IoO ECR Symposium 2026

Tuesday 9 Jun 2026, 08:45 → 18:20 Europe/London

Darwin Lecture Theatre (B40) (Darwin Building)

Giulia De Rossi (University College London), Marc Pabst (University College London)

Join us for the 9th IoO ECR Symposium on 9 June 2026! 

      

A day of engaging discussions and valuable networking with fellow early career researchers!

The ECR Committee warmly invites all researchers, students, and staff to attend this exciting event.

“It was really nice to have a lot of time for poster session, perfect to discuss with everyone!”

Previous ECR presenter

 

"I was really impressed by the diversity of research presented. The friendly, supportive atmosphere made it easy to ask questions and connect with other researchers."

Previous ECR attendee

 

Sponsors:

 

Registration Registration is open to all IoO/Moorfields staff and students. If you have a UCL account, please select EduGain and use your UCL credentials. Registration Deadline: 3 June 2026 (23:59 BST) Register now

 

Abstract Submission If you are an Early Career Researcher at the Institute of Ophthalmology (including students, research assistants/technicians, and postdoctoral researchers), we also strongly encourage you to submit your talk and/or poster abstract! Call for Abstracts Opens: 1 April 2026 Abstract Submission Deadline:  1 May 2026 (23:59 BST) Extended to 15 May (23:59 BST)! Submit abstract

 

1 Welcome

Speakers: Marc Pabst (University College London), Alison Hardcastle (UCL Institute of Ophthalmology)

Talks: Session 1 (2 talks)

2 Characterising neurovascular morphology in human diabetic retinopathy using 3D multiplexed immunohistochemistry

Diabetic retinopathy (DR) is one of the leading causes of irreversible blindness in people of working age. It is characterised by retinal vascular degeneration due to chronically elevated blood glucose levels. As the disease progresses, ischemia causes abnormal neovascularisation leading to retinal detachments, haemorrhage and vision loss.
We characterised pathological retinal vessel morphology in post-mortem diabetic retinopathy samples and healthy controls. We optimised multiplexed immunohistochemistry panels for staining 3D, cleared retinal wholemounts. Optical clearing of the retinal wholemounts with Ce3D was used to visualise the full retinal thickness.
We categorised multiple types of DR-specific vascular abnormalities, such as microaneurysms, venous beading and capillary decellularisation. We also identified rare structural features such as initiating neovascular tufts, identifiable only by imaging large 3D regions. The panel included markers for general vessel structures (Collagen IV, Isolectin-B4, UEA, alpha-SMA) as well as signalling molecules involved in vessel formation and maintenance (VEGFR-2, Iba1, Desmin). We then analysed the datasets using Imaris and Aivia software. We identified a spatial relationship between the frequency of devitalised capillaries, the severity of DR and the distribution of microglia. We also captured the expression of alpha-SMA in the lumen of forming neovascular tufts that could represent pericyte remodelling.
This approach allows us to image DR in an unprecedented way, to identify novel spatial changes in the retinal neurovascular unit, enabled only by 3D multiplexed imaging.

Speaker: Zuzanna Dzieniak (UCL Institute of Ophthalmology)

3 Modelling IFT140-associated Retinal Dystrophy

Ciliopathies are a group of disorders associated with pathogenic variants in genes that result in abnormal formation or dysfunction of cilia. Retinal degeneration is a common feature in ciliopathies. Pathogenic variants in Intraflagellar transport 140 (IFT140) are associated to syndromic and non-syndromic ciliopathies. Yet, the different effects of IFT140 variants in different tissues, especially in the retina, is not fully understood. The lack of appropriate human retinal models limited the investigation of IFT140 function in the retina and the development of targeted therapeutic strategies.

We investigated patient fibroblasts carrying different IFT140 variants, and identified variations in cilia length between these lines, that was accompanied by mislocalisation of IFT components, suggesting defective retrograde transport. These defects were visualised using ultrastructure expansion microscopy (U-ExM), which confirmed disrupted IFT transport.

To investigate the retinal consequences associated with IFT140 dysfunction, we characterized induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) and retinal organoids (ROs) models, from CRISPR/Cas9 IFT140 knockout (IFT140-KO), patient-derived c.1451C>T (p.T484M) and isogenic control (WT) lines. The results showed that loss of IFT140 led to shorter cilia and IFT88 accumulation at the cilia tip in iPSC-RPE. In iPSC-ROs, the IFT140-KO line displayed a thinner brush border corresponding to the photoreceptor outer segments, while the T484M line showed a phenotype intermediate between WT and IFT140-KO.

These results suggested that IFT140 deficiency disrupts cilia trafficking in human retinal cells and impairs photoreceptor development, providing mechanistic insight into retinal degeneration associated with IFT140-related ciliopathies.

Speaker: Yi Jiang

4 Moorfields Eye Charity, our new grants strategy and research opportunities

Speaker: Peter Bloomfield

5 Introduction to the BRC

Speakers: Helen Baker, Helen Khan

10:25 Morning Coffee Break

6 Growing Up in Science with Andrew Dick

Have you ever wondered what your favourite institute director struggled with as a graduate student? What they struggle with now? Growing up in Science is a conversation series featuring personal narratives of becoming and being a scientist.

Speakers: Marc Pabst (University College London), Giulia De Rossi (University College London)

Talks: Session 2 (3 talks)

7 Brainwide encoding of mixed navigational signals

Navigation is a complex goal-directed behaviour that requires the integration of sensation, reward, motion, and internal spatial representations to understand one’s position in the environment. Forms of spatial representations were identified in many brain regions beyond the hippocampal formation. However, these findings arose from a variety of experimental conditions, often not suited to isolate spatial from other navigational signals, such as visual landmarks.
Here we show that navigation recruits a distributed network of highly mixed-selective neurons tuned to combinations of running, sensation, reward, and position in an audiovisual virtual corridor. By decoupling position from external inputs and idiothetic processes, we found that all navigational signals contributed to activity of many of the 20.777 neurons we recorded across the mouse brain. Although the hippocampus showed a modest overrepresentation of spatial processes, like most regions it contained highly mixed-selective neurons.
Our study is the first to systematically demonstrate that spatial navigation engages brainwide, mixed representations of sensation, reward, motion, and position.

Speaker: Enny van Beest (UCL - Institute of Ophthalmology)

8 Developmental Dynamics of the African Turquoise Killifish Retina: From Early Retinogenesis to Rapid Post-Embryonic Expansion

The retina, a light-sensitive tissue at the back of the eye, undergoes neurodegeneration and vision loss with age. Progress in understanding these processes, and in developing treatments for age-related retinal diseases, has been limited by the difficulty of studying aged human tissue and the constraints of traditional animal models. The African turquoise killifish, with its short lifespan of approximately 6 months and conserved retinal structure, provides a powerful alternative model. Previous studies have focused on late-adult stages, showing that killifish recapitulate key features of human retinal ageing. However, understanding how neurons and glia deteriorate with age requires first defining how these cells are established during development. Here, we characterize retinogenesis in killifish from embryonic stages using histology, including the multiplexed immunohistochemistry method we adapted called IBEX. Retinal structure and histogenesis were found to be broadly conserved with zebrafish and mammals. Following hatching, killifish undergo accelerated growth during juvenile stages. We analyzed retinal progenitor proliferation and the ciliary marginal zone (CMZ), identifying enhanced proliferation and expanded stem cell niches during this period. Bulk RNA sequencing of juvenile retinas, combined with pharmacological inhibition of Notch and IGF1R signaling, identified conserved candidate genes and pathways driving this accelerated growth. Our further investigation into the proliferative capacity of the killifish retina has shown that, in addition to lifelong proliferation in the CMZ, the outer nuclear layer of the central retina also continues to proliferate throughout life - a phenomenon not previously described in other model organisms. Single-cell RNA sequencing further revealed genes driving this photoreceptor proliferation in the adult retina. These findings open new avenues for retinal regenerative research by enabling direct investigation of photoreceptor proliferation mechanisms. Together, this work provides the first comprehensive characterization of retinal development in killifish, establishing a foundation for studying retinal neurodegeneration and regeneration across the lifespan.

Speaker: Aleksandra Krzywanska (University College London, Institute of Ophthalmology)

9 Antisense oligonucleotide allele-specific targeting of EFEMP1 in a patient-derived model of Doyne honeycomb retinal dystrophy

Doyne honeycomb retinal dystrophy is an incurable juvenile macular dystrophy that leads to visual impairment by early to mid-adulthood. It is an autosomal dominant disorder caused by a c.1033C>T, p.Arg(345Trp) variant in EFEMP1, and is characterised by the early onset extracellular deposition of drusen between the retinal pigment epithelium basement membrane and underlying layers of Bruch’s membrane. In this study, we developed an antisense oligonucleotide approach to target EFEMP1. We reprogrammed patient-derived renal epithelial cells to induced pluripotent stem cells followed by directed differentiation to retinal pigment epithelium and compared the phenotype to gene-corrected and EFEMP1 knockout patient-derived retinal pigment epithelium. In the patient-derived disease model, remodelling of the extracellular matrix occurred with progressive accumulation of extracellular deposits containing the drusen-associated proteins apolipoprotein E and collagen IV, in addition to EFEMP1. Moreover, the intracellular accumulation of neutral lipids was evident. We developed an allele-specific antisense oligonucleotide which specifically and effectively promoted the clearance of the EFEMP1 c.1033C>T transcript in the patient-derived disease model following assisted or gymnotic delivery. In this disease model, gymnotic delivery led to a decrease in extracellular deposits and cleared the intracellular accumulation of lipids, even after the onset of this disease phenotype, suggesting this could be a practical and effective therapeutic approach.

Speaker: Farah Olivia Rezek (UCL)

10 The Equality Challenge Team

Speaker: Ah-Lai Law

11 Introduction to the IoO Mentoring Scheme

Speaker: Andrea Martello (University College London)

Flash Talks: Session 1

12 cGMP analogues for understanding and targeting early pathological changes in LCA4

Leber congenital amaurosis type 4 (LCA4) is an early-onset inherited retinal disorder caused by pathogenic variants in AIPL1. Dysfunction of AIPL1 compromises the stability of phosphodiesterase 6 (PDE6), leading to non-homeostatic accumulation of cyclic guanosine monophosphate (cGMP), which is strongly implicated in photoreceptor degeneration. Despite this, the initial cellular and molecular disturbances triggered by elevated cGMP remain insufficiently characterised.
To investigate these early disease mechanisms, we have established three-dimensional retinal organoids derived from induced pluripotent stem cells (iPSCs) reprogrammed from an LCA4 patient carrying a homozygous c.834G>A (p.W278X) mutation. CRISPR/Cas9-mediated correction of this variant enabled generation of a genetically matched control line, while targeted disruption of AIPL1 in control iPSCs produced an isogenic knockout model. These complementary systems recapitulate key disease-associated features, including PDE6 depletion and increased intracellular cGMP levels.
We have assessed the capacity of selected cGMP analogues to modulate downstream signalling events and ameliorate molecular and functional abnormalities associated with cGMP dysregulation. Transcriptomic profiling has already identified pronounced alterations in pathways governing ion regulation and intracellular signalling. These findings will be substantiated through quantitative gene expression analysis, immunohistochemical assessment, and in situ enzymatic assays. In parallel, two-photon calcium imaging techniques have been tested, and will be employed to examine functional perturbations in calcium homeostasis.
This work aims to define early pathogenic processes in LCA4, and to establish a foundation for therapeutic strategies targeting disease progression prior to irreversible photoreceptor loss.

Speaker: Erika Aguzzi

13 Local Rules, Global Order: How Müller Glia Self-Organise in the Developing Retina

During development, cells self-organise into functional tissues through local interactions, yet the mechanisms coordinating form with function remain poorly understood. Müller glia (MG), the principal glia of the retina, establish non-overlapping territories that span all retinal layers, providing metabolic support and synaptic regulation. How MG coordinate territorial organisation during morphogenesis is unclear. Here I investigate the cellular dynamics and molecular mechanisms underlying MG territorial development in the zebrafish retina using in vivo imaging. MG morphogenesis was characterised throughout morphogenesis using high-resolution imaging (60-120 hours post-fertilisation), revealing progressive elaboration of processes in the neuropil, which coincided with neuronal synaptic maturation. MG branches become increasingly complex while territories transition from overlapping to spatially organised domains. To identify molecular regulators, I employed single-cell RNA sequencing datasets across these developmental timepoints, revealing dynamic expression of adhesion molecules, cytoskeletal regulators, and signalling pathways. Ligand-receptor interactome analysis identified candidate pathways mediating MG-neuron and MG-MG communication during development. HCR validation of 20 candidate genes confirmed MG expression across morphogenesis, with a subset showing dynamic upregulation coinciding with the onset of territorial establishment.

To evaluate what local cellular interactions are sufficient to generate spatial organisation and guide experimental focus, I developed an agent-based model of MG morphogenesis. Simulations implementing contact-mediated interactions demonstrated that homotypic repulsion alone can produce spatially organised territories, with branching rate emerging as a critical determinant of morphological complexity and territorial stability. Together, this work establishes a framework combining experimental observation with computational modelling to understand glial morphogenesis, revealing how complex tissue architecture can emerge from local perception-action loops in which individual cells sense their environment and adapt their form accordingly.

Speaker: Gina Gilpin (UCL / Francis Crick Institute)

14 Genetic investigation and iPSC-derived corneal cell modelling of familial keratoconus

INTRODUCTION
Keratoconus (KC) is a complex, progressive corneal ectasia and one of the most common causes of visual deterioration in young adults. Genetic risk contributes to the development of KC, but we lack understanding of the aetiology of this condition. Here we aim to complement our GWAS approaches with experimental investigation of familial KC, with the potential to identify rare variants not detected in GWAS and variants with larger effect that confer substantial risk of KC.
METHODS
We recruited a KC pedigree with multiple affected individuals across two generations for whole genome sequencing (WGS) and urine sample collection to generate iPSCs. WGS analysis initially focussed on any shared haplotypes that encompass previously identified GWAS loci and rare variants within these loci were assessed for potential pathogenicity. Candidate genes were assessed for biological relevance and corneal expression. Using a newly developed protocol, we differentiated corneal epithelial-like cells (CEpi) from iPSCs and performed immunocytochemistry to test for expression of corneal epithelial cell markers and bulk RNA sequencing.
RESULTS
Two haplotypes encompassing GWAS loci that fully or partially segregated with KC were identified encompassing the candidate genes RORA, DOCK9, COL4A1 and COL4A2. CEpi were differentiated from iPSCs for one KC patient and one control line as a pilot study. Successful differentiation was demonstrated using ICC for CEpi markers (K14, K3, P63 and PAX6). Bulk RNA sequencing confirmed CEpi differentiation and enabled identification of dysregulated genes and variants in candidate loci. COL4A1 and COL4A2 were downregulated in patient CEpi and rare intronic variants were identified in both genes on chr13q32.1-q34.
CONCLUSION
Integrating genome sequencing with iPSC-derived CEpi models for familial KC holds promise for identifying genes, variants and pathways that confer substantial risk of developing KC. We aim to expand this cohort and perform transcriptomics on a larger scale to validate candidate genes and pathways identified in this pilot study.

Speaker: Dr Freddie L. Braddock (UCL Institute of Ophthalmology, University College London, London, UK)

15 Developing an mRNA therapy for RLBP1-retinopathy

Background: Patients with biallelic mutations in RLBP1 gene develop a spectrum of autosomal recessive rod-cone dystrophies, for which there are currently no approved treatments. RLBP1 encodes cellular retinaldehyde binding protein 1 (CRALBP), a 36-kD protein that regenerates the visual chromophore 11-cis retinal. In this study, we aimed to assess the efficacy of RLBP1-mRNA therapy introduced to a patient induced pluripotent stem cell (iPSC) derived RPE using lipid-based nanoparticles (LNP).
Methods: Fibroblasts from one patient harbouring compound heterozygous variants in RLBP1 c.250del p.(Val84Trpfs*35) and c.361C>T p.(Arg121Trp) were isolated and cultured. RLBP1 and control iPSC-RPE lines were generated by reprogramming and subsequent differentiation. RLBP1-mRNA was transfected to RPE cells with a LNP, and CRALBP protein expression was assessed.
Results: mRNA coupled with LNP was successfully delivered to iPSC-derived mature and polarized RPE cells. CRALBP was not detected in untreated patient RPE cells, but expression levels after RLBP1-mRNA transfection reached ~30-50% compared to control RPE cells.
Conclusions: RLBP1-mRNA-LNP treatment rescues the missing protein in patient cells, making it a promising therapeutic option for individuals carrying variants in the RLBP1 gene. The next step will be to assess the function of the produced CRALBP protein by measuring 11-cis retinal production following transfection.

Speaker: Sara Romero Vázquez (UCL Institute of Ophthalmology, London EC1V 9EL, UK)

12:35 Lunch

Please join us for lunch outside the lecture theatre.

