{"id":{"repo_id":"buffalo","oai_identifier":"oai:ubir.buffalo.edu:10477/78597"},"canonical_url":"https://search.dev.ndltd.org/etd/buffalo/oai:ubir.buffalo.edu:10477/78597","repository":{"repo_id":"buffalo","name":"Buffalo","base_url":"https://ubir.buffalo.edu/oai/request"},"display":{"title":"Studies on the Role of Sterol and Non-Sterol Isoprenoids in Retinal Health and Disease","abstract":"Ph.D.","abstract_html":"Ph.D.","abstract_has_math":false,"creators":["Ramachandra Rao, Sriganesh; 0000-0003-1029-1051"],"institution":"State University of New York at Buffalo","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Fliesler, Steven","Biochemistry"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018-10-26T02:56:16Z","date_published":"2018-10-26T02:56:16Z","updated_at":"2026-07-27T19:05:12Z","subjects":["ophthalmology","cellular biology","genetics"],"languages":["eng"],"rights":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10477/78597","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Fliesler, Steven","Biochemistry"]},{"key":"dc:creator","label":"Author","values":["Ramachandra Rao, Sriganesh; 0000-0003-1029-1051"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2018-10-26T02:56:16Z","2018","2018-08-09 16:14:10"]},{"key":"dc:publisher","label":"Institution","values":["State University of New York at Buffalo"]},{"key":"dc:type","label":"Dc Type","values":["Text","Dissertation"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["ophthalmology","cellular biology","genetics"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/10477/78597"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Ph.D.","The mevalonate pathway generates several biologically important products, including both sterol (e.g., cholesterol (Chol)) and non-sterol (e.g., dolichol) isoprenoids, which are required for a variety of cellular functions. The broad goal of the research described in this thesis is to elucidate the consequences of deletion or inhibition of key enzymatic steps in the synthesis of Chol and dolichol in the neural retina and the retinal pigmented epithelia (RPE). Targeted inhibition of Chol synthesis (at the final step, i.e., NADPH-dependent reduction of 7-dehydrocholesteol (7DHC) to Chol, catalyzed by 7DHC reductase (DHCR7), and dolichol synthesis (at the commitment step, i.e., condensation of farnesyl pyrophosphate and isopentenyl pyrophosphate to form polyprenyl pyrophosphate, catalyzed by dehydrodolichyldiphosphate synthase (DHDDS)) provides an avenue to study the retinal pathology in rare recessive genetic disorders caused by mutations in DHCR7 [Smith-Lemli-Opitz Syndrome (SLOS)] and DHDDS [autosomal recessive retinitis pigmentosa 59 (arRP59)] genes, respectively. This thesis work describes the effects of DHCR7 inhibition on the diurnal heterophagic function of the RPE, as well as the biochemical consequences of targeted deletion of Dhcr7 in the photoreceptor cells. This work also entails generation of a novel animal model of arRP59, created by targeted deletion of Dhdds in the photoreceptor cells. The findings from the studies are briefly summarized below.Treatment of rats with the Chol pathway inhibitor AY9944 produces an animal model of SLOS. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated electrophysiological function deficits and pathological features of the RPE. Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E (a lipofuscin fluorophore) and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)-derived SLOS RPE cells (notably, the first time such cells have been created), we found significantly elevated steady-state levels of 7DHC and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (ROS), as evidenced by persistence of the C-terminal 1D4 epitope of rhodopsin (RHO) in SLOS RPE cells compared to iPSC-derived normal human control RPE cells. SLOS RPE cells exhibited lysosomal pH levels and cathepsin-D (CTSD) activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed. Further attempts to study autophagy in the neural retina and the RPE using the tandem-tag autophagy reporter mouse model (CAG-RFP-GFP-:LC3) necessitated the identification and accounting of technical and biological factors that may affect reliable identification of autophagosomes using laser confocal fluorescence microscopy.Dehydrodolichyldiphosphate synthase (DHDDS), and its dimeric partner Nogo B-Receptor (NUS1), constitute the commitment step in dolichol synthesis: the cis-prenyl chain elongation to produce the polyprenyl backbone of dolichol. Dolichylphosphate (Dol-P) is the obligate glycan carrier essential for N-glycosylation of proteins. We hypothesized that photoreceptor (PR)-specific deletion of Dhdds elicits primary retinal degeneration owing to the photoreceptor requirement for dolichol synthesis and N-glycosylation. A Dhddsflx/flx mouse line was crossed with Rho-iCre75 (rhodopsin promoter-driven Cre recombinase) mice to ultimately generate second filial, homozygous Dhdds knockout mice [Dhddsflx/flx iCre+] (notably, the