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This project explores the use of a novel sustained release mucoadhesive micelle-based drug delivery system in combination with 0.005% latanoprost (LTP) on our partially open angle mouse model of glaucoma (AP-2β TMR-KO). We previously tested for LTP treatment in our model and found a reduction in intraocular pressure (IOP) 20 minutes following treatment. This information led us to investigate the long-term effect of LTP treatment and micelle loaded with LTP (MLTP) treatment in our model. We hypothesized that the MLTP treatment would be more effective in reducing IOP and preventing glaucomatous effects than LTP treatment alone in the AP-2β TMR-KO mice. The MLTP groups of animals (wildtype and mutant) were treated every 3 days, and this was compared with animals treated with LTP daily as well as animals treated every 3 days with LTP alone for comparison’s sake for 60 days. IOP measurements were taken every 3 days. Following long term LTP treatment alone, mutant mice showed a consistent decrease in their baseline IOPs with a significant reduction in baseline IOP at 35 days of treatment across all cohorts (P<0.0001). In comparison, mutants treated with MLTP exhibited an even greater reduction in baseline IOP following long term treatment. After the treatment period, mice were euthanized, and their eyes were enucleated, fixed, sectioned, and stained for retinal ganglion cells (RGCs) using Brn3a. Mutant mice exhibited a significant decrease in RGC cell number when compared to wildtype, and this loss was not rescued by treatment with LTP. However, mutants treated with MLTP demonstrated significant RGC cell protection compared to eyes of untreated mutants, as well as everyday LTP treated mutants. / Thesis / Master of Science (MSc) / An effective treatment strategy is required to prevent irreversible blindness caused from glaucoma. Unfortunately, compliance with current medications is extremely poor, as they require frequent administration due to their low ocular bioavailability and short-term effect. As such, this thesis aims to explore an alternative drug delivery approach in a partially open angle mouse model of glaucoma to prevent the worsening of glaucoma and ultimately improve patient compliance.

Indiana University-Purdue University Indianapolis (IUPUI) / Glaucoma is a leading cause of blindness characterized by the progressive loss of retinal ganglion cells (RGCs), essentially severing the connection between the eye and the brain. Among many underlying causes of the disease, mutations in the Optineurin (OPTN) gene result in severe RGC neurodegeneration in the absence of elevated intraocular pressure, providing a novel opportunity to study molecular mechanisms that lead to RGC neurodegeneration associated with glaucoma. Efforts of this study establishing a human pluripotent stem cell (hPSC)-derived in vitro disease model by inserting OPTN(E50K) mutation via CRISPR/Cas9 genome editing and investigate the cellular mechanisms of RGC neurodegeneration associated with glaucoma. OPTN(E50K) RGCs revealed neurodegeneration phenotypes, including downregulation of RGCs transcription factors, neurite retraction, and hyperexcitability, suggesting that OPTN(E50K) RGCs can serve as an appropriate disease model to study glaucoma-associated neurodegeneration. Since OPTN serves a primary role as an autophagy receptor, we further hypothesized that the OPTN(E50K) mutation disrupts autophagy in RGCs, and modulation of autophagy by mammalian target of rapamycin (mTOR)-independent pathways can preserve RGC phenotypes by maintaining mTOR signaling. OPTN(E50K) RGCs exhibited a higher number of OPTN puncta along with an overall reduced expression of OPTN protein, indicating a gain of toxic protein accumulation or loss of protein function. Furthermore, OPTN(E50K) RGCs revealed an accumulation of the autophagosome protein LC3 in a punctal manner as well as increased expression of lysosomal proteins, suggesting a disruption of degradation pathway in autophagosome and lysosome fusion. As mTOR complex 1 (mTORC1) signaling serves as a negative regulator of autophagy, a downregulation of mTORC1 signaling via activation of stress sensor adenosine monophosphate-activated protein kinase (AMPK) was observed as a possible compensatory mechanism for autophagy deficits in OPTN(E50K) RGCs. Pharmacological inhibition of mTOR in wild-type hRGCs resulted in similar disease-related phenotypes, while preservation of the mTOR pathway in OPTN(E50K) RGCs by treatment with the mTOR-independent autophagy modulator trehalose cleared OPTN accumulated puncta, preserving mTORC1 signaling, as well as rescuing neurodegenerative phenotypes. To further validate