Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition.

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

CCR2-positive monocytes contribute to the pathogenesis of early diabetic retinopathy in mice.

AIMS/HYPOTHESIS: Accumulating evidence suggests that leucocytes play a critical role in diabetes-induced vascular lesions and other abnormalities that characterise the early stages of diabetic retinopathy. However, the role of monocytes has yet to be fully investigated; therefore, we used Ccr2-/- mice to study the role of CCR2+ inflammatory monocytes in the pathogenesis of diabetes-induced degeneration of retinal capillaries. METHODS: Experimental diabetes was induced in wild-type and Ccr2-/- mice using streptozotocin. After 2 months, superoxide levels, expression of inflammatory genes, leucostasis, leucocyte- and monocyte-mediated cytotoxicity against retinal endothelial cell death, retinal thickness and visual function were evaluated. Retinal capillary degeneration was determined after 8 months of diabetes. Flow cytometry of peripheral blood for differential expression of CCR2 in monocytes was assessed. RESULTS: In nondiabetic mice, CCR2 was highly expressed on monocytes, and Ccr2-/- mice lack CCR2+ monocytes in the peripheral blood. Diabetes-induced retinal superoxide, expression of proinflammatory genes Inos and Icam1, leucostasis and leucocyte-mediated cytotoxicity against retinal endothelial cells were inhibited in diabetic Ccr2-deficient mice and in chimeric mice lacking Ccr2 only from myeloid cells. In order to focus on monocytes, these cells were immuno-isolated after 2 months of diabetes, and they significantly increased monocyte-mediated endothelial cell cytotoxicity ex vivo. Monocytes from Ccr2-deficient mice caused significantly less endothelial cell death. The diabetes-induced retinal capillary degeneration was inhibited in Ccr2-/- mice and in chimeric mice lacking Ccr2 only from myeloid cells. CONCLUSIONS/INTERPRETATION: CCR2+ inflammatory monocytes contribute to the pathogenesis of early lesions of diabetic retinopathy.

ICAM-1 on the luminal surface of endothelial cells is induced to a greater extent in mouse retina than in other tissues in diabetes.

AIMS/HYPOTHESIS: Induction of intercellular adhesion molecule-1 (ICAM-1) has been implicated in the development of macrovascular and microvascular diseases such as diabetic retinopathy. Lesions of diabetic retinopathy are unique to the retina but the reason for this is unclear, as all tissues are exposed to the same hyperglycaemic insult. We tested whether diabetes induces ICAM-1 on the luminal surface of endothelial cells to a greater extent in the retina than in other tissues and the role of vision itself in that induction. METHODS: Experimental diabetes was induced in C57Bl/6J, P23H opsin mutant and Gnat1-/- × Gnat2-/- double knockout mice using streptozotocin. The relative abundance of ICAM-1 on the luminal surface of endothelial cells in retina and other tissues was determined by conjugating anti-ICAM-1 antibodies to fluorescent microspheres (2 µm), injecting them intravenously and allowing them to circulate for 30 min. After transcardial perfusion, quantification of microspheres adherent to the endothelium in tissues throughout the body was carried out by fluorescent microscopy or flow cytometry. Mice injected with lipopolysaccharide (LPS) were used as positive controls. The difference in leucostasis between retinal and non-retinal vasculature was evaluated. RESULTS: Diabetes significantly increased ICAM-1-mediated adherence of microspheres to retinal microvessels by almost threefold, independent of sex. In contrast, diabetes had a much smaller effect on endothelial ICAM-1 in other tissues, and more tissues showed a significant induction of endothelial ICAM-1 with LPS than with diabetes. The diabetes-induced increase in endothelial ICAM-1 in retinal vasculature was inhibited by blocking phototransduction in photoreceptor cells. Diabetes significantly increased leucostasis in the retina by threefold compared with a non-ocular tissue (cremaster). CONCLUSIONS/INTERPRETATION: The diabetes-induced upregulation of ICAM-1 on the luminal surface of the vascular endothelium varies considerably among tissues and is highest in the retina. Induction of ICAM-1 on retinal vascular endothelial cells in diabetes is influenced by vision-related processes in photoreceptor cells. The unique presence of photoreceptors in the retina might contribute to the greater susceptibility of this tissue to vascular disease in diabetes.