first time such a mouse line has been generated). Rod PR-specific expression and activity of Cre recombinase and subsequent excision of Dhdds was verified using reporter mice, indirect-tail snip and direct-retina genotyping, and in situ hybridization (ISH). Cre recombinase was active in over 95% of PR cells by postnatal (PN) 4 weeks. Optical coherence tomography (OCT) and histological analysis of Dhddsflx/flx iCre+ mouse retinas showed no appreciable changes in the thickness of outer nuclear layer (ONL) at PN 4 weeks compared to age-matched controls, but exhibited rapid PR dystrophy accompanied by shortening of ROS. By PN 6 weeks, only two rows of PR nuclei remained, and the remnant PRs essentially lacked ROS. Scotopic a-wave and b-wave ERG responses of Dhddsflx/flx iCre+ were significantly reduced at all tested time points [n=28], and were extinct by PN 6 weeks, compared to the robust ERGs of age-matched controls. The photopic ERG responses of Dhddsflx/flx iCre+ mice were comparable to those of controls until PN 5 weeks, but were essentially extinguished at PN 6 weeks. Surprisingly, PR-specific glycoproteins (e.g., RHO) were sensitive to PNGase-F treatment, suggesting successful glycosylation in the PRs of Dhddsflx/flx iCre+ mice. The hypoglycosylation-independent retinal dystrophy observed in the Dhddsflx/flx iCre+ model may provide novel insights into the disease mechanisms underlying arRP59.Cellular Chol homeostasis involves a balance between input--- i.e., endogenous de novo Chol synthesis and/or exogenous Chol uptake--- and export. The input aspect represents the two major mechanisms required to meet cellular Chol requirements. The requirement of PR Chol synthesis was tested by Rho-iCre75-driven deletion of loxP-modified Dhcr7 Exon 8 (Dhcr7flx/flx iCre+), and subsequent analysis of whole retinal 7DHC/Chol mole ratio. Dhcr7flx/flx iCre+ mice did not exhibit significant increase in 7DHC/Chol ratio, as compared to Dhcr7flx/flx iCre- (n=3/group). However, Albumin promoter-driven, hepatocyte-specific Dhcr7 deletion (Dhcr7flx/flx Alb-Cre+, serves as a positive control) altered whole liver 7DHC/Chol mole ratio to 0.15 (n=4). The lack of a biochemical phenotype in Dhcr7flx/flx iCre+ was further accompanied by robust scotopic ERG responses (until PN 10 months), comparable to that of age-matched controls (n=3/group). Scotopic ERG responses in Dhcr7flx/flx iCre+ mice were not altered upon exposure to intense constant light (the “retinal light damage” paradigm), and were comparable to age-matched C57BL6/J controls (n=3), unlike previously reported light-sensitivity of the AY9944-induced SLOS rat model. These findings suggest that endogenous de novo synthesis of Chol by PRs may be a relatively minor contributor to the overall Chol pool utilized by PRs to meet their cellular requirements, implying that exogenous uptake is the dominant source of Chol for PRs."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Studies on the Role of Sterol and Non-Sterol Isoprenoids in Retinal Health and Disease"]}]}],"canonical_facts":{"dc:contributor":["Fliesler, Steven","Biochemistry"],"dc:creator":["Ramachandra Rao, Sriganesh; 0000-0003-1029-1051"],"dc:date":["2018-10-26T02:56:16Z","2018","2018-08-09 16:14:10"],"dc:description":["Ph.D.","The mevalonate pathway generates several biologically important products, including both sterol (e.g., cholesterol (Chol)) and non-sterol (e.g., dolichol) isoprenoids, which are required for a variety of cellular functions. The broad goal of the research described in this thesis is to elucidate the consequences of deletion or inhibition of key enzymatic steps in the synthesis of Chol and dolichol in the neural retina and the retinal pigmented epithelia (RPE). Targeted inhibition of Chol synthesis (at the final step, i.e., NADPH-dependent reduction of 7-dehydrocholesteol (7DHC) to Chol, catalyzed by 7DHC reductase (DHCR7), and dolichol synthesis (at the commitment step, i.e., condensation of farnesyl pyrophosphate and isopentenyl pyrophosphate to form polyprenyl pyrophosphate, catalyzed by dehydrodolichyldiphosphate synthase (DHDDS)) provides an avenue to study the retinal pathology in rare recessive genetic disorders caused by mutations in DHCR7 [Smith-Lemli-Opitz Syndrome (SLOS)] and DHDDS [autosomal recessive retinitis pigmentosa 59 (arRP59)] genes, respectively. This thesis work describes the effects of DHCR7 inhibition on the diurnal heterophagic function of the RPE, as well as the biochemical consequences of targeted deletion of Dhcr7 in the photoreceptor cells. This work also entails generation of a novel animal model of arRP59, created by targeted deletion of Dhdds in the photoreceptor cells. The findings from the studies are briefly summarized below.Treatment of rats with the Chol pathway inhibitor AY9944 produces an animal model of SLOS. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated electrophysiological function deficits and pathological features of the RPE. Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E (a lipofuscin fluorophore) and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)-derived SLOS RPE cells (notably, the first time such cells have been created), we found significantly elevated steady-state levels of 7DHC and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (ROS), as evidenced by persistence of the C-terminal 1D4 epitope of rhodopsin (RHO) in SLOS RPE cells compared to iPSC-derived normal human control RPE cells. SLOS RPE cells exhibited lysosomal pH levels and cathepsin-D (CTSD) activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed. Further attempts to study autophagy in the neural retina and the RPE using the tandem-tag autophagy reporter mouse model (CAG-RFP-GFP-:LC3) necessitated the identification and accounting of technical and biological factors that may affect reliable identification of autophagosomes using laser confocal fluorescence microscopy.Dehydrodolichyldiphosphate synthase (DHDDS), and its dimeric partner Nogo B-Receptor (NUS1), constitute the commitment step in dolichol synthesis: the cis-prenyl chain elongation to produce the polyprenyl backbone of dolichol. Dolichylphosphate (Dol-P) is the obligate glycan carrier essential for N-glycosylation of proteins. We hypothesized that photoreceptor (PR)-specific deletion of Dhdds elicits primary retinal degeneration owing to the photoreceptor requirement for dolichol synthesis and N-glycosylation. A Dhddsflx/flx mouse line was crossed with Rho-iCre75 (rhodopsin promoter-driven Cre recombinase) mice to ultimately generate second filial, homozygous Dhdds knockout mice [Dhddsflx/flx iCre+] (notably, the first time such a mouse line has been generated). Rod PR-specific expression and activity of Cre recombinase and subsequent excision of Dhdds was verified using reporter mice, indirect-tail snip and direct-retina genotyping, and in situ hybridization (ISH). Cre recombinase was active in over 95% of PR cells by postnatal (PN) 4 weeks. Optical coherence tomography (OCT) and histological analysis of Dhddsflx/flx iCre+ mouse retinas showed no appreciable changes in the thickness of outer nuclear layer (ONL) at PN 4 weeks compared to age-matched controls, but exhibited rapid PR dystrophy accompanied by shortening of ROS. By PN 6 weeks, only two rows of PR nuclei remained, and the remnant PRs essentially lacked ROS. Scotopic a-wave and b-wave ERG responses of Dhddsflx/flx iCre+ were significantly reduced at all tested time points [n=28], and were extinct by PN 6 weeks, compared to the robust ERGs of age-matched controls. The photopic ERG responses of Dhddsflx/flx iCre+ mice were comparable to those of controls until PN 5 weeks, but were essentially extinguished at PN 6 weeks. Surprisingly, PR-specific glycoproteins (e.g., RHO) were sensitive to PNGase-F treatment, suggesting successful glycosylation in the PRs of Dhddsflx/flx iCre+ mice. The hypoglycosylation-independent retinal dystrophy observed in the Dhddsflx/flx iCre+ model may provide novel insights into the disease mechanisms underlying arRP59.Cellular Chol homeostasis involves a balance between input--- i.e., endogenous de novo Chol synthesis and/or exogenous Chol uptake--- and export. The input aspect represents the two major mechanisms required to meet cellular Chol requirements. The requirement of PR Chol synthesis was tested by Rho-iCre75-driven deletion of loxP-modified Dhcr7 Exon 8 (Dhcr7flx/flx iCre+), and subsequent analysis of whole retinal 7DHC/Chol mole ratio. Dhcr7flx/flx iCre+ mice did not exhibit significant increase in 7DHC/Chol ratio, as compared to Dhcr7flx/flx iCre- (n=3/group). However, Albumin promoter-driven, hepatocyte-specific Dhcr7 deletion (Dhcr7flx/flx Alb-Cre+, serves as a positive control) altered whole liver 7DHC/Chol mole ratio to 0.15 (n=4). The lack of a biochemical phenotype in Dhcr7flx/flx iCre+ was further accompanied by robust scotopic ERG responses (until PN 10 months), comparable to that of age-matched controls (n=3/group). Scotopic ERG responses in Dhcr7flx/flx iCre+ mice were not altered upon exposure to intense constant light (the “retinal light damage” paradigm), and were comparable to age-matched C57BL6/J controls (n=3), unlike previously reported light-sensitivity of the AY9944-induced SLOS rat model. These findings suggest that endogenous de novo synthesis of Chol by PRs may be a relatively minor contributor to the overall Chol pool utilized by PRs to meet their cellular requirements, implying that exogenous uptake is the dominant source of Chol for PRs."],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/10477/78597"],"dc:language":["eng"],"dc:publisher":["State University of New York at Buffalo"],"dc:rights":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."],"dc:subject":["ophthalmology","cellular biology","genetics"],"dc:title":["Studies on the Role of Sterol and Non-Sterol Isoprenoids in Retinal Health and Disease"],"dc:type":["Text","Dissertation"]},"updated_at":"2026-07-27T19:05:12Z"}