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Indiana University-Purdue University Indianapolis (IUPUI) / Huntington disease (HD) is an autosomal dominant disorder characterized by progressive decline of motor, cognitive, and behavioral function. Saccades (rapid, gaze-shifting eye movements) are affected before a clinical diagnosis of HD is certain (i.e. during the premanifest period of the disease). Fundamental questions remain regarding the neural substrates of abnormal saccades and the course of premanifest disease. This work addressed these questions using magnetic resonance imaging (MRI) and a longitudinal study of premanifest disease progression. Gray matter atrophy is a characteristic of HD that can be reliably detected during the premanifest period, but it is not known how such changes influence saccadic behavior. We evaluated antisaccades (AS) and memory guided saccades (MG) in premanifest and manifest HD, then tested for associations between impaired saccadic measures and gray matter atrophy in brain regions involved in these saccadic tasks. The results suggest that slowed vertical AS responses indicate cortical and subcortical atrophy and may be a noninvasive marker of atrophic changes in the brain. We also investigated the brain changes that underlie AS impairment using an event-related AS design with functional MRI (fMRI). We found that, in premanifest and manifest HD, blood oxygenation level dependent (BOLD) response was abnormally absent in the pre-supplementary motor area and dorsal anterior cingulate cortex following incorrect AS responses. These results are the first to suggest that abnormalities in an error-related response network underlie early disease-related saccadic changes, and they emphasize the important influence of regions outside the striatum and frontal cortex in disease manifestations. Though saccadic abnormalities have been repeatedly observed cross sectionally, they have not yet been studied longitudinally in premanifest HD. We found different patterns of decline; for some measures the rate of decline increased as individuals approached onset, while for others the rate was constant throughout the premanifest period. These results establish the effectiveness of saccadic measures in tracking premanifest disease progression, and argue for their use in clinical trials. Together, these studies establish the utility of saccade measures as a marker of HD neurodegeneration and suggest that they would be a valuable component of batteries evaluating the efficacy of neuroprotective therapies.

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer’s disease (AD) is a neurological disorder defined by the presence of plaques comprised mostly of amyloid-β (Aβ), and neurofibrillary tangles consisting of hyperphosphorylated microtubule associated protein tau (MAPT). AD is also characterized by widespread synapse loss and degeneration followed by death of neurons in the brain. Inflammatory processes, such as glial activation, are also implicated. In order to study mechanisms of neurodegeneration and evaluate potential therapeutic agents that could slow or reverse this process, a tissue culture system was developed based on primary embryonic cerebrocortical neurons. This culture system was observed to exhibit time-dependent neurodegeneration, glial proliferation, and synaptic marker loss consistent with AD-affected brains. The regulatory promoter regions of several genes implicated in AD, including the Aβ precursor protein (APP), β-amyloid cleaving enzyme (BACE1), and MAPT, were studied in this culture model. The MAPT gene promoter activity followed the pattern of neuronal maturation and degeneration quite closely, increasing in the initial phase of the tissue culture, then reducing markedly during neurodegeneration while APP and BACE1 gene promoters remained active. Deletion series of these promoters were tested to give an initial indication of the active regions of the gene promoter regions. Furthermore, the effects of exogenous Aβ and overexpression of p25, which are two possible pathogenic mechanisms of gene regulation in AD, were studied. Response to Aβ varied between the promoters and by length of the Aβ fragment used. Overexpression of p25 increased MAPT, but not APP or BACE1, promoter activity. This neurodegeneration model was also used to study the putative neuroprotective action of the NMDA receptor antagonist memantine. Treatment with memantine prevented loss of synaptic markers and preserved neuronal morphology, while having no apparent effect on glial activation. The protective action on synaptic markers was also observed with two other structurally distinct NMDA receptor antagonists, suggesting that the effects of memantine are produced by its action on the NMDA receptor. It is concluded that this tissue culture model will be useful for the study of gene regulation and therapeutic agents for neurodegeneration, and that the efficacy of memantine may result from preservation of synaptic connections in the brain.