Photoreceptor Cell Calcium Dysregulation and Calpain Activation Promote Pathogenic Photoreceptor Oxidative Stress and Inflammation in Prodromal Diabetic Retinopathy.

This study tested the hypothesis that diabetes promotes a greater than normal cytosolic calcium level in rod cells that activates a Ca2+-sensitive protease, calpain, resulting in oxidative stress and inflammation, two pathogenic factors of early diabetic retinopathy. Nondiabetic and 2-month diabetic C57Bl/6J and calpain1 knockout (Capn1-/-) mice were studied; subgroups were treated with a calpain inhibitor (CI). Ca2+ content was measured in photoreceptors using Fura-2. Retinal calpain expression was studied by quantitative RT-PCR and immunohistochemistry. Superoxide and expression of inflammatory proteins were measured using published methods. Proteomic analysis was conducted on photoreceptors isolated from untreated diabetic mice or treated daily with CI for 2 months. Cytosolic Ca2+ content was increased twofold in photoreceptors of diabetic mice as compared with nondiabetic mice. Capn1 expression increased fivefold in photoreceptor outer segments of diabetic mice. Pharmacologic inhibition or genetic deletion of Capn1 significantly suppressed diabetes-induced oxidative stress and expression of proinflammatory proteins in retina. Proteomics identified a protein (WW domain-containing oxidoreductase [WWOX]) whose expression was significantly increased in photoreceptors from mice diabetic for 2 months and was inhibited with CI. Knockdown of Wwox using specific siRNA in vitro inhibited increase in superoxide caused by the high glucose. These results suggest that reducing Ca2+ accumulation, suppressing calpain activation, and/or reducing Wwox up-regulation are novel targets for treating early diabetic retinopathy.

Studies of ApoD-/- and ApoD-/-ApoE-/- mice uncover the APOD significance for retinal metabolism, function, and status of chorioretinal blood vessels.

Apolipoprotein D (APOD) is an atypical apolipoprotein with unknown significance for retinal structure and function. Conversely, apolipoprotein E (APOE) is a typical apolipoprotein with established roles in retinal cholesterol transport. Herein, we immunolocalized APOD to the photoreceptor inner segments and conducted ophthalmic characterizations of ApoD-/- and ApoD-/-ApoE-/- mice. ApoD-/- mice had normal levels of retinal sterols but changes in the chorioretinal blood vessels and impaired retinal function. The whole-body glucose disposal was impaired in this genotype but the retinal glucose metabolism was unchanged. ApoD-/-ApoE-/- mice had altered sterol profile in the retina but apparently normal chorioretinal vasculature and function. The whole-body glucose disposal and retinal glucose utilization were enhanced in this genotype. OB-Rb, both leptin and APOD receptor, was found to be expressed in the photoreceptor inner segments and was at increased abundance in the ApoD-/- and ApoD-/-ApoE-/- retinas. Retinal levels of Glut4 and Cd36, the glucose transporter and scavenger receptor, respectively, were increased as well, thus linking APOD to retinal glucose and fatty acid metabolism and suggesting the APOD-OB-Rb-GLUT4/CD36 axis. In vivo isotopic labeling, transmission electron microscopy, and retinal proteomics provided additional insights into the mechanism underlying the retinal phenotypes of ApoD-/- and ApoD-/-ApoE-/- mice. Collectively, our data suggest that the APOD roles in the retina are context specific and could determine retinal glucose fluxes into different pathways. APOD and APOE do not play redundant, complementary or opposing roles in the retina, rather their interplay is more complex and reflects retinal responses elicited by lack of these apolipoproteins.

