Biology subjects

Bharti, K.

Publications and source records attributed to Bharti, K..

9 recordsLinked to original sources

bioRxiv2026

A genome-scale ORF screen for resilience factors that protect the RPE and restore vision in aged mice

Aging is a complex phenomenon, yet therapeutic discovery has largely relied on testing individual hypothesis-driven targets. Here, we performed a genome-scale open reading frame (ORF) screen for oxidative-stress resistance in retinal pigment epithelium (RPE) cells, which are vulnerable to oxidative damage during aging. The screen identified nine protective ORFs including antioxidant factors and an epithelial-to-mesenchymal transition regulator. Notably, we also identified three factors that reduce transcriptomic age including the cardiac transcription factor NKX2-5. NKX2-5 conferred robust oxidative resilience in vitro and in vivo. Domain dissection revealed that its homeodomain is dispensable for resilience, enabling development of NKX2-5{Delta}HD, which retained protective activity without detectable toxicity over 14 months. In middle-aged mice, subretinal NKX2-5{Delta}HD restored visual and electrophysiological function, and its systemic delivery improved grip strength and reduced frailty in late-aged mice. These findings highlight the value of unbiased, genome-scale gain-of-function screening, as resilience factors normally expressed in other contexts can be reengineered as potential therapeutics for age-related decline in other tissues.

genetics↗

bioRxiv2026

An Integrated Multi-omics Single Cell Atlas of the Human RPE and Choroid

The retinal pigment epithelium and choroid are critical for supporting the function and maintaining the homeostasis of the outer retina, and their dysfunction underlies a range of inherited and complex ocular diseases. To comprehensively characterize the cellular, transcriptomic, and epigenomic heterogeneity and dynamics within these tissues, we assembled an integrated multi-omics reference atlas comprising 719,813 single-cell/single-nucleus transcriptomes and 234,007 snATAC-seq profiles from 102 ancestrally diverse donors spanning 0 to 99 years of age, including cells from both the macula and periphery. This atlas resolves 48 distinct cell types or states and catalogs 448,567 open chromatin regions. Specifically, we resolved five distinct RPE subpopulations organized along a central-to-peripheral spatial axis, alongside two distinct stress/senescence states. We reconstructed the transcriptomic and epigenetic zonation of endothelial cells and expanded choroidal stromal heterogeneity by characterizing 11 fibroblast and two pericyte types. Age-associated compositional analysis revealed a significant fractional depletion of melanocytes, PI16+ fibroblasts, and venule endothelial cells with age, alongside a modest relative loss of central RPE and a corresponding increase in far-peripheral RPE. Cell-type-specific aging transcriptomics uncovered shared pathways related to inflammatory responses alongside distinct cell-type-specific signatures. Notably, significant age-associated epigenetic changes concentrated in the macula during the transition from early-to-middle adulthood and remained stable into old age, with transcription factors from the AP-1/bZIP family emerging as the dominant enriched motifs. Finally, integrating this atlas with AMD GWAS data provides novel variant-to-gene evidence implicating LIPG and COL4A3 in AMD pathogenesis. Together, this multi-omics atlas serves as both an invaluable community reference and a powerful discovery engine that translates genetic risk signals into localized target cells and candidate mechanisms, laying a foundation for understanding RPE/choroid biology in health and disease.

genomics↗

bioRxiv2026

Nutrient microenvironments reprogram RPE metabolism

Induced pluripotent stem cell-derived retinal pigment epithelium (iPSC RPE) has become a widely used model for studying the mechanisms of age-related macular degeneration (AMD). However, the nutrient composition of currently used RPE culture media is highly variable, posing a major challenge to reproducibility in RPE metabolism and phenotype. We systematically investigate how six distinct nutrient microenvironments shape RPE phenotype, function and metabolism in both iPSC RPE and fetal RPE (fRPE). These included MEM, DMEM-HG/F12 basal media, physiological human plasma-like medium (HPLM) supplemented with FBS or B27, and X-VIVO 10. Although canonical RPE markers were expressed across all conditions, B27 supplementation and X-VIVO 10 increased RPE cell size, hexagonality, and transepithelial resistance. Culture in HPLM+FBS induced accumulation of lipid droplets and sub-RPE deposits, whereas X-VIVO 10 resulted in the formation of large intracellular vacuoles. B27 supplementation enhanced basal respiration, while X-VIVO 10 increased glycolytic capacity. Amino acid consumption was broadly conserved across media types, including complete depletion of proline in all conditions by 48 hours; however, lipid and nucleotide metabolism varied substantially between conditions. Notably, B27 supplementation in specific media types reversed the net direction of several metabolites, with creatine, serine and taurine shifting from consumption to production, while riboflavin and guanine shifted from production to consumption. These findings establish the nutrient environment as a key determinant of RPE phenotype, function and metabolism. Our work provides a valuable resource for media selection and interpretation of cellular and metabolic phenotypes relevant to RPE disease modeling.

cell biology↗

bioRxiv2025

A non-canonical AKT1-TERT pathway coordinates autophagy and ERphagy

The retinal pigment epithelium (RPE) sustains lifelong proteostasis under chronic stress, yet how post-mitotic cells activate autophagy when canonical kinase pathways suppress it remains unresolved. Here we demonstrate that AKT1, conventionally regarded as an autophagy suppressor, drives autophagy through a non-canonical transcriptional mechanism triggered by isoform imbalance. In RPE and age-related macular degeneration (AMD) models, AKT2 hyperactivation destabilizes mTORC2 and engages S6K-mediated IRS-1 inhibition, creating a feedforward autophagic arrest. Compensatory AKT1 activation via mTORC2 phosphorylates telomerase reverse transcriptase (TERT) at Serine 824, driving nuclear translocation that is independent of telomere maintenance. In the nucleus, TERT assembles with FOXO3 and MYC into a tripartite transcriptional complex that occupies the EIF2AK3 promoter, enabling PERK transcriptional activation. This activity converts the unfolded protein response from pro-apoptotic to cytoprotective: PERK-ATF4 signaling drives biogenesis of core autophagy machinery while simultaneously inducing selective ERphagy through the receptors TEX264 and CCPG1, which prevents pathological PERK clustering and preserves tubular ER architecture in diseased RPE. Using C. elegans phosphorylation-deficient mutants, mouse models and AMD patient induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE), we establish that AKT1-mediated TERT phosphorylation is an evolutionarily conserved prerequisite for FOXO/DAF-16 nuclear function and lysosomal homeostasis in post-mitotic cells. Pharmacologic targeting of AKT2 with a first-in-class dual-pocket allosteric inhibitor selectively enhances AKT1 compensation, restoring macroautophagic and ERphagy flux across disease models. These findings reveal a kinase-to-transcription axis that reprograms organelle quality control and identify the AKT1-TERT-PERK-ATF4 pathway as a therapeutic target in proteostasis-driven disease.

cell biology↗

bioRxiv2025

Method for Extracellular Electrochemical Impedance Spectroscopy on Epithelia

Epithelial tissues form barriers to the flow of ions, nutrients, waste products, bacteria, and viruses. The conventional electrophysiology measurement of transepithelial resistance (TER) can quantify epithelial barrier integrity, but does not capture all the electrical behavior of the tissue or provide insight into membrane specific properties. Electrochemical impedance spectroscopy, in addition to measurement of TER, enables measurement of transepithelial capacitance (TEC) and a ratio of electrical time constants for the tissue, which we term membrane ratio. This protocol describes how to perform galvanostatic electrochemical impedance spectroscopy on epithelia using commercially available cell culture inserts and chambers, detailing the apparatus, electrical signal, fitting techniques, and error quantification. The measurement can be performed in approximately one minute using instrumentation capable of galvanostatic sinusoidal signal processing (4 A amplitude, 2 Hz-50 kHz). All fits to the model have less than 10 {Omega} mean absolute error, revealing repeatable values distinct for each cell type. On representative retinal pigment (n=3) and bronchiolar epithelial samples (n=4), we measured TER 500-667 {Omega}.cm2 and 955-1034 {Omega}.cm2, within the expected range, TEC 3.65-4.10 F/cm2 and 1.07-1.10 F/cm2, and membrane ratios 18-22 and 1.9-2.2, respectively.

bioengineering↗

bioRxiv2024

The AMD-associated genetic polymorphism CFH Y402H confers vulnerability to Hydroquinone-induced stress in iPSC-RPE cells

Age-related macular degeneration (AMD), a degenerative disease of the macula, is caused by an interplay of diverse risk factors (genetic predisposition, age and lifestyle habits). One of the main genetic risks includes the Y402H polymorphism in complement Factor H (FH), an inhibitor of complement system activation. There has been, and continues to be, much discussion around the functional consequences of this Y402H polymorphism, whether the soluble FH protein confers its risk association, or if the cells expressing the protein themselves are affected by the genetic alteration. In our study, we examined the cell characteristics of the retinal pigment epithelium (RPE) cells, which play a major role in retinal homeostasis and stability and which are synonymously linked to AMD. Here, we employ RPE cells derived from induced pluripotent stem cells (iPSC) generated from donors, carrying either homozygous 402Y (low risk) or 402H (high risk) variants of the CFH gene. RPE cells were treated with Hydroquinone (HQ), a component of cigarette smoke, to induce oxidative damage. Intriguingly, RPE cells carrying high genetic risk proved more vulnerable to oxidative insult when exposed to HQ, as demonstrated by increased cytotoxicity, caspase activation, compared to the low-risk RPE cells. The exposure of RPE cells to RPE conditioned medium, normal human serum (NHS) and inactivated NHS (iNHS) had minimal impact on cell cytotoxicity and caspase activation, nor did the presence of purified soluble FH rescue the observed effects. This suggests that the degree of cellular susceptibility to oxidative stress is not conferred by soluble FH protein and other complement sources, but intercellularly because of the corresponding genetic risk predisposition. Considering the known connection of oxidative stress to proteotoxic stress and degrading processes, we investigated the unfolded protein response (UPR) and autophagy. When exposed to HQ, RPE cells showed an increase in autophagy markers, however iPSC-RPE cells carrying high genetic risk showed an overall reduced autophagic flux. Our data support the hypothesis that RPE cells carrying high genetic risk are less resilient to oxidative stress.

cell biology↗

bioRxiv2023

The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in atrophic AMD

Introductory paragraphAge-related macular degeneration (AMD), the leading cause of geriatric blindness, is a multi-factorial disease with retinal-pigmented epithelial (RPE) cell dysfunction as a central pathogenic driver. With RPE degeneration, lysosomal function is a core process that is disrupted. Transcription factors EB/E3 (TFEB/E3) tightly control lysosomal function; their disruption can cause aging disorders, such as AMD. Here, we show that induced pluripotent stem cells (iPSC)-derived RPE cells with the complement factor H variant [CFH (Y402H)] have increased AKT2, which impairs TFEB/TFE3 nuclear translocation and lysosomal function. Increased AKT2 can inhibit PGC1, which downregulates SIRT5, an AKT2 binding partner. SIRT5 and AKT2 co-regulate each other, thereby modulating TFEB-dependent lysosomal function in the RPE. Failure of the AKT2/SIRT5/TFEB pathway in the RPE induced abnormalities in the autophagy-lysosome cellular axis by upregulating secretory autophagy, thereby releasing a plethora of factors that likely contribute to drusen formation, a hallmark of AMD. Finally, overexpressing AKT2 in RPE cells in mice led to an AMD-like phenotype. Thus, targeting the AKT2/SIRT5/TFEB pathway could be a potential therapy for atrophic AMD.