Posters: Session 1

16 Developing an mRNA therapy for RLBP1-retinopathy

Background: Patients with biallelic mutations in RLBP1 gene develop a spectrum of autosomal recessive rod-cone dystrophies, for which there are currently no approved treatments. RLBP1 encodes cellular retinaldehyde binding protein 1 (CRALBP), a 36-kD protein that regenerates the visual chromophore 11-cis retinal. In this study, we aimed to assess the efficacy of RLBP1-mRNA therapy introduced to a patient induced pluripotent stem cell (iPSC) derived RPE using lipid-based nanoparticles (LNP).
Methods: Fibroblasts from one patient harbouring compound heterozygous variants in RLBP1 c.250del p.(Val84Trpfs*35) and c.361C>T p.(Arg121Trp) were isolated and cultured. RLBP1 and control iPSC-RPE lines were generated by reprogramming and subsequent differentiation. RLBP1-mRNA was transfected to RPE cells with a LNP, and CRALBP protein expression was assessed.
Results: mRNA coupled with LNP was successfully delivered to iPSC-derived mature and polarized RPE cells. CRALBP was not detected in untreated patient RPE cells, but expression levels after RLBP1-mRNA transfection reached ~30-50% compared to control RPE cells.
Conclusions: RLBP1-mRNA-LNP treatment rescues the missing protein in patient cells, making it a promising therapeutic option for individuals carrying variants in the RLBP1 gene. The next step will be to assess the function of the produced CRALBP protein by measuring 11-cis retinal production following transfection.

ECR symposium poster_2026.pdf

17 Omics data challenge the proposed role for TSAd in vascular permeability

Excessive vascular permeability causes damaging tissue oedema in neovascular and inflammatory diseases and is, in part, driven by the vascular endothelial growth factor VEGF. Several molecular pathways have been implicated in VEGF-induced permeability signal transduction, including recruitment of a SRC family kinase by the VEGF-activated tyrosine kinase receptor VEGFR2 via the T-cell specific adaptor TSAd. To assess the expression of signal transducers in this pathway across organ vasculature in an unbiased manner, we examined bulk and single-cell RNAseq, proteomics and epigenomics EC data. All known signa transducers proposed to act in VEGF-induced permeability signal transduction could be detected, except TSAd, which was not detected or detected at extremely low levels, concordant with known models of leaky transcription. Extremely low or absent TSAd transcripts and/or protein were also found in datasets derived from VEGF-stimulated ECs and ECs of tumours with increased vascular leakiness. Epigenomics data indicated that the TSAd promoter was located in closed chromatin in normal and tumour ECs. Overall, our results suggest that TSAd is unlikely to be a critical signal transducer in VEGF-induced permeability signalling.

ECR_2026_Guillermo_Diez_Pinel_SUBMISSION.pdf

18 Local Rules, Global Order: How Müller Glia Self-Organise in the Developing Retina

During development, cells self-organise into functional tissues through local interactions, yet the mechanisms coordinating form with function remain poorly understood. Müller glia (MG), the principal glia of the retina, establish non-overlapping territories that span all retinal layers, providing metabolic support and synaptic regulation. How MG coordinate territorial organisation during morphogenesis is unclear. Here I investigate the cellular dynamics and molecular mechanisms underlying MG territorial development in the zebrafish retina using in vivo imaging. MG morphogenesis was characterised throughout morphogenesis using high-resolution imaging (60-120 hours post-fertilisation), revealing progressive elaboration of processes in the neuropil, which coincided with neuronal synaptic maturation. MG branches become increasingly complex while territories transition from overlapping to spatially organised domains. To identify molecular regulators, I employed single-cell RNA sequencing datasets across these developmental timepoints, revealing dynamic expression of adhesion molecules, cytoskeletal regulators, and signalling pathways. Ligand-receptor interactome analysis identified candidate pathways mediating MG-neuron and MG-MG communication during development. HCR validation of 20 candidate genes confirmed MG expression across morphogenesis, with a subset showing dynamic upregulation coinciding with the onset of territorial establishment.

To evaluate what local cellular interactions are sufficient to generate spatial organisation and guide experimental focus, I developed an agent-based model of MG morphogenesis. Simulations implementing contact-mediated interactions demonstrated that homotypic repulsion alone can produce spatially organised territories, with branching rate emerging as a critical determinant of morphological complexity and territorial stability. Together, this work establishes a framework combining experimental observation with computational modelling to understand glial morphogenesis, revealing how complex tissue architecture can emerge from local perception-action loops in which individual cells sense their environment and adapt their form accordingly.

19 Celastrol Nanoparticle Exhibits Neuroprotective Effects in a Rat Model of RGC

Purpose: Celastrol, a quinone methide triterpenoid isolated from Tripterygium wilfordii, possesses antioxidant, anti inflammatory and anti apoptotic properties, making it a promising neuroprotective candidate for retinal neurodegenerative diseases such as glaucoma. However, effective therapeutic rescue and preservation of retinal ganglion cells (RGCs) remain an unmet clinical need. The partial optic nerve transection (pONT) model is a well established model of primary and secondary RGC degeneration. This study evaluated the neuroprotective efficacy of a novel nanoformulated Celastrol eye drop in preserving RGCs following pONT injury.

Methods: Celastrol nanoparticles (Cel NPs) were prepared using a thin film rehydration method and characterised by high performance liquid chromatography, dynamic light scattering, and X ray diffraction. In vitro, R28 cells exposed to cobalt chloride were treated with different concentrations of Cel NPs to assess cytoprotective effects. For in vivo evaluation, Dark Agouti rats subjected to pONT received daily topical treatment for 3 weeks with Cel NP, vehicle, Cel only solution, or nerve growth factor (NGF), alongside untreated pONT and healthy control groups. At the study endpoint, retinal apoptosis was assessed using Detection of Apoptosing Retinal Cells (DARC) imaging. Retinal wholemounts were immunostained for RBPMS to quantify RGC survival and IBA 1 to assess microglial reactivity. Superior and inferior retinal quadrants were analysed separately to distinguish effects on primary and secondary degeneration.

Results: Cel NPs showed an encapsulation efficiency greater than 95% and a particle size below 20 nm. In vitro, Cel NPs at an optimal concentration significantly improved cell viability compared with untreated injured cells. In vivo, treatment preserved RGC survival after pONT, with a more pronounced protective effect in the inferior retina, where RGC density was comparable to that of healthy controls. This regional pattern suggests stronger protection against secondary degeneration. Cel NP treatment also reduced hyper ramified and activated microglia in the inferior retina, indicating partial attenuation of injury associated neuroinflammatory responses.

Conclusions: Topical Cel NP demonstrated neuroprotective potential in the pONT model by reducing RGC loss and modulating microglial activation, with particularly strong effects in retinal regions associated with secondary degeneration.

Kun poster.pdf

20 cGMP analogues for understanding and targeting early pathological changes in LCA4

Leber congenital amaurosis type 4 (LCA4) is an early-onset inherited retinal disorder caused by pathogenic variants in AIPL1. Dysfunction of AIPL1 compromises the stability of phosphodiesterase 6 (PDE6), leading to non-homeostatic accumulation of cyclic guanosine monophosphate (cGMP), which is strongly implicated in photoreceptor degeneration. Despite this, the initial cellular and molecular disturbances triggered by elevated cGMP remain insufficiently characterised.
To investigate these early disease mechanisms, we have established three-dimensional retinal organoids derived from induced pluripotent stem cells (iPSCs) reprogrammed from an LCA4 patient carrying a homozygous c.834G>A (p.W278X) mutation. CRISPR/Cas9-mediated correction of this variant enabled generation of a genetically matched control line, while targeted disruption of AIPL1 in control iPSCs produced an isogenic knockout model. These complementary systems recapitulate key disease-associated features, including PDE6 depletion and increased intracellular cGMP levels.
We have assessed the capacity of selected cGMP analogues to modulate downstream signalling events and ameliorate molecular and functional abnormalities associated with cGMP dysregulation. Transcriptomic profiling has already identified pronounced alterations in pathways governing ion regulation and intracellular signalling. These findings will be substantiated through quantitative gene expression analysis, immunohistochemical assessment, and in situ enzymatic assays. In parallel, two-photon calcium imaging techniques have been tested, and will be employed to examine functional perturbations in calcium homeostasis.
This work aims to define early pathogenic processes in LCA4, and to establish a foundation for therapeutic strategies targeting disease progression prior to irreversible photoreceptor loss.

21 Creating a mouse model for a newly discovered autosomal-dominant form of Retinitis Pigmentosa.

Retinitis Pigmentosa (adRP) is the most common Inherited Retinal Dystrophy (IRD). The autosomal-dominant form (adRP) accounts for 25-30% of the cases. adRP primarily affects rod photoreceptors and can progress into the central retina, affecting cone photoreceptor function and survival. We recently identified multiple genomic structural variants (SVs) at the previously unsolved RP17 locus on Chr17q22. These SVs are the cause of IRD in a growing number of families affected by adRP, although the mechanism of disease is currently unknown. These complex SVs have the potential to reorganise the 3D structure of the genome. This study aims to uncover mechanisms of disease for adRP caused by SVs at the RP17 locus by producing and characterising mouse models with similar SVs.
Genetically altered (GA) mice were generated using CRISMERE to recreate a patient SV in the syntenic region of the mouse genome (Chr11qC), that contains conserved genes and regulatory regions, including Gdpd1, Ypel2 and a retinal enhancer. This resulted in several different genomic rearrangements, including deletions, duplications and inversions. The RP17 mice were characterised on a genomic, transcriptomic, proteomic and visual functional level. Optical Genome Mapping (OGM) was used to validate the genomic rearrangements. RT-qPCR, RNAseq and Western Blots were used to create transcriptomic and proteomic profiles on each line, and mice were tested for visual functionality by Electroretinography (ERG) and Optical Coherence Tomography (OCT).
The data show that the mouse model recapitulates key molecular changes observed in patient-derived retinal cells, including dysregulation of genes within the locus. Despite these molecular alterations, however, the mice do not exhibit overt retinal degeneration. Importantly, differences in genomic organisation led to variable effects on Gdpd1 expression. Despite carrying equivalent gene copy numbers, the duplication and duplicated inversion have different levels of Gdpd1 matching the predicted effects on the 3D folding of the genome, repositioning genes relative to their regulatory elements and thereby dysregulating their expression. In contrast, Ypel2 expression remains largely stable. These findings support disrupted TAD boundaries as a potential disease model and provide a framework for designing small molecule therapies to correct aberrant gene expression.

IoO ECR Poster 2026 Sara.pdf

22 Development of Paediatric Isogenic Posterior Ocular Mimetics for Myopia Research

Myopia is a leading global ophthalmologic disorder, with 50% of the population projected to be affected by 2050. Current management focuses on optical correction, neglecting the underlying pathological remodeling of posterior ocular tissues. Furthermore, animal models are limited in translational validity to human outcomes due to interspecies differences.
To overcome these challenges, we developed a paediatric isogenic posterior ocular tissue mimetic (IPOM) using primary scleral and choroidal fibroblasts from paediatric donor sclera and choroid tissues. These were reprogrammed into iPSCs and differentiated into RPE, endothelial cells, and pericytes to ensure a donor-matched background. IPOM was engineered within a tri-layer architecture: a compressed collagen scleral layer mimicking native stiff sclera, a vascularized choroid, and an RPE monolayer. Over 21 days, the IPOM successfully demonstrated matrix remodeling, characterized by significant reductions in both scleral and choroidal thickness as cells actively interacted with the collagen matrix. By Day 21, dense CD31+ vascular networks in choroid layer matured into fenestrated, PLVAP+ capillaries wrapped by PDGFRβ+ iPSC-pericytes, mirroring native choriocapillaris. Simultaneously, the iPSC-RPE layer established hallmarks including apical polarity, basal infoldings and E-cadherin+ junctional complexes.
IPOM is a novel engineered tissue mimetic that reproduces the architecture and vascularization of human posterior ocular tissues. This multi-cell-type IPSC-derived platform enables personalized modelling of posterior ocular pathology including myopia. IPOM is dynamic and exhibits biological responsiveness, establishing it as a robust human system for investigating the mechanisms of myopic progression and advancing personalized medicine.

IoO ECR symposium poster 090626.pdf

23 Distinguishing Stem Cell-Derived Microglia from Macrophages: Marker Identification in iPSC-Derived Immune Cells

Microglia are the resident immune cells of the central nervous system and are thought to play key roles in retinal disease. However, distinguishing microglia from infiltrating macrophages remains a challenge, as both cell types share overlapping transcriptional and functional profiles. This complicates interpretation of immune activation in retinal disorders such as age‑related macular degeneration (AMD) and limits the precision of in‑vitro disease models.
To address this, microglia‑specific markers were identified in induced pluripotent stem cell‑derived microglia (iPSC‑MG) through comparison with iPSC‑macrophages (iPSC‑MP). Bulk RNA sequencing and qPCR were used to compare transcriptional profiles of iPSC‑MG with iPSC‑MP and their precursors. These datasets were also compared with publicly available primary microglia and macrophage datasets. Candidate markers were then assessed for stability across different cell lines and additional cell types.
Commonly used “microglia‑specific” markers showed substantial overlap between iPSC‑MG and iPSC‑MP in both bulk RNA‑seq and qPCR analyses. New microglia‑enriched markers were identified and demonstrated repeated specificity in iPSC‑derived cells, although this specificity was not reflected in immortalised microglia (HMC3) or macrophages (THP‑1‑derived). Despite these differences, comparative analysis of iPSC‑MG with primary microglial datasets showed significant transcriptional overlap, supporting microglia‑like behaviour of the iPSC‑derived cells.
Refining a reliable set of microglial markers contributes to clearer biological definitions of microglial identity and provides practical tools for distinguishing microglia from macrophages in retinal research. Establishing these markers is essential for improving in‑vitro disease models, strengthening interpretation of immune responses, and ultimately supporting more accurate studies of AMD and other neuroinflammatory conditions.

24 Emergence and Compensation of Eye Size Asymmetry During Early Eye Development in Zebrafish

The vertebrate eye originates from a single forebrain-derived eye field, which splits into two optic vesicles during evagination. While this process is generally assumed to generate bilaterally symmetric structures, it remains unclear whether symmetry is established during this initial evagination or is subsequently rectified.
To address this, we used zebrafish as a vertebrate model because their external and transparent embryonic development enables direct observation of early eye morphogenesis. We quantified eye volume in zebrafish using transgenic lines expressing fluorescent proteins in the developing eye. Embryos were imaged using confocal microscopy, and eye volumes were reconstructed in three dimensions using Imaris. This allowed direct comparison of paired eye volumes within individual embryos. Size difference between left and right eyes were quantified as the normalized relative difference between paired eye volumes in individual embryos.
Our data suggest that measurable left–right size asymmetries are present at early developmental stages. Notably, size asymmetries are progressively reduced over time, suggesting that compensatory growth mechanisms that enable bilateral symmetry during eye development.
This study establishes a quantitative framework for assessing bilateral symmetry and provides initial evidence that organ size is actively regulated to ensure symmetry during development. Future work will extend this approach to eye-development mutants with a smaller eye field. This will allow us to test whether developmental perturbations increase left–right eye size asymmetry or impair the ability of compensatory growth to enable bilateral symmetry. Together, this work provides a quantitative framework for understanding how bilateral symmetry is dynamically achieved during organogenesis.

ECR POSTER Byron V4.pdf

25 Enhanced spatial mapping in a mouse model of retinitis pigmentosa

Vision plays a crucial role in navigation, particularly when tasks require spatial awareness and quick decision-making. Following loss of vision, compensatory approaches are needed to navigate, including use of other senses, memory of a journey (path retracing) and calculation of a homing path (path integration). Although it is commonly assumed that memory is too imprecise to serve as a reliable substitute for vision, this assumption has not been rigorously tested. Using mouse models of retinitis pigmentosa (Pde6brd1/rd1) in an open-field arena, we investigated the extent to which memory can compensate for vision loss. Surprisingly, we found that rd1 mice were able to recall the location of an escape door with high precision. Moreover, rd1 mice showed enhanced path retracing and path integration compared to wild-type controls, suggesting not just compensation but strengthening of memory-based navigation strategies. While their behaviour showed some differences to wild-type mice, overall performance indicates that memory can serve as a highly effective, even enhanced, mechanism for navigation in the absence of vision. These findings highlight the remarkable adaptability of navigation strategies despite visual impairment and may have important implications for understanding retinal degeneration in humans and developing interventions to assist visually impaired individuals in navigating complex environments.

ECR_poster.pdf

26 Extracellular matrix gene dysregulation and alternative splicing in Fuchs endothelial corneal dystrophy stratified by TCF4 repeat expansion status.

Title: Extracellular matrix gene dysregulation and alternative splicing in Fuchs endothelial corneal dystrophy stratified by TCF4 repeat expansion status.

Purpose: Fuchs Endothelial Corneal Dystrophy (FECD) is characterised by abnormal extracellular matrix (ECM) protein accumulation and corneal endothelial cell (CEC) loss. Most European cases (~80%) are associated with a pathogenic CTG repeat expansion (≥50 repeats) in the TCF4 gene. Here we aim to define transcriptomic alterations associated with TCF4 expansion status and their contribution to ECM dysregulation.