Retinal Vascular Abnormalities and Microglia Activation in Mice with Deficiency in Cytochrome P450 46A1-Mediated Cholesterol Removal.

CYP46A1 is the cytochrome P450 enzyme that converts cholesterol to 24-hydroxycholesterol, a cholesterol elimination product and a potent liver X receptor (LXR) ligand. We conducted retinal characterizations of Cyp46a1-/- mice that had normal fasting blood glucose levels but up to a 1.8-fold increase in retinal cholesterol. The retina of Cyp46a1-/- mice exhibited venous beading and tortuosity, microglia/macrophage activation, and increased vascular permeability, features commonly associated with diabetic retinopathy. The expression of Lxrα and Lxrß was increased in both the whole Cyp46a1-/- retina and retinal macroglia/macrophages. The LXR-target genes were affected as well, primarily in activated microglial cells and macrophages. In the latter, the LXR-transactivated genes (Abca1, Abcg1, Apod, Apoe, Mylip, and Arg2) were up-regulated; similarly, there was an up-regulation of the LXR-transrepressed genes (Ccl2, Ptgs2, Cxcl1, Il1b, Il6, Nos2, and Tnfa). For comparison, gene expression was investigated in bone marrow-derived macrophages from Cyp46a1-/- mice as well as retinal and bone marrow-derived macrophages from Cyp27a1-/- and Cyp27a1-/-Cyp46a1-/- mice. CYP46A1 expression was detected in retinal endothelial cells, and this expression was increased in the proinflammatory environment. Retinal Cyp46a1-/- phosphoproteome revealed altered phosphorylation of 30 different proteins, including tight junction protein zonula occludens 1 and aquaporin 4. Collectively, the data obtained establish metabolic and regulatory significance of CYP46A1 for the retina and suggest pharmacologic activation of CYP46A1 as a potential therapeutic approach to dyslipidemia-induced retinal damage.

Neutrophil elastase contributes to the pathological vascular permeability characteristic of diabetic retinopathy.

AIMS/HYPOTHESIS: Levels of neutrophil elastase, a serine protease secreted by neutrophils, are elevated in diabetes. The purpose of this study was to determine whether neutrophil elastase (NE) contributes to the diabetes-induced increase in retinal vascular permeability in mice with streptozotocin-induced diabetes, and, if so, to investigate the potential role of IL-17 in this process. METHODS: In vivo, diabetes was induced in neutrophil elastase-deficient (Elane-/-), Il-17a-/- and wild-type mice. After 8 months of diabetes, Elane-/- mice and wild-type age-matched control mice were injected with FITC-BSA. Fluorescence microscopy was used to assess leakage of FITC-BSA from the retinal vasculature into the neural retina. The level of NE in Il-17a-/- diabetic retina and sera were determined by ELISA. In vitro, the effect of NE on the permeability and viability of human retinal endothelial cells and the expression of junction proteins and adhesion molecules were studied. RESULTS: Eight months of diabetes resulted in increased retinal vascular permeability and levels of NE in retina and plasma of wild-type animals. All of these abnormalities were significantly inhibited in mice lacking the elastase. The diabetes-induced increase in NE was inhibited in mice lacking IL-17. In vitro, NE increased retinal endothelial cell permeability, which was partially inhibited by a myeloid differentiation primary response 88 (MyD88) inhibitor, NF-κB inhibitor, and protease-activated receptor (PAR)2 inhibitor. NE degraded vascular endothelial-cadherin (VE-cadherin) in a concentration-dependent manner. CONCLUSIONS/INTERPRETATION: IL-17 regulates NE expression in diabetes. NE contributes to vascular leakage in diabetic retinopathy, partially through activation of MyD88, NF-κB and PAR2 and degradation of VE-cadherin.

Mechanisms that minimize retinal impact of apolipoprotein E absence.