Indiana University-Purdue University Indianapolis (IUPUI) / The main, underlying cause of neurodegenerative disease is the progressive loss of neuronal structure or function, whereby central and/or peripheral nervous system circuitry is severely and irreversibly damaged, resulting in the manifestation of clinical symptoms and signs. Neurodegenerative research has revealed many similarities among these diseases: although their clinical presentation and outcomes may differ, many parallels in their pathological mechanisms can be found. Unraveling these relationships and similarities could provide the potential for the discovery of therapeutic advances such that a treatment for one neurologic disease may also be effective for several other neurodegenerative disorders. There is growing awareness that due to the complexity of pathophysiological processes in human disease, specifically targeting or inactivating a single degenerative process or a discrete cellular molecular pathway may be ineffective in the treatment of these multifaceted disorders. Rather, potential therapeutics with a multi-target approach may be required to successfully and effectively control disease progression. Recent advances in neurodegenerative research involve the creation of animal disease models that closely mimic their human counterparts. The use of both toxin- exposure and genetic animal models in combination may give insight into the underlying pathologic mechanisms of neurodegenerative disorders (target identification) leading to the development and screening of prospective treatments and determination of their neuroprotective mechanism (target validation). Taken together, ideal candidates for the treatment of neurodegenerative disease would need to exert their neuroprotective effect on multiple pathological pathways. Previous studies from this laboratory and collaborators have shown that the naturally-occurring compound, caffeic acid phenethyl ester (CAPE), is efficacious for the treatment against neurodegeneration. Because of its versatile abilities, CAPE was chosen for this study as this compound may be able to target the pathogenic pathways shared by two different animal models of neurodegeneration and may exhibit neuroprotection. In addition, adipose-derived stem cell conditioned media (ASC-CM), a biologically-derived reagent containing a multitude of neuroprotective and neurotrophic factors, was selected as ASC-CM has been previously shown to be neuroprotective by using both animal and cell culture models of neurodegeneration.

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The retinal pigment epithelium (RPE) is a monolayer of cells located directly behind the photoreceptor cells in the retina. These cells are involved in a variety of functions that support the visual process in the eye, namely 1) they form a blood-retina barrier which separates the neural retina from the choroid's blood supply, 2) the apical processes of RPE cells diurnally phagocytose the outer segments of photoreceptor cells, and 3) they participate in the renewal of the photopigment 11-cis retinal. Age-related macular degneration (AMD) is the leading cause of blindness in people over the age of 50 years in North America and other developed countries. AMD involves the death of retinal pigment epithelial (RPE) cells in the macula early in the progress of the disease. Like some other postmitotic cells, the RPE accumulates autofluorescent lysosomal storage bodies (lipofuscin) during senescence. Lipofuscin is reported to begin accumulating in the human RPE around age 20 and continues to accumulate throughout an individual's life. This progressive accumulation of lipofuscin can eventually occupy a substantial fraction of the RPE cytoplasmic volume and may lead to impairment of normal RPE functions, resulting in retinal degeneration and loss of visual function as in AMD. Another autofluorescent granule that accumulates in RPE cells and may contribute to the etiology of AMD is a complex granule exhibiting properties of both melanosomes and lipofuscin granules called melanolipofuscin (MLF). In contrast with the accumulation of LF in the RPE, MLF accumulation has been reported by Feeney-Burns to more closely reflect the onset of AMD. Although there have been significant advances in our understanding of AMD, knowledge of the mechanisms responsible for its progression remain unclear. This dissertation details experiments that were designed to better understand the factors that may play a causal role in AMD as well as the development of methods to assist in AMD research. Specifically, the protein composition of retinal LF was assessed to elucidate its origin. These findings are reported in chapter 2. The accumulation, composition and phototoxicity of MLF were analyzed to assess MLF's origin and possible contribution to AMD. These results are reported in chapter 3. Because protein oxidation is possibly a common posttranslational modification to proteins which accumulate in lipofuscin and melanolipofuscin granules, a method for the detection and analysis of oxidized proteins was developed and is reported in chapter 4. Chapter 5 details the proteomic differences between ARPE-19 cells - the only human RPE cell line available for research - in their differentiated and undifferentiated states and compares these to the proteome of human RPE cells. These results are also compared to the phenotypic difference of these cells as observed by transmission electron microscopy.

