Cholesterol Dyshomeostasis And Age Related Macular Degeneration

This dissertation work focused on retinal modifications that are relevant to Age-related macular degeneration (AMD) in cholesterol-fed rabbit model of Alzheimer\u27s disease (AD), as AD and AMD share common features. It is unknown whether cholesterol-fed rabbit model of AD displays any AMD features in retina. Previous research showed 27-hydroxycholesterol (27-OHC) involvement in AD like pathology in organotypic hippocampal slices of rabbit brain and human SHSY-5Y neuroblastoma cells. The extent to which and the mechanisms by which 27-OHC may also cause pathological hallmarks related to AMD are not known. Various studies suggested estrogen\u27s (E2) role in AMD development. 27-OHC is a ligand for estrogen receptor (ER) and liver X receptor (LXR). 25-hydroxycholesterol (25-OHC) and 7-ketocholesterol (7-KC) are also implicated in AMD development. 25-OHC and 7-KC were shown to be ligands of ER and LXR in various cell types. It is unknown whether 27-OHC, 25-OHC and 7-KC influence ER and LXR transcriptional activity in ARPE-19 cells, a spontaneously arising human RPE cell line with normal karyology. ARPE-19 cells and cholesterol-fed rabbit eyes were used for the study. Paraffin embedded eye cross sections were used for immunohistochemistry. Cholesterol was quantified by cholesterol/cholesteryl ester quantification kit. Oxysterols in the rabbit retinas were measured by mass spectrometry. Western blotting for detecting proteins, CytoTox-ONE homogenous membrane integrity assay for measuring lactate dehydrogenase from cells, ELISA (Enzyme-linked immunosorbent assay) for quantifying amyloid beta 1-42 and 1-40, tumor necrosis factor alpha, DCFH-DA (2\u27,7\u27-dichlorfluorescein-diacetate) assay for measuring reactive oxygen species (ROS), amplex red hydrogen peroxide / peroxidase assay for quantifying hydrogen peroxide and peroxidase activity, JC-1 (5,5\u27,6,6\u27-tetrachloro-1,1\u27,3,3\u27-tetraethylbenzimidazolylcarbocyanine iodide) assay for mitochondrial membrane potential detection, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay for apoptotic cell detection, GSH-Glo assay for glutathione (GSH) quantification, calcium imaging, immunocytochemistry, immunohistochemistry and H&E (hematoxylin and eosin) staining, transfection and dual-luciferase reporter assays were used for this work. This study showed retinal modifications that are relevant to AMD in cholesterol-fed rabbits. Increased abeta levels, decreased apoptosis regulator Bcl-2 levels, increased apoptosis regulator BAX and growth arrest and DNA damage-inducible protein GADD153 proteins, apoptotic cells, and increased generation of ROS were found in retinas from cholesterol-fed rabbit retinas. Furthermore, astrogliosis, drusen-like debris and cholesterol accumulations in retinas from cholesterol-fed rabbits were observed. Oxysterol levels in retinas from cholesterol-fed rabbits were increased. 27-OHC increased abeta peptide production, increased caspase 12 and GADD153, reduced mitochondrial membrane potential, triggered Ca2+ dyshomeostasis, and increased levels of the Nuclear factor NF-kappa-B p65 subunit and Heme oxygenase 1. Additionally, 27-OHC caused GSH depletion, ROS generation, inflammation and apoptotic-mediated cell death in ARPE-19 cells. ARPE-19 cells express ERalpha, ERbeta, LXRalpha and LXRbeta. ER and LXR are transcriptionally active. ER agonist E2 protected cells from 25-OHC and 27-OHC induced cytotoxicity. E2 counteracted 27-OHC and 25-OHC induced mitochondrial membrane potential decline. Taken together, data demonstrate that cholesterol-enriched diets induce pathological hallmarks suggestive of AMD in rabbit retinas. The cholesterol diet substantially increased concentrations of the major oxysterols, accumulation of which is toxic to retinal cells. Incubation of cells with oxysterols also reproduced the majors effects observed in vivo with the diet rich in cholesterol. These results strongly suggest that hypercholesterolemia and subsequent increase in oxysterol formation may contribute to the pathogenesis of AMD. Our data also shows that ER modulation may play an important role in the cholesterol and oxysterol effects. Specifically, ER agonists may provide protection against oxysterol deleterious effects on retinas. As well, reduction of cholesterol plasma levels may prevent excess conversion of cholesterol to oxysterols and precludes the incidence of AMD