Apolipoprotein E (APOE) is a component of lipid-transporting particles and a recognition ligand for receptors, which bind these particles. The APOE isoform ε2 is a risk factor for age-related macular degeneration; nevertheless, APOE absence in humans and mice does not significantly affect the retina. We found that retinal cholesterol biosynthesis and the levels of retinal cholesterol were increased in Apoe-/- mice, whereas cholesterol elimination by metabolism was decreased. No focal cholesterol deposits were observed in the Apoe-/- retina. Retinal proteomics identified the most abundant cholesterol-related proteins in WT mice and revealed that, of these cholesterol-related proteins, only APOA4 had increased expression in the Apoe-/- retina. In addition, there were changes in retinal abundance of proteins involved in proinflammatory and antiinflammatory responses, cellular cytoskeleton maintenance, vesicular traffic, and retinal iron homeostasis. The data obtained indicate that when APOE is absent, particles containing APOA1, APOA4, and APOJ still transport cholesterol in the intraretinal space, but these particles are not taken up by retinal cells. Therefore, cholesterol biosynthesis inside retinal cells increase, whereas metabolism to oxysterols decreases to prevent cells from cholesterol depletion. These and other compensatory changes underlie only a minor retinal phenotype in Apoe-/- mice.

Retinal Hypercholesterolemia Triggers Cholesterol Accumulation and Esterification in Photoreceptor Cells.

The process of vision is impossible without the photoreceptor cells, which have a unique structure and specific maintenance of cholesterol. Herein we report on the previously unrecognized cholesterol-related pathway in the retina discovered during follow-up characterizations of Cyp27a1(-/-)Cyp46a1(-/-) mice. These animals have retinal hypercholesterolemia and convert excess retinal cholesterol into cholesterol esters, normally present in the retina in very small amounts. We established that in the Cyp27a1(-/-)Cyp46a1(-/-) retina, cholesterol esters are generated by and accumulate in the photoreceptor outer segments (OS), which is the retinal layer with the lowest cholesterol content. Mouse OS were also found to express the cholesterol-esterifying enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT1), but not lecithin-cholesterol acyltransferase (LCAT), and to differ from humans in retinal expression of ACAT1. Nevertheless, cholesterol esters were discovered to be abundant in human OS. We suggest a mechanism for cholesterol ester accumulation in the OS and that activity impairment of ACAT1 in humans may underlie the development of subretinal drusenoid deposits, a hallmark of age-related macular degeneration, which is a common blinding disease. We generated Cyp27a1(-/-)Cyp46a1(-/-)Acat1(-/-) mice, characterized their retina by different imaging modalities, and confirmed that unesterified cholesterol does accumulate in their OS and that there is photoreceptor apoptosis and OS degeneration in this line. Our results provide insights into the retinal response to local hypercholesterolemia and the retinal significance of cholesterol esterification, which could be cell-specific and both beneficial and detrimental for retinal structure and function.

Pathways of cholesterol homeostasis in mouse retina responsive to dietary and pharmacologic treatments.

Effects of serum cholesterol on cholesterol content in the retina are currently unknown. It is also unclear how cholesterol levels are controlled in the retina. High-cholesterol diet and oral administrations of simvastatin were used to modulate serum cholesterol in mice. These treatments only modestly affected cholesterol content in the retina and had no significant effect on retinal expression of the major cholesterol- and vision-related genes; the sterol-regulatory element binding protein pathway of transcriptional regulation does not seem to be operative in the retina under the experimental conditions used. Evidence is obtained that posttranslational mechanisms play a role in the control of retinal cholesterol. Retinal genes were only upregulated by oral administrations of TO901317 activating liver X receptors. Three of the upregulated genes could be of particular importance (apoD, Idol, and Rpe65) and have not yet been considered in the context of cholesterol homeostasis in the retina. Collectively, the data obtained identify specific features of retinal cholesterol maintenance and suggest additional therapies for age-related macular degeneration, a blinding disease characterized by cholesterol and lipid accumulations in chorioretinal tissues.

Retinal and nonocular abnormalities in Cyp27a1(-/-)Cyp46a1(-/-) mice with dysfunctional metabolism of cholesterol.