Methods: RNA-Seq data was generated from CECs derived from FECD patients with TCF4 repeat expansion (Exp+, n=3), without TCF4 repeat expansion (Exp-, n=3) and healthy controls (n=4). Differential gene expression analysis was performed using DESeq2 and ECM-related genes were identified though Gene Ontology (GO) enrichment analysis (PANTHER). Alternative splicing events were assessed using rMATS and visualized and mapped using Intergrative Genomics Viewer (IGV) and Sashimi plots.

Results: GO analysis identified 527 ECM-related genes. Differential expression analysis revealed distinct patterns of ECM gene dysregulation between groups. In Exp+ FECD, 63 genes were upregulated and 51 downregulated compared to controls, whereas in Exp- FECD, 23 genes were upregulated and 21 downregulated, with 15 genes shared in each category. rMATS identified 14 ECM-related genes with significant alternative splicing changes unique to Exp+, 4 unique to Exp-, and 14 shared between groups. Notably, in the FN1 gene, inclusion of the EDA and EDB domains, that are critical for FN1 protein function, was increased in the Exp+, while only EDB inclusion was increased in Exp- cases.

Conclusion: TCF4 repeat expansion status is associated with distinct ECM gene expression and splicing alterations patterns, likely contributing to FECD pathogenesis through abnormal ECM accumulation.

Ioannis Pyrousis_ECR_poster.pdf

Ioannis Pyrousis_ECR_poster.pptx

27 Galectin-1 as a pathological inducer of microvascular leakage in diabetic retinopathy

Introduction
Pathological leakage and angiogenesis are hallmark features of diabetic retinopathy (DR), with vascular endothelial growth factor (VEGF) prominently driving disease. However, therapeutic targeting of VEGF is only fully effective for around half of patients, pointing to important yet unknown VEGF-independent mechanisms. Galectin-1, a β-galactoside-binding protein encoded by LGALS1, can activate VEGF receptors and sustain angiogenesis in anti-VEGF-resistant tumors.

Objectives
We aimed to investigate whether galectin-1 is a pathologically relevant leakage inducer in retinopathy, particularly in DR.

Methods
We combined immunostaining, proteomic, and single-cell transcriptomic analyses of human aqueous humour and retinal tissue to define the expression pattern of galectin-1/LGALS1. Rodent ex vivo retinal explants and cultured microvascular endothelial cells were used to measure vascular permeability in response to galectin-1 under various conditions. In vivo, the function of Lgals1 was interrogated in mice carrying knockouts, choroidal neovascularisation lesions, or rendered diabetic by streptozotocin intoxication.

Results
Galectin-1 levels were significantly higher in human aqueous humour from patients with DR compared to those with only diabetes. Immunostaining of retinal sections showed predominant localisation of galectin-1 to blood vessels, consistent with single-cell transcriptomic data showing the strongest expression in vascular and perivascular cells, especially in the context of retinovascular disease. Functionally, galectin-1 induced vascular leakage comparable to VEGF, using the same receptors and triggering similar downstream signalling. Under hypoxic conditions, the response of endothelial cells to galectin-1 was sensitized approximately 30-fold, an effect that could be attributed to differential surface glycosylation of endothelial cells. Validation of galectin-1 as a pathological inducer of retinal vascular leakage in vivo is underway.

Conclusion
Galectin-1 is upregulated in pathological conditions, enriched in the retinal neurovascular unit, and promotes vascular leakage, especially during hypoxia. These findings highlight galectin-1 as a potential therapeutic target in DR.

ECR symposium 3 YX.pdf

28 Genetic investigation and iPSC-derived corneal cell modelling of familial keratoconus

INTRODUCTION
Keratoconus (KC) is a complex, progressive corneal ectasia and one of the most common causes of visual deterioration in young adults. Genetic risk contributes to the development of KC, but we lack understanding of the aetiology of this condition. Here we aim to complement our GWAS approaches with experimental investigation of familial KC, with the potential to identify rare variants not detected in GWAS and variants with larger effect that confer substantial risk of KC.
METHODS
We recruited a KC pedigree with multiple affected individuals across two generations for whole genome sequencing (WGS) and urine sample collection to generate iPSCs. WGS analysis initially focussed on any shared haplotypes that encompass previously identified GWAS loci and rare variants within these loci were assessed for potential pathogenicity. Candidate genes were assessed for biological relevance and corneal expression. Using a newly developed protocol, we differentiated corneal epithelial-like cells (CEpi) from iPSCs and performed immunocytochemistry to test for expression of corneal epithelial cell markers and bulk RNA sequencing.
RESULTS
Two haplotypes encompassing GWAS loci that fully or partially segregated with KC were identified encompassing the candidate genes RORA, DOCK9, COL4A1 and COL4A2. CEpi were differentiated from iPSCs for one KC patient and one control line as a pilot study. Successful differentiation was demonstrated using ICC for CEpi markers (K14, K3, P63 and PAX6). Bulk RNA sequencing confirmed CEpi differentiation and enabled identification of dysregulated genes and variants in candidate loci. COL4A1 and COL4A2 were downregulated in patient CEpi and rare intronic variants were identified in both genes on chr13q32.1-q34.
CONCLUSION
Integrating genome sequencing with iPSC-derived CEpi models for familial KC holds promise for identifying genes, variants and pathways that confer substantial risk of developing KC. We aim to expand this cohort and perform transcriptomics on a larger scale to validate candidate genes and pathways identified in this pilot study.

ECR poster symposium.pdf

29 Identification of a de novo Cyclin-C (CCNC) variant in microphthalmia

Microphthalmia is a rare congenital eye defect characterised by a small, underdeveloped eye. Notably, microphthalmia exhibits genetic heterogeneity, in which mutations in different genes can result in a similar clinical phenotype. Although gene panel testing covering 147 genes is currently used by clinicians in the United Kingdom for the genetic diagnosis of microphthalmia, approximately 70% of patients remain without a molecular diagnosis. This highlights the need to identify additional genes associated with the condition. By examining the de novo variants within the unresolved microphthalmia cohort recorded in Genomics England, we identified an individual with severe microphthalmia who had a heterozygous missense variant in CCNC. Further examination of the variant suggests that this change is rare with predicted pathogenicity. CCNC is a non-cycling cyclin that involves in transcription activity regulation in metazoan. To examine the effect of CCNC on eye development in vivo, we established a ccnc zebrafish mutant line using CRISPR/Cas9-mediated mutagenesis. ccnc disrupted embryos showed significant reduction in eye diameter, resembling the microphthalmia phenotype observed in the human proband. Together, these results support CCNC as a novel microphthalmia-associated gene in vertebrates.

30 In Situ Sequencing characterises ganglion-cell subset loss in a model of Glaucoma

Glaucoma is the leading cause of irreversible blindness. The pathophysiology is characterised by damage to retinal ganglion cells (RGCs), typically associated with raised intra-ocular pressure. An arising theory is that retinal macroglia can contribute to the progression of glaucoma when they become dysregulated. The project aims to better understand the glia and RGC interactions using RNA sequencing and protein imaging in a mouse model of glaucoma.

The silicone oil model was initiated by an intracameral injection into the anterior chamber of the mouse eye, creating a pupil block that disrupts the outflow of aqueous humor. CoppaFISH (COmbinatorial Padlock Probe Amplification Fluorescence In Situ Hybridisation), an RNA sequencing method, and IBEX (Iterative Bleaching Extends multipleXity), a multiplexed immunohistochemistry technique, were combined on the same tissue sections to classify RGC subtypes and changes to glia.

CoppaFISH and IBEX were successfully combined for RNA and protein detection, respectively, on the same mouse retinal tissue. 40 gene probes were designed and imaged, focused on RGC subsets and macroglia activation, identifying changes between silicone-oil injected and contralateral control eyes. Cell calling was attributed by genes spot counts in nuclei alongside protein labelling for the cell type and analysed with Aivia software. Correlations of subtype loss and macroglia activation was measured to explore their relationship to glaucoma progression.

CoppaFISH-IBEX has been successfully optimised to map the spatial landscape of the retina in a glaucoma-induced mouse model. This project will confirm the resiliency of RGC subtypes and relate these changes to macroglia activation, to better understand the progression of Glaucoma.

Lorna Fowler in situ sequencing poster.pdf

31 Influence of culture media on the in vitro functionality of ARPE-19 cellshere

Purpose: Adult retinal pigment epithelial-19 (ARPE-19) cell line is widely used to study RPE physiology. Despite its common use, these cells show poor RPE-like characteristics including limited pigmentation and epithelial morphology. In the present study, we aimed to investigate the impact of the culture media on the morphology, pigmentation, and functional properties of the ARPE-19 cells, assessed by Ca2+ imaging.

Methods: ARPE-19 cells were seeded at a density of 30 000 cells/cm2 on a Matrigel-coated 96 well plates. The cells were cultured for 6 weeks either in DMEM supplemented with 1 % FBS and 1% Pen/Strep or in the commercially available XVIVO-10 medium. For live-cell Ca2+ imaging experiments, indo-1 was used as the Ca2+ indicator dye. Cellular responses were examined following stimulation with 100 µM ATP.

Results: ARPE-19 cells cultured in XVIVO-10 developed an RPE-like cobblestone morphology with evident pigmentation, whereas cells cultured in DMEM lacked both pigmentation and cobblestone morphology. All cells, regardless of culture conditions, exhibited Ca2+ responses following ATP stimulation. However, cells cultured in XVIVO-10 showed overall faster response kinetics, characterised by a more rapid rise and decline. In addition, the duration of the responses was significantly shorter in XVIVO-10-cultured cells. Cells cultured in DMEM recovered to baseline or near baseline Ca2+ levels after ATP stimulation, whereas responses in XVIVO-10-cultured cells plateaued at approximately half of the peak response.

Conclusions: ARPE-19 cells are typically cultured in DMEM. Our results indicate that culturing ARPE-19 cells in XVIVO-10 leads to improved RPE-like characteristics, including morphology and pigmentation. Moreover, Ca2+ responses were faster in X-VIVO10-cultured cells, suggesting an enhanced capacity for Ca2+ release. However, recovery was impaired, as Ca2+ levels failing to return to pre-stimulation baseline.

32 Investigating common risk genes and molecular mechanisms in age-related macular degeneration and Alzheimer’s disease

Age-related macular degeneration (AMD) and Alzheimer’s disease (AD) are both severe age-related disorders that are the leading causes of vision loss and dementia in older adults, respectively. The diseases share similar risk profiles; ageing, smoking, and cardiovascular dysfunction, as well as similar pathologic processes; chronic inflammation, lysosome dysfunction and amyloid accumulation. Because of this, a key research area focuses on the diseases in tandem to identify whether there are common molecular/cellular mechanisms and thus whether treatments could be used to alleviate symptoms of both disorders. Our study extends this research in an unbiased genome-wide approach by directly comparing bulk-RNAseq data derived from AMD iPSC-macrophages with common pathways and genes associated with AD. Key genes identified through network co-expression analysis were then studied further in human microglial cell (HMC3) and iPSC-microglial cultures, to discern the effect of activation via different immunomodulatory regulators that induce AD-related pathological responses. As well as this, we knocked down the immune gene, CD74, to investigate the functional effects of CD74 on expression of key disease-associated genes associated with different immune microglial processes. Our findings implicate lysosome dysfunction and degradation of the extracellular matrix as key drivers in AMD pathology. As well as this, AMD disease-associated genes overlapped with key genes implicated in the disease-associated microglial (DAM) state which has been previously linked to AD, thus highlighting a critical role of immune cell dysfunction in AMD, and pointing to shared pathological drivers. However, we did not find critical overlap between macrophage activation state and disease, despite the association with disease-associated microglia. Finally, key risk genes TNF, CD33 and HEXB were found to functionally rely on the immune response of CD74 to protect against the impact of pro-inflammatory activators. Further studies should make use of CRISPR gene editing to discern the causal directional effect of key gene dysfunction within these AMD and AD pathologies. Collectively this work identifies new genes, pathways and processes involved in AMD, and some shared with AD, which may provide opportunities for new drug discovery research, or alternative methods to develop biomarkers to track disease stages better.

33 Investigating the role of GSK3 in N-Acetyl-L-Leucine (NALL)-mediated effects in cellular models of the lysosomal storage disease Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is a rare lysosomal storage disorder, most often caused by loss-of-function mutations in NPC1, resulting in pathological cholesterol and sphingolipid accumulation within late endosomes/lysosomes (LE/Lys). A recently-approved therapeutic, N-acetyl-L-leucine (NALL), was found to clear lysosomal lipids in NPC patient cells. NALL’s mechanism of action is not fully understood, but recent work from our lab showed that NALL normalises dysregulated inter-organelle membrane contact sites in NPC. The serine/threonine kinase Glycogen Synthase Kinase-3 Beta (GSK3β) has been implicated in contact site regulation and is recruited to the lysosome in response to leucine supplementation under starved conditions (Schwendener Frokel et al., 2024). Preliminary data suggest that NALL-mediated cholesterol clearance is GSK3β-dependent, which potentially implicates GSK3β in NALL's therapeutic effects. Here, NALL-mediated rescue of filipin-stained cholesterol accumulation in NPC patient cells is reproduced in NPC1-knockout/inhibited HeLa cells and the role for GSK3β supported. On GSK3β inhibition, NALL failed to rescue cholesterol accumulation in NPC1-knockout/inhibited cells. Current and future work aims to determine the effect of NALL on GSK3β’s subcellular localisation and if GSK3β activity is required for NALL-mediated normalisation of LE/Lys contacts with mitochondria and ER. Together, these findings will improve understanding of the molecular pathways underpinning NALL's therapeutic mechanism of action.

Research project poster .pdf

34 Measuring Contrast Sensitivity in glaucoma with functional magnetic resonance imaging - a proof-of-concept study

Glaucoma is primarily an ocular disorder, yet damage to the optic nerve causes widespread structural and functional alterations in the visual brain. Prior fMRI work suggests that the visual cortex maintains its coarse-scale retinotopic organization in glaucoma, but shows a significant reduction in BOLD amplitude—an alteration that persists beyond predictions based on perimetry. However, BOLD amplitude reductions alone do not directly quantify how visual processing and function are altered. Here, we address this gap by using fMRI to measure contrast sensitivity in the cortex (the neural contrast sensitivity function - nCSF) in glaucoma patients. We first demonstrate that the nCSF approach accurately predicts visual cortex responses to stimuli varying in spatial frequency and contrast in patients and controls. In five glaucoma patients and six healthy controls we found that patients displayed reduced contrast sensitivity as compared to healthy controls in both neural (nCSF) and behavioral (bCSF) measures, with consistent relationships observed between the two estimates. Notably, these differences were observed in the projection zone of the central visual field (0–5° eccentricity) of patients with early stage glaucoma, a region often considered largely spared by conventional standard automated perimetry. These findings provide preliminary evidence that behavioral contrast sensitivity deficits in glaucoma are reflected in the cortex. This highlights the potential of the nCSF as a tool for quantifying and understanding vision loss allowing the characterization of glaucoma’s impact along the visual pathway beyond the eye.

ecr-glauc-2026-v003.pdf

35 Mitochondrial Dysfunction Triggers Extracellular Matrix Remodelling In Hipsc-derived Retinal Pigment Epithelium

Age-related macular degeneration (AMD) is the primary cause of legal blindness in the developed world. However, AMD remains incompletely understood due to a complex interplay of genetic and environmental factors and currently lacks an effective cure. Central to disease progression is degeneration of the retinal pigment epithelium (RPE), which is essential for photoreceptor homeostasis. Although RPE mitochondrial dysfunction and extracellular matrix (ECM) alterations have been strongly implicated, the pathogenic sequence linking these processes remains unclear. Here, we sought to elucidate AMD pathogenesis by interrogating a hiPSC-derived RPE model of the monogenic mitochondrial disorder caused by the mt.3243A>G mutation, which can manifest with macular degeneration in young patients. We compared isogenic hiPSC-derived RPE clones harbouring low, intermediate, or high levels of mt.3243A>G heteroplasmy, assessing bioenergetics, cell identity, and transcriptomic changes by RNA-seq. Our results demonstrate that RPE bioenergetic and oxygen consumption were inversely correlated with heteroplasmy levels, without affecting typical RPE morphology or marker expression. Notably, transcriptomic analysis revealed significant enrichment of ECM remodelling pathways in mutant RPE in a heteroplasmy-dependent manner, predominantly orchestrated by TGF-β signalling. Integration with human AMD datasets identified shared ECM- and TGF-β–associated pathways. Mechanistically, ROS scavenging or TGF-β inhibition reduced expression of downstream ECM effectors in high-heteroplasmy RPE cells. Collectively, our mt.3243A>G disease model establishes a direct causal link between mitochondrial dysfunction and TGF-β–dependent ECM remodelling in RPE. Given the central role of these processes in AMD and mt.3243A>G-associated macular atrophy, our findings position mitochondrial dysfunction as a potential upstream driver of AMD pathogenesis and a promising therapeutic target.

36 Modelling of RP17 structural variants in retinal organoids reveals upregulation of GDPD1 caused by enhancer hijacking

Introduction
Retinitis pigmentosa (RP) affects approximately 1:4000 people, and 40% of RP cases remain genetically unsolved. We recently discovered complex structural variants (SVs) as the cause of RP17, and our lead hypothesis for the mechanism of disease is the RP17 SVs alter topological associated domain structure and result in ectopic contact of a retinal enhancer with GDPD1. To test this hypothesis, we generated retinal organoid models.

Methods
Patient-derived iPSCs were CRISPR corrected to remove the SV, with synthetic single-stranded DNA templates mediating homology-directed repair. The RP17 patient-derived iPSCs and isogenic control iPSCs were differentiated into retinal organoids alongside an additional control line. ROs were analysed by bulk RNAseq and immunohistochemistry.

Results
RP17 ROs developed normally compared to controls, with lamination and brush borders. Unbiased RNAseq analysis of dysregulated transcripts revealed upregulation of GDPD1 in RP17 ROs compared to the isogenic and independent control ROs. Strikingly, the levels of GDPD1 transcript were reduced to normal levels in the isogenic ROs. Other dysregulated transcripts were also identified as a potential downstream consequence of over expression of GDPD1. Preliminary immunohistochemical analysis indicates a potential cilia phenotype in RP17 ROs, that requires further validation.

Conclusions
RO modelling of RP17 supports the hypothesis that the SVs cause upregulation of GDPD1 by 3D remodeling and retinal enhancer hijacking. The isogenic line, alongside its parental RP17 patient line, provide a robust model with a controlled genetic background to elucidate SV-driven disease mechanisms in RP17.

37 Modelling PCARE-Associated Retinopathy in Human Retinal Organoids and a Mouse Model

Purpose: Retinitis pigmentosa 54 (RP54) is a severe inherited retinal dystrophy caused by pathogenic variants in PCARE, which encodes the photoreceptor cilium actin regulator protein (PCARE), essential for the outer segment (OS) disc formation. To date, PCARE mutations have been reported in ~40 families, and no treatment exists. This study aims to define the pathogenic mechanisms underlying PCARE-related retinal degeneration and to establish patient-derived retinal models and a knockout mouse model (BC -/-) as platforms for future PCARE gene therapy.
Methods: A homozygous PCARE c.946delA (p.Asn237MetfsX5) hiPSC line from a patient with atypical RP54 and a control hiPSC line were differentiated into retinal organoids (ROs) and retinal pigment epithelium (RPE) cells. For in vivo studies, we used the BC027072 knockout mouse (referred to as BC -/-), which lacks the murine pcare homolog.
Results: RP54-hiPSCs maintained pluripotency and differentiated into all germ layers. RP54-RPE cells exhibited normal morphology but displayed dysregulated RPE-specific gene expression, suggesting functional defects. Early-stage RP54 ROs developed normally, but at later stages, ROs displayed aberrant OS structures and a disrupted external limiting membrane, mislocalization of PCARE, and abnormal retinal cell proliferation. BC-/- mice exhibited rapid and severe photoreceptor and retinal degeneration with markedly impaired light responses beginning at one month of age.
Conclusions: PCARE loss leads to profound structural and functional photoreceptor abnormalities in both hiPSC-derived retinal models and BC-/- mice. These models represent robust preclinical platforms for evaluating AAV-mediated PCARE gene therapy, which will be assessed in upcoming studies.

38 Molecular Mechanisms of Müller Glia Morphogenesis and Neuron–Glial Contact in Retinal Development

The contact of glial cells with neurons is necessary for the development of the central nervous system. This process is facilitated by the distinctive morphologies of glial cells that allow precise spatial contact with neurons. Specifically, glial cells and neurons form functional compartments at the synapse where the glial projection ensheathes the pre- and post-synaptic terminals. Among glial cells, the Müller glia (MG) is the primary synaptic astroglia of the retina that plays multiple roles during retinal development. MG extends across the inner plexiform layer (IPL) and forms precise circuits with neurons during retinal development. The formation of such morphology is important since abnormal glial morphology is a consistent characteristic in multiple neurological disorders. However, the exact mechanisms of MG morphology establishment and precise synaptic contacts are not well studied. To further characterize these processes, genes of interest are selected from a zebrafish retinal single-cell RNA sequencing database. Hybridization chain reaction (HCR) and immunohistochemistry are then performed to assess specific expression in MG and contacted neurons, spatially validating the genes of interest. Subsequently, crispant knock-out or knock-down models are generated to assess phenotypic outcomes, thus explaining the roles of these genes in MG morphology and MG-neuron contact. This study aims to define the molecular mechanisms underlying MG morphogenesis and neuron–glial connectivity during retinal development. Clarifying these mechanisms will advance understanding of MG function in retinal development and may also give insights into the contribution of glial dysfunction to neurological diseases.

39 Non-viral mRNA therapy in a retinal organoid model of CRB1 retinopathy.

Background:
Variants in the CRB1 gene cause severe inherited retinal dystrophies, including Leber congenital amaurosis, retinitis pigmentosa, cone-rod dystrophy and macular dystrophy. CRB1-retinopathies represent the 10th most common cause of early-onset vision loss in the UK, yet no clinically approved treatments currently exist. While AAV-mediated gene therapy has shown promise, limitations including restricted cargo capacity and immunogenicity have driven interest in non-viral delivery strategies. mRNA-based therapies represent a potential alternative, however efficient delivery and functional rescue in retinal systems remain largely unexplored.

Methods:
Day 34 human iPSC-derived CRB1 patient and control (WT) retinal organoids were transfected with lipid-peptide nanocomplex with Peptide Y or ME27 peptide at 0.1µg (0.1) or 0.2µg (0.2) EGFP mRNA. Transfected organoids were imaged at day 35 and EGFP-positive cells were manually quantified.

Results:
In both WT and CRB1 organoids, the nanocomplex incorporating the ME27 peptide with 0.1µg EGFP mRNA (ME27-0.1) was the most efficient condition overall (p<0.01), yielding 31.4 ± 5.12 and 25.3 ± 3.26 EGFP-positive cells in WT and CRB1 organoids, respectively. This was higher than Peptide Y with 0.1µg EGFP mRNA (Peptide Y-0.1), which yielded 13.8 ± 1.59 and 12.3 ± 2.00 cells. In contrast, at 0.2µg mRNA, no significant difference was observed between peptides in either model. In WT organoids, ME27 yielded 18.6 ± 2.86 EGFP-positive cells compared with 10.4 ± 1.17 for Peptide Y (p=0.547), while in CRB1 organoids, ME27 yielded 19.0 ± 3.48 cells compared with 8.43 ± 1.85 (p=0.632).

Conclusion:
These findings demonstrate that lipid-peptide nanocomplexes can successfully transfect human iPSC-derived retinal organoid models. Further studies will investigate CRB1 mRNA delivery to evaluate whether nanocomplex-mediated therapy can rescue the CRB1-associated phenotype in a retinal organoid model.

40 Optic nerve microglia in a model of retinal inflammatory disease - Experimental Autoimmune Uveitis (EAU)

Experimental Autoimmune Uveitis (EAU) is a murine model of non-infectious posterior uveitis characterised by inflammation of the posterior segment, including the retina and ON. In this study, we characterised microglial cells within the optic nerve (ON) at early (e; 2–3 weeks post-immunisation, wPI) and late (l; 4–6 wPI) EAU phases using IBA1 as a marker of microglia and P2RY12 as a marker of surveillant microglia. C57Bl/6 mice (5–7 weeks) were immunised with IRBP1–20 in CFA s.c. with pertussis i.p.; controls received CFA/pertussis only. Weekly Micron V fundoscopy (EAU: n = 6–18 eyes; controls: n = 5–15 per time point) revealed significant optic disc inflammation (p < 0.01 at 2 wPI; p < 0.001 at 5 wPI; p < 0.0001 at 3, 4, 6 wPI), retinal vessel inflammation (p < 0.01 at 3–6 wPI), and retinal structural damage (p < 0.01 at 3, 5, 6 wPI; p < 0.001 at 4 wPI) vs. controls. Longitudinal ON cryosections (10 μm) were immunolabelled and analysed (e-SHAM = 8; l-SHAM = 9; e-EAU = 9; l-EAU = 12). IBA1 staining showed a higher number of morphologically activated microglia in e-EAU vs. e-SHAM (p < 0.05) and l-EAU (p < 0.05), suggesting a transient early inflammatory role. P2RY12 expression did not differ across groups, suggesting the surveillant microglia population remains stable. The data suggest that ON microglial activation is an early response of EAU pathogenesis. Future work could investigate the retina to define temporal differences in EAU-induced microglial activation.

ECR poster abstract 59 SChoi_v3.pdf

ECR poster abstract 59 SChoi_v3.pptx

41 Optic nerve regeneration with citicoline treatment

Scientific Abstract

Background – Glaucoma affects more than 60 million people worldwide and is a leading cause of irreversible blindness. While elevated intraocular pressure (IOP) is the primary modifiable risk factor for glaucoma, degeneration of retinal ganglion cell (RGC) continues even after IOP is reduced.This continued apoptosis of RGC is a shared pathway in glaucoma, retinal ischemia, and optic nerve injury. Citicoline, an endogenous mononucleotide derived from choline, has demonstrated neuroprotective effects on RGC morpho-functional integrity. It also has been shown citicoline slows down visual function decline in patients, through an IOP-independent pathway. However, whether this is through its neuroprotective or regenerative effect of damaged optic nerve remains unclear.

Methods- Unilateral optic nerve crush (ONC) was performed on the right eye adult C57BL/6J mice aged 9 weeks. Animals were randomly allocated to either the control group (n=5) or the citicoline treatment group(n=5). The animals in the treatment group received daily intraperitoneal citicoline injections (500 mg/kg), while the animals in the control group received daily saline solution, for 7 days. Intravitreal injections of cholera toxin subunit B (CTB)-555 (1 µL) were performed on all animals two days before tissue collection to assess axonal regeneration.

Results-CTB fluorescence was detected in all animals, however, the signal intensity was variable, indicating inconsistency in intravitreal injections. A distinct crush site was identifiable in only two animals, limiting the ability to reliably assess axonal regeneration. No evidence of axonal regeneration beyond the crush site was observed in either group.

IoO Symposium poster (Nazifa).pdf

IoO Symposium poster (Nazifa).pptx

42 Refining the genetic landscape of anophthalmia and microphthalmia

Disruption of genes essential to early eye development may lead to anophthalmia or microphthalmia (A/M) which are characterised by absent or underdeveloped eyes, respectively. A/M are clinically and genetically heterogeneous, with their aetiology still not fully understood. This results in a low molecular diagnostic rate (~20–30%), limiting clinical management and genetic counselling. Furthermore, pathogenic variants may remain undetected due to incomplete gene panels and variant interpretation deficiencies. This study aimed to increase the yield of A/M pathogenic variant identification by implementing an enhanced variant-investigation framework.

We curated an updated A/M gene panel of 160 genes through a systematic literature review, and screened for rare loss-of-function, missense, splicing, and structural variants, which were annotated using deep-learning tools (AlphaMissense, SpliceAI) and other in silico predictors (REVEL, Missense3D, and molecular dynamics simulations). We identified candidate variants in 35 probands, increasing the candidate detection rate from 15.5% (44/283) to 27.9% (79/283). Across the 160 A/M-associated genes in our panel, variants occurred most frequently in established A/M genes, but we also strengthened genotype-phenotype correlations with weaker A/M genes such as ACTG1, DYRK1A, HDAC6, RERE and SIX3. We also identified multiple variants in syndromic participants in the genes CAMK2B, NR2F1 and TRAF7, which have not previously been associated with A/M. We therefore propose these genes as novel candidate A/M genes, contributing to the congenital eye phenotype within the context of their respective syndromic presentations.

This study increased the yield of A/M pathogenic variants, strengthened genotype-phenotype correlations, by implementing an enhanced variant-investigation framework integrating a curated gene panel, comprehensive variant detection pipelines, and advanced in silico analysis. Furthermore, we propose 3 novel candidate A/M genes. Nonetheless, the modest diagnostic rate highlights the genetic complexity of A/M and the ongoing need for improved functional annotation and discovery of additional disease-relevant loci.

43 RPE photoreceptor phagocytosis in lysosomal storage disease models of AMD

The retinal pigment epithelium (RPE) is essential to photoreceptor homeostasis and visual function. Daily RPE phagocytosis of photoreceptor outer segment tips (OS) is essential for photoreceptor renewal following photooxidative damage. Impaired degradation of phagocytosed OS in the RPE leads to accumulation of lipid-rich lipofuscin and RPE degeneration in the common vision-loss disease Age-related Macular Degeneration (AMD). The complexity of genetic and environmental risk factors makes AMD difficult to model, but the accumulation of poorly digested, lipid-rich material is an early hallmark shared by lysosomal storage diseases including Niemann-Pick disease type C (NPC). Mouse models of NPC bare AMD hallmarks including lipofuscin accumulation, photoreceptor degeneration and vision loss, demonstrating the potential value of NPC for studying lipid storage in AMD. Our lab has identified a fusion defect between OS-containing phagosomes and lysosomes in NPC models as well as aberrant membrane contact sites (MCS), where organelles are tethered together in close apposition, resulting in dysregulated inter-organelle communication.
ER contact sites with phagocytic and endocytic organelles provide platforms for lipid and ion exchange and are implicated in maturation of endocytic and phagocytic pathways and in lysosomal calcium signalling, known to be important for lysosomal fusion. However, the role of MCS in phagosome maturation and phagolysosome fusion in the RPE has not been investigated.
The aims of my project are to characterise the role of MCS in phagosome maturation and lysosome fusion in the RPE and to identify compounds that can reverse defective phagosome-lysosome fusion in NPC as potential therapeutic candidates for AMD.

44 Spatial proteomic profiling of immune cells in archived FFPE human eyes with ocular inflammation

Inflammation of eye known as Uveitis is responsible for 15% of blindness in the western world, particularly affecting the working age population. Uveitis can be induced by infection but often is associated with autoimmunity that is not well understood. Inflammation occurring in the posterior region of the eye may cause blindness as it disrupts the delicate structure of the retina and damages photoreceptors. Biopsies and direct characterisation of the immune cell populations in the retina are rare and difficult to obtain.

Formalin-fixed paraffin embedded (FFPE) archived enucleated and eviscerated human uveitis eyes collected via routine clinical care over the last 30 years at Moorfields Eye Hospital were used for this study. FFPE-chorioretinal biopsies with a histopathological diagnosis of uveitis, were sourced capturing the active inflammatory stage of uveitis. These tissues underwent multiplexed immunohistochemistry using IBEX adapted for FFPE imaging and incorporating autofluorescence quenching. IBEX is an open-source method that incorporates iterative fluorescent immunohistochemical staining. Over 25 various immune cell and structural markers have been imaged on 10 uveitis samples. Tissues were segmented using DeepCell Mesmer and processed using SPACE R package for spatial analysis revealing immune cell populations and their spatial configurations.

Infectious and presumed autoimmune uveitis cases exhibited unique immune cell populations and exhibit spatially distinct features. Equally, active stage uveitis captured in chorioretinal biopsies versus late-stage chronic diseases in eviscerated or enucleated samples also had consistent differences in the immune cell localisation to the retinal and choroidal structures.

Multiplexed immunohistochemistry on these archived ocular samples provides a new understanding of the clinical pathology of uveitis. The immune cell profile and distribution of each of the subtypes can be compared to clinical data, while the spatial configuration of such as tertiary lymphoid structures and granulomas may indicate areas of therapeutic investigation for the development of targeted treatments for uveitis.

Speaker: Jakub Kubiak (Institute of Ophthalmology)

45 Targeted long-read methylation analysis of the TCF4 repeat in Fuchs endothelial corneal dystrophy

Background
Expansion of an intronic CTG repeat within the TCF4 gene (termed CTG18.1) is the most common genetic risk factor for Fuchs endothelial corneal dystrophy (FECD), an age-related corneal-specific eye disease. PureTarget is an amplification-free targeted long-read sequencing approach that enables simultaneous assessment of TCF4 repeat size, sequence composition and 5mC methylation status.

Purpose: To determine whether DNA methylation status is altered between expanded and non-expanded alleles in affected corneal endothelial cells (CECs).

Methods
Ultra-high molecular weight DNA from eight CEC samples derived from patients with monoallelic expansions was investigated with PureTarget. HiFi reads were aligned to hg38, phased into non-expanded and expanded alleles using TRGT, and CpG methylation positions were identified with Fiberseq. Expanded reads with at least 5% CCG content were classified as interruption-containing reads using BBDuk, and methylation at reference CpG sites was quantified using CpGtools and assessed for differential methylation with MethBat.
Methylation was then assessed using two complementary pipelines: within-repeat methylation was summarised by binning per-read CpG methylation positions into 1 kb windows along each molecule, while methylation of the repeat-flanking regions was quantified at reference CpG sites near the repeat.

Results
Within the CTG repeat tract, interrupted expanded reads displayed the greatest levels of methylation and variability, exceeding 300 methylated CpGs per 1 kb bin, while non-expanded reads generally remained below ~50 methylated CpGs per bin. Expanded reads without interruptions showed intermediate levels of methylation.
At reference CpG sites upstream of CTG18.1, non-expanded reads maintained low baseline methylation (<5%). In contrast, interrupted expanded reads reached >60% methylation, compared with ~25% across expanded reads without interruptions, defining a nearby differentially methylated region.

Conclusions
Methylation status at this TCF4 locus is shaped not simply by repeat length, but by repeat sequence composition, with CCG-interrupted expanded alleles forming a distinctly hypermethylated subgroup in CECs. This suggests that repeat interruptions may underlie tissue-specific epigenetic changes relevant to FECD pathogenesis.

HQ_ECR_26.pdf

46 The protective effect of citicoline and vitamin k on visual pathway degeneration following optic nerve damage

Glaucoma is a progressive neuropathy causing retinal ganglion cell loss and visual field defects, with elevated intraocular pressure (IOP) as a major risk factor. Lowering IOP can slow glaucoma progression, but vision loss still occurs in some patients which led to the idea of neuroprotection in glaucoma , to preserve retinal ganglion cells and prevent apoptosis. This project investigates whether 500 mg/kg citicoline and 10 mg/kg vitamin K can protect retinal ganglion cell by altering the relationship between retinal ganglion cell structure and visual function in the mouse model of Optic Nerve Crush. Adult C57BL/6J mice underwent monocular right-eye crush, with the left eye serving as an internal control. Functional outcomes were assessed longitudinally using optomotor response and pattern electroretinography ,to assess how neuroprotection with citicoline and vitamin k alters the relationship between retinal ganglion cell structure and visual function such as contrast sensitivity, visual acuity. PERG amplitude and contrast sensitivity was analysed at multiple time points ,to compare the efficacy of citicoline and vitamin K as neuroprotective strategies. This study addresses a major challenge in glaucoma research about the lack of safe, effective neuroprotective therapies in glaucoma, to support the use of citicoline and vitamin K in ophthalmic pharmacotherapy.

Riya poster.pptx.pdf

47 Understanding and targeting fibrosis in Neovascular Age-Related Macular Degeneration

Neovascular age-related macular degeneration (nvAMD) is a leading cause of irreversible vision loss, with subretinal fibrosis representing a late and vision-threatening stage of disease progression. Although anti-VEGF therapies effectively suppress pathological neovascularisation and vascular leakage, they do not reliably prevent fibrotic scarring, and fibrosis can still develop despite repeated treatment. There are currently no approved therapies that directly target subretinal fibrosis, highlighting a major unmet clinical need.
Subretinal fibrosis is driven by the accumulation of extracellular matrix and contractile myofibroblast-like cells within choroidal neovascular membranes. These myofibroblasts may arise from multiple ocular cell populations, including retinal pigment epithelial cells, pericytes and other stromal cells, but the molecular signals that promote their emergence in nvAMD remain poorly understood.
Leucine-rich α-2 glycoprotein 1 (LRG1) is a secreted glycoprotein known to modulate TGF-β signalling and promote fibrosis in several organs, including the lung and kidney. Elevated LRG1 levels have been reported in ocular fluids and neovascular membranes from patients with nvAMD, suggesting that LRG1 may contribute to the local pro-fibrotic environment. We therefore investigated whether LRG1 acts as a pro-fibrotic factor in choroidal neovascularisation by promoting myofibroblast-like activation of ocular cells.
Using differentiated ARPE-19 cells cultured in Williams’ medium as a model of retinal pigment epithelium, we found that LRG1 increased the expression of pro-fibrotic markers, whereas undifferentiated ARPE-19 cells were unresponsive. LRG1 similarly enhanced fibrotic marker expression in human retinal pericytes. In parallel, we are using an ex vivo mouse choroid explant model in which sprouting CNV-like lesions develop progressive fibrotic features over time and respond to pro-fibrotic stimulation with TGF-β. This platform will enable us to define how LRG1 regulates fibrosis within sprouting neovascular lesions and assess its potential as a therapeutic target in nvAMD.

31.05.26 poster EG 2.pdf

48 "Some days it hits me like a ton of bricks that I can, in fact, not see": A qualitative reflexive thematic analysis study of factors which support and limit mental well-being in young people with vision impairment from inherited eye disease.

Background: Young people with vision impairment (VI) report lower well-being than their peers, but the mechanisms underlying this disparity are not well understood. Evidence for the effectiveness of existing emotional support is limited, and provision is often inconsistent. This study used qualitative interviews to explore factors that hinder or support well-being in adolescents with acquired VI, as well as barriers and facilitators to accessing support, with the aim of informing recommendations, strategies and resources to improve outcomes.
Methods: Purposive sampling recruited adolescents with, and parents of adolescents with, inherited macular disease and VI from a hospital low vision clinic and a vision impairment charity. Participants completed semi-structured interviews with open-ended questions exploring the impact of VI on daily life, particularly at diagnosis, focusing on mental well-being, coping activities and perceptions of available support. Interviews were recorded, anonymised, transcribed and analysed using reflexive thematic analysis. Themes were developed using an a posteriori approach.
Results: Eighteen interviews were analysed. Four overarching themes were identified. “Living in the aftermath of diagnosis” captured the enduring emotional impact of VI. “Fighting the system” described how families navigate complex support structures and the barriers encountered. “Being seen and being misunderstood” reflected how VI shapes relationships, identity and social positioning. “Building a life with vision impairment” focused on everyday functioning, support systems and future aspirations. Together, these themes demonstrate how psychological, social and systemic factors interact to influence well-being and life trajectories.
Conclusions: Participants linked multiple aspects of VI to reduced well-being. These findings will inform the development of targeted interventions, including Acceptance and Commitment Therapy, to better support adolescents with VI.

49 MEC Lay Abstract Award Winner Talk

Talks: Session 3 (3 talks)

50 Unravelling transcriptional dysregulation events that underlie Posterior Polymorphous Corneal Dystrophy

Posterior polymorphous corneal dystrophy (PPCD) is a rare inherited corneal endothelial dystrophy caused by pathogenic variants in the transcription factor encoding genes OVOL2, GRHL2, and ZEB1, key regulators of epithelial-mesenchymal/mesenchymal-epithelial transition (EMT/MET) and cellular differentiation. In PPCD, reduced ZEB1 expression or ectopic expression of OVOL2 or GRHL2 is thought to promote an endothelial-to-epithelial cell fate transition. However, comprehensive transcriptomic studies of human PPCD tissue remain limited, and it is unclear whether distinct genetic subtypes converge on shared disease mechanisms.

Here, we performed the largest transcriptomic study of PPCD to date. Using primary corneal endothelial cell lines derived from five patients harbouring the Czech founder OVOL2 promoter variant, we show widespread transcriptomic dysregulation of EMT/MET-associated pathways, evidence of epithelial differentiation corresponding to disease severity, and dysregulated RNA-binding proteins, including ectopic expression of the epithelial splicing regulator ESRP1, accompanied by extensive alternative splicing changes. Functional modelling in an immortalised human corneal endothelial cell line overexpressing ESRP1 recapitulated multiple transcriptomic alterations observed in patient-derived cells, supporting a mechanistic role for ESRP1-mediated post-transcriptional regulation in PPCD pathogenesis. Analysis of additional cases carrying an independent OVOL2 variant and a ZEB1 variant demonstrated consistent molecular signatures across these distinct PPCD genotypes.

Together, these findings indicate that PPCD pathogenesis converges on a shared mechanism involving disruption of EMT/MET-associated transcriptional and post-transcriptional regulatory networks. This work establishes a disease framework for future mechanistic studies and supports the development of therapeutics targeting convergent downstream pathways across PPCD subtypes.

Speaker: Nihar Bhattacharyya (UCL Institute of Ophthalmology)

51 Early visual cortex predominantly represents visual inputs, not percepts

Top-down signalling across the visual hierarchy is thought to support invariant perception, enabling object recognition despite changes in viewpoint, noise, and ambiguity. A central unresolved question is whether high-level prior knowledge shapes neural representations throughout the visual hierarchy, including early visual cortex, or whether its influence is largely confined to higher-order visual regions. While feedback-related modulation has been demonstrated in primary visual cortex (V1) for simple stimuli, evidence that early representations of complex scenes are reorganised towards high-level priors remains mixed. To address this, we presented ambiguous naturalistic images during functional magnetic resonance imaging (fMRI) and tested how prior-knowledge-driven perceptual reorganisation altered neural representations across the visual hierarchy. We found that perceptual reorganisation was accompanied by representational changes in higher-order visual regions, including posterior fusiform (pFs) and lateral occipital cortex (LOC), such that activity patterns became more similar to those associated with the cued percept. By contrast, early visual areas V1 and V2 remained dominated by the sensory input and did not show significant shifts towards the prior-driven percept. These findings challenge accounts of widespread top-down reorganisation across visual cortex with transformative effects for early visual representations. Crucially, our design controlled for temporal-order confounds that commonly affect perceptual reorganisation paradigms, allowing a stronger attribution of representational change to prior-knowledge-dependent mechanisms. Together, the results reveal a steep hierarchical gradient, from input-dominated encoding in early visual cortex to prior-driven representations in higher-order visual areas.

Speaker: Georgia Milne

52 Dbl3-driven Cdc42 signaling regulates apical membrane trafficking in polarised epithelia

Epithelial polarity is essential for retinal pigment epithelium (RPE) function, yet how polarity signalling regulates membrane trafficking remains poorly understood. We previously identified the apical polarity determinant Dbl3, a Cdc42-specific GEF, and its effector kinase MRCKβ as key regulators of apical actomyosin contractility. Here, we investigate whether this signalling axis coordinates cytoskeletal dynamics with apical membrane trafficking. Using loss-of-function approaches in polarised MDCK and ARPE-19 cells, we combined quantitative fluid-phase uptake assays, antibody-based trafficking reporters, and confocal z-stack imaging to analyse apical endocytosis and recycling. Depletion or chemical inhibition of MRCKβ significantly reduced apical fluid-phase uptake. Using the FcLR 5-22 apical recycling reporter cell line, we showed that disruption of Dbl3-MRCKβ signalling impairs clathrin-mediated endocytosis and perturbs apical recycling, resulting in altered receptor distribution and increased endolysosomal accumulation. Colocalisation analysis further indicates receptor mis-sorting along the apical endosomal axis upon pathway downregulation. Our findings demonstrate that the Dbl3-MRCKβ pathway integrates polarity signalling with actomyosin contractility to regulate apical endocytic trafficking. This work expands the role of polarity-dependent cytoskeletal regulation beyond epithelial polarisation, identifying a mechanistic link between apical polarity and membrane trafficking dynamics. These results provide insights into how spatial signalling networks coordinate membrane organisation and intracellular transport in polarised epithelial cells.

Speaker: Alex He

Flash Talks: Session 2

53 Multimodal in vivo and ex vivo imaging of putative hyalocyte-associated signals at the human vitreoretinal interface

Hyalocytes are macrophage-lineage border immune cells located at the posterior hyaloid and vitreoretinal interface (VRI), a specialised compartment between the vitreous and neural retina. Clinical OCT frequently reveals punctate hyperreflective cell-like signals at this interface, but their cellular identity remains uncertain. These signals are often grouped under broad descriptive terms such as macrophage-like cells or microglia-like cells, although label-free OCT alone cannot assign molecular identity.
This project combines in vivo retinal imaging with ex vivo spatial phenotyping to improve the detection, localisation and interpretation of VRI-associated cell-like signals in health and inflammation. In human participants, swept-source OCT/OCTA was used for same-location imaging of hyperreflective VRI signals. Thin en face structural OCT slabs positioned around the inner limiting membrane and posterior hyaloid were registered to OCTA vascular landmarks to improve spatial localisation and longitudinal comparability. In healthy eyes, VRI-associated objects showed a reproducible parafoveal distribution with relative sparing of the foveal avascular zone. In uveitic eyes, these signals showed focal clustering around inflammatory lesions and increased apparent object area, suggesting inflammation-associated remodelling of the VRI compartment.
Adaptive optics scanning light ophthalmoscopy provided complementary cellular-resolution imaging of VRI-associated structures in living eyes, allowing assessment of morphology and spatial relationships to retinal vasculature. To support cellular interpretation, human donor retinal tissue was analysed using multiplex immunofluorescence. VRI-localised IBA1-positive cells with round-to-amoeboid morphology and enriched CD74 expression were identified at the retinal surface, distinct from ramified parenchymal microglia within deeper retinal layers.
Together, these findings support a translational imaging framework in which SS-OCT provides scalable object-level mapping, AOSLO provides single-cell morphological information, and ex vivo immunofluorescence provides molecular and anatomical validation. This approach may help refine the interpretation of VRI hyperreflective cell-like signals and improve the use of advanced retinal imaging as a window onto immune activity at the retina–vitreous boundary.

Speaker: Yuxuan Meng (UCL IoO and Morfields Eye Hospital)

54 AAV-mediated ocular gene therapy overcomes retinal immune tolerance, to enable cytotoxic T cell-mediated retinal cell loss

Luxturna was the first approved adeno-associated virus (AAV)-based gene therapy, however, patients exhibit progressive retinal atrophy following treatment. One proposed cause is a transgene-specific cytotoxic CD8+ T cell response. Notably, this is not observed in mice suggesting they lack comparable CD8+ responses, limiting their ability to recapitulate clinical observations. This project therefore aims to understand cytotoxic T cell trafficking into the retina and effector function during AAV delivery.

B10.D2 mice received intravitreally injected AAV expressing enhanced Green Fluorescence Protein (eGFP). Activated eGFP-specific CD8+ T cells from Just EGFP Death Inducing (JEDI) mice were adoptively transferred into AAV2-recipients or Cx3Cr1GFPxB10.D2 mice. Post-mortem retinas were analysed using multiplexed immunohistochemistry and spatially analysed using Aivia.

Cx3cr1GFPxB10.D2 mice showed no loss of GFP+ microglia following transfer of GFP specific T cells. In contrast, AAV2 injected recipients exhibited significant loss of GFP+ retinal cells. 3D analysis confirmed entry of CD8+ T cells into AAV-treated retinas of adoptive transfer-recipients and direct interactions with GFP-expressing cells.

This demonstrates that transgene specific CD8+ T cells infiltrate the retina and target antigen presenting retinal cells after AAV delivery, but not when the same antigen is endogenously expressed under healthy conditions, highlighting the critical role of tissue inflammatory context in retinal cytotoxic immune responses.

Speaker: Rose Avient

55 Muller glia: The architects of retinal synapse remodelling

Synaptic pruning is a neurodevelopmental checkpoint that occurs to eliminate excess neurons and refine circuits in the nervous system. The retina consists of highly organised networks of neuronal and glial cells that connect to pass visual information. However, the cellular and molecular processes behind synaptic pruning in the retina remain unclear. Glial cells, like astrocytes, microglia and Müller glia (MG; the principal glia of the retina), contact neurons and synapses to provide a myriad of support functions. Microglia have long been thought to solely undertake retinal synapse pruning due to their widely reported role in the brain. Here, using fixed and live zebrafish retinas, we show that MG carry out phagocytosis of synapses, working alongside, but independently of microglia.

Pruning occurs in two distinct time points during development, with each glial cell type being the principal “pruner” at each stage. We conducted a molecular screen to investigate the molecular mechanisms that regulate these pruning events. We found that two signalling pathways belonging to the complement system, the classical and lectin complement pathways, are expressed in glia at the stages of pruning in the retina. Consistent with the brain, microglia act through the classical complement pathway, whilst MG mainly rely on the lectin system. We are now using CRISPR/Cas9 knockout and cell-specific over-expression to determine the role of these pathways in each glial cell during development. These findings provide new insights into the role of MG in retinal development and provide potential novel molecules to explore in promoting synaptic maintenance and support in ageing and neurodegenerative disease.

Speaker: Roxana Lungu (University College London)

15:25 Afternoon Coffee Break

Posters: Session 2

56 AAV-mediated ocular gene therapy overcomes retinal immune tolerance, to enable cytotoxic T cell-mediated retinal cell loss

Luxturna was the first approved adeno-associated virus (AAV)-based gene therapy, however, patients exhibit progressive retinal atrophy following treatment. One proposed cause is a transgene-specific cytotoxic CD8+ T cell response. Notably, this is not observed in mice suggesting they lack comparable CD8+ responses, limiting their ability to recapitulate clinical observations. This project therefore aims to understand cytotoxic T cell trafficking into the retina and effector function during AAV delivery.

B10.D2 mice received intravitreally injected AAV expressing enhanced Green Fluorescence Protein (eGFP). Activated eGFP-specific CD8+ T cells from Just EGFP Death Inducing (JEDI) mice were adoptively transferred into AAV2-recipients or Cx3Cr1GFPxB10.D2 mice. Post-mortem retinas were analysed using multiplexed immunohistochemistry and spatially analysed using Aivia.

