Selective transcriptomic dysregulation of metabolic pathways in liver and retina by short- and long-term dietary hyperglycemia.

A high glycemic index (HGI) diet induces hyperglycemia, a risk factor for diseases affecting multiple organ systems. Here, we evaluated tissue-specific adaptations in the liver and retina after feeding HGI diet to mice for 1 or 12 month. In the liver, genes associated with inflammation and fatty acid metabolism were altered within 1 month of HGI diet, whereas 12-month HGI diet-fed group showed dysregulated expression of cytochrome P450 genes and overexpression of lipogenic factors including Srebf1 and Elovl5. In contrast, retinal transcriptome exhibited HGI-related notable alterations in energy metabolism genes only after 12 months. Liver fatty acid profiles in HGI group revealed higher levels of monounsaturated and lower levels of saturated and polyunsaturated fatty acids. Additionally, HGI diet increased blood low-density lipoprotein, and diet-aging interactions affected expression of mitochondrial oxidative phosphorylation genes in the liver and disease-associated genes in retina. Thus, our findings provide new insights into retinal and hepatic adaptive mechanisms to dietary hyperglycemia.

Dietary fish oil enriched in very-long-chain polyunsaturated fatty acid reduces cardiometabolic risk factors and improves retinal function.

Very-long-chain polyunsaturated fatty acids (VLCPUFAs; C24-38) constitute a unique class of PUFA that have important biological roles, but the lack of a suitable dietary source has limited research in this field. We produced an n-3 C24-28-rich VLCPUFA-oil concentrated from fish oil to study its bioavailability and physiological functions in C57BL/6J mice. The serum and retinal C24:5 levels increased significantly compared to control after a single-dose gavage, and VLCPUFAs were incorporated into the liver, brain, and eyes after 8-week supplementation. Dietary VLCPUFAs resulted in favorable cardiometabolic changes, and improved electroretinography responses and visual performance. VLCPUFA supplementation changed the expression of genes involved in PPAR signaling pathways. Further in vitro studies demonstrated that the VLCPUFA-oil and chemically synthesized C24:5 are potent agonists for PPARs. The multiple potential beneficial effects of fish oil-derived VLCPUFAs on cardiometabolic risk and eye health in mice support future efforts to develop VLCPUFA-oil into a supplemental therapy.

Network Biology and Medicine to Rescue: Applications for Retinal Disease Mechanisms and Therapy.

Inherited retinal degenerations (IRDs) are clinically and genetically heterogenous blinding diseases that manifest through dysfunction of target cells, photoreceptors, and retinal pigment epithelium (RPE) in the retina. Despite knowledge of numerous underlying genetic defects, current therapeutic approaches, including gene centric applications, have had limited success, thereby asserting the need of new directions for basic and translational research. Human diseases have commonalities that can be represented in a network form, called diseasome, which captures relationships among disease genes, proteins, metabolites, and patient meta-data. Clinical and genetic information of IRDs suggest shared relationships among pathobiological factors, making these a model case for network medicine. Characterization of the diseasome would considerably improve our understanding of retinal pathologies and permit better design of targeted therapies for disrupted regions within the integrated disease network. Network medicine in synergy with the ongoing artificial intelligence revolution can boost therapeutic developments, especially gene agnostic treatment opportunities.

Reserpine maintains photoreceptor survival in retinal ciliopathy by resolving proteostasis imbalance and ciliogenesis defects.