Cholesterol elimination from nonhepatic cells involves metabolism to side-chain oxysterols, which serve as transport forms of cholesterol and bioactive molecules modulating a variety of cellular processes. Cholesterol metabolism is tissue specific, and its significance has not yet been established for the retina, where cytochromes P450 (CYP27A1 and CYP46A1) are the major cholesterol-metabolizing enzymes. We generated Cyp27a1(-/-)Cyp46a1(-/-) mice, which were lean and had normal serum cholesterol and glucose levels. These animals, however, had changes in the retinal vasculature, retina, and several nonocular organs (lungs, liver, and spleen). Changes in the retinal vasculature included structural abnormalities (retinal-choroidal anastomoses, arteriovenous shunts, increased permeability, dilation, nonperfusion, and capillary degeneration) and cholesterol deposition and oxidation in the vascular wall, which also exhibited increased adhesion of leukocytes and activation of the complement pathway. Changes in the retina included increased content of cholesterol and its metabolite, cholestanol, which were focally deposited at the apical and basal sides of the retinal pigment epithelium. Retinal macrophages of Cyp27a1(-/-)Cyp46a1(-/-) mice were activated, and oxidative stress was noted in their photoreceptor inner segments. Our findings demonstrate the importance of retinal cholesterol metabolism for maintenance of the normal retina, and suggest new targets for diseases affecting the retinal vasculature.

Pretreatment with pyridoxamine mitigates isolevuglandin-associated retinal effects in mice exposed to bright light.

The benefits of antioxidant therapy for treating age-related macular degeneration, a devastating retinal disease, are limited. Perhaps species other than reactive oxygen intermediates should be considered as therapeutic targets. These could be lipid peroxidation products, including isolevuglandins (isoLGs), prototypical and extraordinarily reactive γ-ketoaldehydes that avidly bind to proteins, phospholipids, and DNA and modulate the properties of these biomolecules. We found isoLG adducts in aged human retina but not in the retina of mice kept under dim lighting. Hence, to test whether scavenging of isoLGs could complement or supplant antioxidant therapy, we exposed mice to bright light and found that this insult leads to retinal isoLG-adduct formation. We then pretreated mice with pyridoxamine, a B6 vitamer and efficient scavenger of γ-ketoaldehydes, and found that the levels of retinal isoLG adducts are decreased, and morphological changes in photoreceptor mitochondria are not as pronounced as in untreated animals. Our study demonstrates that preventing the damage to biomolecules by lipid peroxidation products, a novel concept in vision research, is a viable strategy to combat oxidative stress in the retina.

Low-Dose Anti-HIV Drug Efavirenz Mitigates Retinal Vascular Lesions in a Mouse Model of Alzheimer's Disease.

A small dose of the anti-HIV drug efavirenz (EFV) was previously discovered to activate CYP46A1, a cholesterol-eliminating enzyme in the brain, and mitigate some of the manifestation of Alzheimer's disease in 5XFAD mice. Herein, we investigated the retina of these animals, which were found to have genetically determined retinal vascular lesions associated with deposits within the retinal pigment epithelium and subretinal space. We established that EFV treatment activated CYP46A1 in the retina, enhanced retinal cholesterol turnover, and diminished the lesion frequency >5-fold. In addition, the treatment mitigated fluorescein leakage from the aberrant blood vessels, deposit size, activation of retinal macrophages/microglia, and focal accumulations of amyloid ß plaques, unesterified cholesterol, and Oil Red O-positive lipids. Studies of retinal transcriptomics and proteomics identified biological processes enriched with differentially expressed genes and proteins. We discuss the mechanisms of the beneficial EFV effects on the retinal phenotype of 5XFAD mice. As EFV is an FDA-approved drug, and we already tested the safety of small-dose EFV in patients with Alzheimer's disease, our data support further clinical investigation of this drug in subjects with retinal vascular lesions or neovascular age-related macular degeneration.

Neutrophil-Derived Proteases Contribute to the Pathogenesis of Early Diabetic Retinopathy.