Cx3cr1GFPxB10.D2 mice showed no loss of GFP+ microglia following transfer of GFP specific T cells. In contrast, AAV2 injected recipients exhibited significant loss of GFP+ retinal cells. 3D analysis confirmed entry of CD8+ T cells into AAV-treated retinas of adoptive transfer-recipients and direct interactions with GFP-expressing cells.

This demonstrates that transgene specific CD8+ T cells infiltrate the retina and target antigen presenting retinal cells after AAV delivery, but not when the same antigen is endogenously expressed under healthy conditions, highlighting the critical role of tissue inflammatory context in retinal cytotoxic immune responses.

Rose_Avient_ECR.pdf

57 Multimodal in vivo and ex vivo imaging of putative hyalocyte-associated signals at the human vitreoretinal interface

Hyalocytes are macrophage-lineage border immune cells located at the posterior hyaloid and vitreoretinal interface (VRI), a specialised compartment between the vitreous and neural retina. Clinical OCT frequently reveals punctate hyperreflective cell-like signals at this interface, but their cellular identity remains uncertain. These signals are often grouped under broad descriptive terms such as macrophage-like cells or microglia-like cells, although label-free OCT alone cannot assign molecular identity.
This project combines in vivo retinal imaging with ex vivo spatial phenotyping to improve the detection, localisation and interpretation of VRI-associated cell-like signals in health and inflammation. In human participants, swept-source OCT/OCTA was used for same-location imaging of hyperreflective VRI signals. Thin en face structural OCT slabs positioned around the inner limiting membrane and posterior hyaloid were registered to OCTA vascular landmarks to improve spatial localisation and longitudinal comparability. In healthy eyes, VRI-associated objects showed a reproducible parafoveal distribution with relative sparing of the foveal avascular zone. In uveitic eyes, these signals showed focal clustering around inflammatory lesions and increased apparent object area, suggesting inflammation-associated remodelling of the VRI compartment.
Adaptive optics scanning light ophthalmoscopy provided complementary cellular-resolution imaging of VRI-associated structures in living eyes, allowing assessment of morphology and spatial relationships to retinal vasculature. To support cellular interpretation, human donor retinal tissue was analysed using multiplex immunofluorescence. VRI-localised IBA1-positive cells with round-to-amoeboid morphology and enriched CD74 expression were identified at the retinal surface, distinct from ramified parenchymal microglia within deeper retinal layers.
Together, these findings support a translational imaging framework in which SS-OCT provides scalable object-level mapping, AOSLO provides single-cell morphological information, and ex vivo immunofluorescence provides molecular and anatomical validation. This approach may help refine the interpretation of VRI hyperreflective cell-like signals and improve the use of advanced retinal imaging as a window onto immune activity at the retina–vitreous boundary.

58 A Comparative Study of Deep Learning-based Retinal Image Registration Methods

Aim: To benchmark three deep learning-based retinal image registration methods RetinaRegNet, EyeLiner, and GeoFormer on the Fundus Image Registration (FIRE) dataset, comparing accuracy and computational efficiency using Mean Landmark Error (MLE) as the primary metric.
Methods: The methods were evaluated under consistent conditions across three overlap-based categories: Class S (71 pairs, >75% overlap without anatomical changes), Class A (14 pairs, >75% overlap with anatomical variations), and Class P (49 pairs, <75% overlap without anatomical changes). RetinaRegNet integrates diffusion features, dual keypoint sampling (SIFT and random), two-stage outlier removal, and a hierarchical registration strategy from homography to polynomial transforms. EyeLiner combines anatomical segmentation with SuperPoint feature extraction, LightGlue matching, and thin-plate spline warping. GeoFormer extends Local Feature Transformers (LoFTR) using cross-attention mechanisms and RANSAC-based refinement. Performance was assessed using MLE.
Results: Across 134 FIRE image pairs, RetinaRegNet achieved the best MLE (3.12 pixels), outperforming EyeLiner (3.81 pixels) and GeoFormer (6.06 pixels).Class-wise, RetinaRegNet performed best in Class S (1.70 pixels) and Class P (4.57 pixels), while EyeLiner showed comparable performance in Class A (4.87 vs 5.24 pixels). GeoFormer exhibited substantially higher errors in Class P (11.20 pixels).In terms of runtime, GeoFormer was fastest (0.32 s), followed by EyeLiner (4.92 s) and RetinaRegNet (31.23 s).
Conclusions: Results indicate a trade-off between accuracy and efficiency: RetinaRegNet provides highest precision, EyeLiner balances both, and GeoFormer prioritizes speed.

Retinal_Image_Registration_Poster_A1.jpg

Retinal_Image_Registration_Poster_A1.pdf

59 A Gelatin-Supported Protocol for Preserving Delicate Retinal Tissue Enables Spatial Analyses

Spatial transcriptomics has become an essential tool in retinal research, yet preservation of both tissue architecture and RNA integrity remains technically challenging for post-mortem human samples. Difficulties in working with enucleated globes include retinal detachment from retinal pigment epithelium layer and retinal folding, which commonly occur during dissection due to the retina’s thin and fragile laminated structure. This becomes particularly challenging when seeking to isolate defined regions of interest such as the macula. We have developed a sucrose-gelatin–based stabilisation and embedding protocol that minimizes retinal distortion, followed by cryopreservation of the entire globe prior to regional dissection. Using methylene blue staining, hematoxylin and eosin (H&E) staining, and immunohistochemistry, we demonstrate preservation of macular architecture and retinal lamination. Fluorescent staining for RNA quality from processed samples suggest compatibility with RNA-targeted probe hybridisation, supporting applicability to downstream spatial transcriptomics workflows. This protocol provides a practical solution for spatial analyses of enucleated globes including human tissue and may facilitate similar studies in other mammalian specimens.

IoO Symposium_2026 poster_GaryC.pdf

60 A novel NRP1 pathway in physiological and ischaemic retinal vascularisation

The retina relies on a dense vascular network to meet its high metabolic demand. Impaired vascular function and insufficient vessel growth are key features of several sight-threatening retinal diseases. Although anti-VEGF therapies are the current gold-standard treatment, a substantial proportion of patients respond poorly, and retinal ischemia remains unresolved due to vessel loss and abnormal vessel regrowth. Neuropilin (NRP) 1, a co-receptor for vascular endothelial growth factor receptor-2 (VEGFR2), is a multifunctional transmembrane receptor protein critical for retinal angiogenesis. However, loss of VEGF binding to NRP1 only mildly impairs retina angiogenesis, suggesting that NRP1 promotes retina vascularisation in a VEGF-independent manner through unidentified ligands. Here, we show that both physiological vascularisation and ischemic neovascularisation are decreased in the retina of mice with a mutation in one of the two adhesion domains in NRP1. This finding suggests that NRP1 can contribute to angiogenesis even under VEGF-neutralising conditions.

61 Arhgef18/p114RhoGEF is a structural component of the outer limiting membrane essential for retinal integrity and mitochondrial homeostasis

Purpose

Biallelic mutations in ARHGEF18 cause adult-onset retinal degeneration, but its retinal function is unknown. Arhgef18/p114RhoGEF is a RhoA exchange factor that regulates the mechanical stability of epithelial cell–cell adhesions. We therefore hypothesized that it supports retinal integrity by stabilizing cell–cell adhesion in specific retinal cell types. We investigated the localization of Arhgef18/p114RhoGEF in the retina and the consequences of genetic knockouts in different retinal cells.

Methods

Arhgef18 in mice was deleted in Müller glial cells using a Pdgfra-Cre driver and in endothelial cells using Tie2-Cre. Retinal structure and function were assessed by SD-OCT, fundus imaging, fluorescein angiography, ERG, and histology. Outer limiting membrane (OLM) integrity and retinal degeneration were evaluated by immunofluorescence and TUNEL assays. Mechanistic studies were performed in cultured human Müller glia (MIO-M1A) using siRNA-mediated ARHGEF18 depletion combined with assays to identify deregulated signaling mechanisms and metabolic dysfunction.

Results

Arhgef18/p114RhoGEF associated with the retinal OLM, colocalizing with ZO-1 and p120-catenin. Müller cell–specific knockout led to OLM disruption and progressive vision loss by P60. Although retinas initially formed normally, retinal rosettes appeared by P8 and were followed by OLM dissociation, retinal thinning, and vascular leakage. ARHGEF18/p114RhoGEF depletion in cultured Müller cells confirmed disruption of junctional recruitment of OLM proteins. The depletion also activated NF-κB and β-catenin signaling, as well as the multifunctional kinase TBK1, while reducing mitochondrial activity. TBK1 inhibition or metabolic support with nicotinamide attenuated NF-κB and β-catenin signaling and restored mitochondrial function.

Conclusions

Arhgef18/p114RhoGEF is a novel component of the OLM required for junctional stability and photoreceptor survival. Our results also identify a druggable pathway that may be exploited to treat ARHGEF18-related and, possibly, other OLM-associated retinal degenerations.

62 Behavioural state-dependent serotonergic modulation of the visual cortex

Individual neurons in the primary visual cortex (V1) respond strongly to visual stimuli. The evoked responses of these neurons can be modulated by the behavioural state of the animal, such as increases in arousal and locomotion. This behavioural state-dependent cortical activity is regulated by neuromodulators, including the well-studied cholinergic and noradrenergic systems. Recent work examining the activity of serotonergic neurons in the dorsal raphe nucleus (DRN) during differing behavioural states, has shown an inverse relationship between serotonin (5HT) activity and locomotion. Furthermore, serotonin modulation of V1 neurons reduces the gain of evoked and spontaneous responses, whereas locomotion enhances visually evoked responses in V1 neurons. However, the relationship between serotonergic and locomotive modulation on V1 neurons remains unexamined.

Moreover, serotonergic neurons in the DRN are the main source of forebrain innervation, yet majority of research investigating serotonin activity has been performed in the DRN. Whether the activity of DRN-5HT neurons seen in relation to behavioural states is also reflected in their projection fibres, requires further investigation. Therefore, using a combination of two-photon imaging and calcium indicators, I can acquire activity of 5HT projection fibres in the V1, with high spatial and temporal resolution. Thus, allowing to examine the relationship of serotonergic activity in the V1 during different behavioural states.

ECR 2026 poster_Rhys Burrows.pdf

63 Building a retina-specific neurovascular unit in vitro

Cerebral blood vessels play a central role in supplying and regulating the disproportionately large amount of energy the brain requires for neurological function. They also protect the central nervous system by forming the blood-brain barrier (BBB), which shields neurons from pathogens and immune attack. This intimate functional interdependency between neurons and their associated blood vessels is referred to as the neurovascular unit (NVU). Dysfunction of the NVU underlies many neurological diseases and has been recognised by the American Heart and Stroke Associations as central to understanding and effectively treating conditions as wide-ranging as stroke, multiple sclerosis, and Alzheimer's disease. An analogous structure exists in the retina, where the NVU forms the blood-retinal barrier (BRB), and its dysfunction frequently contributes to neuronal morbidity and vision loss.

Cell culture models are instrumental for interrogating NVU function, particularly its barrier properties. However, models specific to the retinal NVU do not currently exist. Retinal paradigms are instead inferred from the many models developed for the BBB and cerebral NVU, despite well-established differences between the two systems. For instance, retinal blood vessels exhibit distinct pericyte coverage ratios and interact with retina-specific glial cells rather than the astrocytes that predominate at the BBB.

To address this gap, we are establishing cell culture models specifically tailored to the retinal NVU. We used a combination of established cell lines and primary cells isolated from porcine and rodent brain and retina, including endothelial cells, pericytes, Müller cells, and retinal ganglion cells. These cell types are characterised individually and then integrated into co-culture and tri-culture configurations to recapitulate the multicellular architecture and intercellular signalling of the retinal NVU in vivo. Barrier function is assessed using transendothelial electrical resistance (TEER) and permeability assays, alongside immunofluorescence of tight junction proteins and cell-type-specific markers.

This study contributes to a better understanding of how the retinal NVU differs from that found in the brain and facilitates future approaches to study NVU dysfunction associated with widespread blinding diseases such as diabetic retinopathy.

64 Cellular and extracellular matrix reorganisation in proliferative vitreoretinopathy

Introduction
The commonest cause of failure in retinal reattachment surgery is the anomalous wound healing process called proliferative vitreoretinopathy (PVR). The development of measures to improve outcomes depends on an understanding of the cellular mechanisms involved.

Methods
We imaged retinectomy tissue from eyes of three patients with recurrent retinal detachment associated with PVR grade C using highly multiplexed immunohistochemistry (IBEX). Bright field imaging was utilised to visualise pigmented cells. Equivalent tissue from the retina of an eye unaffected by retinal detachment was used as a normal control.

Results
There was significant disorganisation of retinal structure. Within the retina, there was evidence of ganglion cell neurites extending beyond the outer nuclear layer. The reactive Müller cell and astrocyte marker, GFAP was substantially upregulated in the inner retina with extensive horizontal fibres. Similarly, vimentin also representing Müller glia was upregulated in the inner retina. A discontinuous collagen I/III-positive fibrillary membrane lined the surface of the inner retina, co-localising with the collagen IV-positive internal limiting membrane. There was also evidence of extracellular matrix components, fibronectin and laminin.
Epiretinal membranes had pigmented cells visualised on bright field imaging. These membranes encompassed vimentin-positive and αSMA-positive cells, as well as aforementioned extracellular matrix components.

Conclusion
Epiretinal PVR membranes were comprised of reactive glial cells, fibroblasts and scattered pigment-laden cells alongside extracellular matrix components. IBEX offers a potentially powerful new technique to study PVR in retinectomy tissue.

ECR symposium 2026_Achini M_V4.0.pdf

ECR symposium 2026_Achini M_V4.0.pptx

65 Dbl3–MRCKβ Signalling Couples Epithelial Polarity to Transcriptional Regulation

Dbl3 is an apical guanine nucleotide exchange factor that promotes polarised Cdc42 activation to stimulate the apical effectors MRCKβ and the Par3/6/aPKC polarity complex, and, thereby, organisation of the apical actomyosin cytoskeleton. Its overexpression also rescues apical phagocytosis in diseased retinal pigment epithelial (RPE) cells. Polarisation of epithelia is accompanied by transcriptional changes and the acquisition of cell-type-specific functions. Here, we asked if and how apical Dbl3 signalling regulates such transcriptional changes. Gain- and loss-of-function approaches using the kidney epithelial cell line MDCK and RPE cells (ARPE-19 and RPE cells differentiated from iPSC) were combined with RNA sequencing, assays for specific transcriptional pathways and morphological/cytoskeletal responses. RNA sequencing of iPSC-derived RPE cells treated with either Dbl3- or MRCKβ-targeting siRNAs or, alternatively, an MRCK inhibitor indicated deregulation of genes associated with the differentiated phenotype and genes regulating polarisation, adhesion, cell proliferation, morphological changes, and genes indicative of deregulation of inflammatory signalling and YAP/TAZ activity. Gene reporter assays, immunofluorescence, and immunoblotting confirmed the regulation of YAP/TAZ, NFkB, and STAT3 signalling by the Dbl3/MRCKβ pathway. Our data thus suggest that Dbl3–MRCKβ signalling links epithelial apical polarisation to regulation of transcription, suggesting that approaches to stimulate Dbl3 function in malfunctioning epithelia may be beneficial in disease beyond the rescue of specific apical processes by regulating genes imported for epithelial integrity and function.

66 Deep Learning Segmentation of Retinal Biomarkers in RPE65‑IRD

Background RPE65-biallelic mutation-associated inherited retinal disease (RPE65‑IRD) presents with distinct retinal imaging features that evolve with disease progression, including whitish dots (WD), marble‑like pathology (ML), hypopigmented areas (HA), patchy chorioretinal atrophy (PCA), pigmented spicules (PS), and spotty irregular hyperpigmentation (SIHP). Quantification of these features may provide objective biomarkers for disease monitoring.

Aim To develop and evaluate deep learning models for segmentation of six RPE65‑IRD-associated imaging features.

Method A retrospective cohort of 23 patients (46 eyes) with genetically confirmed RPE65 variants was studied. A total of 178 ultra‑widefield (133°) true‑colour fundus images were graded by a clinician. A feature-specific curation scheme was implemented. Deep learning models were trained using full‑image and patch‑based approaches on the complete and curated datasets with five‑fold cross‑validation. Performance was evaluated using the Sørensen-Dice coefficient, sensitivity, and specificity, with comparisons assessed using the Wilcoxon signed‑rank test. Qualitative validation used the DINOv3 vision foundation model.

Results Patch‑based training significantly improved segmentation of small features (PS, SIHP; p < 0.05). Image‑level classification showed no comparable gains, reflecting sensitivity to annotation noise. DINOv3 analysis confirmed visual similarity between PS and SIHP.

Conclusion Targeted data curation and patch‑based learning mitigate label noise and enable robust segmentation of peripheral RPE65‑IRD biomarkers, supporting objective assessment of disease status.

[poster] IoO ECR Symposium 2026.jpg

[poster] IoO ECR Symposium 2026.pdf

67 Design and Optimisation of pegRNA Strategies for Prime Editing of the IFT140 T484M Variant in an Inherited Retinal Ciliopathy

Genome editing encompasses the targeted modification of DNA, enabling insertions, deletions, or substitutions at defined genomic loci. Conventional approaches, however, rely on the generation of a double-stranded break (DSB), which limits both their precision and efficiency.

