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1.
EBioMedicine ; 104: 105170, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38823088

ABSTRACT

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 Cytotoxicity
2.
iScience ; 26(4): 106417, 2023 Apr 21.
Article in English | MEDLINE | ID: mdl-37153444

ABSTRACT

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.

3.
Prog Retin Eye Res ; 95: 101147, 2023 Jul.
Article in English | MEDLINE | ID: mdl-36402656

ABSTRACT

Retinoschisin (RS1) is a secreted protein that is essential for maintaining integrity of the retina. Numerous mutations in RS1 cause X-linked retinoschisis (XLRS), a progressive degeneration of the retina that leads to vision loss in young males. A key manifestation of XLRS is the formation of cavities (cysts) in the retina and separation of the layers (schisis), disrupting synaptic transmission. There are currently no approved treatments for patients with XLRS. Strategies using adeno-associated viral (AAV) vectors to deliver functional copies of RS1 as a form of gene augmentation therapy, are under clinical evaluation. To improve therapeutic strategies for treating XLRS, it is critical to better understand the secretion of RS1 and its molecular function. Immunofluorescence and immunoelectron microscopy show that RS1 is located on the surfaces of the photoreceptor inner segments and bipolar cells. Sequence homology indicates a discoidin domain fold, similar to many other proteins with demonstrated adhesion functions. Recent structural studies revealed the tertiary structure of RS1 as two back-to-back octameric rings, each cross-linked by disulfides. The observation of higher order structures in vitro suggests the formation of an adhesive matrix spanning the distance between cells (∼100 nm). Several studies indicated that RS1 readily binds to other proteins such as the sodium-potassium ATPase (NaK-ATPase) and extracellular matrix proteins. Alternatively, RS1 may influence fluid regulation via interaction with membrane proteins such as the NaK-ATPase, largely inferred from the use of carbonic anhydrase inhibitors to shrink the typical intra-retinal cysts in XLRS. We discuss these models in light of RS1 structure and address the difficulty in understanding the function of RS1.


Subject(s)
Retina , Retinoschisis , Male , Humans , Molecular Structure , Retina/metabolism , Retinoschisis/genetics , Retinoschisis/metabolism , Mutation , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Eye Proteins/genetics
4.
Invest Ophthalmol Vis Sci ; 63(11): 8, 2022 10 03.
Article in English | MEDLINE | ID: mdl-36227606

ABSTRACT

Purpose: Loss of retinoschisin (RS1) function underlies X-linked retinoschisis (XLRS) pathology. In the retina, both photoreceptor inner segments and bipolar cells express RS1. However, the loss of RS1 function causes schisis primarily in the inner retina. To understand these cell type-specific phenotypes, we decoupled RS1 effects in bipolar cells from that in photoreceptors. Methods: Bipolar cell transgene RS1 expression was achieved using two inner retina-specific promoters: (1) a minimal promoter engineered from glutamate receptor, metabotropic glutamate receptor 6 gene (mini-mGluR6/ Grm6) and (2) MiniPromoter (Ple155). Adeno-associated virus vectors encoding RS1 gene under either the mini-mGluR6 or Ple-155 promoter were delivered to the XLRS mouse retina through intravitreal or subretinal injection on postnatal day 14. Retinal structure and function were assessed 5 weeks later: immunohistochemistry for morphological characterization, optical coherence tomography and electroretinography (ERG) for structural and functional evaluation. Results: Immunohistochemical analysis of RS1expression showed that expression with the MiniPromoter (Ple155) was heavily enriched in bipolar cells. Despite variations in vector penetrance and gene transfer efficiency across the injected retinas, those retinal areas with robust bipolar cell RS1 expression showed tightly packed bipolar cells with fewer cavities and marked improvement in inner retinal structure and synaptic function as judged by optical coherence tomography and electroretinography, respectively. Conclusions: These results demonstrate that RS1 gene expression primarily in bipolar cells of the XLRS mouse retina, independent of photoreceptor expression, can ameliorate retinoschisis structural pathology and provide further evidence of RS1 role in cell adhesion.