Purpose: Previous studies indicate that leukocytes, notably neutrophils, play a causal role in the capillary degeneration observed in diabetic retinopathy (DR), however, the mechanism by which they cause such degeneration is unknown. Neutrophil elastase (NE) is a protease released by neutrophils which participates in a variety of inflammatory diseases. In the present work, we investigated the potential involvement of NE in the development of early DR. Methods: Experimental diabetes was induced in NE-deficient mice (Elane-/-), in mice treated daily with the NE inhibitor, sivelestat, and in mice overexpressing human alpha-1 antitrypsin (hAAT+). Mice were assessed for diabetes-induced retinal superoxide generation, inflammation, leukostasis, and capillary degeneration. Results: In mice diabetic for 2 months, deletion of NE or selective inhibition of NE inhibited diabetes-induced retinal superoxide levels and inflammation, and inhibited leukocyte-mediated cytotoxicity of retinal endothelial cells. In mice diabetic for 8 months, genetic deletion of NE significantly inhibited diabetes-induced retinal capillary degeneration. Conclusions: These results suggest that a protease released from neutrophils contributes to the development of DR, and that blocking NE activity could be a novel therapy to inhibit DR.

Sildenafil-evoked photoreceptor oxidative stress in vivo is unrelated to impaired visual performance in mice.

PURPOSE: The phosphodiesterase inhibitor sildenafil is a promising treatment for neurodegenerative disease, but it can cause oxidative stress in photoreceptors ex vivo and degrade visual performance in humans. Here, we test the hypotheses that in wildtype mice sildenafil causes i) wide-spread photoreceptor oxidative stress in vivo that is linked with ii) impaired vision. METHODS: In dark or light-adapted C57BL/6 mice ± sildenafil treatment, the presence of oxidative stress was evaluated in retina laminae in vivo by QUEnch-assiSTed (QUEST) magnetic resonance imaging, in the subretinal space in vivo by QUEST optical coherence tomography, and in freshly excised retina by a dichlorofluorescein assay. Visual performance indices were also evaluated by QUEST optokinetic tracking. RESULTS: In light-adapted mice, 1 hr post-sildenafil administration, oxidative stress was most evident in the superior peripheral outer retina on both in vivo and ex vivo examinations; little evidence was noted for central retina oxidative stress in vivo and ex vivo. In dark-adapted mice 1 hr after sildenafil, no evidence for outer retina oxidative stress was found in vivo. Evidence for sildenafil-induced central retina rod cGMP accumulation was suggested as a panretinally thinner, dark-like subretinal space thickness in light-adapted mice at 1 hr but not 5 hr post-sildenafil. Cone-based visual performance was impaired by 5 hr post-sildenafil and not corrected with anti-oxidants; vision was normal at 1 hr and 24 hr post-sildenafil. CONCLUSIONS: The sildenafil-induced spatiotemporal pattern of oxidative stress in photoreceptors dominated by rods was unrelated to impairment of cone-based visual performance in wildtype mice.

Successful induction of diabetes in mice demonstrates no gender difference in development of early diabetic retinopathy.

PURPOSE: Female mice have been found to be resistant to streptozotocin (STZ)-induced diabetes, and pre-clinical research related to diabetic complications commonly omits females. The purpose of this study was to develop a method to induce diabetes in female mice, and to determine if retinas of diabetic female mice develop molecular changes and histopathological abnormalities comparable to those which develop in male diabetic mice. METHODS: To induce diabetes, animals of both sexes received daily intraperitoneal (i.p.) injection of STZ for 5 consecutive days at 55 mg/kg BW (a dose that is known to induce diabetes in male mice) or for females, 75 mg/kg BW of STZ. Retinal abnormalities that have been implicated in the development of the retinopathy (superoxide generation and expression of inflammatory proteins, iNOS and ICAM-1) were evaluated at 2 months of diabetes, and retinal capillary degeneration was evaluated at 8 months of diabetes. RESULTS: Daily i.p. injection of STZ for 5 consecutive days at a concentration of 55 mg/kg BW was sufficient to induce diabetes in 100% of male mice, but only 33% of female mice. However, females did become hyperglycemic when the dose of STZ administered was increased to 75 mg/kg BW. The resulting STZ-induced hyperglycemia in female and male mice was sustained for at least 8 months. After induction of the diabetes, both sexes responded similarly with respect to the oxidative stress, expression of iNOS, and degeneration of retinal capillaries, but differed in the limited population evaluated with respect to expression of ICAM-1. CONCLUSIONS: The resistance of female mice to STZ-induced diabetes can be overcome by increasing the dose of STZ used. Female mice can, and should, be included in pre-clinical studies of diabetes and its complications.