Prime editing, characterised as a "search-and-replace" genome editing technology, builds upon earlier CRISPR-based systems to enable all 12 possible transition and transversion mutations, as well as small insertions and deletions, without the requirement for a double-stranded break. This research focuses on the IFT140-associated inherited retinal ciliopathy, specifically targeting the T484M point mutation located on chromosome 16.

Using established in silico design tools, PEgIT and pegFinder, multiple prime editing components have been designed spanning successive prime editor generations, in order to identify the most effective combination for targeting this disorder.

Twelve pegRNAs have been designed, incorporating two secondary nicking sites. Designs include the use of canonical and non-canonical PAM sites, tiling strategies, PE3 secondary nicking guides, and a comparison of tevopreQ1 epegRNAs versus standard pegRNAs. Transfection experiments to assess the efficiency of introducing the T484M point mutation are currently ongoing in HEK293T cells.

This work will provide an optimisation framework of pegRNA designs capable of targeting this mutation within the genome and serve as a proof of concept for the application of prime editing to this disorder. Following HEK293T transfections to establish a cellular disease model, the optimised guides will subsequently be applied to correct the causative mutations in patient-derived iPSCs.

68 Developing a human light damage model of rhodopsin retinitis pigmentosa

Introduction: Retinitis Pigmentosa (RP) is one of the most common forms of inherited retinal dystrophy (IRD) and a leading cause of blindness in the working-age population. Disease progression can be exacerbated by environmental light exposure. Variants in the RHO gene encoding rhodopsin, the light-sensitive G protein-coupled receptor that initiates phototransduction, are a major cause of autosomal dominant RP. The RHOM39R variant is one of the most common in the UK and is associated with sector RP, where degeneration affects regions of the retina most exposed to light. While RhoM39R mouse models confirm strong light-induced degeneration, the lack of appropriate human models has limited mechanistic insight and therapeutic development.

Methods: Human retinal organoids were generated from induced pluripotent stem cells carrying the RHOM39R variants, including patient-derived heterozygous, CRISPR-corrected isogenic controls and gene-edited heterozygous and homozygous lines. iPSC were differentiated to retinal organoids and characterised by immunohistochemistry (IHC). A light damage assay was established using four conditions: ambient light ± 9-cis retinal and bright light ± 9-cis retinal. Cell death was assessed by TUNEL reactivity.

Results: Both heterozygous and homozygous RHOM39R organoids showed phenotypic differences compared to isogenic controls, including altered brush border organisation. Homozygous RHOM39R organoids exhibited rhodopsin mislocalisation, with reduced levels of rhodopsin in the outer segment and retention in the inner segment and outer nuclear layer. Bright light exposure in the presence of 9-cis retinal significantly increased TUNEL-positive nuclei in both patient-derived and gene-edited heterozygous organoids across media conditions, compared to all other conditions. No significant differences were observed in control or homozygous organoids.

Conclusions: These findings demonstrate that human retinal organoids can model key features of RHOM39R associated light-induced degeneration. The degeneration observed in heterozygous organoids highlights the role of light in driving disease pathology. In contrast, the absence of a light damage phenotype in homozygous organoids is consistent with rhodopsin mislocalisation limiting light activation. This model provides a platform to study human photoreceptors responses to light-induced stress and to support the development of strategies to protect photoreceptors.

69 Generation and charactersiation of hiSPC-derived retinal organoids modelling RP1 associated dominant and recessive RP

Heterozygous changes within a mutation hotspot in RP1 are associated with autosomal dominant retinitis pigmentosa (adRP). Homozygous or compound heterozygous variations outside of this region are associated with autosomal-recessive retinitis pigmentosa (arRP). We have developed hiPSC-derived retinal organoid (RO) models of adRP-RP1 harbouring a common variant (p.Gln686*, KI) and an RP1 knock out (KO) model of arRP-RP1 to allow for disease modelling.

CRISPR-Cas9 editing in control hiPSCs was used to generate both models. Homology-directed repair (HDR) was used to knock in the heterozygous adRP associated RP1 variation c.2056C>T p.Gln686 and non-homologous end joining (NHEJ) used to generate the RP1 KO, a homozygous stop-gained frameshift deletion in exon 2 (c.85-88del, p.Thr30Ilefs49) predicted to lead to nonsense-mediated decay. hiPSC lines (WT, KI and KO) were differentiated into ROs using an established protocol. One way ANOVA was used for statistical analysis.

All iPSC cell lines were shown to be pluripotent, able to undergo trilineage differentiation and genomically stable. Both RO models showed shorter photoreceptor outer segments when compared to the isogenic control. The outer segments of the KI iPSC-ROs were significantly shorter at three time points than those of the control ROs. This was also seen in IHC staining of rhodopsin in each line.

Our results show that both the KI and KO organoids show differences to the isogenic control. The phenotype of the KO retinal organoids appears more severe than that of the KI organoids, which aligns with the less severe phenotype of patients with RP1-associated adRP. This is the first time these diseases have been modelled in human retinal organoids and show that these can be a valuable tool to study this disease further.

70 Identification of Candidate Genetic Variants in Unresolved Anophthalmia and Microphthalmia Cases Using Whole Genome Sequencing Data from the 100.000 Genomes Project

Anophthalmia and microphthalmia (A/M) are rare developmental eye disorders characterised by the absence or reduced size of the eye, respectively. These conditions are genetically heterogeneous, with over 150 causative genes identified, yet much of their genetic basis remains unresolved, resulting in only 20-30% of affected individuals receiving a genetic diagnosis. This highlights a critical gap in understanding the genetic basis of A/M and the need to identify additional causal variants to improve diagnosis and understanding of the disease.

We investigated unresolved A/M in participants recruited to the 100,000 Genomes Project to identify novel causal genes and improve diagnostic rates. Analysis focused on families with available trio or extended family data, particularly those with more than one affected individual, enhancing segregation analysis. A broad range of variant types were assessed and prioritised, such as SNVs, Indels and structural variants, based on low allele frequency and predicted functional impact using AlphaMissense and SpliceAI tools. Eight candidate variants were identified in two probands. Notably, a pathogenic variant in KIF17 showed strong segregation with disease and a high predicted impact on splicing. As KIF17 has only been associated with microphthalmia in one reported family, this study strengthens the evidence supporting its role in A/M.

Our ongoing work aims to functionally validate candidate genes by CRISPR knockdown in zebrafish to assess their role in eye development and investigate the impact of the identified variants in vivo, providing further insight into disease mechanisms. Ultimately, this study has the potential to increase molecular diagnosis rates in affected individuals, and improve our understanding of A/M and the developmental mechanisms involved in its aetiology.

ECR Poster Oliwia Sawicz.pdf

71 Improving the molecular diagnostic rate for developmental glaucoma in the 100,000 Genomes Project

Developmental glaucoma is a potentially blinding condition caused by raised intraocular pressure in babies and young children. It may be isolated or syndromic and is known to have a genetic basis, but in the UK the molecular diagnostic rate is less than 25%. Established causes of developmental glaucoma include pathogenic variants in CYP1B1, FOXC1, PAX6 and SLC4A11.

The 100,000 Genomes Project is a national study of families with rare diseases and cancer who underwent whole genome sequencing to aid research into the genetic basis of these conditions. This includes 98 families with developmental glaucoma, of whom 67 remain without a molecular diagnosis. Recruitment finished in 2018, and since then 44 additional genes have been associated with developmental glaucoma in the published literature.

We re-evaluated the whole genome sequencing data for the 67 unsolved families against an updated panel for developmental glaucoma comprising 52 genes. The genetic cause for disease was identified in an additional five families, with variants in SLC4A11, FOXC1, THBS1, GJA8 and CEP164 identified in one family each. This increases the molecular diagnostic rate in this cohort from 31.6% to 36.7%.

SLC4A11, FOXC1 and GJA8 are established causes of developmental glaucoma associated with other ocular and systemic anomalies. However, THBS1 and CEP164 are more recently associated causative genes, and their identification in this independent cohort supports their role in the pathogenesis of this condition. Identification and verification of novel causative genes for developmental glaucoma aids our understanding of the biological pathways that underly anterior segment development and opens potential avenues for therapeutic development.

IoO ECR 2026 Cronbach.pdf

72 Investigating Genetic Variation Affecting Vitamin A Metabolism in Microphthalmia, Anophthalmia and Coloboma

Microphthalmia, anophthalmia, and coloboma (MAC) are developmental eye disorders that remain one of the major causes of childhood blindness, affecting 1 to 4 in every 10,000 live births. While genetic heterogeneity of the MAC clinical spectrum is well recognised, many cases still lack an identifiable genetic diagnosis. Vitamin A deficiency has been highlighted as an environmental cause for MAC in previous cases, yet the effect of vitamin A metabolic gene variants has not been systematically examined.

Using the Genomics England 100,000 Genome Project dataset, this project aims to identify candidate genes linked to both vitamin A pathways and MAC condition and integrate these with patient-level genomic and phenotypic data to uncover novel gene–disease associations. An initial set of 71 genes were prioritised through Gene Ontology-based filtering, while 237 individuals were identified with unresolved MAC spectrum disorders as target cohort. Subsequent variants filtering yielded 34 single-nucleotide variants in candidate genes with a maximum allele frequency <0.05%.

Future work is to systematically filter rare variants for their predicted pathogenicity and evaluate these through a case-to-case analysis. Functional validation of these variants will be done by zebrafish mutagenesis. This project will ultimately provide new insights into the underlying molecular mechanisms of the MAC spectrum.

73 Macrophages and macrophage-conditioned medium activate contraction and migration in Mitomycin C-arrested fibroblasts

Inflammation is a major driver of fibrosis. In the eye, Mitomycin-C (MMC) is used in the clinic to prevent scarring following surgery to manage glaucoma. High inflammation levels before surgery is a significant risk factor for the development of fibrosis and subsequent treatment failure. Furthermore, in the presence of local inflammation, MMC is less efficient in modulating scarring, despite higher doses often used to compensate. We have developed a co-culture system to study how macrophages promote conjunctival fibroblast-mediated matrix contraction, as a model for post-surgical scarring. We show that macrophages stimulate fibroblast-mediated gel contraction through an increase in fibroblast spreading and protrusive activity, leading to a more elongated cell phenotype. Consistent with the clinical phenotype, MMC-arrested fibroblasts still contract gels efficiently in the presence of macrophages, despite being unable to do so following serum stimulation. Macrophage-conditioned medium partially recapitulated the effect of macrophage co-culture on gel contraction. In a wound scratch assay, while both baseline serum-free migration and serum-stimulated migration were inhibited in MMC-treated cells, MMC treatment had little effect on fibroblast migration in the presence of macrophage-conditioned medium. Both EGF and VEGF receptor inhibition reduced fibroblast migration and contraction. Increased Rab5 levels in the presence of macrophage-derived signals suggest enhanced receptor trafficking. These findings suggest that macrophage-derived growth factors promote fibroblast activation through growth factor-dependent signalling, influencing migration, polarity, and contractility independently of MMC arrest.

Conference Poster Mert Karakus ecr.pdf

74 Mitochondrial Dysfunction in Geographic Atrophy

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in adults over 60 years. It can be classified into two main forms: neovascular and dry AMD. The hallmarks of dry AMD are the accumulation of drusen (yellow deposits composed of lipids and protein) and the disruption of the retinal pigmented epithelium (RPE) cells and photoreceptors, leading to cell death. The late-stage, geographic atrophy, is characterised by progressive, expanding lesions in the retina that critically affect the macula, resulting in central vision loss.
Mitochondrial dysfunction in RPE cells is increasingly implicated in retinal degeneration; however, the cellular pathways that determine RPE survival or death under mitochondrial stress remain poorly understood. Interestingly, around 20% of patients with maternally inherited diabetes and deafness (MIDD), caused by the m.3243A>G mitochondrial DNA mutation, develop a geographic atrophy like phenotype. Reports have shown that patients mitochondrial tRNA Leucine (UUR) lacks the tm5U wobble uridine modification, performed by GTPBP3 and MTO1 enzymes.
The aim of this project is to develop a cellular model of mitochondrial dysfunction and use that for a whole-genome CRISPR knockout screen to systematically identify genetic modifiers of RPE vulnerability.
We have successfully knocked out GTPBP3 in ARPE-19 cells using CRISPR-Cas9 and validated at the DNA, RNA and protein level. Functionally, the OXPHOS complex subunits abundance was reduced, particularly of Complex I and Complex IV, in line with published studies. Seahorse XF Mito Stress test demonstrated significantly reduced basal/maximal respiration and ATP production in the knockout compared to the wild type. Finally, XTT metabolic test demonstrated that the knockout metabolic activity is significantly reduced in challenging conditions (galactose). In parallel, the GeCKO v2 whole-genome CRISPR library was successfully amplified and validated, meeting all quality control criteria for downstream screening. We have also successfully completed the pre-screen tests (seeding density, antibiotic resistance and multiplicity of infection) and are currently in screening phase.
Together, these results establish a scalable and phenotypically validated MIDD-like RPE model necessary to conduct an unbiased genome-wide screen for genetic modifiers of mitochondrial dysfunction, with the long-term goal of identifying novel therapeutic entry points for geographic atrophy.

75 Modelling Epigenetic Aging in Retinal Pigment Epithelial Cells to Investigate AMD Pathogenesis

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in high-income countries. Although aging is the strongest known risk factor for AMD, the molecular mechanisms linking aging to disease pathogenesis remain poorly understood.
The Information Theory of Aging proposes that aging is driven, in part, by a progressive loss of epigenetic information, in which DNA double-strand breaks (DSBs) and their repair may erode the epigenetic landscape and disrupt cellular identity. This project aims to establish an in vitro retinal pigment epithelial cell model to investigate whether DNA damage-induced epigenetic changes contribute to AMD-relevant aging phenotypes.
Specifically, this work adapts the Inducible Changes to the Epigenome (ICE) system in ARPE-19 cells through stable expression of inducible I-PpoI, providing a controllable platform for inducing DSBs. To date, plasmid constructs encoding inducible I-PpoI have been generated, and CRISPR/Cas9-mediated knock-in has been used to establish stable ARPE-19 cell lines as a controllable DSB-induction model. Initial validation using γH2AX staining, SA-β-Gal staining, and qPCR analysis of aging-related markers indicates that I-PpoI activation induces nuclear DNA damage and promotes senescence-associated aging phenotypes in ARPE-19 cells.
Overall, this work establishes the foundation for a retinal epithelial model of DNA damage-induced cellular aging. Based on this established system, future work will apply single-cell RNA sequencing, ATAC-seq, and DNA methylation profiling, and integrate these data with AMD patient datasets to identify candidate molecular targets and pathways that link DSB-induced epigenetic aging to AMD-relevant cellular dysfunction. These candidates will then be functionally tested to determine whether targeting them can reverse aging-associated cellular phenotypes relevant to AMD.

76 Muller glia: The architects of retinal synapse remodelling

Synaptic pruning is a neurodevelopmental checkpoint that occurs to eliminate excess neurons and refine circuits in the nervous system. The retina consists of highly organised networks of neuronal and glial cells that connect to pass visual information. However, the cellular and molecular processes behind synaptic pruning in the retina remain unclear. Glial cells, like astrocytes, microglia and Müller glia (MG; the principal glia of the retina), contact neurons and synapses to provide a myriad of support functions. Microglia have long been thought to solely undertake retinal synapse pruning due to their widely reported role in the brain. Here, using fixed and live zebrafish retinas, we show that MG carry out phagocytosis of synapses, working alongside, but independently of microglia.

Pruning occurs in two distinct time points during development, with each glial cell type being the principal “pruner” at each stage. We conducted a molecular screen to investigate the molecular mechanisms that regulate these pruning events. We found that two signalling pathways belonging to the complement system, the classical and lectin complement pathways, are expressed in glia at the stages of pruning in the retina. Consistent with the brain, microglia act through the classical complement pathway, whilst MG mainly rely on the lectin system. We are now using CRISPR/Cas9 knockout and cell-specific over-expression to determine the role of these pathways in each glial cell during development. These findings provide new insights into the role of MG in retinal development and provide potential novel molecules to explore in promoting synaptic maintenance and support in ageing and neurodegenerative disease.

