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Arrigo A, Cremona O, Aragona E, Casoni F, Consalez G, Dogru RM, Hauck SM, Antropoli A, Bianco L, Parodi MB, Bandello F, Grosche A. Müller cells trophism and pathology as the next therapeutic targets for retinal diseases. Prog Retin Eye Res 2025:101357. [PMID: 40254246 DOI: 10.1016/j.preteyeres.2025.101357] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2025] [Revised: 04/14/2025] [Accepted: 04/15/2025] [Indexed: 04/22/2025]
Abstract
Müller cells are a crucial retinal cell type involved in multiple regulatory processes and functions that are essential for retinal health and functionality. Acting as structural and functional support for retinal neurons and photoreceptors, Müller cells produce growth factors, regulate ion and fluid homeostasis, and facilitate neuronal signaling. They play a pivotal role in retinal morphogenesis and cell differentiation, significantly contributing to macular development. Due to their radial morphology and unique cytoskeletal organization, Müller cells act as optical fibers, efficiently channeling photons directly to the photoreceptors. In response to retinal damage, Müller cells undergo specific gene expression and functional changes that serve as a first line of defense for neurons, but can also lead to unwarranted cell dysfunction, contributing to cell death and neurodegeneration. In some species, Müller cells can reactivate their developmental program, promoting retinal regeneration and plasticity-a remarkable ability that holds promising therapeutic potential if harnessed in mammals. The crucial and multifaceted roles of Müller cells-that we propose to collectively call "Müller cells trophism"-highlight the necessity of maintaining their functionality. Dysfunction of Müller cells, termed "Müller cells pathology," has been associated with a plethora of retinal diseases, including age-related macular degeneration, diabetic retinopathy, vitreomacular disorders, macular telangiectasia, and inherited retinal dystrophies. In this review, we outline how even subtle disruptions in Müller cells trophism can drive the pathological cascade of Müller cells pathology, emphasizing the need for targeted therapies to preserve retinal health and prevent disease progression.
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Affiliation(s)
- Alessandro Arrigo
- Ophthalmology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy; Eye Repair Unit, Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy.
| | - Ottavio Cremona
- Vita-Salute San Raffaele University, Milan, Italy; Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy.
| | - Emanuela Aragona
- Ophthalmology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Filippo Casoni
- Vita-Salute San Raffaele University, Milan, Italy; Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Giacomo Consalez
- Vita-Salute San Raffaele University, Milan, Italy; Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Rüya Merve Dogru
- Department of Physiological Genomics, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany
| | - Stefanie M Hauck
- Metabolomics and Proteomics Core, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich 80939, Germany
| | - Alessio Antropoli
- Ophthalmology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Lorenzo Bianco
- Ophthalmology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | | | - Francesco Bandello
- Ophthalmology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | - Antje Grosche
- Department of Physiological Genomics, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.
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Shi G, Nagarajan V, Caspi RR. AI-Driven Analysis Unveils Functional Dynamics of Müller Cells in Retinal Autoimmune Inflammation. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.02.28.640907. [PMID: 40093069 PMCID: PMC11908203 DOI: 10.1101/2025.02.28.640907] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/19/2025]
Abstract
Müller cell is the most common type of glial cell in the human and mouse retina, playing a crucial role in maintaining retinal homeostasis. In addition to providing structural support to the retina, Müller cells can also supply trophic substances to retinal neurons, remove metabolic waste, mitigate oxidative stress, and promote synaptic activities. However, many roles of Müller cells remain largely unknown, particularly for those in the inflamed retina. In this article, we reanalyzed a single cell RNA-seq (scRNA-seq) dataset from Aire-/- mice, which exhibits autoimmune retinal inflammation, specifically focusing on Müller cells and T cells, identifying nine distinct Müller cell subgroups along with five T cell subgroups. Among them, three subgroups of Müller cells are activated Müller cells, representing over 60% Müller cells in the inflamed retina. Using SCassist - an Artificial Intelligence (AI) based workflow assistant for single-cell analysis, we constructed a comparison matrix to quantify the involvement of pathways characterizing the functions of each Müller cell subpopulation. The activated Müller cells primarily present a macrophage-like phenotype with or without augmentation of the known Müller cell functions. Trajectory analysis further identified two paths, validating the presence of these two phenotypes, governed by Neurod1 and Irf family transcription factors (TFs). We further inferred the interactions between Müller cells and T cells and observed that activated Müller cells do not exhibit extra chemoattraction to Th1 cells compared to other Müller cells but display nearly exclusive expression of immune checkpoint molecules, primarily targeting Th1 cells. Our findings open new avenues for understanding the specialized mechanisms of retinal pathogenic autoimmunity and identifying candidates to explore potential inhibitory pathways in the inflamed retina.