Ciliopathies manifest from sensory abnormalities to syndromic disorders with multi-organ pathologies, with retinal degeneration a highly penetrant phenotype. Photoreceptor cell death is a major cause of incurable blindness in retinal ciliopathies. To identify drug candidates to maintain photoreceptor survival, we performed an unbiased, high-throughput screening of over 6000 bioactive small molecules using retinal organoids differentiated from induced pluripotent stem cells (iPSC) of rd16 mouse, which is a model of Leber congenital amaurosis (LCA) type 10 caused by mutations in the cilia-centrosomal gene CEP290. We identified five non-toxic positive hits, including the lead molecule reserpine, which maintained photoreceptor development and survival in rd16 organoids. Reserpine also improved photoreceptors in retinal organoids derived from induced pluripotent stem cells of LCA10 patients and in rd16 mouse retina in vivo. Reserpine-treated patient organoids revealed modulation of signaling pathways related to cell survival/death, metabolism, and proteostasis. Further investigation uncovered dysregulation of autophagy associated with compromised primary cilium biogenesis in patient organoids and rd16 mouse retina. Reserpine partially restored the balance between autophagy and the ubiquitin-proteasome system at least in part by increasing the cargo adaptor p62, resulting in improved primary cilium assembly. Our study identifies effective drug candidates in preclinical studies of CEP290 retinal ciliopathies through cross-species drug discovery using iPSC-derived organoids, highlights the impact of proteostasis in the pathogenesis of ciliopathies, and provides new insights for treatments of retinal neurodegeneration.

Leber congenital amaurosis (LCA) is an inherited disease that affects the eyes and causes sight loss in early childhood, which generally gets worse over time. Individuals with this condition have genetic mutations that result in the death of light-sensitive cells, known as photoreceptors, in a region called the retina at the back of the eye. Patients carrying a genetic change in the gene CEP290 account for 20-25% of all LCA. At present, treatment options are only available for a limited number of patients with LCA. One option is to use small molecules as drugs that may target or bypass the faulty processes within the eye to help the photoreceptors survive in many different forms of LCA and other retinal diseases. However, over 90% of new drug candidates fail the first phase of clinical trials for human diseases. This in part due to the candidates having been developed using cell cultures or animal models that do not faithfully reflect how the human body works. Recent advances in cell and developmental biology are now enabling researchers to use stem cells derived from humans to grow retina tissues in a dish in the laboratory. These tissues, known as retinal organoids, behave in a more similar way to retinas in human eyes than those of traditional animal models. However, the methods for making and maintaining human retinal organoids are time-consuming and labor-intensive, which has so far limited their use in the search for new therapies. To address this challenge, Chen et al. developed a large-scale approach to grow retinal organoids from rd16 mutant mice stem cells (which are a good model for LCA caused by mutations to CEP290) and used the photoreceptors from these organoids to screen over 6,000 existing drugs for their ability to promote the survival of photoreceptors. The experiments found that the drug reserpine, which was previously approved to treat high blood pressure, also helped photoreceptors to survive in the diseased organoids. Reserpine also had a similar effect in retinal organoids derived from human patients with LCA and in the rd16 mice themselves. Further experiments suggest that reserpine may help patients with LCA by partially restoring a process by which the body destroys and recycles old and damaged proteins in the cells. The next steps following on from this work will be to perform further tests to demonstrate that this use of reserpine is safe to enter clinical trials as a treatment for LCA and other similar eye diseases.

Aging of the Retina: Molecular and Metabolic Turbulences and Potential Interventions.

Multifaceted and divergent manifestations across tissues and cell types have curtailed advances in deciphering the cellular events that accompany advanced age and contribute to morbidities and mortalities. Increase in human lifespan during the past century has heightened awareness of the need to prevent age-associated frailty of neuronal and sensory systems to allow a healthy and productive life. In this review, we discuss molecular and physiological attributes of aging of the retina, with a goal of understanding age-related impairment of visual function. We highlight the epigenome-metabolism nexus and proteostasis as key contributors to retinal aging and discuss lifestyle changes as potential modulators of retinal function. Finally, we deliberate promising intervention strategies for promoting healthy aging of the retina for improved vision.

Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration.