Molecular interplay between leptin, insulin-like growth factor-1, and β-amyloid in organotypic slices from rabbit hippocampus

<p>Abstract</p> <p>Background</p> <p>Evidence shows that the insulin-like growth factor-1 (IGF-1) and leptin reduce β-amyloid (Aβ) production and tau phosphorylation, two major hallmarks of Alzheimer's disease (AD). IGF-1 expression involves the JAK/STAT pathway and the expression of leptin is regulated by the mammalian target of rapamycin complex 1 (mTORC1). We have previously shown that Aβ reduces leptin by inhibiting the mTORC1 pathway and Aβ was also suggested to inhibit the JAK/STAT pathway, potentially attenuating IGF-1 expression. As IGF-1 can activate mTORC1 and leptin can modulate JAK/STAT pathway, we determined the extent to which IGF-1 and leptin can upregulate the expression of one another and protect against Aβ-induced downregulation.</p> <p>Results</p> <p>We demonstrate that incubation of organotypic slices from adult rabbit hippocampus with Aβ42 downregulates IGF-1 expression by inhibiting JAK2/STAT5 pathway. Leptin treatment reverses these Aβ42 effects on IGF-1 and treatment with the STAT5 inhibitor completely abrogated the leptin-induced increase in IGF-1. Furthermore, EMSA and ChIP analyses revealed that leptin increases the STAT5 binding to the IGF-1 promoter. We also show that IGF-1 increases the expression of leptin and reverses the Aβ42-induced attenuation in leptin expression via the activation of mTORC1 signaling as the mTORC1 inhibitor rapamycin completely precluded the IGF-1-induced increase in leptin expression.</p> <p>Conclusion</p> <p>Our results demonstrate for the first time that Aβ42 downregulates IGF-1 expression and that leptin and IGF-1 rescue one another from downregulation by Aβ42. Our study provides a valuable insight into the leptin/IGF-1/Aβ interplay that may be relevant to the pathophysiology of AD.</p

Cholesterol-enriched diet causes age-related macular degeneration-like pathology in rabbit retina

<p>Abstract</p> <p>Background</p> <p>Alzheimer's disease (AD) and age-related macular degeneration (AMD) share several pathological hallmarks including β-amyloid (Aβ) accumulation, oxidative stress, and apoptotic cell death. The causes of AD and AMD are likely multi-factorial with several factors such as diet, environment, and genetic susceptibility participating in the pathogenesis of these diseases. Epidemiological studies correlated high plasma cholesterol levels with high incidence of AD, and feeding rabbits with a diet rich in cholesterol has been shown to induce AD-like pathology in rabbit brain. High intake of cholesterol and saturated fat were also long been suspected to increase the risk for AMD. However, the extent to which cholesterol-enriched diet may also cause AMD-like features in rabbit retinas is not well known.</p> <p>Methods</p> <p>Male New Zealand white rabbits were fed normal chow or a 2% cholesterol-enriched diet for 12 weeks. At necropsy, animals were perfused with Dulbecco's phosphate-buffered saline and the eyes were promptly removed. One eye of each animal was used for immunohistochemistry and retina dissected from the other eye was used for Western blot, ELISA assays, spectrophotometry and mass spectrometry analyses.</p> <p>Results</p> <p>Increased levels of Aβ, decreased levels of the anti-apoptotic protein Bcl-2, increased levels of the pro-apoptotic Bax and gadd153 proteins, emergence of TUNEL-positive cells, and increased generation of reactive oxygen species were found in retinas from cholesterol-fed compared to normal chow-fed rabbits. Additionally, astrogliosis, drusen-like debris and cholesterol accumulations in retinas from cholesterol-fed rabbits were observed. As several lines of evidence suggest that oxidized cholesterol metabolites (oxysterols) may be the link by which cholesterol contributes to the pathogenesis of AMD, we determined levels of oxysterols and found a dramatic increase in levels of oxysterols in retinas from cholesterol-fed rabbits.</p> <p>Conclusions</p> <p>Our results suggest that cholesterol-enriched diets cause retinal degeneration that is relevant to AMD. Furthermore, our data suggests high cholesterol levels and subsequent increase in the cholesterol metabolites as potential culprits to AMD.</p

The oxysterol 27-hydroxycholesterol increases β-amyloid and oxidative stress in retinal pigment epithelial cells