these associations in an animal model, the microbead occlusion mouse model was established by injection of magnetic microbeads in the anterior chamber to block aqueous outflow resulting ocular hypertension. In agreement with our findings in hRGCs, a decrease in mTOR signaling associated with an increase in the expression of autophagy-associated proteins was observed in RGCs in the microbead occlusion model. Additionally, these disease-related phenotypes were observed specifically within RGCs but not cortical neurons with an underlying OPTN(E50K) mutation, demonstrating that autophagy represents an essential pathway in RGCs to maintain homeostasis, and selective disrupt of autophagy in RGCs leads to neurodegeneration. Taken together, the results of this study highlight an essential balance between autophagy and mTORC1 signaling that is essential for the homeostasis of RGCs, while disruption to these signaling pathways contributes to neurodegenerative features in glaucoma. These results also demonstrated the ability to pharmacologically intervene to experimentally manipulate these pathways and rescue neurodegenerative phenotypes, providing a potential therapeutic target to prevent glaucoma-associated neurodegeneration.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第25160号 / 医博第5046号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 渡邊 直樹, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Quatre microélectrodes ont été insérées dans le ganglion stellaire gauche (GS) de préparations canines in vivo pour évaluer la décharge des potentiels d’action dans les neurones situés dans ce ganglion périphérique durant un état cardiovasculaire stable et suivant des injections systémiques et locales de nicotine. Durant les périodes de contrôle, des changements mineurs ont été observés dans la pression artérielle systolique, dans le rythme cardiaque et dans le temps de conduction atrio-ventriculaire. L’activité générée par les neurones du GS est demeurée relativement constante à l’intérieure de chaque chien, mais variait entre les préparations. L’administration de nicotine systémique a altéré les variables physiologiques et augmenté l’activité neuronale. Même si différents changements au niveau des variables physiologiques ont été observés entre les animaux, ces changements demeuraient relativement constants pour un même animal. La dynamique de la réponse neuronale était similaire, mais l’amplitude et la durée variaient entre et au sein des chiens. L’injection de nicotine dans une artère à proximité du GS a provoqué une augmentation marquée des potentiels d’action sans faire changer les variables physiologiques. La technique d’enregistrement permet donc de suivre le comportement de multiples populations de neurones intrathoraciques situés dans le GS. La relation entre l’activation neuronale du GS et les changements physiologiques sont stables pour chaque chien, mais varient entre les animaux. Cela suggère que le poids relatif des boucles de rétroaction impliquées dans la régulation cardiovasculaire peut être une caractéristique propre à chaque animal. / Four micro-electrodes were inserted in the left stellate ganglion (SG) of in vivo canine preparations to evaluate the firing of neuronal somata located in this peripheral ganglion during stable cardiovascular state and following local and systemic injection of nicotine. During control periods, minor changes were observed in systolic arterial pressure, the heart rhythm and the atrioventricular conduction time. The activity generated by SG neurons remained relatively constant within each dog, but the firing rate was variable among the preparations. Systemic nicotine administration altered the physiological variables and increased the neuronal activity. Although different patterns of physiological changes were observed among the preparations, it remained invariant upon successive injections in each animal. The behaviour of the neuronal response was similar but varies in amplitude and duration both within and between the dogs. Local injections of nicotine in an artery close to the SG induced a brief and huge burst of neuronal firing, but did not influence the physiological response. The recording technique thus permit to follow the behaviour of multiple intrathoracic neuronal populations located in the SG. The relation between the SG firing and the physiological changes is stable in each dog, but differed between the animals. It suggests that the weight of the different feedback loops involved in the cardiovascular regulation might be a characteristic feature of each animal and/or the position of the electrodes in the SG is critical, since different neuronal populations are present and could react differently.