77 Peroxisomal Dynamics and Organelle Interactions in Retinal Pigment Epithelium

Photoreceptors undergo continuous renewal to maintain visual function and retinal integrity. Each day, neighbouring retinal pigment epithelial (RPE) cells phagocytose the aged and damaged tips of photoreceptor outer segments (OS). Within the RPE, these components are degraded, while essential lipids and metabolites are recycled back to photoreceptors. This tightly regulated process supports photoreceptor survival, retinal homeostasis, and efficient phototransduction. RPE cells manage a remarkable amount of material daily, processing up to 30 OS tips per cell, each representing approximately 10% of the entire OS. Consequently, disruption of lipid homeostasis and lipid accumulation are hallmark features of dysfunctional RPE observed in retinal diseases such as Stargardt disease, choroideremia, and age-related macular degeneration.
Peroxisomes are highly dynamic membrane-bound organelles involved in lipid β-oxidation, lipid biosynthesis, hydrogen peroxide metabolism, and detoxification. Their importance in retinal health is highlighted in patients with Zellweger syndrome, a severe inherited peroxisome biogenesis disorder affecting multiple organs, including the eye. Patients with Zellweger syndrome develop retinopathy that can lead to early-onset blindness, likely caused by lipid accumulation and dysfunction in both RPE cells and photoreceptors. Despite their importance, the role of peroxisomes in RPE cells and the mechanisms linking peroxisomal dysfunction to lipid accumulation remain poorly understood.
This study aims to address this gap by investigating interactions between peroxisomes and other cellular structures in healthy RPE using animal and cellular models. Immunofluorescence imaging revealed an extensive peroxisomal network distributed throughout the cell. Electron microscopy confirmed known interactions between peroxisomes, mitochondria, and the endoplasmic reticulum and identified new type of contacts with the plasma membrane. We also observed interactions with RPE-specific organelles, including melanosomes and phagosomes, which may play important roles in lipid transport. Finally, tomographic analysis resolved tethering structures between selected organelles, providing insights into proteins potentially involved in these interactions. Together, these findings establish a foundation for future studies using induced pluripotent stem cell-derived RPE models carrying pathogenic mutations observed in patients with Zellweger syndrome with retina phenotypes.

78 PLXND1 signalling excludes blood vessels from airway smooth muscle and epithelium

Guided by multiple signalling pathways, vascular endothelial cells (ECs) closely align with alveolar epithelium to mediate gas exchange in the lung. By contrast, the molecular and cellular mechanisms that organise airway vasculature are poorly understood. Here, we have immunostained mouse lungs to reveal that the vascular, bronchiolar smooth muscle and epithelial compartments of the airways adopt a strict zonation that is established during embryonic development when the first bronchi are formed and maintained into adulthood. Airway zonation is disrupted in mice lacking PLXND1 either globally or selectively in ECs, with angiogenic sprouting of lung capillaries first into the airway smooth muscle and then into the subepithelial space. Unexpectedly, single or combined ablation of the PLXND1 ligands with known vascular repulsive activity, SEMA3C and SEMA3E, did not compromise airway zonation to phenocopy PLXND1 loss. Nevertheless, ablating all SEMA3 signalling through PLXND1 and its co-receptors NRP1 and NRP2 caused vascular invasion of the airways, similar to the phenotype caused by PLXND1 loss, suggesting that multiple SEMA3 ligands converge on PLXND1 in an unknown mechanism. We conclude that partial redundancy of SEMA3 signalling through PLXND1 needs to be defined further to understand how airway zonation is established. Moreover, the functional significance of airway zonation needs to be investigated.

79 Retrotransposon insertions upstream of CRX are associated with autosomal dominant cone rod dystrophy

Introduction
A significant proportion of individuals with inherited retinal dystrophies (IRD) remain genetically unsolved. New technologies offer the opportunity to identify and characterise previously inaccessible areas of the genome that could harbour disease-causing variants. We investigated five families affected by autosomal dominant cone-rod dystrophy (adCRD); one family from the UK, which has remained genetically unsolved for decades, and four families originating from a single village in Greece.
Methods
The genetic cause of IRD in these families was investigated using a combination of whole-genome sequencing, optical genome mapping (OGM) and long read Nanopore sequencing.
Results
We identified two distinct mobile element insertions (MEI) ~16Kb upstream of CRX in the UK and Greek families, located ~200bp apart. OGM and long-read sequencing enabled characterization of these MEIs as SINE-VNTR-Alu (SVA) insertions. The UK family carried a ~2.5Kb SVA type D insertion whereas the Greek families carried a ~3Kb SVA type F insertion. Based on retina-specific chromatin architecture and cis-regulatory interactions, we hypothesize that these insertions disrupt CRX expression.
Conclusion
The MEIs we identified represent a new genomic mechanism of disease at this locus. The localisation of these variants within a shared regulatory region upstream of CRX supports a cis-regulatory pathogenic effect.

80 Targeting Semaphorin 3 Signalling Promotes Reparative Angiogenesis in Murine Models of Retinopathy

Retinopathy of prematurity (ROP) is a leading cause of neonatal blindness, characterized by hyperoxia-induced vascular regression followed by pathological neovascularisation. Restoring physiological revascularisation while preventing aberrant neovascular tuft formation remains a major therapeutic challenge. Although anti-VEGF-A therapy is the current standard of care, frequent treatment failure and disease recurrence underscore the need for alternative strategies. Here, we investigate the endogenous role of class 3 semaphorins (SEMA3s) in pathological angiogenesis using genetically modified mouse models of oxygen-induced retinopathy (OIR) and physiological retinal angiogenesis. Inhibition of SEMA3 signalling enhanced retinal revascularisation and remodelled the neovascular niche in hypoxic retinas, shifting angiogenesis from pathological neovascularisation towards vascular repair. Loss of SEMA3E completely suppressed neovascular tuft formation and promoted reparative angiogenesis in hypoxic retinas. SEMA3E loss was associated with enhanced microglial activation and infiltration of the avascular zone in hypoxic retinas. These findings identify SEMA3E as a critical regulator of retinal vascular growth that operates independently of vascular endothelial growth factor (Vegfa) expression in hypoxic retinas. Inhibition of Sema3s led to increased vascular growth, not only under hypoxic conditions but also during development; Loss of SEMA3A, SEMA3E, or SEMA3F increased endothelial cell density during physiological angiogenesis. Mechanistically, SEMA3s crosstalk with VEGF signalling, with Vegfa upregulated during physiological angiogenesis. SEMA3s deficiency also regulated the formation of endothelial tight and adherent junctions. Targeting SEMA3 signalling may provide a novel therapeutic approach to promote reparative angiogenesis while limiting pathological neovascularisation in ROP and other ischaemic retinopathies.

2026_ Targeting Semaphorin 3 Signalling Promotes Reparative Angiogenesis in Retinopathy.pdf

81 The impact of amyloid precursor protein knockout on protein homeostasis in the ageing retina of African turquoise killifish

In the field of ageing, protein aggregation and the downstream pathologies implicated in neurodegenerative diseases have been extensively studied in the brain. The retina, often termed the window to the brain, has been suggested to exhibit maladaptive processes similar to those seen in the brain, however, the extent to which aggregation-prone proteins accumulate in the healthy aged retina remains underexplored. Progress in this area has been limited by long-lived animal models, which hinder experimental throughput, while the use of shorter living models often compromises translational relevance. The African turquoise killifish (Nothobranchius furzeri), a naturally compressed-lifespan vertebrate displaying conserved retinal structure and hallmarks of human ageing, offers a powerful model to address this question. Here, we investigated retinal distribution of aggregation-prone proteins and whether reducing amyloid-precursor protein-a (appa) burden using appa-/- killifish will improve ageing-associated retinal phenotypes. We analysed retinas from young (6-week) and aged (36-week) wild-type killifish and compared these with age-matched appa-/- fish, focusing on amyloid-β, tau, hyperphosphorylated tau (p-Tau) and ⍺-synuclein. Immunohistochemistry analysis showed that protein accumulation was most prominent within outer segments of photoreceptors, amacrine cells and ganglion cells, which are retinal populations known to be vulnerable to degenerative stress. Furthermore, parallel staining on human retinas revealed comparable layer-specific localisation patterns of aggregation-prone proteins. Notably, appa-/- appears to alter retinal p-Tau distribution in aged killifish, potentially due to reduced p-Tau levels or altered cell-type localisation. Future work will determine whether appa KO alters upstream proteostasis pathways, including ubiquitin-proteasome, lysosomal and autophagic systems, as well as downstream mitochondrial dysfunction and cell death. Together, these findings further support the killifish as a tractable model for studying age-related neurodegenerative mechanisms and may aid development of retinal biomarkers for diagnosis, disease staging, and severity assessment.

82 The role of the RNA-binding protein Musashi 1 (MSI1) in retinal development and alternative mRNA splicing

A specialised transcriptome is activated early during retinal development. Importantly, numerous splicing altering variants are associated with Inherited retinal diseases (IRD’s), like Leber Congenital Amaurosis (LCA), therefore, an improved understanding of retinal alternative splicing could help reveal underlying disease mechanisms. The inclusion of retina-specific exons is thought to be driven by a group of RNA-binding proteins, including Musashi 1 (MSI1) and MSI2, which are required for mouse photoreceptor development. Furthermore, MSI1 expression enhanced cryptic exon inclusion in human cells. To investigate MSI1 in human photoreceptors, gene edited induced pluripotent stem cell (iPSC) lines for control, MSI1 knock-out (KO), a homozygous LCA splicing mutant, and LCA-MSI1 KO were differentiated to retinal organoids (ROs). The effect of MSI1 ablation on photoreceptor development, outer segment formation, retinal-specific splicing and CEP290 cryptic exon inclusion was investigated. RO photoreceptor development was delayed, and outer segment density was reduced in the MSI1 KO lines. Immunohistochemistry (IHC) analysis confirmed photoreceptor development in MSI1 KO ROs and showed MSI1/2 localisation to the photoreceptor cytoplasm and nucleus. MSI2 upregulation in MSI1 KO ROs was observed by IHC and RNAseq. These data highlight the complexity of alternative splicing in the human retina and how this could affect splicing dysregulation in IRD.

2026_ECR_Symposium_Poster.pdf

83 The role of VIP interneurons in ocular dominance plasticity

This study investigates the role of vasoactive intestinal peptide (VIP) interneurons in adult ocular dominance plasticity, focusing on how different components of VIP signalling contribute to cortical network reorganisation following monocular deprivation (MD). While plasticity is most prominent during the critical period, the adult visual cortex retains a limited but functionally relevant capacity for experience-dependent change. Locomotion has been shown to enhance this plasticity via a VIP→SST disinhibitory circuit, yet the specific contributions of VIP peptide signalling versus GABAergic output remain unclear.

To address this, two transgenic mouse models were used: VIP knockout (VIPKO) mice, lacking VIP peptide signalling, and VIP-cre;Vgat fl/fl (VIPVGAT) mice, in which GABA release from VIP interneurons is impaired. In vivo two-photon calcium imaging was performed in awake mice to record neuronal activity in layer 2/3 of primary visual cortex across baseline, MD and recovery timepoints. Functional connectivity was assessed using pairwise correlations and then multilayer community analysis. Noise and signal correlations, population coupling was also assessed to help explain potential network configuration.

At the network level, VIPVGAT mice showed trends towards increased modularity, greater community number, and reduced flexibility, consistent with a more segregated and less adaptable network, although these effects were not statistically significant. The most robust finding was a significant change in similarity to baseline community structure, indicating sustained network reorganisation across all groups.

Across all groups, MD induced a reduction in correlations and population coupling, consistent with network reorganisation. Control mice showed partial recovery, particularly during locomotion, suggesting stabilisation of a reorganised network. VIPKO mice exhibited broadly similar network organisation but showed an ipsilateral bias in recovery, indicating asymmetric restoration of connectivity. In contrast, VIPVGAT mice showed weaker recovery overall, with noise correlations and population coupling failing to return towards baseline, especially during running.

Overall, these findings suggest that GABAergic output from VIP interneurons plays a role in coordinating activity-dependent recovery and network restructuring in the adult visual cortex.

ECR symposium poster.pdf

84 Unveiling the role of mitochondria-plasma membrane contacts in healthy vision

Mitochondria play a vital role in the visual system, with functions that include ATP production, to meet the high energy demand of phototransduction in photoreceptors and support the retinal pigment epithelium role in the visual cycle. Other roles include maintenance of the redox balance and calcium/stress homeostasis as well as the organisation of these organelles being proposed to direct light delivery in cone photoreceptors. Previous work from the group identified contacts between mitochondria and the plasma membrane, which are involved in the alignment of mitochondria between adjacent photoreceptors, suggesting a potential role of these contacts in intercellular communication.
This project aims to characterize the architecture of the mitochondria-plasma membrane contacts and determine their functional relevance in photoreceptor health, through both ultrastructural analysis and identification of their molecular regulators. We have used SH-SY5Y neuroblastoma cells as a tractable model, as they have mitochondria-plasma membrane contacts within the neurites.
To identify the proteins involved in these contacts, we have focused on setting up proximity-labelling to identify the tethering proteins at mitochondria-plasma membrane contact sites. These are based on an engineered ascorbate peroxidase (APEX2) and a pair of inactive fragments of a reconstitutable biotin ligase (TurboID), which enable biotinylation of proteins within a 10–20 nm radius of the contact sites. These biotinylated proteins can subsequently be isolated and assessed by mass spectrometry.
We have observed that treatment with FCCP leads to a marked reduction in mitochondria–plasma membrane contact sites. FCCP, a potent mitochondrial uncoupler, collapses the inner membrane proton gradient and induces rapid release of mitochondrial calcium into the cytosol. Therefore, these results raise the possibility that either membrane potential, mitochondrial calcium, or both regulate mitochondria–plasma membrane contacts. Preliminary experiments showed that pharmacological inhibition of the mitochondrial calcium uniporter reduced mitochondria–plasma membrane contacts, suggesting that mitochondrial calcium could be involved in the regulation of these contact sites.
Overall, identifying the proteins and mechanisms that regulate the formation of mitochondria–plasma membrane contacts, as well as their role in photoreceptors, may elucidate how dysfunction of these contacts contributes to disease pathogenesis and vision loss, and can provide molecular targets for future therapeutic interventions.

85 Using whole blood stimulation to identify predictive biomarkers in Birdshot Chorioretinopathy

Birdshot chorioretinopathy (BCR) is a chronic, autoimmune form of posterior uveitis that targets the retina and can result in progressive visual deterioration or permanent blindness. It has a striking genetic association with the HLA A29 molecule, which plays a central role in disease susceptibility. As a result, BCR is widely regarded as a prototypical HLA linked autoimmune ocular disease.
Management of BCR relies on early, aggressive, and sustained immunosuppression. Although, many patients exhibit only a partial response to therapy, and a significant proportion develop treatment resistance over time. Even the most effective immunosuppressant —adalimumab—only has a 60% response rate, highlighting the substantial unmet need in understanding and predicting therapeutic outcomes.
To address this, we applied the TruCulture whole blood stimulation system to a cohort of adalimumab naïve BCR patients in order to investigate their systemic immune response and identify biomarker signatures associated with long term treatment success. Whole blood was collected and incubated for 22 hours in the presence or absence of systemic or targeted immune stimuli, enabling controlled profiling of patient specific inflammatory pathways.
Using the OLINK Reveal proteomic platform, we quantified the expression of over 1,000 proteins across three conditions — Null, LPS, and LPS + Adalimumab. This dataset provides a high resolution view of cytokine responses, inflammatory signalling, and drug modulated pathways. By integrating these findings with each patient’s clinical history and genetic background, we aim to define immune profiles that can guide clinicians toward more targeted and effective treatment strategies for BCR.

86 Validation of GEF-H1 Inhibitors in Preclinical Models of Ocular Disease

Inflammation and fibrosis, which contribute to epithelial and endothelial dysfunction, are common pathological features of ocular diseases such as eye infections and age-related macular degeneration (AMD). Guanine nucleotide exchange factor H1 (GEF-H1) activates RhoA signalling to promote inflammatory and fibrotic responses (PMID: 35681428). Under physiological conditions, GEF-H1 expression is low but is upregulated in patients with ocular diseases (PMID: 20089843), making it a promising therapeutic target.

Using in silico approaches, we have developed peptide- and stapled peptide-based inhibitors (PMID: 35681428; doi.org/10.1101/2024.11.18.624118), as well as more recently identified small-molecule inhibitors targeting GEF-H1. The efficacy of these inhibitors was evaluated using in vitro and in vivo models of ocular disease. In Madin–Darby canine kidney (MDCK) epithelial cells overexpressing GEF-H1, the inhibitors attenuated NF-κB activation and reduced cell elongation. Similarly, they rescued LPS-induced morphological changes in primary human dermal microvascular endothelial cells (HDMECs).

Furthermore, the small molecule SM762 reduced choroidal neovascularization (CNV) lesions in a laser-induced CNV mouse model of AMD, as demonstrated by in vivo imaging, including fundoscopy, spectral-domain optical coherence tomography (SD-OCT), and fundus fluorescein angiography (FFA), as well as RPE–choroid flat-mount staining. In addition, SM762 decreased retinal vascular leakage in oxygen-induced retinopathy (OIR) and Arhgef18 Müller glia-specific knockout (Arhgef18 MG-KO) mouse models, as evidenced by FFA.

Taken together, these findings demonstrate that GEF-H1 inhibitors reduce subretinal neovascularization, fibrosis, and retinal vascular leakage, highlighting their therapeutic potential for ocular diseases associated with inflammation and fibrosis.

87 Awards and Closing Remarks

Speaker: Giulia De Rossi (University College London)

16:50 Networking & Drinks