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Affiliation(s)
- Guangpu Shi
- Laboratory of Immunology, National Eye Institute, NIH, Bethesda 20892, USA
| | | | - Rachel R Caspi
- Laboratory of Immunology, National Eye Institute, NIH, Bethesda 20892, USA
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Rowthorn-Apel N, Vridhachalam N, Connor KM, Bonilla GM, Sadreyev R, Singh C, Gnanaguru G. Microglial depletion decreases Müller cell maturation and inner retinal vascular density. Cell Commun Signal 2025; 23:90. [PMID: 39962511 PMCID: PMC11831819 DOI: 10.1186/s12964-025-02083-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2024] [Accepted: 02/03/2025] [Indexed: 02/21/2025] Open
Abstract
BACKGROUND The neuroretinal vascular system is comprised of three interconnected layers. The initial superficial vascular plexus formation is guided by astrocytes around birth in mice. The formation of the deep and intermediate vascular plexuses occurs in the second postnatal week and is driven by Müller-cell-derived angiogenic signaling. Previously, we reported that microglia play an important role in regulating astrocyte density during superficial vascular plexus formation. Here, we investigated the role of microglia in regulating Müller-cell-dependent inner retinal vascular development. METHODOLOGY In this study, we depleted microglia during retinal development using Csf1R antagonist (PLX5622). We characterized the developmental progression of inner retinal vascular growth, effect of microglial depletion on inner retinal vascular growth and Müller cell marker expressions by immunostaining. Differential expressions of genes in the control and microglia depleted groups were analyzed by mRNA-seq and qPCR. Unpaired t-test was performed to determine the statistical differences between groups. RESULTS This study show that microglia interact with Müller cells and the growing inner retinal vasculature. Depletion of microglia resulted in reduced inner retinal vascular layers densities and decreased Vegfa isoforms transcript levels. RNA-seq analysis further revealed that microglial depletion significantly reduced specific Müller cell maturation markers including glutamine synthetase, responsible for glutamine biosynthesis, necessary for angiogenesis. CONCLUSIONS Our study reveals an important role for microglia in facilitating inner retinal angiogenesis and Müller cell maturation.
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Affiliation(s)
- Nathaniel Rowthorn-Apel
- Department of Ophthalmology, Tufts University School of Medicine, Tufts Medical Center, Boston, MA, 02111, USA
| | - Naveen Vridhachalam
- Department of Ophthalmology, University of North Carolina, Chapel Hill, NC, 27599, USA
| | - Kip M Connor
- Angiogenesis Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA, 02114, USA
| | - Gracia M Bonilla
- Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA
| | - Ruslan Sadreyev
- Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA
- Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA
| | - Charandeep Singh
- Department of Ophthalmology, Tufts University School of Medicine, Tufts Medical Center, Boston, MA, 02111, USA
| | - Gopalan Gnanaguru
- Department of Ophthalmology, Tufts University School of Medicine, Tufts Medical Center, Boston, MA, 02111, USA.
- Vice President of Retinal Strategy, Johnson & Johnson Innovation Center, Cambridge, MA, 02142, USA.
