Biology subjects

Georgiou, M.

Publications and source records attributed to Georgiou, M..

7 recordsLinked to original sources

bioRxiv2026

Lung function candidate genes in Drosophila melanogaster

RationaleChronic obstructive pulmonary disease (COPD) represents a leading cause of global morbidity and mortality. Genome-wide association studies (GWAS) have implicated numerous genetic variants in lung function impairment, yet confidently identifying the underlying genes and pathways, and translating these findings into mechanistic insight, remains a significant challenge. ObjectivesTo leverage the genetic amenability and high-throughput screening capability of Drosophila melanogaster to determine the role of candidate causal genes in epithelial cell homeostasis. MethodsWe performed a loss-of-function analysis of 60 prioritised lung function candidate causal genes implicated from GWAS in two distinct epithelia: the dorsal thorax and trachea. ResultsWe identified 57/60 tested candidate genes that alter at least one aspect of epithelial morphology and behaviour upon knockdown. With a focus on junctional integrity, cell delamination and tissue growth, we identified 11 genes for further study: Sec6, RpS26, pAbp, Arf102f, Riok1, Sra-1, Inpp5e, CG31759, ssh, eIF6 and Rtf1. Further characterisation found a significant reduction in junctional E-Cadherin levels following Arf102F, Rtf1, RioK1 and Sra-1 knockdown. Following a secondary screen in the Drosophila tracheal system for priority candidates, Sec6 and RpS26 were associated with significant airway defects and a reduction in larval body size. 8/11 priority genes exhibited differential lung gene expression between controls and patients with COPD. ConclusionsThese data demonstrate the amenability of Drosophila melanogaster to perform in vivo functional analyses of candidate causal genes at scale. Initial findings implicate several genes in epithelial homeostasis and integrity, providing new mechanistic understanding and potential therapeutic targets for COPD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/731340v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@dbff6dorg.highwire.dtl.DTLVardef@15e84f0org.highwire.dtl.DTLVardef@69d97aorg.highwire.dtl.DTLVardef@144f47e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cell Biology↗

bioRxiv2026

Cell-autonomous restoration of splicing homeostasis and RP11 phenotype in patient-derived RPE and retinal organoids by PRPF31.AAV gene therapy

Mutations in PRPF31 gene cause retinitis pigmentosa type 11 (RP11) through haploinsufficiency, impairing spliceosome assembly and triggering progressive retinal degeneration. While gene augmentation holds therapeutic promise, key questions remain regarding the mechanistic basis of rescue and its therapeutic efficacy across all primarily affected human retinal cell types and disease stages. Here, we utilised patient-specific induced pluripotent stem cells (iPSCs)-derived retinal pigment epithelium (RPE) cells and three-dimensional retinal organoids (ROs) to determine the therapeutic mechanism of AAV-mediated PRPF31 delivery. Using the ShH10(Y445F) serotype to ensure robust dual targeting of RPE and photoreceptors, we demonstrated that PRPF31 transduction restores nuclear localisation, reorganises SC35+ nuclear speckles and enhances p-SF3B1 active spliceosome foci. Transcriptomic and proteomic profiling revealed a global rescue of splicing activity and upregulation of phagocytosis, protein aggregate clearance pathways, and mitochondrial proteins. These molecular shifts facilitated the clearance of proteotoxic cytoplasmic aggregates and reversed key functional deficits; specifically, they reinforced RPE apical-basal polarity, restored phagocytic capacity and normal ciliary morphology and incidence, and boosted light-evoked activity in photoreceptors. Combining gene therapy with rapamycin-mediated-autophagy activation conferred no additive benefit, identifying the restoration of splicing homeostasis as the critical therapeutic driver. Notably, substantial phenotypic rescue is achievable in mature RPE, supporting a broad clinical window for intervention. Collectively, these data provide a systems-level validation of ShH10(Y445F)-PRPF31 gene therapy and establish a mechanistic framework for its clinical translation in RP11.

molecular biology↗

bioRxiv2021

Conjunctival epithelial cells resist productive SARS-CoV-2 infection

Although tropism of SARS-CoV-2 for respiratory tract epithelial cells is well established, an open question is whether the conjunctival epithelium is also a target for SARS-CoV-2. Conjunctival epithelial cells, which express viral entry receptors ACE2 and TMPRSS2, constitute the largest exposed epithelium of the ocular surface tissue, and may represent a relevant viral entry route. To address this question, we generated an organotypic air-liquid-interface model of conjunctival epithelium, composed of progenitor, basal and superficial epithelial cells and fibroblasts, which could be maintained successfully up to day 75 of differentiation. Using single-cell RNA Seq, with complementary imaging and virological assays, we observed that while all conjunctival cell types were permissive to SARS-CoV-2 genome expression, a productive infection did not ensue. The early innate immune response to SARS-CoV-2 infection in conjunctival cells was characterised by a robust autocrine and paracrine NF-K{beta} activity, without activation of antiviral interferon signalling. Collectively, these data enrich our understanding of SARS-CoV-2 infection at the human ocular surface, with potential implications for the design of preventive strategies and conjunctival transplants.

