Nina Riddell

Bidirectional Expression of Metabolic, Structural, and Immune Pathways in Early Myopia and Hyperopia

Myopia (short-sightedness) affects 1.45 billion people worldwide, many of whom will develop sight-threatening secondary disorders. Myopic eyes are characterized by excessive size while hyperopic (long-sighted) eyes are typically small. The biological and genetic mechanisms underpinning the retinas local control of these growth patterns remain unclear. In the present study, we used RNA sequencing to examine gene expression in the retina/RPE/choroid across 3 days of optically-induced myopia and hyperopia induction in chick. Data were analyzed for differential expression of single genes, and Gene Set Enrichment Analysis (GSEA) was used to identify gene sets correlated with ocular axial length and refraction across lens groups. Like previous studies, we found few single genes that were differentially-expressed in a sign-of-defocus dependent manner (only BMP2 at 1 day). Using GSEA, however, we are the first to show that more subtle shifts in structural, metabolic, and immune pathway expression are correlated with the eye size and refractive changes induced by lens defocus. Our findings link gene expression with the morphological characteristics of refractive error, and suggest that physiological stress arising from metabolic and inflammatory pathway activation could increase the vulnerability of myopic eyes to secondary pathologies.

Integrated Comparison of GWAS, Transcriptome, and Proteomics Studies Highlights Similarities in the Biological Basis of Animal and Human Myopia

Purpose To identify commonalities between the genes in close proximity to genome-wide association study (GWAS) refractive error and axial length loci, and the genes and proteins differentially expressed in animal models of optically induced refractive error. Methods The GWAS catalog was searched for loci significantly (P ≤ 5*10-8) associated with refractive error or axial length. PubMed was searched for exploratory animal transcriptome and proteomics studies of optically induced refractive error. A total of 15 GWAS, 7 transcriptome, and 9 proteomics studies met inclusion criteria. Ensembls BioMart was used to identify human orthologs for the differentially expressed genes and proteins from animal studies. These orthologs were then compared to the protein-coding genes within 1 megabase (Mb), 500 kilobases (kb), and 250 kb of human GWAS loci by using the GeneOverlap R package, and Benjamini-Hochberg-adjusted P values and odds ratios (ORs) were calculated for each intersection. Results The genes near human GWAS loci overlapped significantly with the genes downregulated during early myopia induction in animals (1Mb: OR = 1.56, P = 0.025; 500 kb: OR = 1.92, P = 0.010; 250 kb: OR = 2.33, P = 0.010). There was also significant overlap between the genes and proteins differentially expressed in late myopia (OR = 4.12, P = 0.018). When animal study results were segregated by methodologic parameters, GWAS candidate genes overlapped significantly with the genes differentially expressed at early (OR = 1.50, P = 0.010) but not late (OR = 1.04, P = 0.684) induction time-points. Gene and protein expression responses also appeared well conserved across model species, and there was no evidence of greater GWAS-transcriptome concordance in similar species to humans (e.g., primates or mammals). Conclusions These findings suggest that genetic and environmental factors control ocular growth via similar biological pathways across species, and support the continued use of animal models for investigating the biological mechanisms underlying human myopia development.

Novel evidence for complement system activation in chick myopia and hyperopia models: a meta-analysis of transcriptome datasets

Myopia (short-sightedness) and hyperopia (long-sightedness) occur when the eye grows too long or short, respectively, for its refractive power. There are currently approximately 1.45 billion myopes worldwide and prevalence is rising dramatically. Although high myopia significantly increases the risk of developing a range of sight-threatening disorders, the molecular mechanisms underlying ocular growth regulation and its relationship to these secondary complications remain poorly understood. Thus, this study meta-analyzed transcriptome datasets collected in the commonly used chick model of optically-induced refractive error. Fifteen datasets (collected across five previous studies) were obtained from GEO, preprocessed in Bioconductor, and divided into 4 conditions representing early (≤1 day) and late (>1 day) myopia and hyperopia induction. Differentially expressed genes in each condition were then identified using Rank Product meta-analysis. The results provide novel evidence for transcriptional activation of the complement system during both myopia and hyperopia induction, and confirm existing literature implicating cell signaling, mitochondrial, and structural processes in refractive error. Further comparisons demonstrated that the meta-analysis results also significantly improve concordance with broader omics data types (i.e., human genetic association and animal proteomics studies) relative to previous transcriptome studies, and show extensive similarities with the genes linked to age-related macular degeneration, choroidal neovascularization, and cataract.

Pathway analysis identifies altered mitochondrial metabolism, neurotransmission, structural pathways and complement cascade in retina/RPE/ choroid in chick model of form-deprivation myopia

Purpose RNA sequencing analysis has demonstrated bidirectional changes in metabolism, structural and immune pathways during early induction of defocus induced myopia. Thus, the aim of this study was to investigate whether similar gene pathways are also related to the more excessive axial growth, ultrastructural and elemental microanalytic changes seen during the induction and recovery from form-deprivation myopia (FDM) in chicks and predicted by the RIDE model of myopia. Methods Archived genomic transcriptome data from the first three days of induction of monocularly occluded form deprived myopia (FDMI) in chicks was obtained from the GEO database (accession # GSE6543) while data from chicks monocularly occluded for 10 days and then given up to 24 h of normal visual recovery (FDMR) were collected. Gene set enrichment analysis (GSEA) software was used to determine enriched pathways during the induction (FDMI) and recovery (FDMR) from FD. Curated gene-sets were obtained from open access sources. Results Clusters of significant changes in mitochondrial energy metabolism, neurotransmission, ion channel transport, G protein coupled receptor signalling, complement cascades and neuron structure and growth were identified during the 10 days of induction of profound myopia and were found to correlate well with change in axial dimensions. Bile acid and bile salt metabolism pathways (cholesterol/lipid metabolism and sodium channel activation) were significantly upregulated during the first 24 h of recovery from 10 days of FDM. Conclusions The gene pathways altered during induction of FDM are similar to those reported in defocus induced myopia and are established indicators of oxidative stress, osmoregulatory and associated structural changes. These findings are also consistent with the choroidal thinning, axial elongation and hyperosmotic ion distribution patterns across the retina and choroid previously reported in FDM and predicted by RIDE.

An asymmetric outer retinal response to drifting sawtooth gratings.

Electroretinogram (ERG) studies have demonstrated that the retinal response to temporally modulated fast-ON and fast-OFF sawtooth flicker is asymmetric. The response to spatiotemporal sawtooth stimuli has not yet been investigated. Perceptually, such drifting gratings or diamond plaids shaded in a sawtooth pattern appear brighter when movement produces fast-OFF relative to fast-ON luminance profiles. The neural origins of this illusion remain unclear (although a retinal basis has been suggested). Thus we presented toad eyecups with sequential epochs of sawtooth, sine-wave, and square-wave gratings drifting horizontally across the retina at temporal frequencies of 2.5-20 Hz. All ERGs revealed a sustained direct-current (DC) transtissue potential during drift and a peak at drift offset. The amplitudes of both phenomena increased with temporal frequency. Consistent with the human perceptual experience of sawtooth gratings, the sustained DC potential effect was greater for fast-OFF cf. fast-ON sawtooth. Modeling suggested that the dependence of temporal luminance contrast on stimulus device frame rate contributed to the temporal frequency effects but could not explain the divergence in response amplitudes for the two sawtooth profiles. The difference between fast-ON and fast-OFF sawtooth profiles also remained following pharmacological suppression of postreceptoral activity with tetrodotoxin (TTX), 2-amino-4-phosphonobutric acid (APB), and 2,3 cis-piperidine dicarboxylic acid (PDA). Our results indicate that the DC potential difference originates from asymmetries in the photoreceptoral response to fast-ON and fast-OFF sawtooth profiles, thus pointing to an outer retinal origin for the motion-induced drifting sawtooth brightness illusion.