Age-related macular degeneration (AMD) is the leading cause of blindness in the United States of America. The pyridinium bis-retinoid A2-ethanolamine (A2E) has been implicated to play a role in AMD. We have observed novel pyridinium bis-retinoids through melanolipofuscin and human RPE extractions that may also play a role in the pathology of AMD. We have begun the construction of an amino-retinoid library in order to identify these ocular compounds. The compounds from the amino-retinoid library are also used in a targeted and triggered drug delivery system for treating cancer. Folic acid is coupled with the amino-retinoids to specifically target cancer cells. The first two amino-retinoids to be synthesized and characterized were A2-dopamine (A2D) and A2-cadaverine (A2C). Both pyridinium bis-retinoids were shown to generate cytotoxic oxidation products similar to A2E. Successful coupling of folic acid to A2C was achieved to form the folic acid-A2-cadaverine (FA-A2C) product. Preliminary irradiation results suggest that the FA-A2C product may be more photoreactive than initially anticipated. This could mean less drug and light exposure required to induce apoptosis and could eventually lead to a less invasive and toxic cancer treatment.

Photoreceptors are highly specialized neurons within the eye and the key retinal cells sensing light. They are indispensable for our visual perception and loss of photoreceptors consequently leads to loss of vision, a sense that alone is responsible for more than 30% of the input to our brain. Vision impairment and blindness is a leading cause of disability in the industrialized world and is in many cases ultimately due to a degeneration of the photoreceptors, which cannot be halted or reversed. Retinal degenerative diseases encompass a heterogeneous group of etiologies, mainly caused by various mutations in a plethora of proteins involved in the visual process. Currently, several therapeutic options are being explored, with so far one gene therapy for a rare inherited blinding condition being clinically approved. However, the gene therapy approach requires not only the presence of remaining photoreceptors but the tailoring of the therapy to each individual mutation. An alternative, more generally applicable approach is to restore vision through photoreceptor replacement therapy. As such, research on mouse-to-mouse photoreceptor transplantations has been carried out for many years, though with mixed results. In the last decade, it has however also become possible to generate large quantities of human photoreceptors through retinal organoid technology, allowing to instead transplant human cells. While promising, this field is still in development and principal conditions for successful photoreceptor transplantation have yet to be defined. Here, human-to-mouse photoreceptor transplantations were performed and assessed with the aim to receive insights into retinal cell replacement technology with specific focus on photoreceptor maturation, polarization and functional integration. Using a cone-degeneration host line, large-scale incorporation of human photoreceptor grafts into the murine retina was shown for the first time. It was found that for human