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Masri RA, Greferath U, Fletcher EL, Martin PR, Grünert U. Immunohistochemistry and Spatial Density of Müller Cells in the Human Fovea. Invest Ophthalmol Vis Sci 2025; 66:46. [PMID: 39964323 PMCID: PMC11838121 DOI: 10.1167/iovs.66.2.46] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2024] [Accepted: 01/26/2025] [Indexed: 02/21/2025] Open
Abstract
Purpose Previous evidence indicates that molecular properties of foveal Müller cells are different from those in the peripheral retina. Here we aimed to characterize Müller cells in the human fovea (including the foveal floor) with specific focus on their spatial density and immunohistochemistry. Methods Human retinas were obtained postmortem from male and female donors with no known eye disease (aged 31-56 years) or after exenteration (one 75-year-old patient with no retinal disease and one 86-year-old patient with reticular pseudodrusen). Vertical sections through the macula were processed for immunofluorescence using antibodies against cellular retinaldehyde binding protein (CRALBP), glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), transient receptor potential vanilloid 4 (TRPV4), excitatory amino acid transporter 4 (EAAT4), calbindin, and RNA-binding protein with multiple splicing. Sections were imaged using high-resolution, multichannel confocal microscopy. Results Immunofluorescence for CRALBP and GS was found in Müller cells, including their processes throughout the retina. GFAP expression was found in astrocytes outside the fovea and in some foveal somas. Müller cell nuclei had a peak density of about 35,000 cells/mm2 at 500 µm eccentricity. Calbindin was coexpressed with CRALBP in up to 96% of Müller cells in the fovea, but at eccentricities beyond about 1.5 mm calbindin was not expressed by Müller cells. Conversely, calbindin expression in cone photoreceptors was absent in foveal but present in peripheral retina. Conclusions This study supports the hypothesis that Müller cells in the macula have distinct structural, functional, and immunohistochemical properties compared to their peripheral counterparts.
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Affiliation(s)
- Rania A. Masri
- The University of Sydney, Faculty of Medicine and Health, Save Sight Institute, Sydney, NSW, Australia
| | - Ursula Greferath
- The University of Melbourne, Department of Anatomy and Physiology, Melbourne, VIC, Australia
| | - Erica L. Fletcher
- The University of Melbourne, Department of Anatomy and Physiology, Melbourne, VIC, Australia
| | - Paul R. Martin
- The University of Sydney, Faculty of Medicine and Health, Save Sight Institute, Sydney, NSW, Australia
| | - Ulrike Grünert
- The University of Sydney, Faculty of Medicine and Health, Save Sight Institute, Sydney, NSW, Australia
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Grosche A, Grosche J, Verkhratsky A. Physiology and pathophysiology of the retinal neuroglia. HANDBOOK OF CLINICAL NEUROLOGY 2025; 210:239-265. [PMID: 40148047 DOI: 10.1016/b978-0-443-19102-2.00017-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/29/2025]
Abstract
Neuroglia of the retina are represented by Müller glia, parenchymal astrocytes, microglia and oligodendrocytes mainly associated with the optic nerve. Müller glia are the most numerous glia, endowed with multiple homeostatic functions and indispensable for the retinal morphofunctional organization. Müller cells integrate retinal neurons into individual functional units (known as retinal columns) and act as a living light guide, transmitting photons to photoreceptors. In pathology, retinal neuroglia undergo complex changes, which include upregulation of neuroprotection, reactive gliosis, and functional asthenia. The balance between all these changes defines the progression and outcome of retinal disorders.
