Epigenome-metabolism nexus in the retina: implications for aging and disease.

Intimate links between epigenome modifications and metabolites allude to a crucial role of cellular metabolism in transcriptional regulation. Retina, being a highly metabolic tissue, adapts by integrating inputs from genetic, epigenetic, and extracellular signals. Precise global epigenomic signatures guide development and homeostasis of the intricate retinal structure and function. Epigenomic and metabolic realignment are hallmarks of aging and highlight a link of the epigenome-metabolism nexus with aging-associated multifactorial traits affecting the retina, including age-related macular degeneration and glaucoma. Here, we focus on emerging principles of epigenomic and metabolic control of retinal gene regulation, with emphasis on their contribution to human disease. In addition, we discuss potential mitigation strategies involving lifestyle changes that target the epigenome-metabolome relationship for maintaining retinal function.

QTL mapping of human retina DNA methylation identifies 87 gene-epigenome interactions in age-related macular degeneration.

DNA methylation provides a crucial epigenetic mark linking genetic variations to environmental influence. We have analyzed array-based DNA methylation profiles of 160 human retinas with co-measured RNA-seq and >8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 methylation quantitative trait loci and 12,505 expression quantitative trait loci) and 13,747 DNA methylation loci affecting gene expression, with over one-third specific to the retina. Methylation and expression quantitative trait loci show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration. Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of macular degeneration pathology by genotype-environment interaction in retina.

Integrating genetic regulation and single-cell expression with GWAS prioritizes causal genes and cell types for glaucoma.

Primary open-angle glaucoma (POAG), characterized by retinal ganglion cell death, is a leading cause of irreversible blindness worldwide. However, its molecular and cellular causes are not well understood. Elevated intraocular pressure (IOP) is a major risk factor, but many patients have normal IOP. Colocalization and Mendelian randomization analysis of >240 POAG and IOP genome-wide association study (GWAS) loci and overlapping expression and splicing quantitative trait loci (e/sQTLs) in 49 GTEx tissues and retina prioritizes causal genes for 60% of loci. These genes are enriched in pathways implicated in extracellular matrix organization, cell adhesion, and vascular development. Analysis of single-nucleus RNA-seq of glaucoma-relevant eye tissues reveals that the POAG and IOP colocalizing genes and genome-wide associations are enriched in specific cell types in the aqueous outflow pathways, retina, optic nerve head, peripapillary sclera, and choroid. This study nominates IOP-dependent and independent regulatory mechanisms, genes, and cell types that may contribute to POAG pathogenesis.

Whole genome sequencing of 4,787 individuals identifies gene-based rare variants in age-related macular degeneration.

Genome-wide association studies have contributed extensively to the discovery of disease-associated common variants. However, the genetic contribution to complex traits is still largely difficult to interpret. We report a genome-wide association study of 2394 cases and 2393 controls for age-related macular degeneration (AMD) via whole-genome sequencing, with 46.9 million genetic variants. Our study reveals significant single-variant association signals at four loci and independent gene-based signals in CFH, C2, C3, and NRTN. Using data from the Exome Aggregation Consortium (ExAC) for a gene-based test, we demonstrate an enrichment of predicted rare loss-of-function variants in CFH, CFI, and an as-yet unreported gene in AMD, ORMDL2. Our method of using a large variant list without individual-level genotypes as an external reference provides a flexible and convenient approach to leverage the publicly available variant datasets to augment the search for rare variant associations, which can explain additional disease risk in AMD.

QTL mapping of human retina DNA methylation identifies 87 gene-epigenome interactions in age-related macular degeneration.

DNA methylation (DNAm) provides a crucial epigenetic mark linking genetic variations to environmental influence. We analyzed array-based DNAm profiles of 160 human retinas with co-measured RNA-seq and > 8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 mQTLs and 12,505 eQTLs) and 13,747 eQTMs (DNAm loci affecting gene expression), with over one-third specific to the retina. mQTLs and eQTMs show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration (AMD). Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of AMD pathology by genotype-environment interaction in retina.

Ultra-rare complement factor 8 coding variants in families with age-related macular degeneration.

Genome-wide association studies have uncovered 52 independent common and rare variants across 34 genetic loci, which influence susceptibility to age related macular degeneration (AMD). Of the 5 AMD-associated complement genes, complement factor H (CFH) and CFI exhibit a significant rare variant burden implicating a major contribution of the complement pathway to disease pathology. However, the efforts for developing AMD therapy have been challenging as of yet. Here, we report the identification of ultra-rare variants in complement factors 8A and 8B, two components of the terminal complement membrane attack complex (MAC), by whole exome sequencing of a cohort of AMD families. The identified C8 variants impact local interactions among proteins of C8 triplex in vitro, indicating their effect on MAC stability. Our results suggest that MAC, and not the early steps of the complement pathway, might be a more effective target for designing treatments for AMD.

High-resolution genome topology of human retina uncovers super enhancer-promoter interactions at tissue-specific and multifactorial disease loci.

Chromatin organization and enhancer-promoter contacts establish unique spatiotemporal gene expression patterns in distinct cell types. Non-coding genetic variants can influence cellular phenotypes by modifying higher-order transcriptional hubs and consequently gene expression. To elucidate genomic regulation in human retina, we mapped chromatin contacts at high resolution and integrated with super-enhancers (SEs), histone marks, binding of CTCF and select transcription factors. We show that topologically associated domains (TADs) with central SEs exhibit stronger insulation and augmented contact with retinal genes relative to TADs with edge SEs. Merging genome-wide expression quantitative trait loci (eQTLs) with topology map reveals physical links between 100 eQTLs and corresponding eGenes associated with retinal neurodegeneration. Additionally, we uncover candidate genes for susceptibility variants linked to age-related macular degeneration and glaucoma. Our study of high-resolution genomic architecture of human retina provides insights into genetic control of tissue-specific functions, suggests paradigms for missing heritability, and enables the dissection of common blinding disease phenotypes.

Frmpd1 Facilitates Trafficking of G-Protein Transducin and Modulates Synaptic Function in Rod Photoreceptors of Mammalian Retina.

Trafficking of transducin (Gαt) in rod photoreceptors is critical for adaptive and modulatory responses of the retina to varying light intensities. In addition to fine-tuning phototransduction gain in rod outer segments (OSs), light-induced translocation of Gαt to the rod synapse enhances rod to rod bipolar synaptic transmission. Here, we show that the rod-specific loss of Frmpd1 (FERM and PDZ domain containing 1), in the retina of both female and male mice, results in delayed return of Gαt from the synapse back to outer segments in the dark, compromising the capacity of rods to recover from light adaptation. Frmpd1 directly interacts with Gpsm2 (G-protein signaling modulator 2), and the two proteins are required for appropriate sensitization of rod-rod bipolar signaling under saturating light conditions. These studies provide insight into how the trafficking and function of Gαt is modulated to optimize the photoresponse and synaptic transmission of rod photoreceptors in a light-dependent manner.

GATD3A, a mitochondrial deglycase with evolutionary origins from gammaproteobacteria, restricts the formation of advanced glycation end products.

BACKGROUND: Functional complexity of the eukaryotic mitochondrial proteome is augmented by independent gene acquisition from bacteria since its endosymbiotic origins. Mammalian homologs of many ancestral mitochondrial proteins have uncharacterized catalytic activities. Recent forward genetic approaches attributed functions to proteins in established metabolic pathways, thereby limiting the possibility of identifying novel biology relevant to human disease. We undertook a bottom-up biochemistry approach to discern evolutionarily conserved mitochondrial proteins with catalytic potential. RESULTS: Here, we identify a Parkinson-associated DJ-1/PARK7-like protein-glutamine amidotransferase-like class 1 domain-containing 3A (GATD3A), with bacterial evolutionary affinities although not from alphaproteobacteria. We demonstrate that GATD3A localizes to the mitochondrial matrix and functions as a deglycase. Through its amidolysis domain, GATD3A removes non-enzymatic chemical modifications produced during the Maillard reaction between dicarbonyls and amines of nucleotides and amino acids. GATD3A interacts with factors involved in mitochondrial mRNA processing and translation, suggestive of a role in maintaining integrity of important biomolecules through its deglycase activity. The loss of GATD3A in mice is associated with accumulation of advanced glycation end products (AGEs) and altered mitochondrial dynamics. CONCLUSIONS: An evolutionary perspective helped us prioritize a previously uncharacterized but predicted mitochondrial protein GATD3A, which mediates the removal of early glycation intermediates. GATD3A restricts the formation of AGEs in mitochondria and is a relevant target for diseases where AGE deposition is a pathological hallmark.

The Normal Human Adult Hypothalamus Proteomic Landscape: Rise of Neuroproteomics in Biological Psychiatry and Systems Biology.

The human hypothalamus is central to the regulation of neuroendocrine and neurovegetative systems, as well as modulation of chronobiology and behavioral aspects in human health and disease. Surprisingly, a deep proteomic analysis of the normal human hypothalamic proteome has been missing for such an important organ so far. In this study, we delineated the human hypothalamus proteome using a high-resolution mass spectrometry approach which resulted in the identification of 5349 proteins, while a multiple post-translational modification (PTM) search identified 191 additional proteins, which were missed in the first search. A proteogenomic analysis resulted in the discovery of multiple novel protein-coding regions as we identified proteins from noncoding regions (pseudogenes) and proteins translated from short open reading frames that can be missed using the traditional pipeline of prediction of protein-coding genes as a part of genome annotation. We also identified several PTMs of hypothalamic proteins that may be required for normal hypothalamic functions. Moreover, we observed an enrichment of proteins pertaining to autophagy and adult neurogenesis in the proteome data. We believe that the hypothalamic proteome reported herein would help to decipher the molecular basis for the diverse range of physiological functions attributed to it, as well as its role in neurological and psychiatric diseases. Extensive proteomic profiling of the hypothalamic nuclei would further elaborate on the role and functional characterization of several hypothalamus-specific proteins and pathways to inform future research and clinical discoveries in biological psychiatry, neurology, and system biology.

Correction: Targeting focal adhesion kinase overcomes erlotinib resistance in smoke induced lung cancer by altering phosphorylation of epidermal growth factor receptor.

[This corrects the article DOI: 10.18632/oncoscience.395.].

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.

Proteomic and phosphoproteomic profiling of shammah induced signaling in oral keratinocytes.

Shammah is a smokeless tobacco product often mixed with lime, ash, black pepper and flavorings. Exposure to shammah has been linked with dental diseases and oral squamous cell carcinoma. There is limited literature on the prevalence of shammah and its role in pathobiology of oral cancer. In this study, we developed a cellular model to understand the effect of chronic shammah exposure on oral keratinocytes. Chronic exposure to shammah resulted in increased proliferation and invasiveness of non-transformed oral keratinocytes. Quantitative proteomics of shammah treated cells compared to untreated cells led to quantification of 4712 proteins of which 402 were found to be significantly altered. In addition, phosphoproteomics analysis of shammah treated cells compared to untreated revealed hyperphosphorylation of 36 proteins and hypophosphorylation of 83 proteins (twofold, p-value ≤ 0.05). Bioinformatics analysis of significantly altered proteins showed enrichment of proteins involved in extracellular matrix interactions, necroptosis and peroxisome mediated fatty acid oxidation. Kinase-Substrate Enrichment Analysis showed significant increase in activity of kinases such as ROCK1, RAF1, PRKCE and HIPK2 in shammah treated cells. These results provide better understanding of how shammah transforms non-neoplastic cells and warrants additional studies that may assist in improved early diagnosis and treatment of shammah induced oral cancer.

Proteomic Alterations Associated with Oral Cancer Patients with Tobacco Using Habits.

Tobacco abuse is a major risk factor associated with the development of oral squamous cell carcinoma. Differences in molecular aberrations induced by tobacco exposure by chewing or smoking form are not well studied in case of oral cancer. We used tandem mass tag-based quantitative proteomic approach to delineate proteomic alterations in oral cancer patients based on their history of tobacco using habits (patients who chewed tobacco, patients who smoked tobacco, and those with no history of tobacco consumption). Our data identified distinct dysregulation of biological processes and pathways in each patient cohort. Bioinformatics analysis of dysregulated proteins identified in our proteomic study revealed dysregulation of collagen formation and antigen processing/presentation pathway in oral cancer patients who smoked tobacco, whereas proteins associated with the process of keratinization showed enrichment in patients who chewed tobacco. In addition, we identified overexpression of proteins involved in immune pathways and downregulation of muscle contraction-mediated signaling events in all three cohorts, irrespective of tobacco using habits. This study lays the groundwork for identification of protein markers that may aid in identification of high-risk patients for cancer development based on the history of tobacco exposure habits.

Signaling alterations in oral keratinocytes in response to shisha and crude tobacco extract.

BACKGROUND: Tobacco consumption in smoking and non-smoking forms has been consequential in the rise of oral cancer cases. Among different forms, epidemiological studies from Middle Eastern countries and rural parts of northern India have reported increasing association of oral cancer with waterpipe (hookah) smoking. However, molecular mechanisms and role played by waterpipe smoking in the onset of oral carcinogenesis remains unexplored. METHODS: In this study, immortalized normal human oral keratinocytes were chronically treated with extracts of two varieties of waterpipe tobacco-crude tobacco and processed shisha. Phenotypic changes and molecular aberrations were examined using cell culture-based assays and mass spectrometry-based quantitative proteomic analysis, respectively. Bioinformatics analysis was utilized to analyze proteomics data and identify dysregulated pathways. RESULTS: Our data indicate that chronic treatment with waterpipe tobacco extracts increased proliferation, invasion, migration, and significant dysregulation of protein expression in oral keratinocytes. Altered expression of proteins involved in interferon signaling pathway were observed with both varieties of tobacco. Overexpression of cholesterol metabolism and vesicle-mediated transport proteins were identified exclusively in cells treated with crude tobacco extract. Bioinformatics analyses revealed different oncogenic response in oral cells based on the type of waterpipe tobacco used. CONCLUSIONS: This study may serve as a useful resource in understanding the early onset of oral cancer attributed to waterpipe smoking.

JAK-STAT inhibitor as a potential therapeutic opportunity in AML patients resistant to cytarabine and epigenetic therapy.

The prognosis of AML is generally poor, with 5-year survival rate of 25%. There has been substantial progress in identification of new therapeutic targets, along with approval of at least three targeted therapies for AML in recent years. Nevertheless, treatment has largely remained unchanged over couple of decades, with ~40% patients not achieving remission. AML is a highly heterogenous disease and there is a need for a preclinical platform to understand the heterogeneity and tumor microenvironment that can guide therapy selection. In this study, we employed an ex vivo tumor explant model to study tumor microenvironment and to select a treatment course for AML patients. Our data reveal dysregulation of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) in a subset of AML patients. Based on this observation, epigenetic modulators azacitidine and panobinostat alone and in combination, were evaluated as treatment regimens in cytarabine refractory tumors. More than 50% of the treated samples showed response to the combination therapy. In order to explore alternate treatment modalities for tumors refractory to these epigenetic modulators, TCGA data analysis was done which revealed increased expression and hypomethylation of IFNGR1/2, suggesting activation of JAK/STAT pathway in AML. This was further interrogated ex vivo, with p-STAT3 expression in patients' samples. Fedratinib, a JAK/STAT inhibitor was evaluated and 78% tumor efficacy response was achieved. Taken together, our data indicate that ex vivo platform derived from patient samples is capable in guiding optimal therapy selection for various classes of drugs including identification of novel targeted therapies.

An assembly of galanin-galanin receptor signaling network.

The galanin receptor family of proteins is present throughout the central nervous system and endocrine system. It comprises of three subtypes-GalR1, GalR2, and GalR3; all of which are G-protein-coupled receptors. Galanin predominantly acts as an inhibitory, hyper-polarizing neuromodulator, which has several physiological as well as pathological functions. Galanin has a role in mediating food intake, memory, sexual behavior, nociception and is also associated with diseases such as Alzheimer's disease, epilepsy, diabetes mellitus, and chronic pain. However, the understanding of signaling mechanisms of the galanin family of neuropeptides is limited and an organized pathway map is not yet available. Therefore, a detailed literature mining of the publicly available articles pertaining to the galanin receptor was followed by manual curation of the reactions and their integration into a map. This resulted in the cataloging of molecular reactions involving 64 molecules into five categories such as molecular association, activation/inhibition, catalysis, transport, and gene regulation. For enabling easy access of biomedical researchers, the galanin-galanin receptor signaling pathway data was uploaded to WikiPathways ( https://www.wikipathways.org/index.php/Pathway:WP4970 ), a freely available database of biological pathways.

Phosphoproteomic analysis identifies CLK1 as a novel therapeutic target in gastric cancer.

BACKGROUND: Phosphorylation is an important regulatory mechanism of protein activity in cells. Studies in various cancers have reported perturbations in kinases resulting in aberrant phosphorylation of oncoproteins and tumor suppressor proteins. METHODS: In this study, we carried out quantitative phosphoproteomic analysis of gastric cancer tissues and corresponding xenograft samples. Using these data, we employed bioinformatics analysis to identify aberrant signaling pathways. We further performed molecular inhibition and silencing of the upstream regulatory kinase in gastric cancer cell lines and validated its effect on cellular phenotype. Through an ex vivo technology utilizing patient tumor and blood sample, we sought to understand the therapeutic potential of the kinase by recreating the tumor microenvironment. RESULTS: Using mass spectrometry-based high-throughput analysis, we identified 1,344 phosphosites and 848 phosphoproteins, including differential phosphorylation of 177 proteins (fold change cut-off ≥ 1.5). Our data showed that a subset of differentially phosphorylated proteins belonged to splicing machinery. Pathway analysis highlighted Cdc2-like kinase (CLK1) as upstream kinase. Inhibition of CLK1 using TG003 and CLK1 siRNA resulted in a decreased cell viability, proliferation, invasion and migration as well as modulation in the phosphorylation of SRSF2. Ex vivo experiments which utilizes patient's own tumor and blood to recreate the tumor microenvironment validated the use of CLK1 as a potential target for gastric cancer treatment. CONCLUSIONS: Our data indicates that CLK1 plays a crucial role in the regulation of splicing process in gastric cancer and that CLK1 can act as a novel therapeutic target in gastric cancer.

Chronic shisha exposure alters phosphoproteome of oral keratinocytes.

Shisha smoking has been epidemiologically linked to oral cancer. However, few studies have investigated the pathobiology of shisha-induced cellular transformation. We studied the effects of chronic shisha exposure (8 months) in an in vitro model using immortalized, non-neoplastic oral keratinocytes (OKF6/TERT1). Quantitative proteomic and phosphoproteomic analyses were performed on OKF6/TERT1 cells treated with shisha extract for a period of 8 months. Pathway analysis was carried out to identify significantly enriched biological processes in shisha-treated cells. Chronic shisha exposure resulted in increased cell scattering phenomenon in OKF6/TERT1 cells. Data analysis revealed differential phosphorylation of 164 peptides (fold change ≥1.5, p ≤ 0.0.5) corresponding to 136 proteins. Proteins associated with mTORC1 and EIF4F complexes involved in initiating protein translation were seen to be enriched upon shisha treatment. Network analysis also highlighted downregulation of proteins involved in Type I interferon signaling in shisha-treated cells. Quantitative phosphoproteomic approach elucidated global perturbations to the molecular milieu of oral keratinocytes upon shisha exposure. Further studies are needed to validate putative targets in oral cancer patients with shisha smoking history.

Global Proteome Profiling Reveals Drug-Resistant Traits in Elizabethkingia meningoseptica: An Opportunistic Nosocomial Pathogen.

Elizabethkingia meningoseptica is Gram-negative, rod-shaped opportunistic bacterial pathogen increasingly reported in hospital-acquired outbreaks. This bacterium is well known to thrive in the hospital environment. One of the leading causes of meningitis in pediatric and immune-compromised patients, E. meningoseptica has been noted as a "pathogen of interest" in the context of nosocomial diseases associated with device-related infections in particular. This pathogen's multidrug-resistant phenotype and attendant lack of adequate molecular mechanistic data limit the current approaches for its effective management in hospitals and public health settings. This study provides the global proteome of E. meningoseptica. The reference strain E. meningoseptica ATCC 13253 was used for proteomic analysis using high-resolution Fourier transform mass spectrometry. The study provided translational evidence for 2506 proteins of E. meningoseptica. We identified multiple metallo-ß-lactamases, transcriptional regulators, and efflux transporter proteins associated with multidrug resistance. A protein Car D, which is an enzyme of the carbapenem synthesis pathway, was also discovered in E. meningoseptica. Further, the proteomics data were harnessed for refining the genome annotation. We discovered 39 novel protein-coding genes and corrected four existing translations using proteogenomic workflow. Novel translations reported in this study enhance the molecular data on this organism, thus improving current databases. We believe that the in-depth proteomic data presented in this study offer a platform for accelerated research on this pathogen. The identification of multiple proteins, particularly those involved in drug resistance, offers new future opportunities to design novel and specific antibiotics against infections caused by E. meningoseptica.