photoreceptors, the choice of developmental stage strongly affects incorporation and maturation capacity. Furthermore, the results demonstrate the necessity of adequate graft-host interaction for successful transplant maturation and function, suggesting that photoreceptor replacement strategies might benefit from transplantation in earlier rather than late stages of retinal degeneration. Taken together, this thesis lays important groundwork for the further development of human photoreceptor replacement strategies to treat retinal degenerative disease.:ACKNOWLEDGEMENTS I ABSTRACT III ZUSAMMENFASSUNG V PUBLICATIONS VII TABLE OF CONTENTS IX LIST OF FIGURES XIII LIST OF TABLES XIV GENERAL ABBREVIATIONS XV GENE AND PROTEIN ABBREVIATIONS XVII 1 INTRODUCTION 1 1.1 THE RETINA AND LIGHT PERCEPTION 1 1.1.1 General structure of the eye 1 1.1.2 General structure of the retina 1 1.1.3 General photoreceptor structure 3 1.1.4 Phototransduction 4 1.1.5 Signal transmission to the brain 6 1.1.6 Major differences between rods and cones 7 1.1.7 The role of Müller glia in photoreceptor support and light perception 9 1.2 RETINAL DEGENERATION DISEASES AND TREATMENT OPTIONS 11 1.2.1 Retinal degeneration diseases 11 1.2.2 Therapeutic approaches to treat retinal degeneration diseases 12 1.3 CELL REPLACEMENT AS TREATMENT APPROACH FOR RETINOPATHIES 14 1.3.1 Transplantations of rodent retinal tissue and cells 14 1.3.2 Transplantations of human retinal tissue and cells 17 1.4 AIM OF THIS THESIS 22 2 CHARACTERIZATION OF CRX-MCHERRY HUMAN RETINAL ORGANOIDS AS PHOTORECEPTOR CELL SOURCE 23 2.1 AIMS 23 2.2 CHARACTERIZATION OF CRX-MCHERRY REPORTER-EXPRESSING CELLS 23 2.2.1 Crx-mCherry expression overlaps with endogenous CRX expression and increases over time 23 2.2.2 Crx-mCherry organoids contain an outer and an inner nuclear layer 24 2.2.3 Crx-mCherry+ cells express early and mature rod and cone markers 25 2.2.4 Crx-mCherry+ cells do not express proliferation markers 27 2.3 ENRICHMENT AND CHARACTERIZATION OF CRX-MCHERRY+ DONOR CELLS 28 2.3.1 Enrichment of Crx-mCherry+ cells by FACS 28 2.3.2 Characterization of Crx-mCherry enriched cells by single cell sequencing 29 2.3.3 Characterization of D200 Crx-mCherry-enriched cells by immunocytochemistry 30 2.4 SUMMARY 31 3 TRANSPLANTATION OF HUMAN CRX-MCHERRY+ GRAFTS AGED D100, D200 AND D300 INTO CPFL1 MICE 33 3.1 AIMS 33 3.2 CRX-MCHERRY+ CELLS OF ALL AGES CAN BE TRANSPLANTED AND SURVIVE IN THE MURINE RETINA 33 3.2.1 Human grafts can be identified by RCVRN staining 34 3.2.2 D100 Crx-mCherry+ transplants are larger than D200 and D300 grafts 34 3.2.3 Graft volume increase over time is not due to in vivo proliferation 36 3.3 GRAFT MORPHOLOGY DIFFERS WITH DONOR AGES 37 3.3.1 Human grafts can adopt an intraretinal position 37 3.3.2 Graft positioning changes over time 37 3.3.3 Qualitative differences in graft morphology between donor ages 38 3.4 GRAFT MATURATION 41 3.4.1 D200 but not D100 or D300 grafts develop large quantities of inner segments 41 3.4.2 Inner segment development is associated with close proximity to the host retina 42 3.5 HUMAN IDENTITY OF INTRARETINAL GRAFTS 43 3.5.1 Intraretinal Crx-mCherry+ grafts are largely a result of true morphological incorporation 