and the Müller glial cells (MGCs) were examined. Ethanol treated retinas had an expanded CMZ indicated by histology and Alcama, a retinal stem cell marker, immunolabeling, but reduced expression of rx1 and the cell cycle exit marker, cdkn1c. Ethanol treated retinas also showed reduced MGCs. At 72 hpf, ONL of ethanol exposed fish showed fewer photoreceptors expressing terminal differentiation markers. Importantly, these poorly differentiated photoreceptors co-expressed the basic helix-loop-helix (bHLH) proneural differentiation factor, neurod, indicating that ethanol exposure produced immature and undifferentiated photoreceptors. Reduced differentiation along with increased progenitor marker expression and proliferation suggest cell cycle exit failure due to ethanol exposure. These results suggested that ethanol exposure disrupted stem cell differentiation progression. Wnt, Notch and proneural gene expression regulate retinal stem cell proliferation and transition into progenitor cells. Ethanol exposure disrupted Wnt activity in the CMZ as well as Notch activity and neurod gene expression in the retina. RA and FA co-supplementation were able to rescue Wnt activity in the CMZ and rescue downstream Notch activity. To test whether the rescue of these Wnt-active cells could restore the retinal cell differentiation pathways, ethanol treated embryos were treated with Wnt agonist. This treatment could restore Wnt-active cells in the CMZ, Notch-active cells in the retina, proliferation, and photoreceptor terminal differentiation. We conclude that ethanol exposure produced persistent defects in the stem cell Wnt signaling, a critical pathway in retinal cell differentiation. Further analysis of underlying molecular mechanisms will provide insight into the embryonic origins of ethanol-induced retinal defects and potential therapeutic targets to cure this disorder.

Indiana University-Purdue University Indianapolis (IUPUI) / Dry age-related macular degeneration (AMD) is a progressive neurodegenerative disorder characterized by geographical atrophy of the retinal pigment epithelium (RPE), causing irreversible central vision loss. Systemically-delivered bone marrow-derived cells (BMDCs), programmed to RPE-like cells via expression of human RPE65, regenerate damaged RPE and preserve vision in murine models of retinal degeneration. RPE65 rapidly activates adenylate cyclase (AC), which then activates endogenous Rpe65 and RPE-associated marker Cralbp. Previous studies expressed RPE65 from an integrating lentiviral vector (ILV), which is an unnecessary safety risk due to the potential for insertional mutagenesis, as long- term expression of RPE65 is not required for BMDC programming. Here, we developed a 3rd generation integrase-defective lentiviral vector (IDLV) for programming both murine and human BMDCs to RPE-like cells, reducing insertional mutagenesis risk and expanding the protocol to include human cells. We enhanced IDLV3-RPE65 infection of murine and human BMDCs by preloading concentrated vector on RetroNectin at MOI 50, and infecting with low-speed centrifugation, increasing RPE65 mRNA levels from ~12-fold to ~25-fold (p<0.05). IDLV3-RPE65 infection initiates expression of endogenous Rpe65 mRNA expression in murine BMDC and Cralbp/CRALBP mRNA in both murine and human BMDCs, indicating programming to RPE-like cells. Inhibiting AC in RPE65infected BMDCs abrogated expression of the endogenous genes, confirming the role of AC activation in programming. Critically, IDLV3-RPE65-infected murine BMDCs are recruited to and incorporate into to the RPE layer, and preserve vision in murine models of retinal degeneration. We conclude that BMDCs programmed with IDLV3-RPE65 successfully prevent retinal degeneration progression and are appropriate for testing in human cells, with a view to move into human clinical trial for the treatment of dry AMD. This approach significantly increases the safety of the therapy and is, to the best of our knowledge, the first application of a single IDLV in the generation of therapeutic cells from adult stem cells.

En la presente Tesis Doctoral se describen los cambios celulares que ocurren en la retina en la enfermedad de Parkinson y su posible uso como biomarcadores tempranos de la enfermedad. Los pacientes con enfermedad de Parkinson poseen acumulaciones de alfa sinucleína fosforilada en la retina similares a las que se encuentran en el cerebro de los mismos pacientes. De hecho, la cantidad de alfa-sinucleína fosforilada en la retina correlaciona con la cantidad de alfa-sinucleína fosforilada en el cerebro, con el estadio de progresión de la enfermedad y con la severidad de los síntomas motores. Además, en la retina de enfermos de párkinson se describe una degeneración de las células ganglionares melanopsínicas de la retina, lo que podría explicar las alteraciones en los ritmos circadianos y los desórdenes del sueño que aparecen en pacientes. Finalmente, también se muestra la degeneración de las células amacrinas dopaminérgicas, que se reducen en un 45%. Este fallo en el sistema dopaminérgico de la retina provoca alteraciones morfológicas en las células amacrinas AII, sus principales postsinápticas, y podría explicar algunas alteraciones visuales descritas en la enfermedad como la disminución de la sensibilidad al contraste o de la agudeza visual. En global, los resultados muestran que la retina reproduce los procesos degenerativos que ocurren en el cerebro en la enfermedad de Parkinson y, por tanto, que es un tejido idóneo para el estudio de la enfermedad. Además, el estudio de la retina aporta información sobre el estadio de la enfermedad y puede ser empleado como un biomarcador temprano que ayude al diagnóstico y seguimiento de la misma.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12917号 / 論医博第2092号 / 新制||医||1009(附属図書館) / 32127 / 京都大学大学院医学研究科医学専攻 / (主査)教授 河野 憲二, 教授 大森 治紀, 教授 渡邉 大 / 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(医科学) / 甲第18904号 / 医科博第60号 / 新制||医科||4(附属図書館) / 31855 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 小柳 義夫, 教授 吉村 長久, 教授 髙橋 淳 / 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(社会健康医学) / 甲第20057号 / 社医博第75号 / 新制||社医||9(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 山田 亮, 教授 小泉 昭夫, 教授 松田 文彦 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM