Advancing eye research ANNUAL REPORToria.org.au/wp-content/uploads/2012/02/ORIA-Annual... ·...

ANNUAL REPORT 2013

O R I AAdvancing eye research

The Ophthalmic Research Institute of Australia

94-98 Chalmers Street, Surry Hills NSW 2010Tel: 02 8394 5218Fax: 02 9690 1321Email: [email protected] 37 008 393 146

CONTENTS

The ORIA – AdvAncIng eye ReseARch

Introduction ............................. 5

chairman’s report ................... 6

ORIA grants ............................. 8

Progress reports ............. 12-17

directors’ Report ............ 38-41

Financial statements ..... 42-58

Auditors’ Report .............. 59-61


4 | ORIA ANNUAL REPORT 2013

In 60 yeARs, The ORIA hAs PROvIded Funds TO ReseARch All mAjOR eye dIseAses, IncludIng mAculAR degeneRATIOn, glAucOmA, lOw vIsIOn, lens And cATARAcT, And dIAbeTIc ReTInOPAThy

5ORIA ANNUAL REPORT 2013 |

The bOARdProf Stuart Graham, Sydney (Chairman)

Prof Mark Gillies, Sydney (Vice Chairman)

Dr Richard Mills, Adelaide (Honorary Secretary)

Dr W Heriot, Melbourne (Honorary Treasurer)

Dr Fred Chen, Perth

Dr Colin Clement, Sydney

Prof J Crowston, Melbourne

A/Prof Mark Daniell, Melbourne

Dr Paul Healey, Sydney

Dr Anthony Kwan, Brisbane

Prof David Mackey, Perth

Prof Peter McCluskey, Sydney

Dr John Males, Sydney

Dr Andrea Vincent, New Zealand

Dr Stephanie Watson, Sydney

ReseARch AdvIsORy cOmmITTeeFor assessments of funding in 2013

Prof Peter McCluskey, Sydney (Chair)

Dr Richard Mills, Adelaide (Secretary)

Prof Stuart Graham, Sydney (ex officio Chair ORIA)

A/Prof John Foster, Sydney

A/Prof Paul Baird, Melbourne, (co-opted for Prof Crowston)

Dr Kathryn Burdon, Adelaide

Dr Fred Chen, Perth

Dr Alex Hewitt, Melbourne

Prof David Mackey, Perth

Dr Nick Di Girolamo, Sydney


Dr Trevor Sherwin, New Zealand

Prof Jan Provis, Canberra

Save Sight Society NZ rep – Dr Graham Wilson

Anne Dunn Snape (as Executive Officer, ORIA)

hOnORARy secReTARy Dr Richard Mills 94-98 Chalmers Street, Surry Hills NSW 2010

hOnORARy TReAsuReR Dr Wilson Heriot 94-98 Chalmers Street, Surry Hills NSW 2010

AccOunTAnTsUHY Haines Norton Melbourne Pty Ltd Level 8, 607 Bourke Street, Melbourne Vic 3000

AudITORs Orr Martin & Waters 461 Whitehorse Road, Balwyn Vic 3103

TRusTees National Australia Trustees Ltd, Melbourne, Vic

hOn sOlIcITORsKing and Wood Mallesons Advance Bank Centre 60 Marcus Clarke Street, Canberra 2600

InvesTmenT AdvIsORy cOmmITTee Dr Wilson Heriot – Hon Treasurer, ORIA

A/Prof Mark Daniell

Mr Andrew Miller – Director, UBS Wealth Management

Mr Dennis Clarebrough – Director-Equities, Lodge Partners Pty Ltd

Mr William Jones – Principal, Goldman Sachs J B Were Limited

Secretary to the Committee, Mr Matthew Timothee – National Australia Trustees Limited

eXecuTIve OFFIceRMs Anne Dunn Snape, BA (Soc & Pol Phil), (MQ) PostGradC Ethics & Legal Studs.

nOTIce OF meeTIngThe Annual Report will be presented at the Sixty First Annual General Meeting to be held in Hobart, Tasmania on Sunday 3 November 2013


The Ophthalmic Research Institute of Australia is RANZCO’s research arm, and aims to: “Advance Eye Research”. The ORIA held its first AGM just over 60 years ago and since that time has distributed literally millions of dollars to provide funding for medical eye research in Australia.

The ORIA’s activities are co-ordinated and managed by the 16 member Board of the ORIA and Executive Officer, Anne Dunn Snape. Using the income from its investments and donor organisations, the ORIA continued to contribute to funding for research projects throughout Australia. During the year the ORIA’s Research Advisory Committee considered 49 applications for project funding from Australian researchers, a significant increase from 19 assessed in 2004. It also assessed 4 New Zealand applications for funding on behalf of the Save Sight Society of New Zealand. The NZ Branch is represented on the committee via its Save Sight Society.

The ORIA’s Research Advisory Committee is composed of leading research scientists and ophthalmologists from Australia and New Zealand. All applications are independently peer reviewed which forms the basis for discussion and recommendation of funding by the Committee. The recommendations of the Committee are put forward to the Board of the ORIA who then indicate what funds are available for the forthcoming calendar year. This year $868,000 was distributed to fund 18 one year projects throughout Australia. The RANZCO Eye Foundation contributed $165,000 towards co-supporting four projects and Glaucoma Australia Inc. $50,000 to co-support two projects. This is by far the largest amount of funding the ORIA has distributed in one year, and most fitting for its 60th year.

We are most grateful to both organisations for their continuing support along with previous benefactors whose legacies are acknowledged through the naming of individual grants.

The ORIA continued funding a New Investigator category in an endeavour to encourage up and coming researchers; five grants were awarded this year.

Significant projects to receive funding from January 2013 were:

ORIA/W A QUINLIVAN & GLAUCOMA AUSTRALIA GRANT $50,000

Prof Glen Gole, Dr Nigel Barnett & Prof Steven Bottle Evaluating a novel (two-way, ie reversible) technique to measure oxidation (or reduction) in retinal cells in an experimental glaucoma model

ORIA/RANZCO EYE FOUNDATION GRANT $50,000

A/Prof Robyn Jamieson, A/Prof John Grigg & Ms Tina Lamey Using the latest techniques to find genetic causes of retinitis pigmentosa

ORIA/RANZCO EYE FOUNDATION GRANT $50,000

Dr Alice Pébay, A/Prof Ian Trounce & Dr Karina Needham Stem cells for modelling retinal diseases in a dish

ORIA/ESME ANDERSON GRANT $48,500

A/Prof Nick Di Girolamo Factors that support stem cells on the surface of the eye

Details of all other grants awarded can be found on the ORIA website www.oria.org.au and for New Zealand at www.safesightsociety.org.nz.

CHAIRMAN’S REPORT


The ORIA is always mindful of auditing its research funding to assess how well its mission to advance eye research is being achieved. Each year, progress/final reports are provided from researchers funded during the previous year. A financial statement from each project/institution is also secured to ensure funds have been used in the manner previously indicated in the application.

The ORIA organized an interesting segment at RANZCO last year presenting details of publications arising out of previous ORIA funding. The top papers in the last 5 years were presented by; Prof Jamie Craig: “Burdon, KP; Macgregor, S; Hewitt, AW et al. Genome-wide association study identifies susceptibility loci for open angle glaucoma at TMCO1 and CDKN2B-AS1. Nat Genet. 2011;43:574-578.”; Dr Alex Hewitt:

“Hewitt, AW; Sharma, S; Burdon KP, et al. Ancestral LOXL1 variants are associated with pseudoexfoliation in Caucasian Australians but with markedly lower penetrance than in Nordic people. Hum Mol Genet. 2008;17:710-716.”; Dr Nicole Van Bergen:

“Van Bergen, NJ; Wood, JPM; Chidlow, G, et al. Recharacterization of the RGC-5 Retinal Ganglion Cell Line. IOVS. 2009;50(9):4267-4272.”; Dr Xinyuan Zhang:

“Zhang, XY; Bao, SS ; Lai, DN; et al. MC. Intravitreal triamcinolone acetonide inhibits breakdown of the blood-retinal barrier through differential regulation of VEGF-A and its receptors in early diabetic rat retinas. DIABETES. 2008;57(4):1026-1033.

As this year is the 60th anniversary of ORIA we are taking an historical slant.We have searched our archives and come up with names of researchers we have supported from the 1953-1983 period, 47 in total and pulling out their post 1953 publications. At RANZCO we propose to describe the methodology above and show some historical pics and documents. We have chosen some contemporary researchers in the same field and location to the ORIA funded researcher to give a 2-3 min summary of the person, 2-3 min summary of their life work and then the remaining time to 10 minutes on the ORIA papers. It is planned to write up this presentation as a co-authored paper, with the speakers contributing a few paragraphs on their grantee to go to Clin Exp Ophthalmology.

The ORIA has posted a page on its website as a resource for New Scientists. This will be added to when relevant information comes to hand.

The ORIA has continued its support of the Australasian Ophthalmic and Visual Sciences Meeting (AOVSM). The meeting ran concurrently last year at RANZCO in Melbourne and it has been agreed to continue this format with this year’s RANZCO meeting in Hobart. Program details can be accessed via the RANZCO Congress website.

The ORIA also continues its annual support of the Ringland Anderson Chair of Ophthalmology in Victoria.

During the year, Prof Tien Wong decided to step down from the ORIA Board. We are most grateful for his commitment to the organization.

Prof Stuart Graham Chair, ORIA

ORIA Board 1957Back Row:- Hugh Ryan, Kelvin Lidgett, Ron Lowe, Bruce Hamilton, Bill Deane-Buther, Sir Norman GreggFront – Left-Right:- Walter Lockhart Gibson, John Pockling, Dame Ida Mann, Archie Anderson, Arthur Joyce, A. L. Fostevin


ORIA/W A QUINLIVAN & GLAUCOMA AUSTRALIA GRANT $50,000Prof Glen Gole, Dr Nigel Barnett & Prof Steven Bottle Evaluating a novel (two-way, ie reversible) technique to measure oxidation (or reduction) in retinal cells in an experimental glaucoma model

ORIA/RANZCO EYE FOUNDATION GRANT $50,000A/Prof Robyn Jamieson, A/Prof John Grigg & Ms Tina Lamey Using the latest techniques to find genetic causes of retinitis pigmentosa

ORIA/RANZCO EYE FOUNDATION GRANT $50,000Dr Alice Pébay, A/Prof Ian Trounce & Dr Karina Needham Stem cells for modelling retinal diseases in a dish

ORIA/ESME ANDERSON GRANT $48,500A/Prof Nick Di Girolamo Factors that support stem cells on the surface of the eye

ORIA NEW INVESTIGATOR GRANT $49,500Dr Matthew Rutar, Dr Trent Sandercoe & Dr Riccardo Natoli Delaying the Progress of Age-Related Macular Degeneration (AMD)

ORIA/RANZCO EYE FOUNDATION GRANT $43,550Dr Peter Madden, Dr Peter Beckingsale, A/Prof Damien Harkin & Prof Traian Chirila Growing cells on silk film to replace corneal grafting

ORIA/W A QUINLIVAN & GLAUCOMA AUSTRALIA GRANT $49,500Dr Alex Hewitt, Dr Mirella Dottori & Dr Bryony Nayagam Developing a patient-specific model for glaucoma

ORIA/ RENENSSON BEQUEST GRANT $49,500Prof K A Williams, Dr Helen M Brereton & Emeritus Prof Douglas J Coster Reducing Corneal Transplant Failure

ORIA/BRENDA MITCHELL GRANT $48,500Dr Vicki Chrysostomou & Dr Jelena Kezic The role of mitochondrial dysfunction in ocular Diabetic complications

ORIA GRANT $49,500Dr Kathryn Burdon & A/Prof Jamie Craig Characterisation of genes that lead to glaucoma blindness

ORIA NEW INVESTIGATOR GRANT $49,500Dr Vivek Gupta & Dr Yuyi You 7, 8-Dihydroxyflavone as a novel molecule to protect the retina in glaucoma

ORIA PROJECT FUNDING 2013


ORIA NEW INVESTIGATOR GRANT $49,500Dr Hitesh Peshavariya A new drug derived from natural substances to prevent vision loss

ORIA NEW INVESTIGATOR GRANT $48,000Dr Nicole Van Bergen Cellular energy production defects in glaucoma

ORIA GRANT $49,500Dr Guei-Sheung Liu, Prof Gregory Dusting, Dr Bang Bui, Prof Hong Zhang & Prof Ming-Hong Tai Gene delivery of a new drug for aberrant development of blood vessels

ORIA GRANT $48,000Prof J W McAvoy, A/Prof F J Lovicu and Dr L J Dawes Lens regeneration after cataract surgery

ORIA GRANT $48,500Prof Robert Casson Understanding the Warburg Effect in the Retina

ORIA NEW INVESTIGATOR GRANT $42,000Dr Fred Chen Comparative study of two automated microperimeters

ORIA/RANZCO EYE FOUNDATION INDIGENOUS EYE HEALTH GRANT $45,000Dr Stewart Lake, Dr Tim Henderson & A/Prof Henry Newland Population-based study of diabetic vitrectomy indications and outcomes in Indigenous Australians

TOTAl - $868,550


THANK YOU

DONATIONS

The Institute would like to thank our external referees who kindly gave advice which helped with the allocation of the 2013 grants.

Hema Arvind

Alberto Avolio

Tiziano Barberi

Rachel Barnes

Nigel Barnett

Karl Brown

Amanda Carr

Robert Casson

Elsie Chan

Glyn Chidlow

Colin Clement

Ian Constable

Max Conway

Tony Cook

Mark Daniell

Elfride De Baere

Greg Dusting

Rohan Essex

Paul Foster

Samantha Fraser-Bell

Marcus Fruttiger

Mark Gillies

Glen Gole

Colin Green

Vivek Gupta

Nuri Guven

Robyn Guymer

Paul Healey

Thiran Jayasundera

Vishal Jhanji

Kwon Kang

Ryo Kawasaki

Jane Khan

Sonja Klebe

May Lai

Ecosse Lamoureux

John Landers

Lyndell Lim

Ted Maddess

Michele Madigan

Keith Martin

Alessandra Martins

Ian McAllister

John McAvoy

Neville McBrien

Michel Michaelides

Justin Mora

Ian Morgan

Riccardo Natoli

Alice Pebay

Tunde Peto

Tony Poole

Luba Robman

Kathryn Rose

Ilva Rupenthal

Trent Sandercoe

Valerie Saw

Katrina Schmid

Shiwani Sharma

Weiyong Shen

Robyn Tapp

Angus Turner

Rasik Vajpayee

Krisztina Valter-Kocsi

Peter van Wijngaarden

Nitin Verma

Brendan Vote

Anthony Vugler

Jack Wall

Jie Win Wang

David Wechsler

Andrew White

Keryn Williams

Fulton Wong

John Wood

Dao-Yi Yu

Ehud Zamir

Meidong Zhu

Glaucoma Australia Inc

The RANZCO Eye Foundation

The Estate of Dr Grosvenor Williams

The Estate of Mrs Rosemary Jeanette Conn

RANZCO


dR sTewART lAke And dR muRRAy sTAnley wITh FRIends suPPORTIng The FIRsT ORIA/RAnZcO eye FOundATIOn IndIgenOus eye heAlTh gRAnT FOR dR sTewART lAke, dR TIm hendeRsOn & A/PROF henRy newlAnd POPulATIOn-bAsed sTudy OF dIAbeTIc vITRecTOmy IndIcATIOns And OuTcOmes In IndIgenOus AusTRAlIAns.


ORIA/RAnZcO eye FOundATIOn gRAnTDr Alex Hewitt & Dr Stuart McGregor

Methylation Profiles in Patients with Age-Related Macular Degeneration

April 2012 commemorated the centennial anniversary of the sinking of the Titanic. During the one hundred years since the maritime tragedy, advances in the field of molecular biology have seen the discovery of the structure of DNA and sequencing of the human genome. While the unveiling of our genetic blueprint was celebrated as a fundamental milestone in biology, it was acknowledged that it represents but the tip of the iceberg in our understanding of the molecular mechanisms of disease. In essence, publication of the human genome laid bare our biological building blocks, though we were yet to decipher the instruction manual for assembly.

In challenging the gene-centric paradigm that biology has been founded on, it is now generally appreciated that much of our genome can be assigned a regulatory role in moderating the expression levels of coding DNA. In this context the Ishihara test provides a good metaphor, whereby from a holistic viewpoint, a functional understanding of our genome (i.e. visualising the pattern on an Ishihara plate) requires an appreciation not only of the genes which compose it (target dots) but also the bulk of ‘non-gene’ DNA sequences (surrounding dots)(Figure 1).

In the human genome, DNA methylation occurs primarily on cytosines in ‘CpG’ dinucleotides that are found at a low density but may also be in regions enriched for CG dinucleotides, ‘CpG islands,’ which are present at ~60% of gene promoters. Isolated CG dinucleotides are frequently methylated, with dense methylation in promoter regions generally associated with transcriptional silencing. With the support from the ORIA we have developed bioinformatic tools for synthesis of epigenetic data and analysed the methylation profilies of ocular tissue and AMD samples.

With the increasing ability to generate large epigenetic and expression datasets tools for analyzing, interpreting and visualizing these data are of critical importance to researchers everywhere. We developed the The

Expression Methylation Correlator (EMC): a tool for integrating epigenetic and transcriptomic data. EMC is designed to provide a comprehensive, intuitive suite of tools for integrating related results. It can be used to readily annotate, filter and visualize results from differential methylation and expression datasets, in a user-friendly format. Annotation features and reference genomes are customizable, with all computations being performed in real time, even for datasets with millions of markers.

Given the degree of specificity in methylation profiles between different tissues we investigated the whole genome methylation profiles of ocular tissues. In comparing this profile to unfractionated blood samples from the same individuals, we also sought to investigate the potential utility of leucocyte DNA methylation in the study of ocular disease. Whole blood from the subclavian vein and whole eyes (N=8) were obtained post-mortem. DNA was extracted from whole blood as well as neurosensory retina, retinal pigment epithelium (RPE)/choroid and optic nerve tissue. Following bisulfite conversion samples were hybridized to Illumina Infinium HumanMethylation450 BeadChips. Unstructured hierarchical clustering of all CpG sites for each sample revealed well-defined groupings across individual tissue subtypes. Despite this discrete clustering, there was generally a strong correlation between methylation profiles, across all tissues from each individual (median (range) Spearman corr=0.923 (0.851-0.991)). Over 250,000 CpG sites were found to have similar methylation levels (beta <0.2 or beta >0.8) across different tissues in the same individuals, with a further ~18,000 sites having similar methylation profiles in all ocular tissue only. Our results reveal a strong correlation between the methylation status of peripheral blood leukocytes and different ocular tissues, highlighting the utility of using whole blood to study potential epigenetic changes in ophthalmic disease. These results are particularly encouraging for research where non-end organ tissue is difficult to obtain. An improved

PROGRESS REPORTS


understanding of the epigenetic landscape of ocular tissue will have important ramifications for regenerative medicine and ongoing dissection of gene-environment interactions in eye disease.

Figure 1:The role of non-coding DNA in defining cellular function. Analogous to an Ishihara plate, a gene-coding region of DNA (target dots) only comes into context when the non-coding DNA (surrounding dots) is appreciated.

Figure 2:Genome-wide CpG site inter-tissue and inter-sample relationships. A) Hierarchical clustergram across all samples. Individuals are represented by their corresponding code. B) Scatterplot of methylation profiles between ocular tissues and peripheral blood.


ORIA/w A QuInlIvAn & glAucOmA AusTRAlIA Inc gRAnTInvestigations into optic nerve injury in a rat model of glaucoma

Dr G Chidlow

Glaucoma is a progressive, intraocular pressure (IOP)-sensitive optic neuropathy with a poorly understood pathogenesis and limited treatment options. It affects 3% of the Australian population over 49, with the prevalence increasing exponentially thereafter and is the leading cause of irreversible blindness worldwide. We have recently demonstrated that the optic nerve head (ONH), the location where the optic nerve leaves the eye, is the primary site of injury following moderate elevation of ocular pressure in rats. We established that the earliest indication of damage was disruption to orthograde fast axonal transport within axons in the ONH, which subsequently manifested as Wallerian-like degeneration of injured axons. Somato-dendritic injury to the retinal ganglion cells (RGCs) occurred subsequent to axonal injury during experimental glaucoma. The current project sought to better understand the relationship between elevated IOP, axonal transport disruption and optic nerve degeneration.

IS THERE AN ASSOCIATION BETWEEN AxONAL TRANSPORT DISRUPTION AND HYPOxIA/ISCHEMIA?To date, relatively little is known about the molecular pathways involved in the loss of RGCs and their axons. Essentially, two theories have been proposed: “the mechanical theory” and “the vascular theory”. The former hypothesis contends that elevated IOP leads to physical kinking and distortion of axon bundles resulting in disrupted axoplasmic transport, while the latter hypothesis proposes that RGCs undergo hypoxic or ischemic injury as a result of compromised local blood flow, arising from either increased IOP or other risk factors. The principal objective of the current work was to identify whether cellular components in the ONH or retina express markers indicative of hypoxia/ischemia following induction of experimental glaucoma. We were particularly interested in whether any such markers are spatio-temporally co-ordinated with sites of axonal transport disruption.

To examine the distribution of any hypoxia during experimental glaucoma, we utilised hypoxyprobe-1 (pimonidazole), which forms covalent adducts with cells that have an oxygen partial pressure <10 mmHg. The method is appealing as hypoxic regions can be directly visualized in tissue sections. Our results showed that after 1-3 days of chronic ocular hypertension, axonal transport disruption, as visualised by immunolabelling

for APP, was evident in the prelaminar and laminar regions of the ONH (Fig. 1A). Hypoxia, as visualised by staining for pimonidazole, was also discretely localised to the ONH (Fig. 1B).

APP pimonidazole

haemoxygenase-1

Figure.1. Visualisation of axonal transport disruption (A), hypoxia (B) and haemoxygenase-1 expression (C) in the ONH of a rat subjected to 1 day of experimental glaucoma.

No obvious hypoxic staining was apparent within the retina or the myelinated portion of the optic nerve (ON). The abundance of pimonidazole within the ONH of rats was similar after 1, 2 and 3 days of experimental glaucoma, but was no longer evident by 1 week, a time point typically associated with a return to normal ocular pressure. Moreover, the extent of pimonidazole staining was related to the magnitude of IOP elevation, such that higher IOPs (30-40 mmHg), resulted in well-defined staining, while lower IOPs (20-30 mmHg) were associated with negligible hypoxia. These findings correlate well with the abundance of axonal transport disruption at the ONH, both spatially and temporally, suggesting that hypoxic episodes may well play a role in axonal dysfunction.

In order to further our knowledge of the biochemical effects of hypoxia at the ONH during experimental glaucoma, we investigated expression of hypoxia-inducible proteins, such as haemoxygenase-1 and NADPH oxidase. Our results showed overlapping patterns of pimonidazole and haemoxygenase-1 in the


ONH. Haemoxygenase-1 was expressed by astrocytes within the glaucomatous ONH. It was always more widespread than pimonidazole, but importantly was concentrated at the ONH rather than in the retina or myelinated optic nerve, and, was rarely observed in control retinas. It is clear that astrocytes are adversely affected during experimental glaucoma, but our results show that they remain viable: we observed no positive TUNEL labelling (indicative of programmed cell death) or expression of 8-hydroxy-2-deoxyguanosine (indicative of oxidative DNA damage) within astrocytes.

NADPH oxidase, which is responsible for generation of the reactive oxygen free radical superoxide, was expressed at a low level by microglia throughout the visual pathway. Following induction of experimental glaucoma, activated microglia upregulated the catalytic subunit of NADPH oxidase, gp91phox. This phenomenon was principally evident at the ONH. NADPH oxidase has been strongly implicated in oxidative stress-related injury in various neurodegenerative situations. Experiments are currently underway to examine whether blocking NADPH oxidase activity by administration of the inhibitor apocynin leads to reduced injury at the ONH. We are also making use of the rat Oxidative Stress and Antioxidant Defense PCR SuperArray, which allows the analysis of 84 genes related to oxidative stress by conventional real-time qPCR. By comparing results from control and glaucomatous rats, we will shortly be able to highlight which genes are most strongly affected by raised IOP,

and therefore, which genes might be most amenable to intervention from a therapeutic perspective. These results are presently being analysed. Overall, the combined results provide significant, but preliminary, support for the vascular theory of glaucoma.

Two secondary aims of the project were, firstly, to delineate whether short duration axonal transport disruption is reversible, and secondly, to ascertain whether a continuous period of elevated IOP-induced axonal transport disruption results in greater damage than two discrete periods of elevated IOP. These aims will shed light on what duration of axonal transport perturbation can be tolerated by axons before irreversible injury ensues, and, whether axons that survive a defined period of chronic ocular hypertension necessarily continue to survive if the IOP is raised, for the same duration, for a second time. This is an important issue as sporadic patient non-adherence to glaucoma medication regimes is considered a major risk factor for disease progression. With regard to the first of these aims, our preliminary results suggest that if IOP is raised to a pressure of 40mm Hg for as little as 24 hours, then irreversible damage occurs to a subset of RGC axons, irrespective of whether the IOP is then lowered to a normal level. Analysis of results from additional experiments is currently underway, while experiments are ongoing for the second objective, the results of which will be available shortly.


ORIA/wA QuInlIvAn & glAucOmA AusTRAlIA gRAnTInvestigation of methylation status at the 9p21 glaucoma susceptibility locus between glaucoma cases and controls

Prof Jamie Craig and Dr David Dimasi

We recently discovered association of genetic variants on chromosome 9p21 with primary open-angle glaucoma (POAG) in a genome-wide association study. This association has since been found other cohorts from all over the world and it is clear that this genomic region is important in the development of POAG. Interestingly, the association is stronger in patients with normal tension glaucoma, suggesting that this locus contributes to glaucoma pathogenesis independently of elevated intraocular pressure, which is a common feature of POAG. There are multiple genes in the associated genomic region. CDKN2B-AS1 encodes a non-coding RNA, also known as CDKN2BAS or ANRIL, which regulates neighbouring genes at 9p21. The 9p21 region also harbours the tumour suppressor genes CDKN2A and CDKN2B (cyclin dependent kinase inhibitor 2A and 2B). The CDKN2A gene encodes two proteins, CDKN2A and ARF.

It is not clear which of these genes is ultimately responsible for increased POAG susceptibility or if a combination of factors is at play. Our group is actively investigating this locus from a variety of angles to explore gene function specifically in the eye and to link genetic variation to functional effects. In particular we hypothesise that genetic variation in this region controls expression of the nearby genes.

One mechanism of controlling gene expression is through methylation of DNA in “CpG islands”. This chemical modification of the DNA at CG dinucleotides typically results in suppression of gene expression. The normal function of the genes in this region is to control cell cycle. Thus when expression is dysregulated, the cell receives inappropriate control of cell division, which can result in either cellular proliferation (cancer) or cell death (apoptosis) depending on the cell type and context. POAG is characterised by the apoptosis of retinal ganglion cells. As the 9p21 locus appears to act independently of intraocular pressure, we thus hypothesise that genetic variation at this locus increases the tendency for retinal ganglion cells to undergo apoptosis and specifically that this may be in part controlled by methylation of the CpG islands controlling expression of these cell cycle related genes.

To test this hypothesis we have assessed the methylation of the CpG island upstream of CDKN2B in patients with advanced normal tension POAG and age and gender matched controls. We have found that several CG dinucleotides are under methylated in

normal tension POAG patients compared to controls. These dinucleotides are located in a cluster at one end of the CpG island (see Figure 1). We have also identified a second region at the other end of the island that appears to be more methylated in cases compared to controls, and interestingly, the methylation level in this region appears to be correlated with the nearby genetic variation that is associated with POAG. Individuals carrying 2 copies of the POAG risk alleles in this region have higher methylation levels than those with no copies. This suggests that the genetic variation at this locus may be involved in glaucoma in part through affecting methylation of this region. We have also seen a striking effect of gender, in that the association of methylation level with glaucoma is much more profound in females and is almost non-existent in males.

Taken together, these data indicate that methylation of this particular CpG island is likely to be involved in determining susceptibility to POAG. We are now expanding this work to look at this in a larger cohort and also in patients with high tension POAG which is more common. We are also exploring methylation at the CpG island that is thought to control expression of both CDKN2B-AS1 and ARF.

Figure 1:Schematic showing the CDKN2B gene and the location of the CpG island thought to control expression of the gene and the position of a genetic variant (rs1061392) associated with POAG. The relative locations of the differentially methylated CG dinucleotides are indicated. Not to scale.


ORIA/RAnZcO eye FOundATIOn gRAnTTitle: Axonal Regeneration In Experimental Glaucoma

Dr John P. M. Wood

ORIGINAL HYPOTHESIS AND AIMSLoss of retinal ganglion cells (RGCs) and their axons underlies the pathogenic tissue events occurring in glaucoma. We proposed to build on current strategies, which attempt to minimise or decelerate retinal damaging processes in diagnosed patients, by using a combination of cell culture and animal models to investigate the stimulation of regrowth of damaged cell processes which could potentially restore vision to glaucoma patients.

We have, in our lab, an established experimental rodent paradigm of glaucoma. We hypothesised that in our model, surviving, but compromised, RGCs possessed the ability to regenerate their damaged axons. The aim of the project, therefore, was to explore the potential for long-distance regeneration of axons in our model of experimental glaucoma. Specific aims were to compare the regenerative capacity of some previously-described treatment strategies for axonal regeneration in our model of experimental glaucoma with a model of optic nerve crush (1). Furthermore, exciting data which we had found in preliminary studies, indicated that the cytokine, IL-6, was able to play a role in the regenerative process of damaged RGCs; we therefore aimed to characterise the role of this factor in our models of retinal damage (2).

ExPERIMENTAL PROGRESS FOR AIM 1In this part of the project we aimed to compare the axonal regenerative potential of some previously published regimen in both our classical model of RGC axon injury (optic nerve crush) and our glaucoma model. In order to validate our systems and our methods for the analysis of axonal regeneration we used a procedure termed “lens injury”, whereby 3 days subsequent to induction of each injury model we used a 30G needle to puncture the lens. This induces a macrophage response known to stimulate RGCs to regrow damaged axons through the site of, for example, optic nerve crush injury at the nerve head. We have now shown, using the lens injury paradigm, that we have succeeded in reproducibly enhancing axonal regeneration in animals subjected to nerve crush. Excitingly, we have also shown that lens injury can also induce axonal regrowth through the damaged area of the optic nerve head in our experimental glaucoma model. Our latest studies are investigating the effect of the proposed treatments (Zymosan A, CNTF plus CPT-cAMP, the Rho/Rho kinase inhibitor Y-27632) in our models. We are also investigating the potential for the

immunomodulatory glycoprotein, osteopontin, in which we have a long-standing interest, and which we have shown to promote RGC axonal regeneration in culture, to cause axonal regrowth in vivo.

ExPERIMENTAL PROGRESS FOR AIM 2In preliminary investigations we had determined that the cytokine, IL-6, was elevated in the retina subsequent to experimentally induced glaucoma. Upon further investigation, we confirmed that this was indeed the case and also that this elevation was not a generalised cytokine response, since two other known retinal neuromodulatory cytokine factors, IL-1β and TNFα were not affected in this manner. Using detailed immunohistochemistry we then identified that IL-6 was synthesised by injured RGCs in our experimental glaucoma model, being specifically localised to damaged axons and being transported orthogradely, accumulating at the site of axonal damage itself. These findings from our glaucoma model were replicated in optic nerve infarct sites in two other, non-vascular paradigms of RGC axonal damage. These data prove that the production of IL-6 is a general feature of RGCs subjected to axonal damage, rather than a facet of decreased vascular perfusion specifically resultant from our experimental glaucoma model.

Figure 1:

Increased IL-6 expression within damaged axons in the optic nerve head region as demonstrated by the brown labelling (ON, optic nerve; NFL, GCL, INL, ONL, retinal layers).

We found that the induced IL-6 expression was co-localised with the axonally-transported regenerative protein, growth associated membrane phosphoprotein-43 (GAP-43), in a large proportion of


regenerating axons. These data implied that IL-6 may itself play a role in the early regenerative response of damaged axons to injury. To test this hypothesis we produced cultures of rat RGCs, which were grown under normal conditions with or without added exogenous CNTF, a factor which acts as an exogenous initiator and propagator of GC axons in vitro. The IL-6 clearly and reproducibly enhanced axon branching and outgrowth rates in the presence and absence of additional CNTF.

Figure 2 (below): Stimulation of neurite outgrowth and branching of cultured adult rat RGCs by IL-6.

We are currently investigating the effect of IL-6 application to the eye before induction of either experimental glaucoma or optic nerve crush as we believe that the exciting data outlined above mean that this cytokine will be able to promote RGC survival and regeneration of damaged axons.

REFERENCES PERTAINING TO THE STUDYChidlow G, Wood JPM, Ebneter A, Casson RJ (2012). IL-6 is an efficacious marker of axonal transport disruption during experimental glaucoma and stimulates neuritogenesis in cultured retinal ganglion cells. Neurobiol Dis 48:568-581.

Wood JPM, Mammone T, Chidlow G, Greenwell T, Casson RJ (2012). Mitochondrial inhibition in rat retinal cell cultures as a model of metabolic compromise: Mechanisms of injury and neuroprotection. Invest Ophthalmol Vis Sci 53:4897-4909.

Dr John Wood, Ophthalmic Research Laboratories, Hanson Institute, Adelaide Hospital, received funding through the ORIA in 2012 for Axonal Regeneration in Experimental Glaucoma.


ORIA/esme AndeRsOn gRAnTDr Shiwani Sharma, Dr Kathryn Burdon & Prof Jozef Gécz

Identification of the genetic causes of congenital cataract using massively parallel DNA sequencing

OVERVIEW

Congenital cataract is the leading cause of childhood blindness in the world. Early diagnosis and successful treatment can result in good visual outcomes. Our overall objective is to identify the genetic causes of inherited congenital cataract in affected families. The aim of this project is to identify causative genes in congenital cataract families by employing the “next-generation” DNA sequencing methodology.

BACKGROUND Cataract, opacity of the ocular lens that impairs vision, is common among the elderly (age-related cataract) but rarely also occurs in babies and children (congenital cataract). Twenty-five to 50% of congenital cataracts are inherited. Inherited congenital cataract can occur in isolation (isolated congenital cataract) or in association with other systemic features (syndromic congenital cataract). It displays autosomal dominant, autosomal recessive or X-linked modes of inheritance. High penetrance and monogenic inheritance of the disease facilitates causative gene identification using genetic approaches. More than 25 genes with diverse functions have been reported to cause isolated congenital cataract. Causative genes for many syndromic congenital cataracts have been also described. Additionally, in several families with isolated and some with syndromic congenital cataract, the disease has been mapped to chromosomal regions, but the causative genes have not yet been identified. This suggests the involvement of additional genes in congenital cataract.

As inherited congenital cataract is phenotypically and genotypically a heterogeneous disease, this poses a challenge in accurately predicting the genetic cause based solely on clinical appearance of the cataract. Our collaborative group has a large repository of congenital cataract cases from South-Eastern Australia. The cause of the disease in many families in this repository is not known. Although the conventional genetic approach has led to the identification of the known congenital cataract causing genes, it is laborious and time-consuming as only a few genes in the disease-linked genomic region can be evaluated for causative mutations at a time. The latest sequencing technique, “next-generation” DNA sequencing, however allows evaluation of all the genes in the genome at the same time; thus it is a more cost-effective and efficient approach for causative gene identification. Thus, we employed this approach in this project.

PROGRESS TO DATE We performed next-generation DNA sequencing in three congenital cataract families in our repository for identification of the causative genes. The coding exons, exon-intron boundaries and 5’ and 3’ UTRs of all the genes (exome) in the human genome were sequenced in selected individuals in these families using the Illumina TrueSeq exome capture kit and Illumina HiSeq2000 sequencing platform.

Family CSA69: In this Australian family of Caucasian-Aboriginal descent, congenital cataract is inherited as an autosomal dominant trait and occurs in association with mental retardation, skeletal, skin, and hair defects. The disease in this family was previously mapped to human chromosome 1 at 1p35.3-p36.32 (Hattersley et al., 2010. BMC Med Genet 11: 165). We performed whole exome sequencing on genomic DNA from two affected members of the family. Upon data analysis, seven novel single nucleotide variants in the protein coding regions of six genes (ACOT7, FBXO42, NBPF1, HSPG2, SEPN1 and YTHDF2) in the critical region were shared between these individuals. All these variants were verified by Sanger sequencing; they would lead to missense changes in the respective proteins. Of these, three variants, c.A769G (R257W) in HSPG2, c.G1352A (V451A) in FBXO42, and c.T910C (P305S) in YTHDF2, segregated with the disease in the family. The R257W substitution in HSPG2 is not conserved among mammals and thus was considered less likely to be pathogenic. The amino acid substitution V451A in FBXO42 and P305S in YTHDF2 are highly conserved across species and were predicted to be pathogenic by the online prediction tools, SIFT and PolyPhen2. The corresponding DNA changes were absent in, respectively, 269 and 267 unaffected and unrelated Caucasian controls. However, these changes were present in, respectively, 2 and 7 out of 64 unaffected and unrelated Aboriginal controls. Therefore, they are unlikely to be disease causing. Messenger RNA expression analysis revealed expression of both the FBXO42, and YTHDF2 genes in human lens, retina, brain, skin and hair follicle and in developing mouse embryo, supporting their roles in these tissues and during development. This is consistent with the organs affected in the genetic disorder in Family CSA69. However, these genes are unlikely to be causative in this family. Further, we are investigating an additional six novel variants in non-protein coding regions of the genes in the critical region to determine if they are disease-causing.


Family CSA92: This Caucasian family has isolated congenital cataract with varying disease severity; the disease is thought to be inherited in an autosomal dominant fashion. We performed whole exome sequencing in five members of the family - three affected, one unaffected and one with possibly very mild disease. Several novel nucleotide variants in multiple genes in the genome are shared among the three affected individuals. These variants are being prioritised for validation by Sanger sequencing. The validated variants will be assessed for segregation with the disease.

Family CSA100: Isolated congenital cataract in this Caucasian family is inherited as a recessive trait. Thus we hypothesise the presence of homozygous recessive or compound heterozygous disease-causing mutations in this family. We performed whole exome sequencing in two affected individuals. After data analysis, one novel candidate single nucleotide variant in the ANKRD20A1 gene on chromosome 9 was found in both the affected individuals. In addition, two heterozygous variants, one each in PABPC1 and ESRRA genes, respectively,

on chromosome 8 and 11 were shared by the affected individuals and were candidate compound heterozygous changes. However, none of these variants were found to validate by Sanger sequencing and thus were not pursued further. Furthermore, two rare heterozygous variants one each in PLXNA2 and CXCR7 genes validated by Sanger sequencing. However, neither was found to segregate with the disease in the family. Linkage analysis is being performed in this family to map the chromosomal region linked with the disease. Exome sequencing data in the disease-linked region will be analysed to identify the disease causing gene.

RESEARCH TRAINING ACHIEVED 1.A Masters student pursued a part of this research

project and successfully completed her degree in 2012.

2.A PhD student has been pursuing parts of this research; her research training is continuing.

We are always grateful to our peer reviewers. As a token of our appreciation, Dr Kathryn Burdon from Flinders Medical Centre is presenting Dr Glyn Chidlow from the Hanson Institute, Royal Adelaide Hospital with a bottle of Grange for his assistance with the 2013 round of funding applications.


ORIA/RAnZcO eye FOundATIOn gRAnTA Prospective cohort study evaluating the predictors of outcome in macular hole surgery

Dr Rohan Essex, Dr Willie Campbell, Dr Alex Hunyor Jr & Dr Paul Connell

1. TO EVALUATE THE PREDICTORS OF OUTCOME IN MACULAR HOLE SURGERY.This aspect of the project focuses on OCT-based features, and their correlation with clinical outcome. A post-graduate student was engaged, and she has taken it on as part of a PhD. There are other aspects to her PhD, and this has pushed back the timeline on this aspect of the project by approximately 6 months.

Thus far a complete single surgeon cohort has been analysed, and the results were presented at ARVO, 2013, Seattle. The meeting poster is attached. OCT scans have been collected from one other surgeon (Alex Hunyor Jr.), and this is presently underway for a third (Tony Kwan). The plan is to validate the observations found in the single surgeon series.

The results of the single surgeon series are presently being prepared for publication, and the multi surgeon series will be written up also. A third paper is to be produced which involves re-grading all the OCT images using a different grader to assess inter-user variability of our OCT grading.

There is also a clinical component to this project. We have thus far gathered a cohort of 1,500 macular hole procedures and their outcomes. The target is approximately 2000, at which time we will have sufficient power to determine whether face-down posturing influences macular hole surgery outcomes. Recruitment has slowed over the last 2 years (from 400/year to less than 200), however the launch of the on-line data collection system (see below) should improve this. Publication of any results for this part of the project is therefore delayed.

2. TO ESTABLISH AND EVALUATE A TABLET COMPUTER-BASED DATA ENTRY SYSTEM FOR SURGEONS TO USE AT THE TIME OF SURGERY.This aspect of the project has evolved well, and the fully functional web-based data entry system was launched at the ANZSRS mid-year meeting on June 1st and 2nd. Screen shots of the “front page”, and of the data entry screen for macular hole are shown as an attachment.

There is presently a consultation process underway for the members of the ANZSRS to determine the best dataset for retinal detachment surgery, and this will shortly be incorporated into the on-line system too. The next task will be epiretinal membrane surgery. No further surgical procedures are planned for this audit presently.


ORIA/w A QuInlIvAn & glAucOmA AusTRAlIA gRAnTDoes loss of the ‘good’ Alzheimer’s protein in older eyes contribute to glaucoma?

A/Prof Ian Trounce

SUMMARY: BACKGROUND AND AIMSThere is a growing recognition that similar neurodegenerative processes may be at play in both Alzheimer’s disease and glaucoma. We believe that mitochondrial oxidative stress may underlie both diseases, and have developed surprising preliminary data showing that the key Alzheimer’s protein, the amyloid precursor protein (APP), may protect neurons from mitochondrial oxidative stress.

Mitochondrial oxidative phosphorylation (OXPHOS) complex I impairment is an established cause of some genetic optic neuropathies, including Leber’s Hereditary Optic Neuropathy (LHON). As such, the complex I-specific inhibitor rotenone has been used to model optic neuropathy in rodents. We have found that full-length soluble amyloid precursor protein alpha (sAPPα) protects cultured neurons from rotenone toxicity. Further, we demonstrate that this protection is mediated, in vitro, by a receptor-driven stimulation of the PI3K-AKT pathway.

In work recently completed from a previous ORIA grant, we have established a significant deficiency of OXPHOS complex I in lymphoblast cell lines from primary open angle glaucoma subjects compared with age-matched controls (Lee et al IOVS 2012). This establishes for the first time that primary mitochondrial defects may contribute to the disease, and invites exploration of whether APP-mediated protection may be a new therapeutic target for glaucoma neuroprotection. We have confirmed that APP is highly expressed in mouse and human retinal ganglion cells (RGCs). We have also made a novel finding that sAPPα is secreted into the vitreous fluid at high levels. This suggests that APP may be an important neurotrophic factor protecting retinal neurons from mitochondrial oxidative stress, and casts new light on potential protective functions of the vitreous fluid. These results argue that sAPPα is a neuroprotective factor against mitochondrial complex I-linked oxidative stress. The proposed experiments aimed to establish whether there is an age-related decline in retinal APP in human eyes, and secondly in experimental glaucoma in mice. Demonstration of a loss of APP protective function in these settings would establish the PI3K-AKT pathway as a novel therapeutic target for glaucoma neuroprotection.

HYPOTHESIS: A loss of sAPPα contributes to age-related vulnerability of retinal ganglion cells to mitochondrial oxidative stress in glaucoma

THE SPECIFIC AIMS OF THIS STUDY WERE:a) Determine retinal and vitreal levels of APP and it’s cleavage products in human ageing

b) Compare ageing changes of APP metabolism in healthy and glaucomatous eyes

Determine retinal and vitreal APP levels and metabolism in experimental glaucoma in 3 and 18 month-old C57Bl/6 mice.

RESULTS:Aim 1: A total of 83 human retinal samples, and 41 vitreous samples were analysed. Table 1 shows the ages and gender of the donors. A wide age range allowed the planned examination of APP and sAPPα levels with ageing in the human eye. Patient characteristics of the donors are shown in Table 1 below, and results of western blot quantitation of APP and sAPPα are shown in Figure 1.

Table 1. Characteristics of donor retina and vitreous. Retina and vitreous were collected from eyes donated to research through the Lions Eye Donation Service (LEDS). The number, sex and age of donors are shown both as a whole group and broken down into ageing categories analysed. These categories were under 40 years of age (<40), between 40 and 70 years of age (40-70) and above 70 years of age (>70). Sex is presented as number and percentage of female donors within each category while age is presented as mean ± SD years.

Sample type

Subset N Sex, female

N (%) Age (years)

Retina Total 83 38 (45.8) 59.2 ± 18.9, Range 5- 91

<40 years 16 7 (43.8) 27.9 ± 10.6

40-70 years 37 18 (48.6) 58.8 ± 8.2

>70 years 30 13 (43.3) 76.4 ± 4.9

Vitreous Total 41 18 (43.9) 62.4 ± 16.5, Range 22-91

<40 years 6 2 (33.3) 32 ± 5.3

40-70 years 18 8 (44.4) 59.2 ± 7.4

>70 years 17 8 (47.1) 76.6 ± 6.2


Figure 1. APP metabolism in the ageing human retina. (A) Total APP decreases with ageing in the retina while (B) levels of sAPPα remain unchanged. (C) A proportional increase in alpha cleavage products is demonstrated when the steady state of detected sAPPα is normalised to the decreasing amount of total APP. (D) Representative immunoblots are shown for each antibody at each age group. Immunoblot membranes were probed with 22C11 antibody (Millipore) to detect total APP or sAPPα antibody (IBL) specific for sAPPα. Values presented are mean ± SD units. (*, p<0.05, Student’s t-Test).

Figure 2. Soluble APPα levels do not change with ageing. In this series of vitreous samples (N=41) there was no difference in sAPPα levels with ageing. Representative immunoblots are shown for each age group. Membranes were probed with WO2 antibody (Millipore) to detect sAPPα. Values presented are mean ± SD.

Figure 1 shows that we found that total APP levels did in fact decrease significantly with normal human ageing, while the levels of sAPPα were not changed. When expressed as a ratio of sAPPα/total APP, there was an increase in the relative level of sAPPα in older eyes

(Figure 1c) suggesting an increased non-amyloidogenic cleavage of APP to the neuroprotective sAPPα with age. Figure 2 shows that in the vitreous there was no change in levels of sAPPα with age.

As planned, we also examined APP levels in a small number of glaucoma eyes donated to the Lions Eye Bank. Figure 3 shows that a trend for increased APP, and decreased sAPPα was evident, although with the small number of glaucoma samples (N=4) this did not reach statistical significance. While showing that no major change in APP metabolism was evident in glaucomatous eyes, this does suggest that further work is warranted to see whether sAPPα levels decrease in glaucoma. It is interesting that the control series showed a relative increase of sAPPα/APP (see Figure 1c), while this small set of glaucoma samples shows the opposite trend.

Figure 3. APP and sAPPα in glaucomatous retina. APP and sAPPα levels were unchanged in glaucomatous retinas (N=4) compared to age matched controls (N=28). Immunoblot membranes were probed with 22C11 antibody (Millipore) to detect total APP or sAPPα antibody (IBL) specific for sAPPα. Values presented are mean ± SD units.

AIM 2:We intended to examine APP metabolism in the mouse eye using a chronic increased ocular pressure model induced by injection of microbeads into the anterior chamber. Despite repeated attempts we were unable to achieve sustained increases in ocular pressure using this method. We therefore chose to use another model of retinal ganglion cell stress directly related to mitochondrial impairment. This involved injection of the oxidative phosphorylation complex I inhibitor rotenone into the vitreous, which had previously been shown to induce death of retinal ganglion cells and some inner nuclear layer cells. The advantage of using this model here is that our earlier work had demonstrated that sAPPα was protective against rotenone insult of cultured neurons. Complex I impairment is a well-established cause of retinal ganglion cell loss in mitochondrial optic neuropathies such as Leber’s Hereditary Optic Neuropathy, and as mentioned above we have also established a defect in this enzyme complex in some glaucoma patients. Figure 4 shows the results of these experiments, where some mice


were co-injected with recombinant human sAPPα along with rotenone. Remarkably we found that exogenous sAPPα was indeed protective against rotenone-induced retinal ganglion cell and inner nuclear cell loss.

Figure 4.

Apoptosis in the C57BL/6J mouse retina after rotenone toxicity and sAPPα rescue. Apoptosis was quantified by counting the number of TUNEL+ cells in both the RGC and INL of the mouse retina. There were significantly more TUNEL+ cells in the rotenone treated retinas in both the (A) GCL and (B) INL. This was significantly reduced in the rotenone treated eyes rescued with sAPPα where there was also no difference between treated and vehicle eyes. GCL, ganglion cell layer; INL, inner nuclear layer. Entire retinas were counted (where possible) and in either case counts were normalised to the length (mm) of retina analysed. For each group, counts were obtained from a minimum of 2 retinal sections separated by >50 µM retinal depth. N=5 per group. (*, p<0.05, **, p<0.001, Student’s t-Test). Data is presented as mean ± SD.

In summary, the project has exceeded our expectations by showing for the first time that exogenous sAPPα can provide significant protection from mitochondrial inhibition by rotenone. We are now preparing two manuscripts detailing this work, one describing the human APP ageing study, and the second the in vivo protection. We expect these papers to generate considerable interest in further elucidating the cellular pathways of this protection to define new therapeutic targets for glaucoma and possibly mitochondrial optic neuropathies. The abundance of APP in the retina, and sAPPα in the vitreous, is a major finding that suggests this protein to be an important neurotrophic factor in the retina. We speculate that increased levels of amyloid beta as detected in human glaucoma eyes is merely a marker of increased APP metabolism, as indeed it may be in Alzheimer’s disease, rather than a pathogenic player.

MANUSCRIPTS IN PREPARATION THAT INCLUDE ASPECTS OF THIS ORIA-FUNDED WORK:Amyloid precursor protein in the human retina and vitreous with aging Hayley S. Waugh, Vicki Chrysostomou, Jonathan G.Crowston, Ian A. Trounce

Soluble amyloid precursor protein alpha protects inner retinal neurons against rotenone induced cell death Hayley S. Waugh, Vicki Chrysostomou, James Duce, Jonathan G. Crowston, Ian A. Trounce

PRESENTATIONSAustralian Neuroscience Society annual conference, Gold Coast, January 2012: Poster presentation

International Society for Eye Research biennial conference, Berlin, Germany, July 2012: Poster presentation

AussieMit biennial conference, Melbourne, December 2012: abstract accepted for poster presentation

duRIng The lAsT Ten yeARs, The ORIA, TOgeTheR wITh glAucOmA AusTRAlIA, hAs PROvIded OveR $1.35 mIllIOn TOwARds suPPORTIng ReseARch InTO glAucOmA.


ORIA/g j wIllIAms gRAnTIntravitreal Tenecteplase (metalyse) (TNK) for acute management of retinal vein occlusions.

Prof Ian McAllister, Assoc Prof Sarojini Vijayasekaran, Professor Mariapia Degli-Esposti and Paula Yu

Treatments for RVOs continue to evolve with recent trials showing some success of intravitreal agents in resolving the macular oedema associated with this condition at least in the short-term. Agents to address the occlusion to venous outflow which is a thrombus in branch retinal vein occlusion (BRVO) such as Tissue Plasminogen Activator (tPA) have been tried without success. We have previously proven the third generation thrombolytic TNK to be non-toxic to the retina and able to penetrate the retina (unlike tPA) to potentially reach the intravascular space. The aim of this study was to investigate if intravitreal TNK may have a role in the acute management of retinal vein occlusions by effecting a rapid resolution of the occluding thrombus and if it can penetrate the retinal vein with and without a thrombus and is non-toxic to the retina in a pig model.

In group 1pigs, photo thrombotic branch retinal vein occlusions were created in one eye. Immediately after creation of a venous occlusion, 100 µg of fluorescence conjugated TNK (custom synthesis by Invitrogen Eugene, OR) per 100µl saline intravitreal injections was delivered to both the occluded and non occluded eye. At 24 hours, each eye was enucleated, fixed overnight in 4% paraformaldehyde cut and viewed on a fluorescence microscope to confirm penetration of TNK into the lumen of the retinal veins.

In group 2 pigs, a BRVO was created in both eyes. A week after creation of the occlusion, TNK (without conjugation) 100 µg per 100µl saline was injected into one eye, the fellow eye was used as control (no injection). One week later the animals were sacrificed and the eyes enucleated. The area of the lasered site and an area away from the burn were dissected and processed in epoxy resin, stained and light and/or transmission electron microscopy performed. The percentage blockage, clot volume, extent of fibrinolysis and toxicity of TNK was assessed.

TNK penetrated the veins in both eyes, with more intense staining in the eyes with the occlusion. Thrombolysis was significant in the eyes injected with TNK (P=0.03) in which blockage was seen in all untreated eye and one treated eye. Clot volume was significantly higher in untreated eyes (P=0.028). Percentage blockage varied from 8.5% to 83.9%. Damage by TNK to the neural retina was not seen.

It was concluded that TNK penetrated the retinal veins with and without an occlusion, effected lysis of BRVO and did not cause damage to the retinal tissue.

Intravitreal TNK may be useful as an acute treatment for RVOs of recent onset.

Figure 1 (A): Penetration of fluorescence conjugated TNK in to the

retinal vein.

(B): Photothrombotic branch retinal vein occlusion.

(C): Photothrombotic branch retinal vein occlusion after treatment with TNK.

This study has now been completed. A manuscript (Intravitreal Tenecteplase (metalyse) (TNK) for acute management of retinal vein occlusions) has been accepted for publication in Investigative Ophthalmology and Visual Science.


ORIA/RenenssOn beQuesT gRAnT Target Müller cell dysfunction for treatment of retinal diseases

Drs Weiyong Shen and Dr Ling Zhu

The role of the Müller cell, a specialized glial cell that serves numerous functions essential to retinal homeostasis, in the pathogenesis of retinal diseases is very poorly understood. Through the extensive arborisation of their processes, Müller cells provide nutritional and regulatory support to both neurons and vascular cells. They play a central role in retinal glucose metabolism, are intimately connected with cone photoreceptorsand involved in the formation and maintenance of the blood–retinal barrier (BRB). Therefore Müller cells are both a target and a potential key player in retinal diseases such as Diabetic Retinopathy and Macular Telangiectasia Type 2.1-4 We hypothesise that Müller cell dysfunction is a major contributor to neuronal injury and BRB breakdown in retinal diseases.

We have recently generated an inducible transgenic model of selective ablation of Müller cells using a Müller cell-specific promoter to drive the expression of an attenuated form of diphtheria toxin fragment A (DTA176), which results in selective disruption of Müller cells followed by retinal neuronal damage, BRB breakdown and deep retinal neovascularisation. Using the specialised experience of the investigators in modelling retinal diseases, this study aimed to test strategies for treating the consequences of Müller cell dysfunction: neural apoptosis, BRB breakdown and angiogenesis.

1. EFFECTS OF INTRAVITREAL INJECTION OF RECOMBINANT CILIARY NEUTROPHIC FACTOR (CNTF) ON PHOTORECEPTOR INJURY AND BRB BREAKDOWNSince Müller cells are a significant source of neurotrophic factors,5-7 we reasoned that Müller cell ablation would cause photoreceptor apoptosis as a result of the loss of support from trophic factors. We tested this hypothesis by performing intravitreal injection of recombinant CNTF. Intravitreal CNTF had no effect on wild type retinas (Fig. 1, A and B) but significantly reduced both the area of cone photoreceptor outer segment loss and the number of apoptotic cells in the outer nuclear layer between 7 and 10 days after tamoxifen (TMX)-induced Muller cell ablation (Fig. 1, C-F) in transgenic mice. We further examined whether CNTF treatment also inhibited BRB breakdown. Quantitative measurement of retinal permeability to FITC-dextran and fundus fluorescein angiography showed that CNTF treatment had no effect on BRB breakdown when administrated either soon after Müller

cell ablation (4 days after TMX, Fig. 1, 8G), or later after lesions of focal intense vascular leak had developed (4 months after TMX, Fig. 1, 8H).

Figure 1: Effects of intravitreal injection of CNTF on photoreceptor injury (A-F) and BRB breakdown (G and H). A-D, CNTF (0.5µg) was injected 4 days (4d) after TMX treatment in one eye and the contralateral eye received balanced salt solution (BSS) containing the same amount of bovine serum albumin in each mouse. Peanut agglutinin (PNA) staining was performed 6d later. E, Quantitative analysis of PNA-stained retinal wholemounts. *P<0.01, CNTF vs BSS in TG mice. F, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) for photoreceptor apoptosis. CNTF or BSS was injected 1d after TMX treatment and TUNEL was performed 6d after intravitreal injection. †P<0.05, CNTF vs BSS. G, Quantitative analysis of retinal permeability to fluorescence labelled dextran. CNTF was injected 4d after TMX treatment and permeability was measured 7d later. *P<0.01, WT vs TG. H, Effects of CNTF on established retinal vascular leakage. Fluorescein angiography (FA) was performed to confirm the development of intense focal vascular leakage 4 months (4m) after Müller cell ablation and CNTF was injected 1 week later. Retinal vascular leakage was monitored by FA at 5d and 12d after CNTF injection. Scale bars: 100µm (A-D).


2. IMBALANCE BETWEEN VEGF-A AND PEDF ExPRESSION AFTER MüLLER CELL ABLATIONSince an imbalance between angiogenic and angiostatic factors is associated with BRB breakdown and retinal angiogenesis, we tested whether altered expression of VEGF-A and PEDF occurred after TMX induced Müller cell ablation. Upregulation of VEGF-A was observed as early as 7d after TMX treatment and persisted at 4m but significant reduction in PEDF expression was only detected 4m after TMX when lesions of intense focal vascular leakage had developed (Fig. 2, A and B). IHC showed persistent upregulation of VEGF-A around deep but not superficial retinal vessels 7d and 4m after TMX treatment (Fig. 2, C), which was accompanied by reduction of PEDF expression in areas of Müller cell ablation (Fig. 2, D). Double label IHC indicated that the increased expression of VEGF-A around deep retinal vessels was more likely from pericytes than from vascular endothelial cells (Fig. 2, E and F).

Figure 2 (below)Imbalanced expression of vascular endothelial growth factor A (VEGF-A) and pigment epithelium derived factor (PEDF) after Müller cell ablation. A, Western blot showed upregulation

of VEGF-A while the expression of PEDF remained relatively unchanged 7 days (7d) after Müller cell ablation. *P<0.01, TG vs WT, n=8 in each group. B, By 4 months (4m) after Müller cell ablation, western blot revealed significant upregulation of VEGF-A and down-regulation of PEDF. †P<0.05, TG vs WT controls, n=7~8 in each group. TG mice showed persistent upregulation of glial fibrillary acidic protein (GFAP) 7d and 4m after Müller cell ablation, indicating that the survival Müller cells were at a stage of reactive gliosis. C, Double label immunohistochemistry (IHC) revealed that VEGF-A was weakly expressed in the superficial retina in WT but strong immunoreactivity was detected around deep retinal vessels (arrows) in TG retinas 7d and 4m after Müller cell ablation. D, Reduced expression of PEDF in areas of Müller cell ablation. E and F, Double label IHC using CD31 for the vascular endothelium and smooth muscle actin (SMA) for pericytes in combination an antibody to VEGF-A suggested that overexpression of VEGF-A was more likely from pericytes (F, small arrows) than from the vascular endothelium (E, small arrow). Note: the superficial retinal vessels did not overexpress VEGF-A (large arrows in E and F). GS=glutamine synthetase. GCL=ganglion cell layer, INL=inner nuclear layer, ONL=outer nuclear layer. Scale bars: 50µm (C-F); 30µm (the squared area in E); 20µm (the squared area in F).


3. EFFECTS OF INTRAVITREAL VEGFB20-4.1.1 ON BRB BREAKDOWN AND PHOTORECEPTOR INJURYWe next tested whether inhibition of VEGF-A activity reduced retinal vascular leakage. Intravitreal injection of VEGFB20-4.1.1, a novel VEGF antibody which binds both human and murine VEGF-A,8 inhibited BRB breakdown when the injection was performed 4d after TMX treatment and evaluations were conducted 6d later (Fig. 3A). After injecting into eyes where intense focal retinal vascular leakage had developed, VEGFB20-4.1.1 dramatically inhibited the established vascular leakage when examined 3, 7 and 35d after injection while lesions in eyes receiving vehicle remained relatively unchanged (Fig. 3, B and C). However, anti-VEGF-A treatment did not prevent photoreceptor loss in TG mice when VEGFB20-4.1.1 was injected 4d after TMX treatment and the loss of photoreceptor outer segments was evaluated 6d later (Fig. 3, D and E).

Figure 3. Effects of anti-VEGF-A therapy with VEGFB20-4.1.1 on BRB breakdown (A-C) and photoreceptor injury (D and E). VEGFB20-4.1.1 (16.38µg) was injected in one eye and the contralateral eye received BSS containing the same amount of bovine serum albumin in each mouse. A, Quantitative measurement of retinal permeability to fluorescence labelled dextran. VEGFB20-4.1.1 was injected 4d after TMX treatment and retinal permeability was measured 7d later. *P<0.01 and †P<0.05, both TG vs WT. B, Fluorescein angiography (FA) was performed to confirm the development of intense focal vascular leakage 4m after Müller cell ablation and VEGFB20-4.1.1 was injected 1 week later. Retinal vascular leakage was monitored by fundus fluorescein angiography at various time points after intravitreal injection. C, Graded fluorescein leakage 3d, 7d and 35d after injection of VEGFB20-4.1.1 or BSS. *P<0.01, vs before injection at day 0; †P<0.01 and ‡P<0.05, anti-VEGF-A (VEGFB20-4.1.1) vs BSS respectively; n=18 at each time point. D and E, Analysis of peanut agglutinin (PNA) staining of retinal wholemounts showed anti-VEGF-A action had no effect on protecting photoreceptor injury (*P<0.01, WT vs TG). Scale bars: 100µm (D).


In summary, we have shown that selective Müller cell ablation is sufficient to induce photoreceptor degeneration, vascular telangiectasis, BRB breakdown and intraretinal neovascularization, which are major features shared by a number of retinal diseases such as Diabetic retinopathy and Macular Telangiectasis Type 2. Our findings indicate that Müller glial deficiency may have a so far unappreciated role and be a mechanistic link between neuronal and vascular pathology in retinal diseases. Our data demonstrate that Müller glial deficiency may be one of the upstream causes of retinal neural and vascular pathologies in retinal diseases. A sustained-release formulation of CNTF using encapsulated cell technology has shown encouraging results in protecting photoreceptors in humans with retinal pigmentosa and age-related macular degeneration.9, 10 Anti-VEGF-A therapy has been widely used to treated retinal vascular diseases. The clinical implication of our findings is that a combined therapeutic approach that targets the neuropathy as well as the vasculopathy may be required to treat diseases caused by dysfunction of Müller cells and, potentially, other glial cells in the central nervous system.

REFERENCES:1. Barber AJ, Antonetti DA, Gardner TW: Altered expression of retinal

occludin and glial fibrillary acidic protein in experimental diabetes. The Penn State Retina Research Group, Invest Ophthalmol Vis Sci 2000, 41:3561-3568

2. Fletcher EL, Phipps JA, Ward MM, Puthussery T, Wilkinson-Berka JL: Neuronal and glial cell abnormality as predictors of progression of diabetic retinopathy, Curr Pharm Des 2007, 13:2699-2712

3. Powner MB, Gillies MC, Tretiach M, Scott A, Guymer RH, Hageman GS, Fruttiger M: Perifoveal muller cell depletion in a case of macular telangiectasia type 2, Ophthalmology 2010, 117:2407-2416

4. Sallo FB, Leung I, Chung M, Wolf-Schnurrbusch UE, Dubra A, Williams DR, Clemons T, Pauleikhoff D, Bird AC, Peto T: Retinal crystals in type 2 idiopathic macular telangiectasia, Ophthalmology 2011, 118:2461-2467

5. Cayouette M, Gravel C: Adenovirus-mediated gene transfer of ciliary neurotrophic factor can prevent photoreceptor degeneration in the retinal degeneration (rd) mouse, Human gene therapy 1997, 8:423-430

6. LaVail MM, Unoki K, Yasumura D, Matthes MT, Yancopoulos GD, Steinberg RH: Multiple growth factors, cytokines, and neurotrophins rescue photoreceptors from the damaging effects of constant light, Proceedings of the National Academy of Sciences of the United States of America 1992, 89:11249-11253

7. Walsh N, Valter K, Stone J: Cellular and subcellular patterns of expression of bFGF and CNTF in the normal and light stressed adult rat retina, Experimental eye research 2001, 72:495-501

8. Liang WC, Wu X, Peale FV, Lee CV, Meng YG, Gutierrez J, Fu L, Malik AK, Gerber HP, Ferrara N, Fuh G: Cross-species vascular endothelial growth factor (VEGF)-blocking antibodies completely inhibit the growth of human tumor xenografts and measure the contribution of stromal VEGF, J Biol Chem 2006, 281:951-961

9. Sieving PA, Caruso RC, Tao W, Coleman HR, Thompson DJ, Fullmer KR, Bush RA: Ciliary neurotrophic factor (CNTF) for human retinal degeneration: phase I trial of CNTF delivered by encapsulated cell intraocular implants, Proceedings of the National Academy of Sciences of the United States of America 2006, 103:3896-3901

10.Zhang K, Hopkins JJ, Heier JS, Birch DG, Halperin LS, Albini TA, Brown DM, Jaffe GJ, Tao W, Williams GA: Ciliary neurotrophic factor delivered by encapsulated cell intraocular implants for treatment of geographic atrophy in age-related macular degeneration, Proceedings of the National Academy of Sciences of the United States of America 2011, 108:6241-6245

SUCCESSFUL NHMRC GRANT APPLICATIONData produced from this study have been used to attract a NHMRC project grant in 2013: APP1050373, $541,967 for 2013-2015. Title: The contribution of aberrant Wnt signalling to neuronal and vascular pathology in retinal disease.

PUBLICATIONS AND CONFERENCE PRESENTATIONS1. Shen WY, Fruttiger M, Zhu L, Chung SH, Barnett NL, Kirk JK,

Lee SR, Coorey NC, Killingsworth M, Sherman LS, Gillies MC. Conditional Müller cell ablation causes independent neuronal and vascular pathologies in a novel transgenic model. J Neurosci 2012; 45: 15715-15727.

2. Chung SH, Shen WY, Jayawardana K, Wang P, Yang J, Shackel N, Gillies MC. Differential Gene Expression Profiling After Conditional Müller Cell Ablation In A Novel Transgenic Model. Invest Ophthalmol Vis Sci. 2013;54:2142–2152.

3. Chung SH, Shen WY 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 (in revision).

4. Shen WY, Fruttiger M, Zhu L, Chung SH, Barnett N, Kirk J, Coorey N, Murray Killingsworth M, Capecchi MR and Gillies MC. Neuroprotection and inhibition of blood-retinal barrier breakdown in a transgenic model of conditional Müller cell ablation. The Association for Research in Vision and Ophthalmology, Fort Lauderdale, USA, 2012.

5. Chung SH, Shen WY, Jayawardana K, Wang P, Yang J, Shackel N, Gillies MC. Global Genomic Analysis of Retinae from Selective Müller Cell Knockout Mice. The Association for Research in Vision and Ophthalmology, Fort Lauderdale, USA, 2012.

6. Shen WY, Zhu L, Lee SR, Chung SH and Gillies MC. Modulation of Muller glial-neuronal cell interactions attenuates photoreceptor damage in a novel transgenic model of conditional Muller cell ablation. The Association for Research in Vision and Ophthalmology, Seattle, USA, 2013.


ORIA new InvesTIgATOR gRAnTWestern Australian Pregnancy Cohort 20-year follow up (Raine) Eye Health Study: Ocular biometry and Ultraviolet exposure.

Dr Hannah Forward, Dr Charlotte McKnight & Dr Alexander Tan

Visual impairment is one of the leading causes of morbidity and poor quality of life. It is generally caused by disease seen in children and the elderly. Many population studies have been conducted targeting these age groups to identify trends of ocular disease and allocate relevant resources. Population-based data of prevalence of ocular disease and distribution of ocular biometry in young adults is not a research/public health priority and therefore remains undefined.1 To fill this knowledge gap, over a 24-month period from March 2010 to February 2012, we examined 1344 participants as a part of 20-year follow-up of Western Australian Birth (Raine) Cohort. Prevalence of ophthalmic disease in the cohort is shown in Table 1. The most common ophthalmic condition was myopia. A total of 5.5 % participants had spherical equivalent of less than or equal to -3 diopters. No participant had been diagnosed with retinopathy of prematurity, retinal dystrophy or glaucoma.2

Complex diseases associated with quantitative traits that have a statistical normal distribution will have multiple genes that contribute small effect sizes on the phenotype. Investigating such continuous traits in healthy individuals, for instance, through genome-wide approaches, allow us to identify the genetic loci and the associated environmental factors for disease. Keeping this hypothesis, we have collected detailed phenotypic data during that resulted in a database of over 300 variables being available on each member of the cohort. This provided us the possibility to study multiple outcomes, allowing identification of potential associations and risk factors in multiple ocular disease processes. Some of our findings as follow:

REFRACTIVE ERRORMyopia is the leading eye health problem worldwide, with a major epidemic in Asia. We found comparable amounts of myopia in our Raine cohort as seen in other Western populations. The CREAM consortium that we are part of, conducted genome-wide meta-analyses, including 37,382 individuals from 27 studies of European ancestry and 8,376 from 5 Asian cohorts. We identified 16 new loci for refractive error in individuals of European ancestry, of which 8 were shared with Asians. Combined analysis identified 8 additional associated loci. The new loci include candidate genes with functions in neurotransmission (GRIA4), ion transport (KCNQ5), retinoic acid metabolism (RDH5), extracellular matrix remodeling (LAMA2 and BMP2) and eye development (SIX6 and PRSS56). We also confirmed previously reported associations with GJD2 and RASGRF1 genes. We found a tenfold increased risk of myopia for individuals carrying the highest genetic risk scores.3

Figure 1 (over page)Manhattan plot of the GWAS meta-analysis for refractive error in the combined analysis (n = 45,758). The plot shows −log10-transformed P values for all SNPs. The upper horizontal line represents the genome-wide significance threshold of P < 5.0 × 10−8; the lower line indicates P value of 1 × 10−5. Previously reported genes are shown in gray. The RBFOX1 gene is also known as A2BP1

CENTRAL CORNEAL THICKNESSCentral corneal thickness (CCT) is associated with eye conditions including keratoconus and glaucoma. In a large meta-analysis in which our cohort was included, 16 new loci associated with CCT were identified at genome-wide significance level (P < 5 × 10−8). Two CCT-associated loci, FOXO1 and FNDC3B, were conferred relatively large risks for keratoconus in 874 cases and 6,085 controls. Additionally, FNDC3B was found to be associated with primary open-angle glaucoma. Further analyses indicated involvement of the collagen and extracellular matrix pathways in the regulation of CCT.4

CORNEAL CURVATURECorneal curvature (CC) is another quantitative trait that is important in characterisation of diseases including keratoconus, refractive error and Marfan’s syndrome. We aimed to identify genes underlying corneal curvature and to test whether variants in PDGFRA and FRAP1


that are associated in Asians also determine CC in Australians of north European ancestry. We conducted a meta-analysis by combining data of 1013 individuals from our cohort and 1788 Australian twins. We replicated the association between the SNP rs2114039 near PDGFRA and corneal curvature (P = 0.005) and confirmed that PDGFRA gene has a significant role in determining corneal curvature in the Australian population. The other gene reported to be associated with CC in Asian populations, FRAP1, did not show a significant effect in our samples. Although our study was underpowered to detect novel loci, we found some evidence that SNPs near TRIM29 may play a role in determining CC. Our findings of SNPs at TRIM29 and other regions should be replicated in further studies, with meta-analyses likely to prove important in further dissecting this important quantitative trait.5

CORNEAL ASTIGMATISMPDGFRA locus was also found to be associated with corneal astigmatism in a Singaporean Asian population. We found no strong evidence for replication or transferability of the previously reported association between the rs7677751 variant, at the PDGFRA locus, and corneal astigmatism in our Australian cohorts of Northern European ancestry. However we identified several putative loci, which clearly require replication in ongoing genetic or functional studies.6

Identified genetic factors behind normal ocular variation enabled us to gain greater insight into the possible pathways involved in disease processes. Aforementioned works were published in various academic journals and presented in multiple national and international conferences. The funding provided by ORIA enabled us to collate and analyse the data included in these works. With over 300

ophthalmological variables collected and also being analysed there will be many further publications arising from this work.

PUBLICATIONS TO DATE:1. Forward H, Hewitt AW, Mackey DA. Missing X and Y: a review of

participant ages in population-based eye studies. Clin Experiment Ophthalmol 2011.

2. Yazar S, Forward H, McKnight CM, et al. Raine Eye Health Study: Design, Methodology and Baseline Prevalence of Ophthalmic Disease in a Birth-cohort Study of Young Adults. Ophthalmic Genetics 2013:1–34.

3. Verhoeven VJM, Hysi PG, Wojciechowski R, et al. Genome-wide meta-analyses of multiancestry cohorts identify multiple new susceptibility loci for refractive error and myopia. Nature Publishing Group 2013;45:314–318.

4. Lu Y, Vitart V, Burdon KP, et al. Genome-wide association analyses identify multiple loci associated with central corneal thickness and keratoconus. Nature Publishing Group 2013;45:155–163.

5. Mishra A, Yazar S, Hewitt AW, et al. Genetic variants near PDGFRA are associated with corneal curvature in Australians. Invest Ophthalmol Vis Sci 2012;53:7131–7136.

6. Yazar S, Mishra A, Ang W, et al. Interrogation of the platelet-derived growth factor receptor alpha locus and corneal astigmatism in Australians of Northern European ancestry: Results of a genome-wide association study. Mol Vis 2013;19:1238–1246.


ORIA new InvesTIgATOR gRAnTMacrophages in the Ageing Eye

Dr Jelena Kezic

BACKGROUND Ageing is a major risk factor for the development of neurodegenerative diseases affecting the central nervous system, including the retina and optic nerve. Increasing evidence from both clinical and experimental studies supports a prominent role for the immune system, and more specifically, resident ocular immune cells including retinal microglia, in the pathogenesis of age-associated diseases such as age-related macular degeneration (AMD) and glaucoma. Retinal microglia are a specialized population of macrophages which in resting conditions, perform surveillance, scavenging and homeostatic functions associated with host defense and tissue repair. In an activated state, such as in response to injury or inflammation, microglia can generate reactive oxygen species, increase their phagocytic activity and produce a number of pro-inflammatory cytokines including TNFα, IL-6, IL-1β and IL-12p40. Recent studies have shown that with increasing age, microglia may change their inflammatory profile and functional phenotype, and thus may switch from being protective to pathogenic. An over-responsive or ‘primed’ microglial phenotype during ageing may have implications for microglial responses to various tissue insults or injury. In this study, we set out to investigate whether the macrophage response to retinal injury differs in the ageing mouse, and whether irradiation and/or bone marrow therapy could alter this response.

AIMS:1. To characterize the macrophage response to retinal

injury in the young and ageing eye.

2. To determine whether the introduction of young bone marrow into old mice can supplement the ageing macrophage populations in the eye and alter the course of pathological changes that occur in the ageing retina following injury.

PROGRESS AND RESULTS 1. Characterization of the macrophage response to retinal injury in the young and ageing eye.

Baseline studies were conducted to characterize the macrophage response to an acute form of retinal injury. To induce retinal injury, the anterior chamber of the mouse eye was cannulated and intraocular pressure (IOP) elevated to 50 mm Hg for 30 minutes. Fellow (sham/control) eyes were cannulated and IOP maintained at 12 mmHg. Retinal function was assessed

1 week after injury, followed by analysis of retinal tissue by immunofluorescence staining with various macrophage and immune activation markers. This study showed that cannulation of the anterior chamber alone (without acute pressure increase) induced a marked retinal macrophage response, which was similar to macrophage changes observed following intraocular pressure increase. These macrophage changes included activation, an increase in hyalocyte (vitreal macrophages) density and macrophage accumulation in the subretinal space (Fig 1A-D). Interestingly, these macrophage responses did not appear to correlate with changes to retinal function, as functional impairments were only observed in eyes that had been exposed to intraocular pressure elevation (data not shown). Similarly, Müller cell activation, as indicated by the upregulation of glial fibrillary acidic protein (GFAP), was only observed in eyes that had been subject to acute elevation of IOP (Fig 1E).

This work was recently published: Kezic JM, Chrysostomou V, Trounce IA, McMenamin PG, Crowston JG. Effect of anterior chamber cannulation and acute IOP elevation on retinal macrophages in the adult mouse. Invest Ophthalmol Vis Sci, 2013. April 30,54(4): 3028-36.

A second manuscript examining whether age influences the macrophage response to injury is currently in preparation for publication: Kezic JM, Chrysostomou V, Trounce IA, Crowston JG. The effects of age and mitochondrial dysfunction on the macrophage response to acute retinal injury.

Figure 1 (top next page): A, Vitreal hyalocyte density in naïve eyes and 1 week after cannulation or IOP elevation. B, Percentage of rounded hyalocytes, indicative of cell activation. C, Upregulation of CD68 and Isolectin- B4 on hyalocytes in IOP elevated eye. D, Subretinal macrophage density in naïve eyes and 1 week after cannulation or IOP elevation. E, GFAP expression in cannulated or IOP elevated eyes.

2. Irradiation and bone marrow therapy alters the macrophage response to acute retinal injury

12 month old and 18 month old C57BL/6J mice received lethal irradiation (11 Gy) and injection of bone marrow from 8 week old or 12 month old donors (8 wk → 12 mo; 8 wk → 18 mo; 12 mo → 12 mo). Eight weeks later, all eyes were subjected to acute elevation of IOP (50 mmHg for 30 minutes). Eyes were assessed 1 week after IOP elevation. The acute elevation of


IOP in naïve 12 month old mice led to an increase in subretinal macrophage density and the upregulation of GFAP expression 1 week post injury. Subretinal macrophage accumulation was reduced in mice that had received lethal irradiation and bone marrow transfer 8 weeks earlier (Fig 2). The age of donor bone marrow was not important in this attenuated response, as 12 month old mice that had received bone marrow from 12 month old donor mice also showed a reduction in subretinal macrophage density (Fig 2, 12mo → 12 mo). The upregulation of GFAP expression on Muller cells following IOP elevation was diminished in all mice that had received irradiation and bone marrow treatment 8 weeks prior to IOP elevation (Fig 3).

Figure 2: Subretinal macrophage numbers in non-irradiated mice and 1 week after irradiation and bone marrow treatment.

Figure 3 (below):GFAP expression in retinal sections from non-irradiated 12 month old mice (C57 12 mo), or mice that had been irradiated and received either young bone marrow (8 wk → 12 mo) or bone marrow from 12 month old mice (12 mo → 12 mo). Red = GFAP, Blue = Hoescht.

Further studies are ongoing to determine how irradiation alone (sublethal dose) alters macrophage responses in the normal ageing eye and in response to acute injury. Cytokine studies are currently in progress. Eye and brain tissue have been collected from normal mice of varying ages (8 weeks, 12 months, 18 months) in order to assess the inflammatory profile of the retina with ageing. Eye tissue has also been collected from irradiation experiments to determine whether irradiation and/or bone marrow therapy alters the inflammatory profile in the ageing retina.


ORIA/w A QuInlIvAn/glAucOmA AusTRAlIA gRAnTProf Robert Casson

Can Glucose Eye Drops Improve Vision in Glaucoma?

The study was highly successful and the data are currently being prepared as a manuscript and were presented at ARVO Seattle in May 2013.

BACKGROUNDPrimary open–angle glaucoma (POAG) is a chronic optic neuropathy and the world’s leading cause of irreversible blindness. Converging evidence indicates that energy insufficiency is involved in the pathogenesis in at least some individuals with POAG. Elevated vitreal glucose levels protect neurones in experimental glaucoma.

METHODSWe conducted a double–blind, randomized, first–in–man study of the effect of topical glucose on visual parameters in individuals with POAG. We first demonstrated that 50% topical glucose reached the vitreous in pseudophakic individuals. We then recruited 29 eyes of 16 individuals with POAG, who were randomly assigned to receive 50% glucose then 0.9% saline or vice versa in a crossover design. Change in contrast sensitivity at a spatial frequency of 12 cycles/degree was the primary outcome. We then conducted a follow–up study of 14 eyes of seven individuals including responders from the first group and matched the osmolarity of the saline (8%) to the 50% glucose. The study was registered online with the Australian and New Zealand Clinical Trial Registry (ACTRN12612001134819).

FINDINGS50% glucose reached the vitreous in pseudophakic but not phakic individuals. Glucose significantly improved the mean contrast sensitivity at 12 cycles/degree compared to 0.9% saline by 0·26 (95% CI: 0·13 – 0·38) log units (p < 0.001; Fig. 1 & 2); and in the follow–up study: 0·39 log units (95% CI: 0·20 – 0.60; p = 0·014). Neither the IOP, refraction, nor central corneal thickness were affected by glucose; age was not a significant predictor of the response.

INTERPRETATIONTopical glucose temporarily improves psychophysical visual parameters in some individuals with POAG, suggesting that neuronal energy substrate delivery to the vitreous reservoir may recover function of “sick” retinal neurons, providing a novel treatment strategy for this blinding disease.

Figure 1:Glucose significantly increased contrast sensitivity at 12 cycles/degree by 0·26 log units (*** p < 0.001; error bars show 95% CIs from GEE regression analysis

FIGURE 2:Log contrast sensitivity at various spatieal frequencies at baseline and after glucose or saline control drops.

Professor Robert Casson from the Ophthalmic Research Laboratories, Hanson Institute, Royal Adelaide Hospital with his patient”.


ORIA new InvesTIgATOR gRAnT (2011)A National Registry of Thyroid Eye Disease For Genomic And Transcriptomic Studies

Dr J J Khong

Thyroid orbitopathy is a complex disorder that developed in 25 percent of patients with Graves’ disease. The genetic risk factors for developing thyroid eye disease are unclear, we hypothesized that genetic risk factors exist for the development of thyroid eye disease and aim to elucidate the genetic susceptibility through genetic and transcriptomic approaches.

Since ORIA granted this research initiative, the Flinders University department of ophthalmology research team has secured an NHMRC project grant (App ID 11031362) for genome wide association studies to identify major genetic determinants of 5 blinding eye diseases using pooled DNA, including thyroid eye disease.

SUMMARY OF RESEARCH PROJECTAim 1: Repository of Graves’ disease patients with and without thyroid orbitopathy for genetic studies

872 patients with Graves’ disease and thyroid orbitopathy were recruited from South Australia, Victoria, Tasmania and New South Wales. 532(61%) cases have thyroid orbitopathy and 340(39%) participants have Graves’ disease without thyroid orbitopathy.

This is the largest repository of thyroid orbitopathy cases in Australia, which we have used for genome wide association studies to compare the genetic variation between thyroid orbitopathy cases and Graves’ without orbitopathy, and to determine the genetic variation of Graves’ disease cases compared with normal healthy controls.

Every participant that attended their research appointments had blood taken for DNA stored in whole blood samples, 90% had serum extracted for future research. We have also comprehensively documented their clinical history including known risk factors for developing thyroid eye disease, including smoking status and cigarette pack year which allow dosage response correlation, radioactive iodine treatment exposure, gender, age, ethnicity such that potential confounding factors could be stratified and factored into the genetic data analysis. We have methodically documented ophthalmic findings using the VISA classification, which produced one of the best cohorts of thyroid eye disease cases and Graves’ controls for comparison.

Aim 2: Recruitment of patients for RNA microarray study

The research project collected 101 orbital fat samples from participants including 66 thyroid eye disease cases

and 35 normal controls. This collection has exceeded the projected total orbital fat samples for the study. The repertoire is the largest collection of human orbital fat samples for RNA microarray study for thyroid eye disease reported in the literature so far, with only 3-4 papers on RNA microarray study with less than 10 samples in every study. We will have large sample size for replication study in the future.

Aim 3: Analysis of RNA expression profile in thyroid orbitopathy

Total RNA were extracted from 66 samples using Qiagen RNeasy microarray kit. The quality of RNA extract was confirmed on Agilent bioanalyzer 2100 before hybridization of cDNA on Illumina Human HT12 high throughput RNA microarrays. RNA expression profiles were compared between thyroid orbitopathy cases and normal controls, active thyroid orbitopathy with inactive thyroid orbitopathy, and changes in RNA expression profile were detailed in a thyroid orbitopathy case as the disease progressed from inflammatory phase to burnout phase.

SUMMARY OF FINDINGS:The RNA expression profiles of 29 thyroid orbitopathy cases were compared with 20 healthy controls. The overall differential expression of RNA ranged from 1.25 to 2.45 fold difference.19 genes were differentially expressed with a modest fold change (1.25 to 1.55, p <0.001) Gene PPM1B on chromosome 2p21 was over-expressed in thyroid orbitopathy by 1.28 fold, which reached statistical significance after multiple testing p value correction using the Bonferroni method. PPM1B is a member of the ser/Thr protein phosphatase that is known as a negative regulator of cell stress response pathway. Over-expression of the gene was reported to cause cell death. This phosphatase has been shown to dephosphorylate cyclin dependent kinases. Further work is required to understand the function of the gene in the pathogenesis of thyroid orbitopathy.

The RNA expression profiles of 31 active thyroid orbitopathy cases were compared with 19 inactive thyroid orbitopathy. The overall differential expression of RNA ranged from 1.25 to 2.46 fold difference. Gene GZMH on chromosome 14q12a was over-expressed in active thyroid orbitopathy compared with inactive thyroid orbitopathy (fold change 1.37, p<0.001). GZMH codes for a protein in the granzyme family. It is a protease that eliminate transformed cell and virus


infected cell, the gene is reported to be expressed in natural killer cell and may play a role in the cytotoxic arm of the innate immunity causing cell death.

We followed the RNA expression profile of a case of thyroid orbitopathy as his disease progressed from inflammatory phase to burnout phase. The study revealed marked difference in gene expression profile with 10 genes reaching statistical significance on multiple testing correction using Bonferroni method: FHOD3, TAGLN, SCD5, DEFA1B, SEMA5B, ADAM19, DKK3, VWA5B1, DEFA1 and FAM5C showing fold difference ranging from 1.22 to 10.63. FHOD3 gene was downregulated in inflammatory phase of thyroid orbitopathy and codes for protein forming that regulate actin cytoskeleton. TAGLN was upregulated, codes a protein in actin cross-linking found in fibroblasts and smooth muscle. SCD5 was upregulated, it is an integral membrane protein of the endoplastic reticulum that catalyzes the formation of monounsaturated fatty acids from saturated fatty acids which may have a role in adipogenesis. DEFA1B and DEFA 1 were upregulated, they are genes that encode cytotoxic peptides in neutrophils and likely play a role in phagocyte-mediated host defense. SEMA5B was upregulated, encodes semaphorin protein family which regulates axon growth during development of the nervous system. ADAM19 was upregulated, encodes a disintegrin and metalloprotease domain that is involved in cell migration, cell adhesion and cell-matrix interactions and is proposed to play a role in pathological processes such as cancer and inflammatory disease. DKK3 was downregulated, encodes a protein that is a member of the dickkopf family canonical WNT receptor signaling pathway inhibitor. VWA5B1 was downregulated, it is von Willebrand factor A domain containing 5B1 that is protein encoding.

In summary, the RNA expression study suggests genes involving in regulating cell death possibly by cell stress response pathway or cytotoxic arm of innate immunity may be important in the onset of thyroid orbitopathy and inflammatory phase of the disease. Genes involving actin cross linking, lipid metabolism, innate immunity, cell migration and adhesion, WNT receptor signaling pathway inhibitor were upregulated in the inflammatory phase of thyroid orbitopathy compared to burnout out phase of the disease. The functions of these genes in the pathogenesis of thyroid orbitopathy will require further evaluation.

We would like to thank ORIA for supporting research initiatives in thyroid eye disease.


dR jelenA keZIc, FROm ceRA, melbOuRne whO ReceIved new InvesTIgATOR FundIng ThROugh The ORIA duRIng 2012 FOR mAcROPhAges In The AgeIng eye


DIRECTORS’REPORT

1. dIRecTORsThe names of the Directors of the company in office at the date of this report are:

Professor Stuart L Graham, Sydney (Chairman)

Professor Mark Gillies, Sydney (Vice Chairman)


Dr Wilson Heriot, Melbourne (Honorary Treasurer)

Dr Fred Chen, Perth


Professor Jonathon Crowston, Melbourne

A/Prof Mark D Daniell, Melbourne



Professor David Mackey, Perth

Professor Peter J McCluskey, Sydney




2. InFORmATIOn On dIRecTORsThe names, qualifications and period membership commenced and position held are as follows:

Dr Fred Chen MB BS (Hons), PhD (London), FRANZCO, CSA (Cert) 2011

Dr Colin Clement BSc (Hons), MB BS PhD, FRANZCO 2011

Professor J Crowston BSc, MB BS, FRCOphth, FRANZCO, PhD 2008

A/Prof Mark D Daniell MB BS, MS, FRACS, FRANZCO 2001

Professor Mark Gillies MB BS, PhD, FRANZCO Vice Chairman 2004

Professor Stuart L Graham MB BS, MS, FRANZCO, FRACS Chairman 2001

Dr Paul Healey MB BS (Hons), B (Med) Sc, MMed PhD, FRANZCO 2011

Dr Wilson Heriot MB BS, FRANZCO, FRACS Honorary Treasurer 2009

Dr Anthony Kwan MBChB (UK), MD (London), FRCOphth (UK), FRANZCO 2007

Professor David Mackey MB BS, MD, FRANZCO, FRACS 2005

Professor Peter J McCluskey MB BS, FRANZCO, FRACS 1984

Dr John Males MB BS, M Med, FRANZCO 2009

Dr Richard Mills MB BS, FRCS, FRACS, FRANZCO, PhD Honorary Secretary 2003

Dr Andrea Vincent MBChB, FRANZCO 2008

Dr Stephanie Watson BSc, MB BS, FRANZCO, PhD 2006

Professor Tien Wong MB BS, MPG, PhD, FRANZCO (retired November 2012) 2008

No shares are held by Directors.


3. meeTIngs OF dIRecTORsDuring the financial year three meetings of directors were held. Attendances were:

Number Eligible Number to Attend Attended

Dr Fred Chen 3 3

Dr Colin Clement 3 3

Prof J Crowston, Melbourne 3 2

A/Prof M Daniell, Melbourne 3 2

Prof Mark Gillies, Sydney 3 2

Prof S Graham, Sydney 3 3

A/Prof Paul Healey, Sydney 3 1

Dr Wilson Heriot, Melbourne 3 3

Dr Anthony Kwan, Brisbane 3 2

Prof David Mackey, Perth 3 3

Prof P J McCluskey, Sydney 3 1

Dr J Males, Sydney 3 3

Dr Richard Mills, Adelaide 3 3

Dr A Vincent, New Zealand 3 3

Prof Tien Wong, Melbourne 2 0

Dr Stephanie Watson, Sydney 3 3

4. IndemnIFyIng OFFIceR OR AudITORThe company has not during or since the financial year in respect of any person who is or has been an officer or auditor of the company or a related body corporate indemnified or made any relevant agreement for indemnifying against a liability incurred as an officer including costs and expenses in successfully defending legal proceedings or paid or agreed to pay a premium in respect of a contract of insurance against a liability incurred as an officer for the costs or expenses to defend legal proceedings.

5. PRIncIPAl AcTIvITIesThe principal activity of the company in the course of the financial period was to provide funds for ophthalmic research. There has been no significant change in the nature of this activity during that period.


6. shORT-TeRm And lOng-TeRm ObjecTIvesThe company’s short-term objectives are to:

•continuetofundresearchintoalltypesofeyediseasesannuallyinAustralia

•continuetobeintheforefrontofadvancingeyeresearchinAustralia

•continuetosupportthepresentationofresearchandthepublicationoftheresultsofresearchforvisionscientistsandophthalmologists for the benefit of all Australians.

•continuetosupportnewscientistsbyprovidingapercentageofitsannualfundingtosupportthiscategory

The company’s long-term objectives are to:

•increasethefundsavailablefortheprovisionofresearchfundinginordertoachieveitsmissionstatementofadvancing eye research in Australia.

•ensurethatthefundingitprovidesleadstoresearchersgainingatrackrecordtoenablethemtosecurelargergrantstowards bigger and successful projects.

7. sTRATegIesTo achieve its stated objectives, the company has adopted the following strategies:

- The company is partnered with the RANZCO Eye Foundation who are now primarily responsible for raising additional funding towards the ORIA’s research projects and to raise awareness generally of eye health within Australia.

- The company’s Investment Advisory Committee monitors and works towards successfully managing the company’s invested funds, the profits from which are used annually for research funding.

- The company connects with other vision related organisations in Australia to support funding of projects for specific diseases.

- The company strives to attract, support and retain quality staff who are committed to the work of the organization.

- The company conducts audits on its previously funded research to ensure the funding it provides is meeting its objectives.

- The company’s Board and Research Advisory Committee is made up of leading vision scientists and ophthalmologists within Australia.

8. OPeRATIng ResulTs(1) OPERATING REVENUERevenue is mainly derived from investing in shares and interest bearing securities.

2013 2012 Decrease %

Net dividend, interest and trust distribution income

$ 583,793 $ 620,024 $36,231 5.84

Less Expenses 38,149 33,549

545,644 586,475

(2) OPERATING SURPLUSThe surplus of the company before other comprehensive income for the year ended 30 June 2013 was $758,528 (2012 $622,876). This amount is comprised of the following:

2013 2012

Trust Fund $ 757,703 $ 613,881

Administration 825 8,995

758,528 622,876

Other comprehensive income before grants and Director of Research allocation amounted to a surplus of $1,230,852 (2012: loss of $566,289) and included a profit on re arrangement of investments of $244,636 (2012: loss of $265,020) and valuation gain on available-for-sale financial assets of $983,173 (2012: loss of $301,269).


9. RevIew OF OPeRATIOns The surplus for the year was $758,528 compared to $622,876 in 2012. The income of the trust fund has increased by $135,652 primarily due to an increase of $210,084 in legacies and donations received and a decrease of $36,231 in dividends, interest and distributions received. Distributions from legacies and donations increased to $210,084 from $26,624 in 2012. This increase is mainly due to a large donation received on the winding up of Dr Grosvenor John Williams Endowment Fund. The administrative operations of the institute for the year resulted in a surplus of $825 compared with a surplus of $8,995 in 2012.

10. dIvIdendsThe company’s Articles of Association preclude the payment of dividends to any of its members.

11. sTATe OF AFFAIRsThere has been no significant change in the state of affairs of the company occurring during the year.

12. lIkely develOPmenTsAt the date of this report, there are no known unusual developments that will affect the results of the company’s operations in subsequent financial years.

13. shARe OPTIOnsNo share options were issued during the year.

14. dIRecTORs’ beneFITsWith the exception of the grants made or allocated to Dr Fred Chen no director of the company has since the end of the previous financial year, received or become entitled to receive a benefit not disclosed in the accounts as directors’ emoluments by reason of a contract made by the company or a related corporation with the directors, or with a firm in which he or she has a substantial financial interest.

15. AudITOR’s IndePendence declARATIOnA copy of the auditor’s independence declaration as required under Section 307C of the Corporations Act 2001 is set out at page 25.

For and on behalf of the Board.

Prof Stuart Graham Dr Wilson Heriot Director Director


sTATemenT OF FInAncIAl POsITIOnAS AT 30 JUNE 2013

Note2013

$2012

$

Current Assets

Cash and Cash Equivalents 3 1,093,773 957,903

Receivables 4 183,648 144,665

Investments 5 8,839,231 7,626,966

10,116,652 8,729,534

Non-Current Assets

Plant & Equipment 6 4,284 5,546

Total Assets 10,120,936 8,735,080

Current Liabilities

Payables 7 701,919 481,817

Provisions 8 19,689 19,515

721,608 501,332

Net Assets 9,399,328 8,233,748

Equity

General Fund 13 (a) - -

Capital Funds

Research Fund 9 982,057 941,118

Settled Funds 10 472,556 472,556

Financial Assets Reserve 11 1,026,872 43,699

Capitalised Profit on Re-arrangement of Investments & Capital Distributions

12 6,571,278 6,323,599

9,052,763 7,780,972

Retained Income- Available for grants 13 (b) 346,565 452,776

Total Equity 9,399,328 8,233,748

The accompanying Notes form part of these financial statements.

FINANCIAL STATEMENTS


TRusT Fund sTATemenT OF cOmPRehensIve IncOmeFOR THE YEAR ENDED 30 JUNE 2013

Note2013

$2012

$

Income

Dividends received from:

Other Corporations 482,826 504,348

Total Dividends 482,826 504,348

Interest received from:

Other Entities 78,717 88,303

Trust distributions received from:

Other Entities 22,250 27,373

583,793 620,024

Legacies - Anselmi Estate 43,781 19,556

- Ivy May Stephenson 4,828 5,495

- G. J. Williams 150,000 -

Other Donations and Legacies Received 11,475 1,573

Sundry Income 1,975 782

Total Income for the Year 795,852 647,430

Expenses

Commission Paid 38,149 33,549

38,149 33,549

Surplus For The Year 757,703 613,881

Other Comprehensive Income

Special Dividends and associated imputation credits 3,043 -

Valuation Gains/(Losses) on available-for-sale financial assets 983,173 (301,269)

Profit/(Loss) on Re-arrangement of Investments 244,636 (265,020)

Total other comprehensive income 1,230,852 (566,289)

Surplus for the year before allocation 1,988,555 47,592

Grants Allocated/made during the year 14 648,800 342,690

Allocation to Director of Research - Victoria 15 175,000 186,000

823,800 528,690

Total Comprehensive Income/(Loss) 1,164,755 (481,098)

Profit Attributable to Members of the Entity (66,097) 85,191

Total Other Comprehensive Income Attributable to Members of the Entity 1,230,852 (566,289)



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AdmInIsTRATIOn sTATemenT OF cOmPRehensIve IncOmeFOR THE YEAR ENDED 30 JUNE 2013

Note2013

$2012

$

Income

Membership Fees - RANZCO 137,470 130,162

Total Income 137,470 130,162

Expenses

Accountancy Fees 20,901 22,108

Auditors' Remuneration 16 4,950 4,950

Bank Charges 195 124

Depreciation 1,929 689

General Expenses 6,467 3,576

IT & Webpage Expenses - 392

Insurance 4,306 4,159

Printing & Stationery 6,622 7,482

Staff Salaries 65,157 53,550

Superannuation Contribution 8,323 6,600

Salary Sacrificed Benefits 4,200 1,450

Provision Employee Benefits 174 3,908

Meeting and Travelling Expenses 13,421 12,179

Total Expenses 136,645 121,167

Surplus/(Deficit) For The Year 13 (a) 825 8,995

Other Comprehensive Income - -

Total Comprehensive Income 825 8,995



sTATemenT OF cAsh FlOwsFOR THE YEAR ENDED 30 JUNE 2013

Note2013

$2012

$

Cash Flows From Operating Activities

Receipts

Dividends Received 488,536 712,934

Interest Received 78,717 88,303

Trust Distributions 22,250 27,373

Legacies 4,828 25,051

Other Revenue 7,024 5,454

RANZCO - Reimbursement of membership fees 137,470 130,162

Contributions from Ranzco Eye Foundation 170,000 120,000

Contribution from Glaucoma Australia Inc 49,750 72,005

Donations Received 161,475 1,573

Payments

Commissions (38,149) (33,549)

Research Grants Paid (633,098) (619,997)

Payments to Director of Research - Victoria (186,000) (171,000)

Other Grants & Contributions - -

Other (143,940) (120,480)

Net Cash (Used in)/Provided by Operating Activities 17 118,863 237,830

Cash Flows From Investing Activities

Payments for Property, Plant & Equipment (667) (6,085)

Special Dividends - Capital Reduction - -

Proceeds from Re-arrangement of Investments 2,386,810 5,049,368

Payments for Investments (2,369,136) (5,183,409)

Net Cash Used in Investing Activities 17,007 (140,126)

Net Increase/(Decrease) in Cash and Cash Equivalents 135,870 97,704

Cash and Cash Equivalents at 1 July 2012 957,903 860,199

Cash and cash equivalents at 30 June 2013 3 1,093,773 957,903



1 sTATemenT OF AccOunTIng POlIcIesThe financial statements are for the Ophthalmic Research Institute of Australia, incorporated and domiciled in Australia. The Ophthalmic Research Institute of Australia is a company limited by guarantee.

(A) BASIS OF PREPARATIONThe financial statements are general purpose financial statements that have been prepared in accordance with Australian Accounting Standards (including Australian Accounting Interpretations) and the Corporations Act 2001.

The accounting policies set out below have been consistently applied to all years presented, unless otherwise stated. The financial report has been prepared on an accruals basis and is based on historical costs and does not take into account changing money values or, except where stated, current valuations of non current assets. Cost is based on the fair values of the consideration given in exchange for assets.

The following is a summary of the significant accounting policies adopted by the company in the preparation of the financial report.

(B) INCOME TAxThe company is an approved research institute and is exempt from income tax.

(C) TRANSFERS TO CAPITAL FUNDS(i) Capital profits and losses on disposal of investments & capital distributions.

Realised capital profits and losses on disposal of investments are brought to account in the trust fund as profit/(loss) on rearrangement of investments, however, these amounts are transferred to capital funds and do not form part of retained income available for grants.

Capital Distributions and special dividends together with associated imputation credits recognised in the statement of comprehensive income are also transferred to the capital fund and do not form part of retained income available for grants.

(ii) General Research Capital Fund

Five percent of the net surplus of the General Fund including imputation credits are transferred to the General Research Capital Fund this financial year.

(iii) Allocation of Income to Each Fund

During the year ended 30 June 1993, the investments of the company were separated into the D.W. Research Fund and the General Fund in the ratio of 72% and 28% respectively. As the flow of investment and donation income to and from the two funds does not occur in the same proportion, the ratio of the D.W. Research Fund and the General Fund is no longer at 72% and 28%.

NOTES TO AND FORMING PART OF THE FINANCIAL STATEMENTSFOR THE YEAR ENDED 30 JUNE 2013


Income from the General Fund which comprises of all funds except the D.W. Research Fund, is allocated as follows:

Research Fund 10.0%

Esme Anderson 51.4%

G.J.Williams 8.9%

B. Mitchell 8.9%

Dame Ida Mann 12.5%

R. & L. Lowe Research 8.3%

If and when further donations are received by specific fund(s) the allocation of future income will be distributed to each fund in accordance with its revised proportion to the General Fund.

Fifty percent of the income derived from the D.W. Research Fund and its investments is allocated to the Director of Research Victoria.

(D) CASH AND CASH EQUIVALENTSFor the purpose of the statement of cash flows, cash and cash equivalents include cash on hand and at call deposits with banks.

(E) INVESTMENTSInvestments are carried at fair value. Changes in fair value will be held in an equity reserve until the asset is disposed, at which time the changes in fair value will be brought to account through the statement of comprehensive income.

(F) REVENUEInterest and dividends are recognised when received.

Grants, donations and distributions income are recognised when received.

(G) GOODS AND SERVICES TAx (GST)All revenue, expenses and assets are recognised net of the amount of goods and services tax (GST), except where the amount of GST incurred is not recoverable from the Australian Tax Office. In these circumstances the GST is recognised as part of the cost of acquisition of the asset or as part of an item of the expense. Receivables and payables in the statement of financial position are shown inclusive of GST.

(H) FINANCIAL INSTRUMENTS

Recognition and Initial MeasurementFinancial instruments, incorporating financial assets and financial liabilities, are recognised when the entity becomes a party to the contractual provisions of the instrument.

Financial instruments are initially measured at fair value plus transactions costs where the instrument is not classified as at fair value through profit or loss. Financial instruments are classified and measured as set out below.

Classification and Subsequent Measurement(i) Loans and receivables Loans and receivables are non-derivative financial assets with fixed or determinable payments that are not quoted

in an active market and are subsequently measured at amortised cost using the effective interest rate method.

(ii) Held-to-maturity investments Held-to-maturity investments are non-derivative financial assets that have fixed maturities and fixed or determinable

payments, and it is the entity’s intention to hold these investments to maturity. They are subsequently measured at amortised cost using the effective interest rate method.

(iii) Available-for-sale financial assets Available-for-sale financial assets are non-derivative financial assets that are either designated as such or that are

not classified in any of the other categories. They comprise investments in the equity of other entities where there is neither a fixed maturity nor fixed or determinable payments.


(iv) Financial liabilities Non-derivative financial liabilities (excluding financial guarantees) are subsequently measured at amortised cost

using the effective interest rate method.

Fair valueFair value is determined based on current bid prices for all quoted investments. Valuation techniques are applied to determine the fair value for all unlisted securities, including recent arm’s length transactions, reference to similar instruments and option pricing models.

ImpairmentAt each reporting date, the entity assesses whether there is objective evidence that a financial instrument has been impaired. In the case of available-for-sale financial instruments, a prolonged decline in the value of the instrument is considered to determine whether an impairment has arisen. Impairment losses are recognised in the statement of comprehensive income.

(i) Impairment of Assets At each reporting date, the entity reviews the carrying values of its assets to determine whether there is any

indication that those assets have been impaired. If such an indication exists, the recoverable amount of the asset, being the higher of the asset’s fair value less costs to sell and value in use, is compared to the asset’s carrying value. Any excess of the asset’s carrying value over its recoverable amount is expensed to the statement of comprehensive income.

Where it is not possible to estimate the recoverable amount of an individual asset, the entity estimates the recoverable amount of the cash-generating unit to which the asset belongs.

2 membeRs’ guARAnTeeIf the company is wound up the Memorandum of Association states that each member is required to contribute a maximum of $ 2.00 each towards meeting any outstanding obligations of the company.

3 cAsh And cAsh eQuIvAlenTs

2013 $

2012 $

General Account 722,852 809,193

Donations Account 10,425 29,565

D.W. Research Fund Account 360,496 119,145

1,093,773 957,903

4 ReceIvAbles

Sundry Debtors 183,648 144,665

183,648 144,665

5 InvesTmenTs

Shares in Listed Corporations & Other Securities 8,839,231 7,391,966

Total Available-for-sale Financial Assets 8,839,231 7,391,966

Held-to-maturity Investments

Bank Bills - Cost - 235,000

Total Held-to-Maturity Investments - 235,000

Total Investments 8,839,231 7,626,966


6 PlAnT And eQuIPmenT

2013 $

2012 $

Office Equipment - at cost 9,040 8,373

Less: Accumulated Depreciation (4,756) (2,827)

4,284 5,546

Reconciliation

Reconciliation of the carrying amount of plant and equipment at the beginning and end of the current and previous financial year:

Carrying amount at beginning of year 5,546 150

Additions 667 6,085

Less: Depreciation expense (1,929) (689)

Carrying amount at end of year 4,284 5,546

7 PAyAbles

Creditors and Accruals 24,119 28,469

Grants Payable 502,800 267,348

Director of Research - Victoria (refer note 15) 175,000 186,000

701,919 481,817

8 PROvIsIOns

Employee Benefits 19,689 19,515

9 ReseARch cAPITAl Fund

General

Balance 1 July 2012 619,763 607,202

Allocation to Capital:

- 10% Surplus & Imputation Credits & Other Legacies 40,939 12,561

Balance 30 June 2013 660,702 619,763

Anselmi Estate 290,979 290,979

Balance 1 July 2012 - -

Allocation during year - -

Transfer during year 290,979 290,979

Balance 30 June 2013 660,702 619,763

Ivy May Stephenson Estate

Balance 1 July 2011 30,376 30,376

Allocation during the year - -

Transfer during year - -

Balance 30 June 2012 30,376 30,376

Total 982,057 941,118


10 seTTled Funds

2013 $

2012 $

D.W. Research Funds 200,000 200,000

Esme Anderson 124,326 124,326

G.J. Williams 25,500 25,500

B. Mitchell 26,023 26,023

Dame Ida Mann (Est. 31/03/84) 56,707 56,707

Ronald & Lois Lowe 40,000 40,000

472,556 472,556

11 FInAncIAl AsseTs ReseRve

Balance 1 July 2012 43,699 344,968

Revaluation increment/(decrement) 983,173 (301,269)

Balance 30 June 2013 1,026,872 43,699

Financial assets reserve records unrealised gains on revaluation of financial assets to fair value.

12 cAPITAlIsed PROFIT On Re-ARRAngemenT OF InvesTmenTs & cAPITAl dIsTRIbuTIOns

Balance 30/6/12

$

Allocation of Realised Profit/(Loss) on Rearrangement

of INvestments & Capital Distributions

$

Balance 30/6/13

$

Research Fund

General 119,187 5,191 124,378

Anselmi Estate 42,691 1,859 44,550

Ivy May Stephenson 110 5 115

D.W. Research Funds 4,707,386 177,123 4,884,509

Esme Anderson 840,610 36,265 876,875

G.J. Williams 144,263 6,280 150,543

B. Mitchell 142,327 6,280 148,607

Dame Ida Mann 201,142 8,819 209,961

Ronald & Lois Lowe 125,883 5,857 131,740

6,323,599 247,679 6,571,278


13 AccumulATed Funds

Note2013

$2012

$

(a) Administration

Accumluated Deficits - 1 July 2012 - -

Total Comprehensive Income 825 8,995

Total available for appropriation 825 8,995

Aggregate of amounts transferred from Administration 13 (b) (825) (8,995)

Accumulated Deficits - 30 June 2013 - -

(b) Trust Fund

Retained income - 1 July 2012 452,776 371,151

Total Comprehensive Income (66,097) 85,191

Total available for appropriation 386,679 456,342

Aggregate of amounts transferred to General/Capital Funds

Administration 825 8,995

Research Trust 13 (a) (40,939) (12,561)

Retained income - 30 June 2013 346,565 452,776

14 gRAnTs AllOcATed / mAde duRIng The yeAR

2013 $

2012 $

Dr Alex Hewitt & Dr Stuart Macgregor - 46,270.00

Dr Glyn Chidlow - 49,960.00

A/Prof Jamie Craig & Dr David Dimasi - 49,250.00

Dr John Wood - 49,725.00

Dr Shiwani Sharma, Dr Kathryn Burdon & prof Jozef Gecz - 50,000.00

Dr Rohan Essex, Fr Willie Campbell, Dr Alex Hunyor Jr & Dr Paul Connell

- 48,755.00

A/Prof Ian Trounce - 44,800.00

Prof Ian McAllister, A/Pfor LRS Vijayasekaran, Prof Degli-Esposti & A/Prof Yu

- 49,995.00

Dr Weiyong Shen & Dr Ling Zu - 50,000.00

Dr Hannah Forward, Dr Charlotte McKnight & Dr Alexander Tan - 50,000.00

Dr Jelena Kezic - 45,940.00

Prof Glen Gole, Dr Nigel Barnett & Prof Steve Bottle 50,000.00 -

A/Prof Robyn Jamieson, A/Prof John Grigg & Ms Tina Lamey 50,000.00 -

Dr Alice Pebay, A/Prof Ian Trounce & Dr Karina Needham 50,000.00 -

A/Prof Nick Di Girolamo 48,500.00 -

Dr Matthew Rutar, Dr Trent Sandercoe & Dr Riccardo Natoli 49,500.00 -

Dr Peter Madden, Dr Peter Beckingsale, A/Prof Damien Harkin & Prof Traian Chirila

43,550.00 -

Dr Alex Hewitt, Dr Mirella Dottori & Dr Bryony Nayagam 49,500.00 -


Prof Keryn Williams, Dr Helen Brereton & Emeritus Prof Doug Coster 49,500.00 -

Dr Vicki Chrysostomou & Dr Jelena Kezic 48,500.00 -

Dr Kathryn Burdon & Prof Jamie Craig 49,500.00 -

Dr Vivek Gupta & Dr Yuyi You 49,500.00 -

Dr Hitesh Peshavariya 49,500.00 -

Dr Nicole Van Bergen 48,000.00 -

Dr Guei-Sheung Liu, Prof Gregory Dusting, Dr Bang Bui, Prof Hong Zhang & Prof Ming-Hong Tai

49,500.00 -

Prof John McAvoy, A/Prof Frank J Lovicu & Dr L J Dawes 48,000.00 -

Prof Robert Casson 48,500.00 -

Dr Fred Chen 42,000.00 -

Dr Stewart Lake, Dr Tim Henderson & A/Prof Henry Newland 45,000.00 -

868,550 534,695

Deduct Contribution from:

Glaucoma Foundation Australia Inc 49,750 72,005

RANZCO Eye Foundation 170,000 120,000

219,750 192,005

648,800 342,690

15 Funds AllOcATed TO dIRecTOR OF OPhThAlmIc ReseARch - vIcTORIA

Balance as at 1 July 2012 186,000 171,000

Interest for the year 2,196 2,530

Allocation for year 175,000 186,000

363,196 359,530

Payment made to Director of Research 188,196 173,530

Balance as at 30 June 2013 175,000 186,000

16 AudITORs RemuneRATIOn

Auditing accounts 4,950 4,950

Other services - -

4,950 4,950


17 RecOncIlIATIOn OF neT cAsh PROvIded by OPeRATIng AcTIvITIes TO ResulTs FOR yeAR

Net Surplus/(Deficit)

- Trust Fund 1,164,755 (481,098)

- Administration 825 8,995

1,165,580 (472,103)

Depreciation 1,929 689

Provision for Employee Benefits 174 3,908

(Increase)/Decrease in Receivables (38,983) 208,586

Increase/(Decrease) in Creditors and Accrued Expenses (4,349) 763

Increase/(Decrease) in Grants Payable 235,452 (85,302)

Increase/(Decrease) in allocation to Director of Research - Victoria (11,000) 15,000

Valuation Gains on available-for-sale financial assets (983,173) 301,269

Imputation Credit on WOW in-specie distribution 912 -

(Profit)/Loss on Rearrangement of Investments (247,679) 265,019

Net Cash Provided by /(used in) Operating Activities 118,863 237,830

18 dIsclOsuRes On dIRecTORs And OTheR key mAnAgemenT PeRsOnnelDIRECTORSThe following director received grants during the year. These are detailed at note 14.

Dr Fred Chen

The names of the directors who have held office during the financial year are:

Professor Stuart L Graham, Sydney (Chairman)

Professor Mark Gillies, Sydney (Vice Chairman)


Dr Wilson Heriot, Melbourne (Honorary Treasurer)

Dr Fred Chen, Perth


Professor J Crowston, Melbourne

A/Prof Mark D Daniell, Melbourne



Professor David Mackey, Perth

Professor Peter J McCluskey, Sydney




Professor Tien Wong, Melbourne


KEY MANAGEMENT PERSONNELOther Key Management Personnel include Executive Officer, Anne Dunn Snape.

Key management personnel are those persons having authority and responsibility for planning, directing and controlling the activities of the entity, directly or indirectly, including any director (whether executive or otherwise) of that entity. Control is the power to govern the financial and operating policies of an entity so as to obtain benefits from its activities.

KEY MANAGEMENT PERSONNEL COMPENSATIONKey Management Personnel has been taken to comprise the directors and one member of the executive management responsible for the day to day financial and operational management of the entity.

2013 $

2012 $

(a) Short-term employee benefits 69,531 58,908

(b) Post-employment benefits 8,323 6,600

(c) Other long-term benefits - -

(d) Termination benefits - -

(e) Share-based payment - -

77,854 65,508

19 FInAncIAl InsTRumenTs

(A) FINANCIAL RISK MANAGEMENT POLICIESThe entity’s financial instruments consist mainly of deposits with banks, local money market instruments, short-term investments, accounts receivable and payable.

The entity does not have any derivative instruments at 30 June 2013.

(i) Treasury Risk Management An investment committee consisting of Board members of the entity meet on a regular basis to analyse financial

risk exposure and to evaluate treasury management strategies in the context of the most recent economic conditions and forecasts.

The committee’s overall risk management strategy seeks to assist the entity in meeting its financial targets, whilst minimising potential adverse effects on financial performance.

Risk management policies are approved and reviewed by the Board on a regular basis. These include credit risk policies and future cash flow requirements.

(ii) Financial Exposures and Management Risk The main risks the entity is exposed to through its financial instruments are interest rate risk, liquidity risk and credit risk.

Interest rate risk Interest rate risk is managed with a mixture of fixed and floating rates on investments.

Foreign currency risk The entity is not exposed to fluctuations in foreign currencies.

Liquidity risk The entity manages liquidity risk by monitoring forecast cash flows.

Credit risk The maximum exposure to credit risk, excluding the value of any collateral or other security, at balance date to recognised financial assets, is the carrying amount, net of any provisions for impairment of those assets, as disclosed in the statement of financial position and notes to the financial statements.

The entity does not have any material credit risk exposure to any single receivable or group of receivables under financial instruments entered into by the entity.

Price risk The group is not exposed to any material commodity price risk.


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,116

,652

8,

729,

534

Fina

ncia

l Lia

bili

ties

Pay

able

s-

--

--

--

-70

0,48

748

0,62

070

0,48

748

0,62

0

Tota

l Fin

anci

al L

iabi

litie

s-

--

--

-70

0,48

748

0,62

070

0,48

748

0,62

0

Net

Fin

anci

al A

sset

s1,

093,

773

957,

903

-23

5,00

0-

-8,

322,

392

7,05

6,01

19,

416,

165

8,24

8,91

4


(C) NET FAIR VALUESThe net fair values of listed investments have been valued at the quoted market bid price at balance date. For other assets and other liabilities the net fair value approximates their carrying value. No financial assets and financial liabilities are readily traded on organised markets in standardised form other than listed investments.

The aggregate net fair values and carrying amounts of financial assets and financial liabilities are disclosed in the statement of financial position and in the notes to and forming part of the financial statements.

(D) SENSITIVITY ANALYSIS

Interest Rate RiskThe entity has performed a sensitivity analysis relating to its exposure to interest rate risk at balance date. This sensitivity analysis demonstrates the effect on the current year results and equity which could result from a change in this risk.

Interest Rate Sensitivity Analysis:At 30 June 2013, the effect on profit and equity as a result of changes in the interest rate, with all other variables remaining constant, would be as follows:

2013Carrying Amount

Interest Rate Risk

$ -1% Profit -1% Profit -1% Equity -1% Equity

Financial Assets

Cash and Cash Equivalents 1,093,773 (10,938) 10,938 (10,938) 10,938

2012Carrying Amount

Interest Rate Risk

$ -1% Profit -1% Profit -1% Equity -1% Equity

Financial Assets

Cash and Cash Equivalents 957,903 (9,579) 9,579 (9,579) 9,579


dIRecTORs’ declARATIOn

The directors of the company declare that:

1. the financial statements and notes as set out on pages 5 - 21:

(a) comply with Accounting Standards and Corporations Act 2001; and

(b) give a true and fair view of the financial position as at 30 June 2013 and performance for the

year ended on that date of the company.

2. In the directors’ opinion there are reasonable grounds to believe that the company will be able to pay its debts as and when they become due and payable.

The declaration is made in accordance with a resolution of the board of Directors.

On behalf of the Board.

Prof Stuart Graham Dr Wilson Heriot Director Director

Sydney, this 7 day of September 2013


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