cell biology↗

bioRxiv2022

Zfp503/Nlz2 is Required for RPE Differentiation and Optic Fissure Closure

PurposeUveal coloboma is a congenital eye malformation caused by failure of the optic fissure to close in early human development. Despite significant progress in identifying genes whose regulation is important for executing this closure, mutations are detected in a minority of cases using known gene panels, implying additional genetic complexity. We have previously shown knock down of znf503 (the ortholog of mouse Zfp503) in zebrafish causes coloboma. Here we characterize Zfp503 knock out (KO) mice and evaluate transcriptomic profiling of mutant vs. wild-type (WT) retinal pigment epithelium (RPE)/Choroid. MethodsZfp503 KO mice were generated by gene targeting using homologous recombination. Embryos were characterized grossly and histologically. Patterns and level of developmentally relevant proteins/genes were examined with immunostaining/in situ hybridization. The transcriptomic profile of E11.5 KO RPE/choroid was compared to that of WT. ResultsZfp503 is dynamically expressed in developing mouse eyes and that loss of its expression results in uveal coloboma. KO embryos exhibit altered mRNA levels and expression patterns of several key transcription factors involved in eye development, including Otx2, Mitf, Pax6, Pax2, Vax1 and Vax2, resulting in reduced melanin pigmentation in the presumptive RPE and its differentiation into neural-retina-like lineages. Comparison of RNA-Seq data from wild type and KO E11.5 embryos demonstrated reduced expression of melanin-related genes and significant overlap with genes known to be dynamically regulated at the optic fissure. ConclusionsThese results demonstrate a critical role of Zfp503 in RPE differentiation and in optic fissure closure.

developmental biology↗

bioRxiv2019

Choroid-resident macrophages maintain local vasculature and RPE integrity and spontaneously regenerate following depletion

The choroid of the eye provides necessary vascular supply to the outer retina across the retinal pigment epithelial (RPE) layer and is a locus of progressive degenerative change in aging and age-related macular degeneration (AMD). Cellular mechanisms that maintain or compromise choroidal vasculature homeostasis are not well understood, complicating therapeutic efforts. We discover here that pharmacological ablation of macrophages normally resident in the adult mouse choroid via blockade of the CSF1 receptor resulted in progressive thinning of the choroid layer and atrophy of the choriocapillaris. Concurrently, choroidal macrophage ablation also induced structural disorganization of the RPE cell layer, downregulation of RPE visual cycle genes, and altered RPE angiogenic factor expression. Suspension of CSF1R blockade following ablation conversely enabled spontaneous regeneration of the choroidal macrophage population, restoring original macrophage distribution and morphological features. Macrophage repopulation significantly ameliorated the ablation-induced changes in RPE structure and angiogenic factor expression and arrested choroidal vascular atrophy. These findings reveal a previously unsuspected trophic function of resident choroidal macrophages in the maintenance of choroidal vasculature and the RPE layer, suggesting that insufficiency of macrophage function may contribute to age- and AMD-associated pathology. Modulating choroidal macrophage support function can constitute a strategy for therapeutic preservation of the choroid and RPE in AMD prevention and treatment. Significance StatementChoroidal atrophy is associated with normal aging and progression in age-related macular degeneration (AMD), a leading cause of blindness. Herein, we identify the choroidal macrophage as a critical player in the homeostatic maintenance of choroid-RPE complex of the eye. Depletion of choroidal macrophages resulted in progressive vascular atrophy, altered structure of retinal pigment epithelial (RPE) cells, and dysregulated RPE expression of visual cycle protein and angiogenic factors, VEGF and PEDF. We discovered that choroidal macrophages can regenerate and repopulate the choroid following depletion, ameliorating RPE alterations and arresting vascular atrophy. Our results suggest that insufficiency of macrophage function may contribute to age- and AMD-associated pathology, and modulation of macrophage support functions may be therapeutically useful in AMD prevention and treatment.

immunology↗