<p>Abstract</p> <p>Background</p> <p>Alzheimer's disease (AD) and age-related macular degeneration (AMD) share several pathological features including β-amyloid (Aβ) peptide accumulation, oxidative damage, and cell death. The causes of AD and AMD are not known but several studies suggest disturbances in cholesterol metabolism as a culprit of these diseases. We have recently shown that the cholesterol oxidation metabolite 27-hydroxycholesterol (27-OHC) causes AD-like pathology in human neuroblastoma SH-SY5Y cells and in organotypic hippocampal slices. However, the extent to which and the mechanisms by which 27-OHC may also cause pathological hallmarks related to AMD are ill-defined. In this study, the effects of 27-OHC on AMD-related pathology were determined in ARPE-19 cells. These cells have structural and functional properties relevant to retinal pigmented epithelial cells, a target in the course of AMD.</p> <p>Methods</p> <p>ARPE-19 cells were treated with 0, 10 or 25 μM 27-OHC for 24 hours. Levels of Aβ peptide, mitochondrial and endoplasmic reticulum (ER) stress markers, Ca²⁺homeostasis, glutathione depletion, reactive oxygen species (ROS) generation, inflammation and cell death were assessed using ELISA, Western blot, immunocytochemistry, and specific assays.</p> <p>Results</p> <p>27-OHC dose-dependently increased Aβ peptide production, increased levels of ER stress specific markers caspase 12 and gadd153 (also called CHOP), reduced mitochondrial membrane potential, triggered Ca²⁺dyshomeostasis, increased levels of the nuclear factor κB (NFκB) and heme-oxygenase 1 (HO-1), two proteins activated by oxidative stress. Additionally, 27-OHC caused glutathione depletion, ROS generation, inflammation and apoptotic-mediated cell death.</p> <p>Conclusions</p> <p>The cholesterol metabolite 27-OHC is toxic to RPE cells. The deleterious effects of this oxysterol ranged from Aβ accumulation to oxidative cell damage. Our results suggest that high levels of 27-OHC may represent a common pathogenic factor for both AMD and AD.</p

Reducible PEG-POD/DNA Nanoparticles for Gene Transfer In Vitro and In Vivo: Application in a Mouse Model of Age-Related Macular Degeneration

Non-viral gene delivery systems are being developed to address limitations of viral gene delivery. Many of these non-viral systems are modeled on the properties of viruses including cell surface binding, endocytosis, endosomal escape, and nuclear targeting. Most non-viral gene transfer systems exhibit little correlation between in vitro and in vivo efficiency, hampering a systematic approach to their development. Previously, we have described a 3.5 kDa peptide (peptide for ocular delivery [POD]) that targets cell surface sialic acid. When functionalized with polyethylene glycol (PEG) via a sulfhydryl group on the N-terminal cysteine of POD, PEG-POD could compact plasmid DNA, forming 120- to 180-nm homogeneous nanoparticles. PEG-POD enabled modest gene transfer and rescue of retinal degeneration in vivo. Systematic investigation of different stages of gene transfer by PEG-POD nanoparticles was hampered by their inability to deliver genes in vitro. Herein, we describe functionalization of POD with PEG using a reducible orthopyridyl disulfide bond. These reducible nanoparticles enabled gene transfer in vitro while retaining their in vivo gene transfer properties. These reducible PEG-POD nanoparticles were utilized to deliver human FLT1 to the retina in vivo, achieving a 50% reduction in choroidal neovascularization in a murine model of age-related macular degeneration. Keywords: nanoparticles, age-related macular degeneration, PEGylation, cell penetrating peptide, gene transfer, reducible particles, laser-induced choroidal neovascularizatio

2nd PSL Chemical Biology Symposium (2019): At the Crossroads of Chemistry and Biology

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Abstracts of National Conference on Research and Developments in Material Processing, Modelling and Characterization 2020

This book presents the abstracts of the papers presented to the Online National Conference on Research and Developments in Material Processing, Modelling and Characterization 2020 (RDMPMC-2020) held on 26th and 27th August 2020 organized by the Department of Metallurgical and Materials Science in Association with the Department of Production and Industrial Engineering, National Institute of Technology Jamshedpur, Jharkhand, India. Conference Title: National Conference on Research and Developments in Material Processing, Modelling and Characterization 2020Conference Acronym: RDMPMC-2020Conference Date: 26–27 August 2020Conference Location: Online (Virtual Mode)Conference Organizer: Department of Metallurgical and Materials Engineering, National Institute of Technology JamshedpurCo-organizer: Department of Production and Industrial Engineering, National Institute of Technology Jamshedpur, Jharkhand, IndiaConference Sponsor: TEQIP-