cell biology↗

bioRxiv2021

Translational readthrough as a potential therapeutic for AIPL1-associated Leber Congenital Amaurosis in a patient-derived iPSC-retinal organoid model

Leber Congenital Amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterised by severe sight impairment in infancy and rapidly progressive degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cGMP phosphodiesterase (PDE6) compared to control or CRISPR corrected organoids. Moreover, increased levels of cGMP were detected. The translational readthrough inducing drug (TRID) PTC124 was investigated as a potential therapeutic. LCA4 retinal organoids exhibited rescue of AIPL1 and PDE6; however, the level of full-length, functional AIPL1 induced through PTC124 treatment was insufficient to reduce cGMP levels and fully rescue the LCA4 phenotype. LCA4 retinal organoids are a valuable platform for the in vitro investigation of the molecular mechanisms that drive photoreceptor loss and for the evaluation of novel therapeutics.

cell biology↗

bioRxiv2021

Progressive protein aggregation in PRPF31 patient retinal pigment epithelium cells: the mechanism and its reversal through activation of autophagy

Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. Here, we used iPSC technology to generate retinal organoids and RPE models from three patients with severe and very severe PRPF31-adRP, normal individuals and a CRISPR/Cas9-corrected isogenic control. To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells was carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle proteins, which accumulated progressively with time. Mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wildtype levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells with reduced U4/U6 snRNPs and accumulation of U5, smaller nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin resulted in reduction of cytoplasmic aggregates and improved cell survival. Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients. HighlightsO_LIPRPF31 RP mutations lead to formation of insoluble aggregates containing the mutant PRPF31 and misfolded, ubiquitin conjugated proteins including key visual cycle proteins (e.g. RLBP1) in RPE cells, which accumulate progressively with time and affect tight junctions and cell survival. C_LIO_LIMutant PRPF31 is predominantly localised in cytoplasmic aggregates of patient specific RPE and retinal cells and is not able to be incorporated into splicing complexes to cause direct mis-splicing. C_LIO_LIHigh-throughput quantitative proteomics identifies significantly altered RNA splicing, visual perception, retinoid metabolism, waste disposal and unfolded protein response pathways in patient RPE cells, and autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways in photoreceptor cells. C_LIO_LIAccumulation of PRPF31 mutant variant as cytoplasmic aggregates reduces wildtype PRPF31 in the nucleus leading to tri-snRNP assembly defects, characterised by accumulation of U5 and reduction of U4/U6 snRNPs in Cajal bodies, altered morphology of nuclear speckles and consequently downregulation of active spliceosomes (Bact and C complexes) in PRPF31 patient RPE and retinal cells. C_LIO_LIProteomic study of insoluble aggregates identifies other RP-linked splicing factors and multiple key retinal-specific proteins, whose variants are linked to retinitis pigmentosa, within the aggregates of patient RPE cells. C_LIO_LIPRPF31 patient RPE cells have impaired waste disposal and proteasome mediated degradation, which together with the impaired autophagy pathway, further exacerbate aggregate formation. C_LIO_LIPhagocytosis of photoreceptor outer segment fragments (POS) shed daily by RPE cells accelerates aggregation of key proteins indicating enhanced cytoplasmic aggregate formation under physiological conditions in patient RPE cells. C_LIO_LIActivation of autophagy via administration of rapamycin results in reduction of cytoplasmic aggregates in RPE cells, correct localisation of mislocated and misfolded proteins to the nucleus, thereby improving cell survival. C_LI

cell biology↗

bioRxiv2020

CG7379/ING1 suppress cancer cell invasion by maintaining cell-cell junction integrity

Approximately 90% of cancer related deaths can be attributed to a tumours ability to spread. We have identified CG7379, the fly orthologue of human ING1, as a potent invasion suppressor. ING1 is a type II tumour suppressor with well-established roles in the transcriptional regulation of genes that control cell proliferation, response to DNA damage, oncogene-induced senescence and apoptosis. Recent work suggests a possible role for ING1 in cancer cell invasion and metastasis, but the molecular mechanism underlying this observation is lacking. Our results show that reduced expression of CG7379 promotes invasion in vivo in Drosophila, reduces the junctional localisation of several adherens and septate junction components, and severely disrupts cell-cell junction architecture. Similarly, ING1 knockdown significantly enhances invasion in vitro and disrupts E-cadherin distribution at cell-cell junctions. A transcriptome analysis reveals that loss of ING1 affects the expression of several junctional and cytoskeletal modulators, confirming ING1 as an invasion suppressor and a key regulator of cell-cell junction integrity.

cancer biology↗

bioRxiv2020

Targeting lateral inhibition to improve vision following macular degeneration

Macular degeneration is the leading cause of blindness in the developed world. Whilst most patients lose sight owing to atrophic changes, no treatments currently exist that improve the vision deficit due to atrophy. Here, we identify loss of lateral inhibition as a specific mechanism by which photoreceptor degeneration reduces visual function beyond the atrophic area. We find that this inhibition is adaptive, and that if modulated can improve visual function, making inhibitory circuits an unexpected therapeutic target for age related macular degeneration and related disorders.

neuroscience↗