Subject(s)
Cysts , Retinoschisis , Animals , Mice , Cysts/metabolism , Cysts/pathology , Electroretinography , Eye Proteins/genetics , Eye Proteins/metabolism , Retina/metabolism , Retina/pathology , Retinal Bipolar Cells/metabolism , Retinoschisis/genetics , Retinoschisis/metabolism
5.
Commun Biol ; 5(1): 893, 2022 09 13.
Article in English | MEDLINE | ID: mdl-36100689

ABSTRACT

Choroideremia is an X-linked, blinding retinal degeneration with progressive loss of photoreceptors, retinal pigment epithelial (RPE) cells, and choriocapillaris. To study the extent to which these layers are disrupted in affected males and female carriers, we performed multimodal adaptive optics imaging to better visualize the in vivo pathogenesis of choroideremia in the living human eye. We demonstrate the presence of subclinical, widespread enlarged RPE cells present in all subjects imaged. In the fovea, the last area to be affected in choroideremia, we found greater disruption to the RPE than to either the photoreceptor or choriocapillaris layers. The unexpected finding of patches of photoreceptors that were fluorescently-labeled, but structurally and functionally normal, suggests that the RPE blood barrier function may be altered in choroideremia. Finally, we introduce a strategy for detecting enlarged cells using conventional ophthalmic imaging instrumentation. These findings establish that there is subclinical polymegathism of RPE cells in choroideremia.


Subject(s)
Choroideremia , Retinal Degeneration , Choroid/diagnostic imaging , Choroideremia/genetics , Choroideremia/pathology , Female , Humans , Male , Optics and Photonics , Retinal Cone Photoreceptor Cells , Retinal Degeneration/pathology
6.
Biomed Opt Express ; 13(5): 3042-3055, 2022 May 01.
Article in English | MEDLINE | ID: mdl-35774328

ABSTRACT

Adaptive optics reflectance-based retinal imaging has proved a valuable tool for the noninvasive visualization of cells in the living human retina. Many subcellular features that remain at or below the resolution limit of current in vivo techniques may be more easily visualized with the same modalities in an ex vivo setting. While most microscopy techniques provide significantly higher resolution, enabling the visualization of fine cellular detail in ex vivo retinal samples, they do not replicate the reflectance-based imaging modalities of in vivo retinal imaging. Here, we introduce a strategy for imaging ex vivo samples using the same imaging modalities as those used for in vivo retinal imaging, but with increased resolution. We also demonstrate the ability of this approach to perform protein-specific fluorescence imaging and reflectance imaging simultaneously, enabling the visualization of nearly transparent layers of the retina and the classification of cone photoreceptor types.

7.
Hum Gene Ther ; 32(13-14): 667-681, 2021 07.
Article in English | MEDLINE | ID: mdl-33019822

ABSTRACT

To understand RS1 gene interaction networks in the X-linked retinoschisis (XLRS) mouse retina (Rs1-/y), we analyzed the transcriptome by RNA sequencing before and after in vivo expression of exogenous retinoschisin (RS1) gene delivered by AAV8. RS1 is a secreted cell adhesion protein that is critical for maintaining structural lamination and synaptic integrity of the neural retina. RS1 loss-of-function mutations cause XLRS disease in young boys and men, with splitting ("schisis") of retinal layers and synaptic dysfunction that cause progressive vision loss with age. Analysis of differential gene expression profiles and pathway enrichment analysis of Rs1-KO (Rs1-/y) retina identified cell surface receptor signaling and positive regulation of cell adhesion as potential RS1 gene interaction networks. Most importantly, it also showed massive dysregulation of immune response genes at early age, with characteristics of a microglia-driven proinflammatory state. Delivery of AAV8-RS1 primed the Rs1-KO retina toward structural and functional recovery. The disease transcriptome transitioned toward a recovery phase with upregulation of genes implicated in wound healing, anatomical structure (camera type eye) development, metabolic pathways, and collagen IV networks that provide mechanical stability to basement membrane. AAV8-RS1 expression also attenuated the microglia gene signatures to low levels toward immune quiescence. This study is among the first to identify RS1 gene interaction networks that underlie retinal structural and functional recovery after RS1 gene therapy. Significantly, it also shows that providing wild-type RS1 gene function caused the retina immune status to transition from a degenerative inflammatory phenotype toward immune quiescence, even though the transgene is not directly linked to microglia function. This study indicates that inhibition of microglial proinflammatory responses is an integral part of therapeutic rescue in XLRS gene therapy, and gene therapy might realize its full potential if delivered before microglia activation and photoreceptor cell death. Clinical Trials. gov Identifier NTC 02317887.


Subject(s)
Retinoschisis , Animals , Electroretinography , Eye Proteins/genetics , Gene Regulatory Networks , Genetic Therapy , Genetic Vectors/genetics , Mice , Microglia , Retina , Retinoschisis/genetics , Retinoschisis/therapy
8.
Front Cell Dev Biol ; 9: 810020, 2021.
Article in English | MEDLINE | ID: mdl-35096838

ABSTRACT

Purpose: We constructed and characterized knockout and conditional knockout mice for KCNJ13, encoding the inwardly rectifying K+ channel of the Kir superfamily Kir7.1, mutations in which cause both Snowflake Vitreoretinal Degeneration (SVD) and Retinitis pigmentosa (RP) to further elucidate the pathology of this disease and to develop a potential model system for gene therapy trials. Methods: A Kcnj13 knockout mouse line was constructed by inserting a gene trap cassette expressing beta-galactosidase flanked by FRT sites in intron 1 with LoxP sites flanking exon two and converted to a conditional knockout by FLP recombination followed by crossing with C57BL/6J mice having Cre driven by the VMD2 promoter. Lentiviral replacement of Kcnj13 was driven by the EF1a or VMD2 promoters. Results: Blue-Gal expression is evident in E12.5 brain ventricular choroid plexus, lens, neural retina layer, and anterior RPE. In the adult eye expression is seen in the ciliary body, RPE and choroid. Adult conditional Kcnj13 ko mice show loss of photoreceptors in the outer nuclear layer, inner nuclear layer thinning with loss of bipolar cells, and thinning and disruption of the outer plexiform layer, correlating with Cre expression in the overlying RPE which, although preserved, shows morphological disruption. Fundoscopy and OCT show signs of retinal degeneration consistent with the histology, and photopic and scotopic ERGs are decreased in amplitude or extinguished. Lentiviral based replacement of Kcnj13 resulted in increased ERG c- but not a- or b- wave amplitudes. Conclusion: Ocular KCNJ13 expression starts in the choroid, lens, ciliary body, and anterior retina, while later expression centers on the RPE with no/lower expression in the neuroretina. Although KCNJ13 expression is not required for survival of the RPE, it is necessary for RPE maintenance of the photoreceptors, and loss of the photoreceptor, outer plexiform, and outer nuclear layers occur in adult KCNJ13 cKO mice, concomitant with decreased amplitude and eventual extinguishing of the ERG and signs of retinitis pigmentosa on fundoscopy and OCT. Kcnj13 replacement resulting in recovery of the ERG c- but not a- and b-waves is consistent with the degree of photoreceptor degeneration seen on histology.

9.
PLoS Genet ; 16(12): e1009259, 2020 12.
Article in English | MEDLINE | ID: mdl-33362196

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 , Transcriptome
10.
Mol Vis ; 26: 705-717, 2020.
Article in English | MEDLINE | ID: mdl-33088174

ABSTRACT

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 , Software
11.
JCI Insight ; 4(6)2019 03 21.
Article in English | MEDLINE | ID: mdl-30895942

ABSTRACT

The heterogeneity of individual cells in a tissue has been well characterized, largely using ex vivo approaches that do not permit longitudinal assessments of the same tissue over long periods of time. We demonstrate a potentially novel application of adaptive optics fluorescence microscopy to visualize and track the in situ mosaicism of retinal pigment epithelial (RPE) cells directly in the human eye. After a short, dynamic period during which RPE cells take up i.v.-administered indocyanine green (ICG) dye, we observed a remarkably stable heterogeneity in the fluorescent pattern that gradually disappeared over a period of days. This pattern could be robustly reproduced with a new injection and follow-up imaging in the same eye out to at least 12 months, which enabled longitudinal tracking of RPE cells. Investigation of ICG uptake in primary human RPE cells and in a mouse model of ICG uptake alongside human imaging corroborated our findings that the observed mosaicism is an intrinsic property of the RPE tissue. We demonstrate a potentially novel application of fluorescence microscopy to detect subclinical changes to the RPE, a technical advance that has direct implications for improving our understanding of diseases such as oculocutaneous albinism, late-onset retinal degeneration, and Bietti crystalline dystrophy.


Subject(s)
Microscopy, Fluorescence/methods , Mosaicism , Neuroimaging/methods , Ophthalmology/methods , Retinal Pigment Epithelium/diagnostic imaging , Retinal Pigment Epithelium/pathology , Animals , Female , Genetic Diseases, Inborn/diagnostic imaging , Genetic Diseases, Inborn/pathology , Humans , Indocyanine Green , Mice , Mice, Inbred BALB C
12.
Hum Mol Genet ; 27(20): 3555-3567, 2018 10 15.
Article in English | MEDLINE | ID: mdl-30084954

ABSTRACT

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/genetics
13.
Sci Adv ; 4(3): eaap8492, 2018 03.
Article in English | MEDLINE | ID: mdl-29750189

ABSTRACT

Microglia have been discovered to undergo repopulation following ablation. However, the functionality of repopulated microglia and the mechanisms regulating microglia repopulation are unknown. We examined microglial homeostasis in the adult mouse retina, a specialized neural compartment containing regular arrays of microglia in discrete synaptic laminae that can be directly visualized. Using in vivo imaging and cell-fate mapping techniques, we discovered that repopulation originated from residual microglia proliferating in the central inner retina that subsequently spread by centrifugal migration to fully recapitulate pre-existing microglial distributions and morphologies. Repopulating cells fully restored microglial functions including constitutive "surveying" process movements, behavioral and physiological responses to retinal injury, and maintenance of synaptic structure and function. Microglial repopulation was regulated by CX3CL1-CX3CR1 signaling, slowing in CX3CR1 deficiency and accelerating with exogenous CX3CL1 administration. Microglial homeostasis following perturbation can fully recover microglial organization and function under the regulation of chemokine signaling between neurons and microglia.


Subject(s)
CX3C Chemokine Receptor 1/metabolism , Chemokine CX3CL1/metabolism , Microglia/metabolism , Retina/cytology , Aging/physiology , Animals , Calcium-Binding Proteins/metabolism , Cell Movement , Cell Proliferation , Mice, Transgenic , Microfilament Proteins/metabolism , Microglia/ultrastructure , Signal Transduction
14.
mBio ; 9(1)2018 01 30.
Article in English | MEDLINE | ID: mdl-29382731

ABSTRACT

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease. C. trachomatis isolates are classified into 2 biovars-lymphogranuloma venereum (LGV) and trachoma-which are distinguished biologically by their natural host cell infection tropism. LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculo-urogenital epithelial cells and are noninvasive. The C. trachomatis plasmid is an important virulence factor in the pathogenesis of these infections. Central to its pathogenic role is the transcriptional regulatory function of the plasmid protein Pgp4, which regulates the expression of plasmid and chromosomal virulence genes. As many gene regulatory functions are post-transcriptional, we employed a comparative proteomic study of cells infected with plasmid-cured C. trachomatis serovars A and D (trachoma biovar), a L2 serovar (LGV biovar), and the L2 serovar transformed with a plasmid containing a nonsense mutation in pgp4 to more completely elucidate the effects of the plasmid on chlamydial infection biology. Our results show that the Pgp4-dependent elevations in the levels of Pgp3 and a conserved core set of chromosomally encoded proteins are remarkably similar for serovars within both C. trachomatis biovars. Conversely, we found a plasmid-dependent, Pgp4-independent, negative regulation in the expression of the chlamydial protease-like activity factor (CPAF) for the L2 serovar but not the A and D serovars. The molecular mechanism of plasmid-dependent negative regulation of CPAF expression in the LGV serovar is not understood but is likely important to understanding its macrophage infection tropism and invasive infection nature.IMPORTANCE The Chlamydia trachomatis plasmid is an important virulence factor in the pathogenesis of chlamydial infection. It is known that plasmid protein 4 (Pgp4) functions in the transcriptional regulation of the plasmid virulence protein 3 (Pgp3) and multiple chromosomal loci of unknown function. Since many gene regulatory functions can be post-transcriptional, we undertook a comparative proteomic analysis to better understand the plasmid's role in chlamydial and host protein expression. We report that Pgp4 is a potent and specific master positive regulator of a common core of plasmid and chromosomal virulence genes shared by multiple C. trachomatis serovars. Notably, we show that the plasmid is a negative regulator of the expression of the chlamydial virulence factor CPAF. The plasmid regulation of CPAF is independent of Pgp4 and restricted to a C. trachomatis macrophage-tropic strain. These findings are important because they define a previously unknown role for the plasmid in the pathophysiology of invasive chlamydial infection.


Subject(s)
Chlamydia trachomatis/genetics , Chlamydia trachomatis/metabolism , Endopeptidases/biosynthesis , Gene Expression Regulation, Bacterial , Plasmids , Transcription Factors/metabolism , Chlamydia trachomatis/chemistry , Epithelial Cells/microbiology , HeLa Cells , Humans , Proteome/analysis
15.
Sci Rep ; 7(1): 8433, 2017 08 16.
Article in English | MEDLINE | ID: mdl-28814744

ABSTRACT

Age-related macular degeneration (AMD), a leading contributor of vision loss, currently lacks comprehensive treatment. While AMD histopathology involves retinal pigment epithelium (RPE) injury associated with immune cell infiltration, the nature of immune cell responses to RPE injury remains undefined. We induced RPE injury pharmacologically and genetically in transgenic mouse models in which microglia and systemic monocytes were separately tagged, enabling a spatial and temporal dissection of the relative contributions of microglia vs. monocytes to post-injury changes. We found that myeloid cell responses to RPE injury occur in stages: (1) an early mobilization of endogenous microglia from the inner retina to the RPE layer, followed by (2) subsequent monocyte infiltration from the retinal vasculature into the inner retina that replenishes the local myeloid cell population in a CCR2-regulated manner. These altered distributions of myeloid cells post-injury were long-lived, with recruited monocytes acquiring the distribution, markers, and morphologies of neighboring endogenous microglia in a durable manner. These findings indicate the role played by infiltrating monocytes in maintaining myeloid cell homeostasis in the retina following AMD-relevant RPE injury and provide a foundation for understanding and therapeutically modulating immune aspects in retinal disease.


Subject(s)
Cell Proliferation , Epithelial Cells/metabolism , Monocytes/metabolism , Myeloid Cells/metabolism , Retina/metabolism , Retinal Pigment Epithelium/metabolism , Animals , Epithelial Cells/drug effects , Epithelial Cells/pathology , Homeostasis , Iodates/toxicity , Macular Degeneration/metabolism , Macular Degeneration/pathology , Mice, Inbred C57BL , Mice, Transgenic , Microglia/metabolism , Microscopy, Confocal , Receptors, CCR2/genetics , Receptors, CCR2/metabolism , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/pathology
16.
Hum Mol Genet ; 26(12): 2218-2230, 2017 06 15.
Article in English | MEDLINE | ID: mdl-28369466

ABSTRACT

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/metabolism
17.
J Autoimmun ; 81: 13-23, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28325644

ABSTRACT

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 , Transcriptome
18.
Brain Struct Funct ; 222(6): 2759-2771, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28213784

ABSTRACT

Microglia, the principal resident immune cell in the retina, play constitutive roles in immune surveillance and synapse maintenance, and are also associated with retinal disease, including those occurring in the macula. Perspectives on retinal microglia function have derived largely from rodent models and how these relate to the macula-bearing primate retina is unclear. In this study, we examined microglial distribution and cellular morphology in the adult rhesus macaque retina, and performed comparative characterizations in three retinal locations along the center-to-periphery axis (parafoveal, macular, and the peripheral retina). We found that microglia density peaked in the parafoveal retina and decreased in the peripheral retina. Individual microglial morphology reflected macular specialization, with macular microglia demonstrating the largest and most complex dendritic arbors relative to other retinal locations. Comparing retinal microglia between young and middle-aged animals, microglial density increased in the macular, but not in the peripheral retina with age, while microglial morphology across all locations remained relatively unchanged. Our findings indicate that microglial distribution and morphology demonstrate regional specialization in the retina, correlating with gradients of other retinal cell types. As microglia are innate immune cells implicated in age-related macular diseases, age-related microglial changes may be related to the increased vulnerability of the aged macula to immune-related neurodegeneration.


Subject(s)
Aging , Cell Shape , Macaca mulatta , Microglia/cytology , Retina/cytology , Age Factors , Animals , Biomarkers/analysis , Eye Proteins/analysis , Female , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Immunohistochemistry , Microglia/chemistry , Microscopy, Confocal , Retina/chemistry
19.
Exp Eye Res ; 159: 132-146, 2017 06.
Article in English | MEDLINE | ID: mdl-27865785

ABSTRACT

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 3B
20.
mBio ; 7(5)2016 Sep 27.
Article in English | MEDLINE | ID: mdl-27677792

ABSTRACT

The chlamydial protease-like activity factor (CPAF) is hypothesized to be an important secreted virulence factor; however, challenges in denaturing its proteolytic activity have hampered attempts to identify its legitimate targets. Here, we use a genetic and proteomic approach to identify authentic CPAF targets. Human epithelial cells infected with CPAF-sufficient and CPAF-deficient chlamydiae were lysed using known CPAF-denaturing conditions. Their protein profiles were analyzed using isobaric mass tags and liquid chromatography-tandem mass spectrometry. Comparative analysis of CPAF-sufficient and CPAF-deficient infections identified a limited number of CPAF host and chlamydial protein targets. Host targets were primarily interferon-stimulated gene products, whereas chlamydial targets were type III secreted proteins. We provide evidence supporting a cooperative role for CPAF and type III secreted effectors in blocking NF-κB p65 nuclear translocation, resulting in decreased beta interferon and proinflammatory cytokine synthesis. Genetic complementation of null organisms with CPAF restored p65 nuclear translocation inhibition and proteolysis of chlamydial type III secreted effector proteins (T3SEs). We propose that CPAF and T3SEs cooperate in the inhibition of host innate immunity. IMPORTANCE: Chlamydia trachomatis is an important human pathogen responsible for over 100 million infections each year worldwide. Its success as an intracellular pathogen revolves around its ability to evade host immunity. The chlamydial protease-like activity factor (CPAF) is a conserved serine protease secreted into the host cytosol of infected cells that is thought to play an important role in immune evasion. Currently, CPAF's authentic in situ target(s) and mechanism of action in immune evasion are poorly characterized. Using a CPAF-deficient strain and high-throughput proteomics, we report novel CPAF host and chlamydial targets. Host targets were primarily interferon-stimulated genes, whereas chlamydial targets were exclusively type III secreted proteins. We propose a novel mechanism for CPAF and type III secreted proteins in the evasion of host innate immune responses. These findings provide new insights into CPAF's function as a virulence factor and a better understanding of how chlamydiae evade host immunity.

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