The laboratory unit of the Macular Research Group focuses on studies to understand how the outer retinal metabolism contributes to retinal health, with a particular interest in how Müller cell dysfunction affects photoreceptor cells. The team has generated transgenic mice in which key metabolic genes can be selectively manipulated in Müller cells, photoreceptors and the retinal pigment epithelium. The unit also tries to understand whether Müller cells in the central and peripheral human retina function differently.
NanoVision project: Transform treatment of blindness by harnessing advances in nanomedicine, engineering and synthetic biology to manipulate the genetic code to prevent and treat loss of vision
Using nanoparticles as carriers of drugs or genes is one promising way to treat macular diseases. Nanoparticles, especially lipid nanoparticles, are less immunogenic and less cytotoxic than other carriers such as viruses. They can carry significantly larger payloads than viruses. They are eco-friendly since they are made from natural materials (fats) with very low toxicity that can be digested by the human body.
We are using a unique human macula explant system that allows us to study human retinas that were donated by people who died. We are combining state-of-art single-cell RNA sequencing (scRNAseq) with a specially designed DNA barcode library to perform high-throughput screening for the lipid nanoparticles that we will test for their ability to deliver genes or drugs to the retina. The system is cost-effective because it can screen hundreds of lipid nanoparticles with diverse structures using a limited amount of post-mortem human macula.
This study is an innovative approach to identify new cell-specific carriers to deliver genes or drugs to the human macula. These identified carriers will be used as vectors to introduce the gene constructs or drugs specifically into the target cells. The most efficient and specific nanoparticles will be considered for testing in clinical trials.
The system may narrow the translational gap between laboratory studies and the clinic because of the advantages of using human macula to identify drug carriers for further testing in clinical trials.
Targeting a novel pathogenic glia-neuronal pathway in retinal diseases
We propose a novel glia-neuronal network in the retina that stressed Müller cells release signal(s) which downregulate the expression of Interphotoreceptor Retinoid-Binding protein (IRBP) in photoreceptors. This will lead to photoreceptor degeneration. This network may be common to the development of many retinal diseases. We also hypothesize that the restoration of IRBP expression in the stressed retina will prevent or delay photoreceptor degeneration. Our preliminary studies suggest stressed Müller cells regulate the expression of IRBP in photoreceptors, possibly through the MAPK-TNFa signaling pathway, but the specific molecular mechanisms remain unclear. A better understanding of this process is important to identify new leads to treat the prevalent retinal diseases leading to blindness.
We are now using small molecules to interfere MAPK signaling in light-induced retinal degeneration mice and assessing the expression of IRBP and photoreceptor degeneration. We are also profiling gene expression in Muller cells and photoreceptors in human retinal explants with or without a MAPK signaling inhibitor through single-cell RNA sequencing. The RNA sequencing outcome will be further validated in human AMD specimens and compared with healthy controls. Meanwhile, we are also evaluating the therapeutic effectiveness of reversing IRBP deficiency under retinal stresses using lentivirus-mediated CRISPR gene therapy. The progress of photoreceptor degeneration and visual function will be evaluated after the treatments.
This study will result in a better understanding of the critical role of IRBP dysregulation in the pathogenesis of retinal diseases. It will also provide molecular insights into how Müller cells interact with photoreceptors under pathologic conditions. The Macular Research Group includes a clinical research unit that is well-positioned to test clinically-relevant novel approaches for retinal diseases.
Retinal health relies on proper glucose metabolism to produce energy and metabolites to support different populations of retinal cells including various neurons, Müller cells (a supportive glial cell in the retina) and the retinal pigment epithelium (RPE). Photoreceptors (photo-detecting cells) have high demands for energy to maintain normal vision. Metabolic derangement is likely involved in photoreceptor degeneration, a major feature of macular diseases such as age-related macular degeneration, diabetic macular edema and macular telangiectasia. However, how metabolic dysfunction affects photoreceptor health remains largely unknown.
Müller cells and the RPE play important roles in transporting and metabolising glucose to support photoreceptors. We have generated a unique transgenic mouse in which a Müller cell-specific promoter along with the Cre/Lox-P approach was used for Müller cell-specific gene targeting. We also have transgenic lines carrying cell-specific promoters which allow us to manipulate gene expression in photoreceptors and the RPE. We are currently using these unique cell-specific approaches to study the consequences of selectively disrupting various metabolic pathways in Müller cells, rod photoreceptors and the RPE in the intact retina. This will allow us to precisely dissect the contribution of metabolic derangement in Müller cells, photoreceptors and the RPE to photoreceptor degeneration in retinal diseases.