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Affiliation(s)
- Antje Grosche
- Department of Physiological Genomics, Ludwig-Maximilians-Universität München, München, Germany.
| | | | - Alexei Verkhratsky
- Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom; Department of Neurosciences, University of the Basque Country UPV/EHU and CIBERNED, Leioa, Bizkaia, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
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Qian Z, Jiao M, Zhang N, Tang X, Liu S, Zhang F, Wang C, Zheng F. The IL-33/ST2 Axis Protects Retinal Ganglion Cells by Modulating the Astrocyte Response After Optic Nerve Injury. Neurosci Bull 2025; 41:61-76. [PMID: 39190095 PMCID: PMC11748692 DOI: 10.1007/s12264-024-01279-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2023] [Accepted: 04/29/2024] [Indexed: 08/28/2024] Open
Abstract
IL-33 and its receptor ST2 play crucial roles in tissue repair and homeostasis. However, their involvement in optic neuropathy due to trauma and glaucoma remains unclear. Here, we report that IL-33 and ST2 were highly expressed in the mouse optic nerve and retina. Deletion of IL-33 or ST2 exacerbated retinal ganglion cell (RGC) loss, retinal thinning, and nerve fiber degeneration following optic nerve (ON) injury. This heightened retinal neurodegeneration correlated with increased neurotoxic astrocytes in Il33-/- mice. In vitro, rIL-33 mitigated the neurotoxic astrocyte phenotype and reduced the expression of pro-inflammatory factors, thereby alleviating the RGC death induced by neurotoxic astrocyte-conditioned medium in retinal explants. Exogenous IL-33 treatment improved RGC survival in Il33-/- and WT mice after ON injury, but not in ST2-/- mice. Our findings highlight the role of the IL-33/ST2 axis in modulating reactive astrocyte function and providing neuroprotection for RGCs following ON injury.
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Affiliation(s)
- Zhigang Qian
- Department of Immunology, School of Basic Medicine, Tongji Medical College and State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Huazhong University of Science and Technology, Wuhan, 430030, China
- Department of Ophthalmology, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, 441000, China
| | - Mengya Jiao
- Department of Immunology, School of Basic Medicine, Tongji Medical College and State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Na Zhang
- Department of Immunology, School of Basic Medicine, Tongji Medical College and State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Xuhuan Tang
- Department of Immunology, School of Basic Medicine, Tongji Medical College and State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Shiwang Liu
- Department of Immunology, School of Basic Medicine, Tongji Medical College and State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Feng Zhang
- Department of Immunology, School of Basic Medicine, Tongji Medical College and State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Chenchen Wang
- National Demonstration Center for Experimental Basic Medical Education, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Fang Zheng
- Department of Immunology, School of Basic Medicine, Tongji Medical College and State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Huazhong University of Science and Technology, Wuhan, 430030, China.
- Key Laboratory of Organ Transplantation, Ministry of Education, NHC Key Laboratory of Organ Transplantation, Key Laboratory of Organ Transplantation, Chinese Academy of Medical Sciences, Wuhan, 430030, China.
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Stone ML, Lee HH, Levine EM. Agarose hydrogel-mediated electroporation method for retinal tissue cultured at the air-liquid interface. iScience 2024; 27:111299. [PMID: 39628577 PMCID: PMC11612790 DOI: 10.1016/j.isci.2024.111299] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2024] [Revised: 05/29/2024] [Accepted: 10/29/2024] [Indexed: 12/06/2024] Open
Abstract
It is advantageous to culture the ex vivo retina and other tissues at the air-liquid interface to allow for more efficient gas exchange. However, gene delivery to these cultures can be challenging. Electroporation is a fast and robust method of gene delivery, but typically requires submergence in liquid buffer for electrical current flow. We have developed a submergence-free electroporation technique that incorporates an agarose hydrogel disk between the positive electrode and retina. Inner retinal neurons and Müller glia are transfected with increased propensity toward Müller glia transfection after extended time in culture. We also observed an increase in BrdU incorporation in Müller glia following electrical stimulation, and variation in detection of transfected cells from expression vectors with different promoters. This method advances our ability to use ex vivo retinal tissue for genetic studies and should be adaptable for other tissues cultured at an air-liquid interface.
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Affiliation(s)
- Megan L. