No description available.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [92]-96).

<p>The complex process of visually interpreting our environment begins with the task of detecting the light that enters our eyes. This task is performed by the rod and cone photoreceptors, which both contain a highly evolved sensory cilium called the outer segment. The outer segment is a specialized cellular compartment that contains all of the protein machinery involved in converting the initial light signal into an electrical signal that can be ultimately transmitted to the brain. Outer segments are cylindrical structures that envelop an array of individual, densely packed membrane discs. Discs are renewed throughout the lifetime of a photoreceptor, with older material being shed at the tip and new material added at the base of the outer segment. Many studies conducted over the past 35 years conclude that disc formation starts with evagination of the plasma membrane at the outer segment base, followed by membrane expansion and, in the case of rods, subsequent disc enclosure. Despite the intense interest in the topic, the molecular mechanisms governing how outer segment discs are formed and renewed are not well understood. </p><p>The focus of this dissertation centers on elucidating the molecular role of peripherin/retinal degeneration slow (rds) in outer segment disc morphogenesis, including study of peripherin/rds trafficking from its site of synthesis in the endoplasmic reticulum to its site of function in the outer segment. Peripherin/rds is expressed specifically in photoreceptor outer segments, where it fulfills a critical role in assembling and/or maintaining the structure of this organelle. Mutation or loss of peripherin/rds in humans is often associated with visual impairments, and its knockout in mice results in rudimentary ciliary stumps completely lacking disc structures. </p><p>We found that early outer segment morphogenesis steps in mice lacking peripherin/RDS proceed normally for the first week. However, in the second week of postnatal development at the onset of disc formation, mice lacking peripherin/RDS produce extracellular vesicles next to their connecting cilia rather than discs. We characterized these vesicles and determined that they are enriched in outer segment proteins, are ~230 nm in size, and are formed as outward buds of the plasma membrane. These characteristics allowed us to classify these extracellular vesicles as ciliary ectosomes. Furthermore, we determined that ectosome shedding is arrested upon expression of the peripherin/rds C-terminal cytoplasmic sequence, which allows for the accumulation of excessive membranous material. Thus, we conclude that peripherin/rds transforms the functional dynamics of photoreceptor primary cilium from shedding massive amounts of ectosomes to retaining these membranes in the outer segment to eventually become photoreceptor discs. This novel function of peripherin is performed by its C-terminal cytoplasmic sequence and represents the first step in disc morphogenesis. </p><p>Finally, the morphogenesis study of peripherin/rds is complemented by a study of its trafficking. Understanding how peripherin is delivered from the site of its synthesis is critical for its function at the outer segment. We show that the peripherin/rds targeting sequence is confined within ten amino acid residues, which do not overlap with the putative fusogenic domain, and that only a single amino acid within this region is irreplaceable, a highly conserved valine at position 332.</p><p>Collectively, these studies shed considerable light on the molecular role played by peripherin. Peripherin is a photoreceptor specific protein that transforms the primary sensory cilium into a specialized sensory cilium capable of building the discs required for efficient photon capture. While work in this direction provides a significant advance in our understanding of peripherin’s role in disc morphogenesis, questions such as whether peripherin participates in disc enclosure, remain to be solved.</p> / Dissertation

Wang Zhi-Jun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 54-63). / ABSTRACT --- p.VI / Chapter I. --- INTRODUCTION --- p.1 / Chapter A. --- Literature review --- p.1 / Chapter 1. --- Retinal iron toxicity --- p.2 / Clinical siderotic retinopathy --- p.2 / Experimental siderotic retinopathy --- p.4 / Free radical involvement in siderotic retinopathy --- p.5 / Chapter 2. --- Experimental photic retinopathy in rats --- p.8 / Morphologic features --- p.8 / Free radical involvement in photic retinopathy --- p.9 / Chapter 3. --- Mechanisms of cell death --- p.9 / Necrosis --- p.10 / Apoptosis --- p.10 / Chapter B. --- Statement of the problems --- p.15 / Chapter II. --- MATERIALS AND METHODS --- p.17 / Chapter A. --- Siderotic retinopathy model --- p.17 / Animals --- p.17 / Reagents and equipment --- p.18 / Surgical procedures --- p.18 / Chapter B. --- Histochemical methods --- p.18 / Reagents and equipment --- p.19 / Paraffin sections --- p.19 / H&E staining --- p.19 / TUNEL technique --- p.20 / Schmeltzer's iron staining --- p.21 / Chinoy's ascorbic acid staining --- p.21 / Chapter C. --- Biochemical methods --- p.21 / Reagents and equipment --- p.22 / DNA gel electrophoresis --- p.22 / Analysis of ascorbic acid and uric acid --- p.23 / Chapter III. --- RESULTS --- p.24 / Chapter A. --- Observations in rats --- p.24 / Morphologic changes after H&E staining --- p.24 / Visualization of apoptosis by TUNEL technique --- p.25 / Internucleosomal DNA fragmentation --- p.26 / Negative staining of iron in the ONL --- p.27 / Positive staining of ascorbic acid in the ONL --- p.27 / Chapter B. --- Observations in rabbits --- p.27 / Positive staining of ascorbic acid in all retinal layers --- p.27 / Apoptosis occurred in all retinal layers --- p.28 / Changes of ascorbic acid and uric acid after iron implantatio --- p.28 / Chapter IV. --- DISCUSSION --- p.48 / Chapter V. --- CONCLUSION --- p.53 / References --- p.54

Abstract Photoreceptors convert the energy of light into an electric signal to be processed by the visual system. Photoreceptors of nocturnal insects are adapted for night vision by sacrificing spatial and temporal resolution for improved sensitivity. While the sensitivity-increasing optical adaptations and the temporal properties of light responses have been studied earlier, the intermediate biophysical mechanisms responsible for shaping the captured light into voltage responses were previously not known in detail in any nocturnal species. Using electrophysiological tools and computer simulations the photoreceptors of the nocturnal cockroach (Periplaneta americana) were studied by characterising 1) the electrical properties responsible for shaping the light responses, 2) the properties of light responses at different stages of light and dark adaptation and 3) properties of low-intensity light stimuli and how they are processed by the photoreceptors. The high input resistance and whole-cell capacitance were typical for a nocturnal insect, but the two voltage-dependent potassium conductances were closer to those found in diurnal species. The dominant sustained conductance typically associated with day-light vision activated during simulated light responses whereas the lesser transient conductance previously linked to low-light vision did not. Light responses were persistently slow regardless of the adapting light level and saturated at low intensities, indicating a strong adaptation to vision in dim light. Simulations showed that at such low light levels the physical noise caused by random photons determines the information rate and the biological noise, caused by random latency and amplitude of single photon responses, has only a minor effect. At higher intensities the latency variability degraded the information rates but the amplitude variability did not. Thus, photoreceptors of nocturnal animals can sacrifice phototransduction precision in their natural illumination without compromising their coding performance.

The nervous system is required for human cognition, motor function, and sensory interaction. A complex network of neuronal connections, or synapses, carries out these behaviors, and defects in neural connectivity can result in developmental and degenerative diseases. In vertebrate nervous systems, synapses most commonly occur at axon terminals. Upon reaching their synaptic targets, growth cones lose their motility and become boutons specialized for neurotransmitter release. I am studying this process in R7 photoreceptors in the