We have generated an inducible transgenic line which allows us to specifically manipulate gene expression in Müller cells (Figure 1A). We have crossed this transgenic line with transgenic mice carrying an attenuated form of the diphtheria toxin gene and found that selective Müller cell ablation leads to photoreceptor degeneration, retinal vessel leak, and later, intraretinal neovascularisation (Figure 1B-H). These changes are also accompanied by reactive activation of retinal glia including astrocytes, surviving Müller cells and microglia. These features make our transgenic mice very useful for studying the cellular and molecular mechanisms underlying Müller glia-neuron-vascular interactions. Our transgenic mice can also be used to test novel strategies for neuroprotection, inhibition of retinal vessel leak and prevention of retinal fibrosis.
Why the central retina, or macula, is so susceptible to disease is a major unresolved issue in ophthalmic research?
We believe that one of the important differences between the macula and the rest of the retina is the nature of the Müller cells, the retina’s main glial cell, in the 2 regions. We have found significant differences between Müller cells from the macula and peripheral retina at the transcriptional level. Of note are differences in the de novo serine synthesis pathway, which controls cell susceptibility to stress. We believe Müller cells derived from the central and peripheral retina have different susceptibility to stress due to the differences in this pathway. We aim to compare de novo serine synthesis in central and peripheral Müller cells as well as to understand the pathological consequences of disturbing this pathway in Müller cells. We will further test compounds that can compensate for derangement of Müller cell de novo serine synthesis. This will be the first study to compare Müller cells from human macula and peripheral retina as well as to investigate the role of de novo serine synthesis in Müller cells in health and disease. The significance of this research is that it may provide insights into a major unresolved question in vision research: why is the macula so susceptible to some of the commonest blinding diseases such as age-related macular degeneration and diabetic macular oedema.
Professor Mark Gillies
Phone: (02) 9382 7309
Dr Ling Zhu
Phone: (02) 9283 7270
Zhang T, Zhu L, Madigan MC, Liu W, Shen W, Cherepanoff S, Zhou F, Zeng S, Du J, and Gillies MC. Human macular Müller cells rely more on serine biosynthesis to combat oxidative stress than those from the periphery. Elife. 2019;8.https://www.ncbi.nlm.nih.gov/pubmed/31036157
Zhang T, Gillies M, Wang Y, Shen W, Bahrami B, Zeng S, Zhu M, Yao W, Zhou F, Murray M, Wang K, and Zhu L. Simvastatin protects photoreceptors from oxidative stress induced by all-trans-retinal, through the up-regulation of interphotoreceptor retinoid binding protein. Br J Pharmacol. 2019;176(12):2063-78.https://www.ncbi.nlm.nih.gov/pubmed/30825184
You Y, Zhu L, Zhang T, Shen T, Fontes A, Yiannikas C, Parratt J, Barton J, Schulz A, Gupta V, Barnett MH, Fraser CL, Gillies M, Graham SL, and Klistorner A. Evidence of Müller Glial Dysfunction in Patients with Aquaporin-4 Immunoglobulin G-Positive Neuromyelitis Optica Spectrum Disorder. Ophthalmology. 2019;126(6):801-810. https://www.ncbi.nlm.nih.gov/pubmed/30711604
Zhu L, Shen W, Wang Y, Zhang T, Bahrami B, Zhou F, Gillies MC. Characterization of canonical Wnt signalling changes after induced Müller cell disruption in murine retina. Exp Eye Res. 2018 Oct;175:173-180. https://www.ncbi.nlm.nih.gov/pubmed/29913166
Irhimeh MR, Hamed M, Barthelmes D, Gladbach Y, Helms V, Shen W and Gillies MC. Identification of novel diabetes impaired miRNA-transcription factor co-regulatory networks in bone marrow-derived endothelial progenitor cells. PLOS One 2018; 13 (7), e0200194
Zhang T, Gillies MC, Madigan MC, Shen W, Du J, Grünert U, Zhou F, Yam M, Zhu L. Disruption of De Novo Serine Synthesis in Müller Cells Induced Mitochondrial Dysfunction and Aggravated Oxidative Damage. Mol Neurobiol. 2018;55(8): 7025–7037.
Shen W, Yau B, Lee SR, Zhu L, Yam M, Gillies MC. Effects of ranibizumab and aflibercept on human Müller cells and photoreceptors under stressed conditions. Int J Mol Sci 2017, 18(533):1-16.
Baumann B, Sterling J, Song Y, Song D, Fruttiger M, Gillies M, Shen W and Dunaief JL. Conditional Müller cell ablation leads to retinal iron accumulation. Invest Ophthalmol Vis Sci 2017;58:4223–4234.
Chung SH, Gillies MC, Yam M, Wang , Shen W.
Differential expression of microRNAs in retinal vasculopathy caused by selective Müller cell disruption. Scientific Report 2016;6: 28993.