Transducin1, Phototransduction and the Development of Early Diabetic Retinopathy.

Purpose: Recent evidence suggests that retinal photoreceptor cells have an important role in the pathogenesis of retinal microvascular lesions in diabetes. We investigated the role of rod cell phototransduction on the pathogenesis of early diabetic retinopathy (DR) using Gnat1-/- mice (which causes permanent inhibition of phototransduction in rod cells without degeneration). Methods: Retinal thickness, oxidative stress, expression of inflammatory proteins, electroretinograms (ERG) and optokinetic responses, and capillary permeability and degeneration were evaluated at up to 8 months of diabetes. Results: The diabetes-induced degeneration of retinal capillaries was significantly inhibited in the Gnat1-/- diabetics. The effect of the Gnat1 deletion on the diabetes-induced increase in permeability showed a nonuniform accumulation of albumin in the neural retina; the defect was inhibited in diabetic Gnat1-/- mice in the inner plexiform layer (IPL), but neither in the outer plexiform (OPL) nor inner nuclear (INL) layers. In Gnat1-deficient animals, the diabetes-induced increase in expression of inflammatory associated proteins (iNOS and ICAM-1, and phosphorylation of IĸB) in the retina, and the leukocyte mediated killing of retinal endothelial cells were inhibited, however the diabetes-mediated induction of oxidative stress was not inhibited. Conclusions: In conclusion, deletion of transducin1 (and the resulting inhibition of phototransduction in rod cells) inhibits the development of retinal vascular pathology in early DR.

The Interplay between Retinal Pathways of Cholesterol Output and Its Effects on Mouse Retina.

In mammalian retina, cholesterol excess is mainly metabolized to oxysterols by cytochromes P450 27A1 (CYP27A1) and 46A1 (CYP46A1) or removed on lipoprotein particles containing apolipoprotein E (APOE). In contrast, esterification by sterol-O-acyltransferase 1 (SOAT) plays only a minor role in this process. Accordingly, retinal cholesterol levels are unchanged in Soat1-/- mice but are increased in Cyp27a1-/-Cyp46a1-/- and Apoe-/- mice. Herein, we characterized Cyp27a1-/-Cyp46a1-/-Soat1-/- and Cyp27a1-/-Cyp46a1-/-Apoe-/- mice. In the former, retinal cholesterol levels, anatomical gross structure, and vasculature were normal, yet the electroretinographic responses were impaired. Conversely, in Cyp27a1-/-Cyp46a1-/-Apoe-/- mice, retinal cholesterol levels were increased while anatomical structure and vasculature were unaffected with only male mice showing a decrease in electroretinographic responses. Sterol profiling, qRT-PCR, proteomics, and transmission electron microscopy mapped potential compensatory mechanisms in the Cyp27a1-/-Cyp46a1-/-Soat1-/- and Cyp27a1-/-Cyp46a1-/-Apoe-/- retina. These included decreased cholesterol biosynthesis along with enhanced formation of intra- and extracellular vesicles, possibly a reserve mechanism for lowering retinal cholesterol. In addition, there was altered abundance of proteins in Cyp27a1-/-Cyp46a1-/-Soat1-/- mice that can affect photoreceptor function, survival, and retinal energy homeostasis (glucose and fatty acid metabolism). Therefore, the levels of retinal cholesterol do not seem to predict retinal abnormalities, and it is rather the network of compensatory mechanisms that appears to determine retinal phenotype.

