Loss of paired immunoglobin-like type 2 receptor B gene associated with age-related macular degeneration impairs photoreceptor function in mouse retina.

Genome-wide association studies have uncovered mostly non-coding variants at over 60 genetic loci linked to susceptibility for age-related macular degeneration (AMD). To ascertain the causal gene at the PILRB/PILRA locus, we used a CRISPR strategy to produce germline deletions in the mouse paired immunoglobin-like type 2 receptor (Pilr) genes that encode highly related activating (PILRB) and inhibitory (PILRA) receptors. We show that a combined loss of Pilrb1 and Pilrb2, but not Pilra, leads to an early but relatively stationary defect as the electroretinography (ERG) amplitudes of Pilrb1/2-/- mice exhibit a marked reduction as early as postnatal day 15 and do not show additional significant decrease at 3 and 12-months.

Maf-family bZIP transcription factor NRL interacts with RNA-binding proteins and R-loops in retinal photoreceptors.

RNA-binding proteins (RBPs) perform diverse functions including the regulation of chromatin dynamics and the coupling of transcription with RNA processing. However, our understanding of their actions in mammalian neurons remains limited. Using affinity purification, yeast-two-hybrid and proximity ligation assays, we identified interactions of multiple RBPs with NRL, a Maf-family bZIP transcription factor critical for retinal rod photoreceptor development and function.

Stage-specific dynamic reorganization of genome topology shapes transcriptional neighborhoods in developing human retinal organoids.

We have generated a high-resolution Hi-C map of developing human retinal organoids to elucidate spatiotemporal dynamics of genomic architecture and its relationship with gene expression patterns. We demonstrate progressive stage-specific alterations in DNA topology and correlate these changes with transcription of cell-type-restricted gene markers during retinal differentiation. Temporal Hi-C reveals a shift toward A compartment for protein-coding genes and B compartment for non-coding RNAs, displaying high and low expression, respectively.

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.

Macrophages play a leading role in determining the direction of astrocytic migration in spinal cord injury via ADP-P2Y1R axis.

After spinal cord injury (SCI), inflammatory cells such as macrophages infiltrate the injured area, and astrocytes migrate, forming a glial scar around macrophages. The glial scar inhibits axonal regeneration, resulting in significant permanent disability. However, the mechanism through which glial scar-forming astrocytes migrate to the injury site has not been clarified.

Macrophages play a leading role in determining the direction of astrocytic migration in spinal cord injury via ADP-P2Y1R axis.

After spinal cord injury (SCI), inflammatory cells such as macrophages infiltrate the injured area, and astrocytes migrate, forming a glial scar around macrophages. The glial scar inhibits axonal regeneration, resulting in significant permanent disability. However, the mechanism by which glial scar-forming astrocytes migrate to the injury site has not been clarified.

Pretreatment of human retinal pigment epithelial cells with sterculic acid forestalls fenretinide-induced apoptosis.

The ratio of saturated to monounsaturated fatty acids, thought to play a critical role in many cellular functions, is regulated by stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. Previously, we observed a decrease in both SCD protein and enzymatic activity in apoptosis induced by fenretinide, a synthetic analog of retinoic acid, in the human retinal pigment epithelial (RPE) cell line ARPE-19. Here, we investigated the effect of pretreating ARPE-19 with sterculic acid, a cyclopropenoic fatty acid inhibitor of SCD, on preventing fenretinide-induced apoptosis, given the role of SCD in cell proliferation and apoptosis.

Protein kinase CK2 modulates the activity of Maf-family bZIP transcription factor NRL in rod photoreceptors of mammalian retina.

Maf-family basic motif leucine zipper protein NRL specifies rod photoreceptor cell fate during retinal development and, in concert with homeodomain protein CRX and other regulatory factors, controls the expression of most rod-expressed genes including the visual pigment gene Rhodopsin (Rho). Transcriptional regulatory activity of NRL is modulated by post-translational modifications, especially phosphorylation, and mutations at specific phosphosites can lead to retinal degeneration. During our studies to elucidate NRL-mediated transcriptional regulation, we identified protein kinase CK2 in NRL-enriched complexes bound to Rho promoter-enhancer regions and in NRL-enriched high molecular mass fractions from the bovine retina.

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.

In vitro modeling and rescue of ciliopathy associated with IQCB1/NPHP5 mutations using patient-derived cells.

Mutations in the IQ calmodulin-binding motif containing B1 (IQCB1)/NPHP5 gene encoding the ciliary protein nephrocystin 5 cause early-onset blinding disease Leber congenital amaurosis (LCA), together with kidney dysfunction in Senior-Løken syndrome. For in vitro disease modeling, we obtained dermal fibroblasts from patients with NPHP5-LCA that were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into retinal pigment epithelium (RPE) and retinal organoids. Patient fibroblasts and RPE demonstrated aberrantly elongated ciliary axonemes.

Developmental genome-wide occupancy analysis of bZIP transcription factor NRL uncovers the role of c-Jun in early differentiation of rod photoreceptors in the mammalian retina.

The basic motif-leucine zipper (bZIP) transcription factor neural retina leucine zipper (NRL) determines rod photoreceptor cell fate during retinal development, and its loss leads to cone-only retina in mice. NRL works synergistically with homeodomain protein Cone-Rod Homeobox and other regulatory factors to control the transcription of most genes associated with rod morphogenesis and functional maturation, which span over a period of several weeks in the mammalian retina. We predicted that NRL gradually establishes rod cell identity and function by temporal and dynamic regulation of stage-specific transcriptional targets.

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.

Multiomics analyses reveal early metabolic imbalance and mitochondrial stress in neonatal photoreceptors leading to cell death in Pde6brd1/rd1 mouse model of retinal degeneration.

Retinal diseases exhibit extensive genetic heterogeneity and complex etiology with varying onset and severity. Mutations in over 200 genes can lead to photoreceptor dysfunction and/or cell death in retinal neurodegeneration. To deduce molecular pathways that initiate and/or drive cell death, we adopted a temporal multiomics approach and examined molecular and cellular events in newborn and developing photoreceptors before the onset of degeneration in a widely-used Pde6brd1/rd1 (rd1) mouse, a model of autosomal recessive retinitis pigmentosa caused by PDE6B mutations.

Rapid characterization of adeno-associated virus (AAV) gene therapy vectors by mass photometry.

Recombinant adeno-associated viruses (rAAV) are used extensively as gene delivery vectors in clinical studies, and several rAAV based treatments have already been approved. Significant progress has been made in rAAV manufacturing; however, better and more precise capsid characterization techniques are still needed to guarantee the purity and safety of rAAV preparations. Current analytical techniques used to characterize rAAV preparations are susceptible to background signals, have limited accuracy, or require a large amount of time and material.

HiCRes: a computational method to estimate and predict the genomic resolution of Hi-C libraries.

Three-dimensional (3D) conformation of the chromatin is crucial to stringently regulate gene expression patterns and DNA replication in a cell-type specific manner. Hi-C is a key technique for measuring 3D chromatin interactions genome wide. Estimating and predicting the resolution of a library is an essential step in any Hi-C experimental design.

Genetics and therapy for pediatric eye diseases.

Ocular morphogenesis in vertebrates is a highly organized process, orchestrated largely by intrinsic genetic programs that exhibit stringent spatiotemporal control. Alternations in these genetic instructions can lead to hereditary or nonhereditary congenital disorders, a major cause of childhood visual impairment, and contribute to common late-onset blinding diseases. Currently, limited treatment options exist for clinical phenotypes involving eye development.

The yeast ISW1b ATP-dependent chromatin remodeler is critical for nucleosome spacing and dinucleosome resolution.

Isw1 and Chd1 are ATP-dependent nucleosome-spacing enzymes required to establish regular arrays of phased nucleosomes near transcription start sites of yeast genes. Cells lacking both Isw1 and Chd1 have extremely disrupted chromatin, with weak phasing, irregular spacing and a propensity to form close-packed dinucleosomes. The Isw1 ATPase subunit occurs in two different remodeling complexes: ISW1a (composed of Isw1 and Ioc3) and ISW1b (composed of Isw1, Ioc2 and Ioc4).

Gene Therapy of Dominant CRX-Leber Congenital Amaurosis using Patient Stem Cell-Derived Retinal Organoids.

Mutations in the photoreceptor transcription factor gene cone-rod homeobox (CRX) lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs48 mutation, we established an in vitro model of CRX-LCA in retinal organoids that showed defective photoreceptor maturation by histology and gene profiling, with diminished expression of visual opsins. Adeno-associated virus (AAV)-mediated CRX gene augmentation therapy partially restored photoreceptor phenotype and expression of phototransduction-related genes as determined by single-cell RNA-sequencing.

The microglia response to electrical overstimulation of the retina imaged under a transparent stimulus electrode.

We investigated using the morphological response of retinal microglia as indicators of tissue damage from electrical overstimulation by imaging them through an optically transparent stimulus electrode.To track the microglia, we used a transgenic mouse where the microglia expressed a water soluble green fluorescent protein. The clear stimulus electrode was placed epiretinally on the inner limiting membrane and the microglia layers were imaged using time-lapse confocal microscopy.

Primary cilia biogenesis and associated retinal ciliopathies.

The primary cilium is a ubiquitous microtubule-based organelle that senses external environment and modulates diverse signaling pathways in different cell types and tissues. The cilium originates from the mother centriole through a complex set of cellular events requiring hundreds of distinct components. Aberrant ciliogenesis or ciliary transport leads to a broad spectrum of clinical entities with overlapping yet highly variable phenotypes, collectively called ciliopathies, which include sensory defects and syndromic disorders with multi-organ pathologies.

Ocular delivery of CRISPR/Cas genome editing components for treatment of eye diseases.

A variety of inherited or multifactorial ocular diseases call for novel treatment paradigms. The newly developed genome editing technology, CRISPR, has shown great promise in treating these diseases, but delivery of the CRISPR/Cas components to target ocular tissues and cells requires appropriate use of vectors and routes of administration to ensure safety, efficacy and specificity. Although adeno-associated viral (AAV) vectors are thus far the most commonly used tool for ocular gene delivery, sustained expression of CRISPR/Cas components may cause immune reactions and an increased risk of off-target editing.

Rapid adaptive evolution of the diapause program during range expansion of an invasive mosquito.

In temperate climates, the recurring seasonal exigencies of winter represent a fundamental physiological challenge for a wide range of organisms. In response, many temperate insects enter diapause, an alternative developmental program, including developmental arrest, that allows organisms to synchronize their life cycle with seasonal environmental variation. Geographic variation in diapause phenology contributing to local climatic adaptation is well documented.