Xu C, Zhu L, Chan T, Lu X, Shen W, Madigan MC, Gillies MC and Zhou F. Chloroquine and Hydroxychloroquine Are Novel Inhibitors of Human Organic Anion Transporting Polypeptide 1A2. J Pharma Sci. 2016, 105 884-890.
Coorey NJ, Shen W, Zhu L and Gillies MC. Differential Expression of IL-6/gp130 Cytokines, Jak-STAT Signaling and Neuroprotection After Müller Cell Ablation in a Transgenic Mouse Model. Invest Ophthalmol Vis Sci. 2015;56:2151-2161.
Burdon KP, Fogarty RD, Shen W, Abhary S, Kaidonis G, Appukuttan B, Hewitt AW, Sharma S, Daniell M, Essex RW, Chang JH, Klebe S, Lake SR, Pal B, Jenkins A, Govindarjan G, Sundaresan P, Lamoureux EL, Ramasamy K, Pefkianaki M, Hykin PG, Petrovsky N, Brown MA, Gillies MC and Craig JE. Genome-wide association study for sight-threatening diabetic retinopathy reveals association with genetic variation near the GRB2 gene. Diabetologia 2015;58:2288-2297.
Zhu L, Shen W, Lyons B, Wang Y, Zhou F and Gillies MC. Dysregulation of inter-photoreceptor retinoid-binding protein (IRBP) after induced Müller cell disruption. J Neurochem 2015;133:909-918.
Chung SH, Gillies M, Sugiyama Y, Zhu L, Lee SR and Shen W. Profiling of microRNAs involved in retinal degeneration caused by selective Müller cell ablation. PLOS One 2015;10:e0118949.
Chung SH, Shen W and Gillies M. Genomic analysis using Affymetrix standard microarray genechips (169 format) in degenerate murine retina. Methods in Molecular Biology. 2015;1254:129-140.
Xu C, Zhu L, Chan T, Lu X, Shen W, Gillies MC and Zhou F. 2015. The altered renal and hepatic expression of solute carrier transporters (SLCs) in type 1 diabetic mice. PLOS One 2015;10:e0120760.
Chan T, Zhu L, Madigan MC, Wang K, Shen W, Gillies MC and Zhou F. Human organic anion transporting polypeptide 1A2 (OATP1A2) mediates cellular uptake of all-trans-retinol in human retinal pigmented epithelial cells. Br J Pharmacol 2015;172:2343-2353.
Shen W, Chung SH, Irhimeh MR, Li S, Lee SR and Gillies MC. Systemic Administration of Erythropoietin Inhibits Retinopathy in RCS Rats. PLoS ONE 2014;9(8):e104759.
Shen W, Lee SR, Araujo J, Chung SH, Zhu L, and Gillies MC. Effect of glucocorticoids on neuronal and vascular pathology in a transgenic model of selective Müller cell ablation. Glia. 2014;62:1110-24.
Barthelmes D, Zhu L, Shen W, Gillies MC, and Irhimeh MR. Differential gene expression in Lin-/VEGF-R2+ bone marrow-derived endothelial progenitor cells isolated from diabetic mice. Cardiovasc Diabetol. 2014;13:42
Chung SH, Shen W, Gillies MC. Identification of a novel miRNA targeting CD146 for suppression of angiogenesis. Non-coding RNAs in Endocrinology 2014;1:28-30.
Zhu L, Shen W, Zhu M, Coorey NJ, Nguyen AP, Barthelmes D, and Gillies MC. Anti-retinal antibodies in patients with macular telangiectasia type 2. Invest Ophthalmol Vis Sci. 2013;54:5675-83.
Shen W, Zhu L, Lee SR, Chung SH, and Gillies MC. Involvement of NT3 and P75(NTR) in photoreceptor degeneration following selective Müller cell ablation. J Neuroinflammation. 2013;10:137.
Chung SH, Shen W, Jayawardana K, Wang P, Yang J, Shackel N, and Gillies MC. Differential gene expression profiling after conditional Müller-cell ablation in a novel transgenic model. Invest Ophthalmol Vis Sci. 2013;54:2142-52.
Chung SH, Shen W, and Gillies MC. Laser capture microdissection-directed profiling of glycolytic and mTOR pathways in areas of selectively ablated Müller cells in the murine retina. Invest Ophthalmol Vis Sci. 2013;54:6578-85.
Barthelmes D, Irhimeh MR, Gillies MC, Zhu L, and Shen W. Isolation and characterization of mouse bone marrow-derived Lin(-)/VEGF-R2(+) progenitor cells. Ann Hematol. 2013;92:1461-72
Barthelmes D, Irhimeh MR, Gillies MC, Karimipour M, Zhou M, Zhu L, and Shen WY. Diabetes impairs mobilization of mouse bone marrow-derived Lin(-)/VEGF-R2(+) progenitor cells. Blood Cells Mol Dis. 2013;51:163-73.
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