Introduction Retinitis Pigmentosa affects 1 in 3000 people in the UK, causing photoreceptor degeneration and premature blindness. Mutations in the X-linked RPGR gene cause 20% of all disease and result in a particularly severe form of disease. The function of RPGR is unknown and it has no treatment. Methods Conflicting evidence from animal studies led me to develop a novel model for human disease with which to test the hypothesis that RPGR acts to regulate actin turnover in the photoreceptor connecting cilium. Skin biopsies were performed on patients with RPGR mutations and unaffected relatives. Subsequent fibroblast cultures were reprogrammed to generate induced pluripotent stem cell (iPSC) lines. A retinal differentiation protocol was optimised, resulting in healthy and RPGR-mutant in vitro photoreceptor cultures. Results Cultures were compared. RPGR-mutant iPSC-derived photoreceptors had increased actin polymerisation compared to wild-type control. Unbiased and hypothesis-driven experiments highlighted dysregulation of several key phospho-proteins involved in regulating actin turnover. Notably the RAC-PAK-LIMK-COFILIN pathway was dysregulated in RPGR-mutant cultures. A regulator of this pathway is the actin binding and severing protein, GELSOLIN. GELSOLIN activity was found to be perturbed in RPGR-mutant cultures. Examination of bovine retinal lysate showed an interaction between Rpgr and Gelsolin. Subsequent examination of iPSC-derived human photoreceptor cultures showed compromised interaction in RPGR-mutant cultures compared to controls. An Rpgr knock out mouse was obtained and characterised. Increased actin polymerisation in the connecting cilium and rhodopsin mislocalisation to the inner segment was seen prior to retinal degeneration. Gelsolin activity was perturbed. A Gelsolin knock out mouse was obtained and characterized. It, too, showed rhodopsin mislocalisation and retinal degeneration. Conclusion Results in this thesis confirm the hypothesis that RPGR acts to regulate actin turnover in the photoreceptor. Further, it suggests a mechanism through which this occurs. Further work is required to assess the extent of RPGR’s role in actin regulation in vivo.

No description available.

Abstract In dim light, reliable coding of visual information becomes compromised, unless the sensitivity of the visual system to light is improved by structural and functional adaptations. Thus far, many adaptations for night vision in the compound eyes of nocturnal insects have been described, but little is known about the mechanisms underlying the electrochemical signalling in their photoreceptors. In this thesis, whole-cell patch-clamp and mathematical modelling are utilised to study basic electrical properties and ionic currents in photoreceptors of two nocturnal insects, the American cockroach Periplaneta americana and the field cricket Gryllus bimaculatus. Photoreceptors in both species showed large input resistance, membrane capacitance and phototransduction gain (large single photon responses) compared with most studied diurnal insects, providing improved sensitivity to light. The photoreceptors also expressed two voltage-sensitive outward currents: a transient current and a sustained current. The cricket photoreceptor expressed a dominating transient current, which is a typical characteristic for insects adapted for slow vision in dim light. By contrast, in the majority of cockroach photoreceptors the sustained current dominated, which is more common among fast diurnal species. Model simulations indicated that the sustained current is necessary for improved photoreceptor dynamics. Examination of light-induced currents suggested that the functional variability in cockroach photoreceptors is in part derived from variations in the total area of the photosensitive membrane. Recordings of light-induced currents also revealed that the cockroach light-gated channels are only moderately Ca2+-selective and that the polarisation-sensitive photoreceptors of the cricket may utilise phototransduction machinery in some details different from that in regular photoreceptors. Furthermore, the dynamics and information transfer rates of polarisation-sensitive photoreceptors in the cricket were clearly inferior to their regular counterparts, suggesting that they are not necessary for image formation.

Final publication is available at http://dx.doi.org/10.1016/j.exer.2012.08.013. Congrong Guo, Atsushi Otani, Akio Oishi, Hiroshi Kojima, Yukiko Makiyama, Satoko Nakagawa, Nagahisa Yoshimura, Knockout of ccr2 alleviates photoreceptor cell death in a model of retinitis pigmentosa, Experimental Eye Research, Volume 104, November 2012, Pages 39-47, ISSN 0014-4835 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18680号 / 医博第3952号 / 新制||医||1007(附属図書館) / 31613 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長澤 丘司, 教授 伊藤 壽一, 教授 長田 重一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM