ABSTRACT
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.
Subject(s)
Autophagy , Endocytosis , Guanine Nucleotide Exchange Factors/genetics , Neurogenesis , Photoreceptor Cells/metabolism , Retinal Degeneration/metabolism , Animals , Female , Guanine Nucleotide Exchange Factors/metabolism , Male , Mice , Mice, Inbred BALB C , Photoreceptor Cells/cytology , Retinal Degeneration/genetics , TranscriptomeABSTRACT
In the mammalian retina, rod and cone photoreceptors transmit the visual information to bipolar neurons through highly specialized ribbon synapses. We have limited understanding of regulatory pathways that guide morphogenesis and organization of photoreceptor presynaptic architecture in the developing retina. While neural retina leucine zipper (NRL) transcription factor determines rod cell fate and function, cone-rod homeobox (CRX) controls the expression of both rod- and cone-specific genes and is critical for terminal differentiation of photoreceptors. A comprehensive immunohistochemical evaluation of Crx-/- (null), CrxRip/+ and CrxRip/Rip (models of dominant congenital blindness) mouse retinas revealed abnormal photoreceptor synapses, with atypical ribbon shape, number and length. Integrated analysis of retinal transcriptomes of Crx-mutants with CRX- and NRL-ChIP-Seq data identified a subset of differentially expressed CRX target genes that encode presynaptic proteins associated with the cytomatrix active zone (CAZ) and synaptic vesicles. Immunohistochemistry of Crx-mutant retina validated aberrant expression of REEP6, PSD95, MPP4, UNC119, UNC13, RGS7 and RGS11, with some reduction in Ribeye and no significant change in immunostaining of RIMS1, RIMS2, Bassoon and Pikachurin. Our studies demonstrate that CRX controls the establishment of CAZ and anchoring of ribbons, but not the formation of ribbon itself, in photoreceptor presynaptic terminals.
Subject(s)
Cell Differentiation , Eye Proteins/genetics , Homeodomain Proteins/metabolism , Leber Congenital Amaurosis/metabolism , Retina/metabolism , Signal Transduction , Trans-Activators/metabolism , Animals , Disease Models, Animal , Gene Expression Profiling , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Leber Congenital Amaurosis/genetics , Leber Congenital Amaurosis/physiopathology , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , Presynaptic Terminals , Retina/physiopathology , Retinal Cone Photoreceptor Cells/metabolism , Retinal Cone Photoreceptor Cells/physiology , Retinal Rod Photoreceptor Cells/metabolism , Retinal Rod Photoreceptor Cells/physiology , Trans-Activators/geneticsABSTRACT
Purpose: Single-cell RNA sequencing (scRNA-seq) is a powerful technique used to explore gene expression at the single cell level. However, appropriate preparation of samples is essential to obtain the most information out of this transformative technology. Generating high-quality single-cell suspensions from the retina is critical to preserve the native expression profile that will ensure meaningful transcriptome data analysis. Methods: We modified the conditions for rapid and optimal dissociation of retina sample preparation. We also included additional filtering steps in data analysis for retinal scRNA-seq. Results: We report a gentle method for dissociation of the mouse retina that minimizes cell death and preserves cell morphology. This protocol also results in detection of higher transcriptional complexity. In addition, the modified computational pipeline leads to better-quality single-cell RNA-sequencing data in retina samples. We also demonstrate the advantages and limitations of using fresh versus frozen retinas to prepare cell or nuclei suspensions for scRNA-seq. Conclusions: We provide a simple yet robust and reproducible protocol for retinal scRNA-seq analysis, especially for comparative studies.
Subject(s)
Gene Expression Profiling/methods , Retina/cytology , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Animals , Cell Nucleus , Computational Biology , Mice , Mice, Inbred C57BL , Retina/metabolism , SoftwareABSTRACT
In retinal photoreceptors, vectorial transport of cargo is critical for transduction of visual signals, and defects in intracellular trafficking can lead to photoreceptor degeneration and vision impairment. Molecular signatures associated with routing of transport vesicles in photoreceptors are poorly understood. We previously reported the identification of a novel rod photoreceptor specific isoform of Receptor Expression Enhancing Protein (REEP) 6, which belongs to a family of proteins involved in intracellular transport of receptors to the plasma membrane. Here we show that loss of REEP6 in mice (Reep6-/-) results in progressive retinal degeneration. Rod photoreceptor dysfunction is observed in Reep6-/- mice as early as one month of age and associated with aberrant accumulation of vacuole-like structures at the apical inner segment and reduction in selected rod phototransduction proteins. We demonstrate that REEP6 is detected in a subset of Clathrin-coated vesicles and interacts with the t-SNARE, Syntaxin3. In concordance with the rod degeneration phenotype in Reep6-/- mice, whole exome sequencing identified homozygous REEP6-E75K mutation in two retinitis pigmentosa families of different ethnicities. Our studies suggest a critical function of REEP6 in trafficking of cargo via a subset of Clathrin-coated vesicles to selected membrane sites in retinal rod photoreceptors.
Subject(s)
Membrane Transport Proteins/metabolism , Retinal Rod Photoreceptor Cells/metabolism , Animals , Clathrin-Coated Vesicles/metabolism , Eye Proteins/genetics , Light Signal Transduction , Membrane Proteins , Membrane Transport Proteins/genetics , Mice , Mice, Knockout , Mutation , Photoreceptor Cells, Vertebrate/metabolism , Protein Isoforms/metabolism , Protein Transport , Qa-SNARE Proteins/metabolism , Retinal Degeneration/metabolism , Retinitis Pigmentosa/genetics , SNARE Proteins/metabolismABSTRACT
Microglia, the principal resident immune cell of the CNS, exert significant influence on neurons during development and in pathological situations. However, if and how microglia contribute to normal neuronal function in the mature uninjured CNS is not well understood. We used the model of the adult mouse retina, a part of the CNS amenable to structural and functional analysis, to investigate the constitutive role of microglia by depleting microglia from the retina in a sustained manner using genetic methods. We discovered that microglia are not acutely required for the maintenance of adult retinal architecture, the survival of retinal neurons, or the laminar organization of their dendritic and axonal compartments. However, sustained microglial depletion results in the degeneration of photoreceptor synapses in the outer plexiform layer, leading to a progressive functional deterioration in retinal light responses. Our results demonstrate that microglia are constitutively required for the maintenance of synaptic structure in the adult retina and for synaptic transmission underlying normal visual function. Our findings on constitutive microglial function are relevant in understanding microglial contributions to pathology and in the consideration of therapeutic interventions that reduce or perturb constitutive microglial function. SIGNIFICANCE STATEMENT: Microglia, the principal resident immune cell population in the CNS, has been implicated in diseases in the brain and retina. However, how they contribute to the everyday function of the CNS is unclear. Using the model of the adult mouse retina, we examined the constitutive role of microglia by depleting microglia from the retina. We found that in the absence of microglia, retinal neurons did not undergo overt cell death or become structurally disorganized in their processes. However, connections between neurons called synapses begin to break down, leading to a decreased ability of the retina to transmit light responses. Our results indicate that retinal microglia contribute constitutively to the maintenance of synapses underlying healthy vision.
Subject(s)
Microglia/physiology , Neurons/physiology , Retina/cytology , Synapses/physiology , Animals , Cell Death/genetics , Disease Models, Animal , Dopamine Plasma Membrane Transport Proteins/metabolism , Eye Proteins/metabolism , Female , Gene Expression/genetics , Gene Expression Regulation/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Nystagmus, Optokinetic/genetics , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , Receptors, Interleukin-8A/genetics , Receptors, Interleukin-8A/metabolism , Synapses/genetics , Vision Disorders/genetics , Vision Disorders/pathology , Vision Disorders/physiopathology , Visual Pathways/physiologyABSTRACT
Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for >70% of X-linked retinitis pigmentosa (XLRP) and 15-20% of all inherited retinal degeneration. Gene replacement therapy for RPGR-XLRP was hampered by the relatively slow disease progression in mouse models and by difficulties in cloning the full-length RPGR-ORF15 cDNA that includes a purine-rich 3'-coding region; however, its effectiveness has recently been demonstrated in four dogs with RPGR mutations. To advance the therapy to clinical stage, we generated new stable vectors in AAV8 or AAV9 carrying mouse and human full-length RPGR-ORF15-coding sequence and conducted a comprehensive long-term dose-efficacy study in Rpgr-knockout mice. After validating their ability to produce full-length proteins that localize to photoreceptor connecting cilia, we evaluated various vector doses in mice during a 2-year study. We demonstrate that eyes treated with a single injection of mouse or human RPGR-ORF15 vector at an optimal dose maintained the expression of RPGR-ORF15 throughout the study duration and exhibited higher electroretinogram amplitude, thicker photoreceptor layer and better targeting of opsins to outer segments compared with sham-treated eyes. Furthermore, mice that received treatment at an advanced age also showed remarkable preservation of retinal structure and function. Retinal toxicity was observed at high vector doses, highlighting the importance of careful dose optimization in future clinical experiments. Our long-term dose-efficacy study should facilitate the design of human trials with human RPGR-ORF15 vector as a clinical candidate.
Subject(s)
Carrier Proteins/genetics , Eye Proteins/genetics , Genetic Therapy , Retinitis Pigmentosa/genetics , Animals , Carrier Proteins/metabolism , Dependovirus/genetics , Disease Models, Animal , Electroretinography , Exons , Eye Proteins/metabolism , Genetic Vectors/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , Open Reading Frames , Retina/metabolism , Retinitis Pigmentosa/metabolismABSTRACT
Distinct mutations in the centrosomal-cilia protein CEP290 lead to diverse clinical findings in syndromic ciliopathies. We show that CEP290 localizes to the transition zone in ciliated cells, precisely to the region of Y-linkers between central microtubules and plasma membrane. To create models of CEP290-associated ciliopathy syndromes, we generated Cep290(ko/ko) and Cep290(gt/gt) mice that produce no or a truncated CEP290 protein, respectively. Cep290(ko/ko) mice exhibit early vision loss and die from hydrocephalus. Retinal photoreceptors in Cep290(ko/ko) mice lack connecting cilia, and ciliated ventricular ependyma fails to mature. The minority of Cep290(ko/ko) mice that escape hydrocephalus demonstrate progressive kidney pathology. Cep290(gt/gt) mice die at mid-gestation, and the occasional Cep290(gt/gt) mouse that survives shows hydrocephalus and severely cystic kidneys. Partial loss of CEP290-interacting ciliopathy protein MKKS mitigates lethality and renal pathology in Cep290(gt/gt) mice. Our studies demonstrate domain-specific functions of CEP290 and provide novel therapeutic paradigms for ciliopathies.
Subject(s)
Cilia/metabolism , Hydrocephalus/genetics , Kidney Diseases, Cystic/genetics , Nuclear Proteins/genetics , Animals , Antigens, Neoplasm , Cell Cycle Proteins , Cilia/genetics , Cytoskeletal Proteins , Disease Models, Animal , Female , Humans , Hydrocephalus/metabolism , Kidney Diseases, Cystic/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/metabolism , Organ SpecificityABSTRACT
Genetic and genomic studies have enhanced our understanding of complex neurodegenerative diseases that exert a devastating impact on individuals and society. One such disease, age-related macular degeneration (AMD), is a major cause of progressive and debilitating visual impairment. Since the pioneering discovery in 2005 of complement factor H (CFH) as a major AMD susceptibility gene, extensive investigations have confirmed 19 additional genetic risk loci, and more are anticipated. In addition to common variants identified by now-conventional genome-wide association studies, targeted genomic sequencing and exome-chip analyses are uncovering rare variant alleles of high impact. Here, we provide a critical review of the ongoing genetic studies and of common and rare risk variants at a total of 20 susceptibility loci, which together explain 40-60% of the disease heritability but provide limited power for diagnostic testing of disease risk. Identification of these susceptibility loci has begun to untangle the complex biological pathways underlying AMD pathophysiology, pointing to new testable paradigms for treatment.
Subject(s)
Complement Factor H/genetics , Genetic Predisposition to Disease , Macular Degeneration/genetics , Polymorphism, Single Nucleotide/genetics , Genome-Wide Association Study , Haplotypes , Humans , Macular Degeneration/pathology , Mutation , Risk FactorsABSTRACT
TREX1/DNASE III, the most abundant 3'-5' DNA exonuclease in mammalian cells, is tail-anchored on the endoplasmic reticulum (ER). Mutations at the N-terminus affecting TREX1 DNase activity are associated with autoimmune and inflammatory conditions such as Aicardi-Goutières syndrome (AGS). Mutations in the C-terminus of TREX1 cause loss of localization to the ER and dysregulation of oligosaccharyltransferase (OST) activity, and are associated with retinal vasculopathy with cerebral leukodystrophy (RVCL) and in some cases with systemic lupus erythematosus (SLE). Here we investigate mice with conditional expression of the most common RVCL mutation, V235fs, and another mouse expressing a conditional C-terminal mutation, D272fs, associated with a case of human SLE. Mice homozygous for either mutant allele express the encoded human TREX1 truncations without endogenous mouse TREX1, and both remain DNase active in tissues. The two mouse strains are similar phenotypically without major signs of retinal, cerebral or renal disease but exhibit striking elevations of autoantibodies in the serum. The broad range of autoantibodies is primarily against non-nuclear antigens, in sharp contrast to the predominantly DNA-related autoantibodies produced by a TREX1-D18N mouse that specifically lacks DNase activity. We also found that treatment with an OST inhibitor, aclacinomycin, rapidly suppressed autoantibody production in the TREX1 frame-shift mutant mice. Together, our study presents two new mouse models based on TREX1 frame-shift mutations with a unique set of serologic autoimmune-like phenotypes.
Subject(s)
Autoimmunity/genetics , Autoimmunity/immunology , Exodeoxyribonucleases/genetics , Frameshift Mutation , Phosphoproteins/genetics , Aclarubicin/analogs & derivatives , Aclarubicin/pharmacology , Amino Acid Substitution , Animals , Apoptosis/genetics , Apoptosis/immunology , Autoantibodies/immunology , Autoimmunity/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Enzyme Activation , Exodeoxyribonucleases/chemistry , Exodeoxyribonucleases/metabolism , Gene Expression , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Mice , Mice, Transgenic , Phenotype , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Retina/immunology , Retina/metabolism , Retina/pathology , Thymocytes/immunology , Thymocytes/metabolism , TranscriptomeABSTRACT
Characterizing the role of epigenetic regulation in the mammalian retina is critical for understanding fundamental mechanisms of retinal development and disease. DNA methylation, an epigenetic modifier of genomic DNA, plays an important role in modulating networks of tissue and cell-specific gene expression. However, the impact of DNA methylation on retinal development and homeostasis of retinal neurons remains unclear. Here, we have created a tissue-specific DNA methyltransferase (Dnmt) triple mutant mouse in an effort to characterize the impact of DNA methylation on retinal development and homeostasis. An Rx-Cre transgene was used to drive targeted mutation of all three murine Dnmt genes in the mouse retina encoding major DNA methylation enzymes DNMT1, DNMT3A and DNMT3B. The triple mutant mice represent a hypomorph model since Dnmt1 catalytic activity was still present and excision of Dnmt3a and Dnmt3b had only about 90% efficiency. Mutation of all three Dnmts resulted in global genomic hypomethylation and dramatic reorganization of the photoreceptor and synaptic layers within retina. Transcriptome and proteomic analyses demonstrated enrichment of dysregulated phototransduction and synaptic genes. The 5 mC signal in triple mutant retina was confined to the central heterochromatin but reduced in the peripheral heterochromatin region of photoreceptor nuclei. In addition, we found a reduction of the 5 mC signal in ganglion cell nuclei. Collectively, this data suggests cooperation of all three Dnmts in the formation and homeostasis of photoreceptors and other retinal neurons within the mammalian retina, and highlight the relevance of epigenetic regulation to sensory retinal disorders and vision loss.
Subject(s)
DNA (Cytosine-5-)-Methyltransferases/genetics , DNA/genetics , Mutation , Photoreceptor Cells, Vertebrate/metabolism , Animals , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation , DNA Methyltransferase 3A , DNA Mutational Analysis , Immunoblotting , Immunohistochemistry , Mice , Mice, Mutant Strains , Microscopy, Electron , Models, Animal , Photoreceptor Cells, Vertebrate/ultrastructure , Real-Time Polymerase Chain Reaction , Retinal Neurons/metabolism , Retinal Neurons/ultrastructure , DNA Methyltransferase 3BABSTRACT
Retinitis pigmentosa (RP), a disease characterized by the progressive degeneration of mutation-bearing photoreceptors, is a significant cause of incurable blindness in the young worldwide. Recent studies have found that activated retinal microglia contribute to photoreceptor demise via phagocytosis and proinflammatory factor production, however mechanisms regulating these contributions are not well-defined. In this study, we investigate the role of CX3CR1, a microglia-specific receptor, in regulating microglia-mediated degeneration using the well-established rd10 mouse model of RP. We found that in CX3CR1-deficient (CX3CR1(GFP/GFP) ) rd10 mice microglial infiltration into the photoreceptor layer was significantly augmented and associated with accelerated photoreceptor apoptosis and atrophy compared with CX3CR1-sufficient (CX3CR1(GFP/+) ) rd10 littermates. CX3CR1-deficient microglia demonstrated increased phagocytosis as evidenced by (1) having increased numbers of phagosomes in vivo, (2) an increased rate of phagocytosis of fluorescent beads and photoreceptor cellular debris in vitro, and (3) increased photoreceptor phagocytosis dynamics on live cell imaging in retinal explants, indicating that CX3CR1 signaling in microglia regulates the phagocytic clearance of at-risk photoreceptors. We also found that CX3CR1 deficiency in retinal microglia was associated with increased expression of inflammatory cytokines and microglial activation markers. Significantly, increasing CX3CL1-CX3CR1 signaling in the rd10 retina via exogenous intravitreal delivery of recombinant CX3CL1 was effective in (1) decreasing microglial infiltration, phagocytosis and activation, and (2) improving structural and functional features of photoreceptor degeneration. These results indicate that CX3CL1-CX3CR1 signaling is a molecular mechanism capable of modulating microglial-mediated degeneration and represents a potential molecular target in therapeutic approaches to RP. GLIA 2016;64:1479-1491.
Subject(s)
CX3C Chemokine Receptor 1/metabolism , Chemokine CX3CL1/metabolism , Microglia/metabolism , Phagocytosis/physiology , Photoreceptor Cells/metabolism , Retinitis Pigmentosa/metabolism , Animals , Disease Models, Animal , Macrophages/metabolism , Mice, Transgenic , Neuroprotective Agents/pharmacology , Receptors, Chemokine/metabolism , Retina/metabolism , Signal Transduction/drug effectsABSTRACT
Neurodegenerative diseases affecting the macula constitute a major cause of incurable vision loss and exhibit considerable clinical and genetic heterogeneity, from early-onset monogenic disease to multifactorial late-onset age-related macular degeneration (AMD). As part of our continued efforts to define genetic causes of macular degeneration, we performed whole exome sequencing in four individuals of a two-generation family with autosomal dominant maculopathy and identified a rare variant p.Glu1144Lys in Fibrillin 2 (FBN2), a glycoprotein of the elastin-rich extracellular matrix (ECM). Sanger sequencing validated the segregation of this variant in the complete pedigree, including two additional affected and one unaffected individual. Sequencing of 192 maculopathy patients revealed additional rare variants, predicted to disrupt FBN2 function. We then undertook additional studies to explore the relationship of FBN2 to macular disease. We show that FBN2 localizes to Bruch's membrane and its expression appears to be reduced in aging and AMD eyes, prompting us to examine its relationship with AMD. We detect suggestive association of a common FBN2 non-synonymous variant, rs154001 (p.Val965Ile) with AMD in 10 337 cases and 11 174 controls (OR = 1.10; P-value = 3.79 × 10(-5)). Thus, it appears that rare and common variants in a single gene--FBN2--can contribute to Mendelian and complex forms of macular degeneration. Our studies provide genetic evidence for a key role of elastin microfibers and Bruch's membrane in maintaining blood-retina homeostasis and establish the importance of studying orphan diseases for understanding more common clinical phenotypes.
Subject(s)
Genetic Association Studies , Genetic Variation , Macular Degeneration/genetics , Microfilament Proteins/genetics , Adult , Aged , Amino Acid Sequence , Bruch Membrane/metabolism , DNA Mutational Analysis , Exome , Extracellular Matrix/metabolism , Fibrillin-2 , Fibrillins , High-Throughput Nucleotide Sequencing , Humans , Macular Degeneration/diagnosis , Male , Meta-Analysis as Topic , Microfilament Proteins/metabolism , Middle Aged , Models, Molecular , Molecular Sequence Data , Mutation , Pedigree , Protein Conformation , Protein Stability , Retina/metabolism , Retina/pathology , Sequence AlignmentABSTRACT
Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice. Conditional Dnmt1 knockdown in early eye development mediated by Rx-Cre did not produce lamination or cell fate defects, except in cones; however, the photoreceptors completely lacked outer segments despite near normal expression of phototransduction and cilia genes. We also identified disruption of RPE morphology and polarization as early as E15.5. Defects in outer segment biogenesis were evident with Dnmt1 exon excision only in RPE, but not when excision was directed exclusively to photoreceptors. We detected a reduction in DNA methylation of LINE1 elements (a measure of global DNA methylation) in developing mutant RPE as compared with neural retina, and of Tuba3a, which exhibited dramatically increased expression in mutant retina. These results demonstrate a unique function of DNMT1-mediated DNA methylation in controlling RPE apicobasal polarity and neural retina differentiation. We also establish a model to study the epigenetic mechanisms and signaling pathways that guide the modulation of photoreceptor outer segment morphogenesis by RPE during retinal development and disease.
Subject(s)
Cell Membrane Permeability/physiology , DNA (Cytosine-5-)-Methyltransferases/genetics , Morphogenesis/genetics , Retinal Photoreceptor Cell Outer Segment/physiology , Retinal Pigment Epithelium/physiology , Animals , Cell Membrane Permeability/genetics , Cell Polarity/genetics , DNA (Cytosine-5-)-Methyltransferase 1 , DNA Methylation/genetics , Embryo, Mammalian , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Mice , Mice, Transgenic , Microarray Analysis , Morphogenesis/physiology , Organ Specificity/genetics , Retinal Photoreceptor Cell Outer Segment/metabolism , Retinal Pigment Epithelium/embryology , Retinal Pigment Epithelium/growth & development , Retinal Pigment Epithelium/metabolism , TranscriptomeABSTRACT
Chronic retinal inflammation in the form of activated microglia and macrophages are implicated in the etiology of neurodegenerative diseases of the retina, including age-related macular degeneration, diabetic retinopathy, and glaucoma. However, molecular biomarkers and targeted therapies for immune cell activation in these disorders are currently lacking. To address this, we investigated the involvement and role of translocator protein (TSPO), a biomarker of microglial and astrocyte gliosis in brain degeneration, in the context of retinal inflammation. Here, we find that TSPO is acutely and specifically upregulated in retinal microglia in separate mouse models of retinal inflammation and injury. Concomitantly, its endogenous ligand, diazepam-binding inhibitor (DBI), is upregulated in the macroglia of the mouse retina such as astrocytes and Müller cells. In addition, we discover that TSPO-mediated signaling in microglia via DBI-derived ligands negatively regulates features of microglial activation, including reactive oxygen species production, TNF-α expression and secretion, and microglial proliferation. The inducibility and effects of DBI-TSPO signaling in the retina reveal a mechanism of coordinated macroglia-microglia interactions, the function of which is to limit the magnitude of inflammatory responses after their initiation, facilitating a return to baseline quiescence. Our results indicate that TSPO is a promising molecular marker for imaging inflammatory cell activation in the retina and highlight DBI-TSPO signaling as a potential target for immodulatory therapies.
Subject(s)
Cell Communication/physiology , Microglia/metabolism , Receptors, GABA/physiology , Retina/metabolism , Signal Transduction/physiology , Amino Acid Sequence , Animals , Astrocytes/metabolism , Cells, Cultured , Female , Inflammation/metabolism , Inflammation/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , Receptors, GABA/geneticsABSTRACT
The present study examined the consequences of eliminating horizontal cells from the outer retina during embryogenesis upon the organization and assembly of the outer plexiform layer (OPL). Retinal horizontal cells exhibit a migration defect in Lim1-conditional knock-out (Lim1-CKO) mice and become mispositioned in the inner retina before birth, redirecting their dendrites into the inner plexiform layer. The resultant (mature) OPL, developing in the absence of horizontal cells, shows a retraction of rod spherules into the outer nuclear layer and a sprouting of rod bipolar cell dendrites to reach ectopic ribbon-protein puncta. Cone pedicles and the dendrites of type 7 cone bipolar cells retain their characteristic stratification and colocalization within the collapsed OPL, although both are atrophic and the spatial distribution of the pedicles is disrupted. Developmental analysis of Lim1-CKO retina reveals that components of the rod and cone pathways initially co-assemble within their normal strata in the OPL, indicating that horizontal cells are not required for the correct targeting of photoreceptor terminals or bipolar cell dendrites. As the rod spherules begin to retract during the second postnatal week, rod bipolar cells initially show no signs of ectopic growth, sprouting only subsequently and continuing to do so well after the eighth postnatal week. These results demonstrate the critical yet distinctive roles for horizontal cells on the rod and cone pathways and highlight a unique and as-yet-unrecognized maintenance function of an inhibitory interneuron that is not required for the initial targeting and co-stratification of other components in the circuit.
Subject(s)
Neuronal Plasticity/physiology , Retinal Horizontal Cells/physiology , Retinal Neurons/physiology , Visual Pathways/growth & development , Animals , Female , Mice , Mice, Transgenic , Synapses/metabolism , Visual Pathways/physiologyABSTRACT
Congenital cataracts (CCs), responsible for about one-third of blindness in infants, are a major cause of vision loss in children worldwide. Autosomal-recessive congenital cataracts (arCC) form a clinically diverse and genetically heterogeneous group of disorders of the crystalline lens. To identify the genetic cause of arCC in consanguineous Pakistani families, we performed genome-wide linkage analysis and fine mapping and identified linkage to 3p21-p22 with a summed LOD score of 33.42. Mutations in the gene encoding FYVE and coiled-coil domain containing 1 (FYCO1), a PI(3)P-binding protein family member that is associated with the exterior of autophagosomes and mediates microtubule plus-end-directed vesicle transport, were identified in 12 Pakistani families and one Arab Israeli family in which arCC had previously been mapped to the overlapping CATC2 region. Nine different mutations were identified, including c.3755 delC (p.Ala1252AspfsX71), c.3858_3862dupGGAAT (p.Leu1288TrpfsX37), c.1045 C>T (p.Gln349X), c.2206C>T (p.Gln736X), c.2761C>T (p.Arg921X), c.2830C>T (p.Arg944X), c.3150+1 G>T, c.4127T>C (p.Leu1376Pro), and c.1546C>T (p.Gln516X). Fyco1 is expressed in the mouse embryonic and adult lens and peaks at P12d. Expressed mutant proteins p.Leu1288TrpfsX37 and p.Gln736X are truncated on immunoblots. Wild-type and p.L1376P FYCO1, the only missense mutant identified, migrate at the expected molecular mass. Both wild-type and p. Leu1376Pro FYCO1 proteins expressed in human lens epithelial cells partially colocalize to microtubules and are found adjacent to Golgi, but they primarily colocalize to autophagosomes. Thus, FYCO1 is involved in lens development and transparency in humans, and mutations in this gene are one of the most common causes of arCC in the Pakistani population.
Subject(s)
Cataract/congenital , Cataract/genetics , DNA-Binding Proteins/genetics , Genes, Recessive , Transcription Factors/genetics , Amino Acid Sequence , Cataract/pathology , Genetic Testing , Genome-Wide Association Study , Humans , Microtubule-Associated Proteins , Molecular Sequence Data , Mutation , Pakistan , PedigreeABSTRACT
BACKGROUND: Ebola virus disease (EVD) survivors experience ocular sequelae including retinal lesions, cataracts, and vision loss. While monoclonal antibodies targeting the Ebola virus glycoprotein (EBOV-GP) have shown promise in improving prognosis, their effectiveness in mitigating ocular sequelae remains uncertain. METHODS: We developed and characterized a BSL-2-compatible immunocompetent mouse model to evaluate therapeutics targeting EBOV-GP by inoculating neonatal mice with vesicular stomatitis virus expressing EBOV-GP (VSV-EBOV). To examine the impact of anti-EBOV-GP antibody treatment on acute retinitis and ocular sequelae, VSV-EBOV-infected mice were treated with polyclonal antibodies or monoclonal antibody preparations with antibody-dependent cellular cytotoxicity (ADCC-mAb) or neutralizing activity (NEUT-mAb). FINDINGS: Treatment with all anti-EBOV-GP antibodies tested dramatically reduced viremia and improved survival. Further, all treatments reduced the incidence of cataracts. However, NEUT-mAb alone or in combination with ADCC-mAb reduced viral load in the eyes, downregulated the ocular immune and inflammatory responses, and minimized retinal damage more effectively. INTERPRETATION: Anti-EBOV-GP antibodies can improve survival among EVD patients, but improved therapeutics are needed to reduce life altering sequelae. This animal model offers a new platform to examine the acute and long-term effect of the virus in the eye and the relative impact of therapeutic candidates targeting EBOV-GP. Results indicate that even antibodies that improve systemic viral clearance and survival can differ in their capacity to reduce acute ocular inflammation, and long-term retinal pathology and corneal degeneration. FUNDING: This study was partly supported by Postgraduate Research Fellowship Awards from ORISE through an interagency agreement between the US DOE and the US FDA.
Subject(s)
Antibodies, Viral , Disease Models, Animal , Ebolavirus , Hemorrhagic Fever, Ebola , Animals , Mice , Ebolavirus/immunology , Ebolavirus/pathogenicity , Hemorrhagic Fever, Ebola/virology , Hemorrhagic Fever, Ebola/drug therapy , Hemorrhagic Fever, Ebola/immunology , Antibodies, Viral/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal/pharmacology , Humans , Viral Load , Glycoproteins/immunology , Glycoproteins/metabolism , Viral Envelope Proteins/immunology , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibodies, Neutralizing/therapeutic use , Antibody-Dependent Cell CytotoxicityABSTRACT
Cone photoreceptors are the primary initiator of visual transduction in the human retina. Dysfunction or death of rod photoreceptors precedes cone loss in many retinal and macular degenerative diseases, suggesting a rod-dependent trophic support for cone survival. Rod differentiation and homeostasis are dependent on the basic motif leucine zipper transcription factor neural retina leucine zipper (NRL). The loss of Nrl (Nrl(-/-)) in mice results in a retina with predominantly S-opsin-containing cones that exhibit molecular and functional characteristics of wild-type cones. Here, we report that Nrl(-/-) retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. However, cone degeneration stabilizes by 4 months of age, resulting in a thinner but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic electroretinogram. At this stage, microglia translocate back to the inner retina and reacquire a quiescent morphology. Gene profiling analysis during the period of transient degeneration reveals misregulation of genes related to stress response and inflammation, implying their involvement in cone death. The Nrl(-/-) mouse illustrates the long-term viability of cones in the absence of rods and retinal pigment epithelium defects in a rodless retina. We propose that Nrl(-/-) retina may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula.
Subject(s)
Apoptosis/genetics , Basic-Leucine Zipper Transcription Factors/genetics , Eye Proteins/genetics , Retina/abnormalities , Retina/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Retinal Degeneration/physiopathology , Animals , Basic-Leucine Zipper Transcription Factors/deficiency , Disease Models, Animal , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Retinal Cone Photoreceptor Cells/pathology , Retinal Degeneration/genetics , Retinal Degeneration/pathology , Retinal Detachment/genetics , Retinal Detachment/pathology , Retinal Detachment/physiopathologyABSTRACT
Inward rectifying potassium (Kir) channels participate in regulating potassium concentration (K(+)) in the central nervous system (CNS), including in the retina. We explored the contribution of Kir channels to retinal function by delivering Kir antibodies (Kir-Abs) into the rat eye in vivo to interrupt channel activity. Kir-Abs were coupled to a peptide carrier to reach intracellular epitopes. Functional effects were evaluated by recording the scotopic threshold response (STR) and photopic negative response (PhNR) of the electroretinogram (ERG) noninvasively with an electrode on the cornea to determine activity of the rod and cone pathways, respectively. Intravitreal delivery of Kir2.1-Ab coupled to the peptide carrier diminished these ERG responses equivalent to dimming the stimulus 10- to 100-fold. Immunohistochemistry (IHC) showed Kir2.1 immunostaining of retinal bipolar cells (BCs) matching the labeling pattern obtained with conventional IHC of applying Kir2.1-Ab to fixed retinal sections postmortem. Whole-cell voltage-clamp BC recordings in rat acute retinal slices showed suppression of barium-sensitive Kir2.1 currents upon inclusion of Kir2.1-Ab in the patch pipette. The in vivo functional and structural results implicate a contribution of Kir2.1 channel activity in these electronegative ERG potentials. Studies with Kir4.1-Ab administered in vivo also suppressed the ERG components and showed immunostaining of Müller cells. The strategy of administering Kir antibodies in vivo, coupled to a peptide carrier to facilitate intracellular delivery, identifies roles for Kir2.1 and Kir4.1 in ERG components arising in the proximal retina and suggests this approach could be of further value in research.
Subject(s)
Potassium Channels/metabolism , Retina/metabolism , Animals , Antibodies/metabolism , Antibodies/pharmacology , Antibodies/physiology , Barium/metabolism , Barium/pharmacology , Barium/physiology , Central Nervous System/metabolism , Central Nervous System/physiology , Cytoplasm/metabolism , Cytoplasm/physiology , Electroretinography , Nerve Degeneration/metabolism , Neuroglia/metabolism , Neuroglia/physiology , Patch-Clamp Techniques , Potassium/metabolism , Potassium/pharmacology , Potassium/physiology , Potassium Channels/pharmacology , Potassium Channels/physiology , Potassium Channels, Inwardly Rectifying , Rats , Rats, Mutant Strains , Retina/drug effects , Retina/physiology , Retinaldehyde/metabolism , Retinaldehyde/pharmacology , Retinaldehyde/physiologyABSTRACT
The importance of identifying VEGF-independent pathways in pathological angiogenesis is increasingly recognized as a result of the emerging drug resistance to anti-VEGF therapies. PDGF-CC is the third member of the PDGF family discovered after more than two decades of studies on PDGF-AA and PDGF-BB. The biological function of PDGF-CC and the underlying cellular and molecular mechanisms remain largely unexplored. Here, using different animal models, we report that PDGF-CC inhibition by neutralizing antibody, shRNA, or genetic deletion suppressed both choroidal and retinal neovascularization. Importantly, we revealed that PDGF-CC targeting acted not only on multiple cell types important for pathological angiogenesis, such as vascular mural and endothelial cells, macrophages, choroidal fibroblasts and retinal pigment epithelial cells, but also on the expression of other important angiogenic genes, such as PDGF-BB and PDGF receptors. At a molecular level, we found that PDGF-CC regulated glycogen synthase kinase (GSK)-3beta phosphorylation and expression both in vitro and in vivo. Activation of GSK3beta impaired PDGF-CC-induced angiogenesis, and inhibition of GSK3beta abolished the antiangiogenic effect of PDGF-CC blockade. Thus, we identified PDGF-CC as an important candidate target gene for antiangiogenic therapy, and PDGF-CC inhibition may be of therapeutic value in treating neovascular diseases.