43 3.5.2 Rare indications of potential human-to-mouse material transfer 45 3.6 SUMMARY 47 4 IN DEPTH CHARACTERIZATION OF TRANSPLANTED D200 CRX-MCHERRY+ CELLS 49 4.1 AIMS 49 4.2 EARLY POST TRANSPLANTATION DYNAMICS IN GRAFT POSITIONING AND GRAFT-HOST INTERACTIONS 49 4.2.1 Intraretinal and proximal D200 grafts interact with the host retina while isolated and distal clusters show only little interaction 49 4.2.2 Incorporation of D200 grafts is first evident at 8 weeks post transplantation 50 4.2.3 Host Müller glia extend processes into the graft before host bipolar cells 51 4.2.4 MG staining in D200 grafts originates from host MG 51 4.3 INCORPORATING D200 GRAFTS POLARIZE AND FORM STRUCTURES OF MATURE PHOTORECEPTORS 53 4.3.1 Grafts and host form an outer limiting membrane (OLM)-like structure 53 4.3.2 Inner segment formation occurs where an OLM is formed 54 4.3.3 Incorporating grafts form outer segment-like structures 55 4.3.4 Incorporating grafts form synaptic structures 57 4.3.5 Transplanted Crx-mCherry+ cells become enriched for cones 58 4.3.6 Higher levels of mature photoreceptor markers in ex vivo compared to in vitro cones 60 4.4 INCORPORATION AND MATURATION CAPACITY DEPEND ON THE HOST ENVIRONMENT 63 4.4.1 Graft morphology and maturation in C57BL/6JRj recipients resembles that in Cpfl1 hosts 63 4.4.2 Graft morphology and maturation in highly degenerated rd1 and tgCR host lines differs strongly from the outcome in models with an ONL 63 4.5 SUMMARY 67 5 FUNCTIONAL ASSESSMENT OF TRANSPLANTED CRX-MCHERRY+ CELLS 69 5.1 AIMS 69 5.2 HIGH-LEVEL FUNCTION 69 5.2.1 Light-Dark Box 69 5.3 TISSUE-LEVEL FUNCTION 71 5.3.1 Multi-electrode array assessment of D200+26w grafts in Cpfl1 mice 71 5.3.2 Isolation of cone-mediated RGC response through photopic light stimulation and L-AP4 addition 71 5.3.3 Graft-containing retinal portions exhibit cone-mediated light responses 72 5.4 SUMMARY 74 6 DISCUSSION AND FUTURE PERSPECTIVES 75 6.1 HUMAN GRAFTS CAN MORPHOLOGICALLY INCORPORATE INTO THE MODERATELY DEGENERATED MOUSE RETINA 75 6.2 INTRARETINAL GRAFTS MOSTLY REPRESENT TRUE INCORPORATION EVENTS, NOT MATERIAL TRANSFER 76 6.3 GRAFT MATURATION DEPENDS ON GRAFT-HOST INTERACTION 77 6.4 ESTABLISHMENT OF GRAFT-HOST INTERACTION AND GRAFT INCORPORATION 78 6.5 D200 CRX-MCHERRY+ CELLS ARE THE PREFERABLE DONOR POPULATION COMPARED TO D100 AND D300 80 6.6 CONES SHOW PREFERENTIAL SURVIVAL POST GRAFTING 81 6.7 FUNCTIONAL ANALYSES OF TRANSPLANTED ANIMALS 82 6.8 FUTURE CLINICAL TRANSLATION 85 6.9 MAJOR CONTRIBUTION TO OTHER WORK 88 7 FINAL CONCLUSION 89 8 MATERIALS AND METHODS 91 8.1 STUDY APPROVAL 91 8.2 MATERIALS 91 8.2.1 Materials and Chemicals 91 8.2.2 Cell Line 92 8.2.3 Mouse Lines 92 8.2.4 Antibodies 93 8.3 METHODS 95 8.3.1 Cell culture 95 8.3.2 Transplantations 96 8.3.3 Functional analyses 98 8.3.4 Immunohistochemistry and Immunocytochemistry 100 8.3.5 Imaging and image processing 103 8.3.6 Statistics 106 8.3.7 Single cell sequencing 107 8.3.8 Bioinformatic analysis 108 9 BIBLIOGRAPHY 111 10 APPENDIX 128 10.1 APPENDIX 1: ERKLÄRUNGEN ZUR ERÖFFNUNG DES PROMOTIONSVERFAHRENS 128 10.2 APPENDIX 2: BESTÄTIGUNG ÜBER EINHALTUNG DER AKTUELLEN GESETZLICHEN VORGABEN 129

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