N,N-Dimethyl-3ß-hydroxycholenamide Reduces Retinal Cholesterol via Partial Inhibition of Retinal Cholesterol Biosynthesis Rather Than its Liver X Receptor Transcriptional Activity.

N,N-dimethyl-3ß-hydroxycholenamide (DMHCA) is an experimental pharmaceutical and a steroidal liver X receptor (LXR) agonist, which does not induce undesired hepatic lipogenesis. Herein, DMHCA was evaluated for its retinal effects on normal C57BL/6J and Cyp27a1-/-Cyp46a1-/- mice; the latter having higher retinal total and esterified cholesterol in addition to retinal vascular abnormalities. Different doses and two formulations were used for DMHCA delivery either via drinking water (C57BL/6J mice) or by oral gavage (Cyp27a1-/-Cyp46a1-/- mice). The duration of treatment was 1 week for C57BL/6J mice and 2 or 4 weeks for Cyp27a1-/-Cyp46a1-/- mice. In both genotypes, the higher DMHCA doses (37-80 mg/kg of body weight/day) neither increased serum triglycerides nor serum cholesterol but altered the levels of retinal sterols. Total retinal cholesterol was decreased in the DMHCA-treated mice, mainly due to a decrease in retinal unesterified cholesterol. In addition, retinal levels of cholesterol precursors lanosterol, zymosterol, desmosterol, and lathosterol were changed in Cyp27a1-/-Cyp46a1-/- mice. In both genotypes, DMHCA effect on retinal expression of the LXR target genes was only moderate and gender-specific. Collectively, the data obtained provide evidence for a decrease in retinal cholesterol as a result of DMHCA acting in the retina as an enzyme inhibitor of cholesterol biosynthesis rather than a LXR transcriptional activator. Specifically, DMHCA appears to partially inhibit the cholesterol biosynthetic enzyme Δ24-dehydrocholesterol reductase rather than upregulate the expression of LXR target genes involved in reverse cholesterol transport. The identified DMHCA dosages, formulations, and routes of delivery as well as the observed effects on the retina should be considered in future studies using DMHCA as a potential therapeutic for age-related macular degeneration and diabetic retinopathy.

Cholesterol-metabolizing enzyme cytochrome P450 46A1 as a pharmacologic target for Alzheimer's disease.

Cytochrome P450 46A1 (CYP46A1 or cholesterol 24-hydroxylase) controls cholesterol elimination from the brain and plays a role in higher order brain functions. Genetically enhanced CYP46A1 expression in mouse models of Alzheimer's disease mitigates the manifestations of this disease. We enhanced CYP46A1 activity pharmacologically by treating 5XFAD mice, a model of rapid amyloidogenesis, with a low dose of the anti-HIV medication efavirenz. Efavirenz was administered from 1 to 9 months of age, and mice were evaluated at specific time points. At one month of age, cholesterol homeostasis was already disturbed in the brain of 5XFAD mice. Nevertheless, efavirenz activated CYP46A1 and mouse cerebral cholesterol turnover during the first four months of administration. This treatment time also reduced amyloid burden and microglia activation in the cortex and subiculum of 5XFAD mice as well as protein levels of amyloid precursor protein and the expression of several genes involved in inflammatory response. However, mouse short-term memory and long-term spatial memory were impaired, whereas learning in the context-dependent fear test was improved. Additional four months of drug administration (a total of eight months of treatment) improved long-term spatial memory in the treated as compared to the untreated mice, further decreased amyloid-ß content in 5XFAD brain, and also decreased the mortality rate among male mice. We propose a mechanistic model unifying the observed efavirenz effects. We suggest that CYP46A1 activation by efavirenz could be a new anti-Alzheimer's disease treatment and a tool to study and identify normal and pathological brain processes affected by cholesterol maintenance.