Rab-GTPases and associated effectors mediate cargo transport through the endomembrane system of eukaryotic cells, regulating key processes such as membrane turnover, signal transduction, protein recycling and degradation. Using developmental transcriptome data, we identified Rabgef1 (encoding the protein RabGEF1 or Rabex-5) as the only gene associated with Rab GTPases that exhibited strong concordance with retinal photoreceptor differentiation. Loss of Rabgef1 in mice (Rabgef1-/-) resulted in defects specifically of photoreceptor morphology and almost complete loss of both rod and cone function as early as eye opening; however, aberrant outer segment formation could only partly account for visual function deficits. RabGEF1 protein in retinal photoreceptors interacts with Rabaptin-5, and RabGEF1 absence leads to reduction of early endosomes consistent with studies in other mammalian cells and tissues. Electron microscopy analyses reveal abnormal accumulation of macromolecular aggregates in autophagosome-like vacuoles and enhanced immunostaining for LC3A/B and p62 in Rabgef1-/- photoreceptors, consistent with compromised autophagy. Transcriptome analysis of the developing Rabgef1-/- retina reveals altered expression of 2469 genes related to multiple pathways including phototransduction, mitochondria, oxidative stress and endocytosis, suggesting an early trajectory of photoreceptor cell death. Our results implicate an essential role of the RabGEF1-modulated endocytic and autophagic pathways in photoreceptor differentiation and homeostasis. We propose that RabGEF1 and associated components are potential candidates for syndromic traits that include a retinopathy phenotype.

Genome-wide Profiling Identifies DNA Methylation Signatures of Aging in Rod Photoreceptors Associated with Alterations in Energy Metabolism.

Aging-associated functional decline is accompanied by alterations in the epigenome. To explore DNA modifications that could influence visual function with age, we perform whole-genome bisulfite sequencing of purified mouse rod photoreceptors at four ages and identify 2,054 differentially methylated regions (DMRs). We detect many DMRs during early stages of aging and in rod regulatory regions, and some of these cluster at chromosomal hotspots, especially on chromosome 10, which includes a longevity interactome. Integration of methylome to age-related transcriptome changes, chromatin signatures, and first-order protein-protein interactions uncover an enrichment of DMRs in altered pathways that are associated with rod function, aging, and energy metabolism. In concordance, we detect reduced basal mitochondrial respiration and increased fatty acid dependency with retinal age in ex vivo assays. Our study reveals age-dependent genomic and chromatin features susceptible to DNA methylation changes in rod photoreceptors and identifies a link between DNA methylation and energy metabolism in aging.

Improved Retinal Organoid Differentiation by Modulating Signaling Pathways Revealed by Comparative Transcriptome Analyses with Development In Vivo.

Stem cell-derived retinal organoids recapitulate many landmarks of in vivo differentiation but lack functional maturation of distinct cell types, especially photoreceptors. Using comprehensive temporal transcriptome analyses, we show that transcriptome shift from postnatal day 6 (P6) to P10, associated with morphogenesis and synapse formation during mouse retina development, was not evident in organoids, and co-expression clusters with similar patterns included different sets of genes. Furthermore, network analysis identified divergent regulatory dynamics between developing retina in vivo and in organoids, with temporal dysregulation of specific signaling pathways and delayed or reduced expression of genes involved in photoreceptor function(s) and survival. Accordingly, addition of docosahexaenoic acid and fibroblast growth factor 1 to organoid cultures specifically promoted the maturation of photoreceptors, including cones. Our study thus identifies regulatory signals deficient in developing retinal organoids and provides experimental validation by producing a more mature retina in vitro, thereby facilitating investigations in disease modeling and therapies.

Western Indian Rural Gut Microbial Diversity in Extreme Prakriti Endo-Phenotypes Reveals Signature Microbes.

Heterogeneity amidst healthy individuals at genomic level is being widely acknowledged. This, in turn, is modulated by differential response to environmental cues and treatment regimens, necessitating the need for stratified/personalized therapy. We intend to understand the molecular determinants of Ayurvedic way (ancient Indian system of medicine) of endo-phenotyping individuals into distinct constitution types termed "Prakriti," which forms the basis of personalized treatment. In this study, we explored and analyzed the healthy human gut microbiome structure within three predominant Prakriti groups from a genetically homogenous cohort to discover differentially abundant taxa, using 16S rRNA gene based microbial community profiling. We found Bacteroidetes and Firmicutes as major gut microbial components in varying composition, albeit with similar trend across Prakriti. Multiple species of the core microbiome showed differential abundance within Prakriti types, with gender specific signature taxons. Our study reveals that despite overall uniform composition of gut microbial community, healthy individuals belonging to different Prakriti groups have enrichment of specific bacteria. It highlights the importance of Prakriti based endo-phenotypes to explain the variability amongst healthy individuals in gut microbial flora that have important consequences for an individual's health, disease and treatment.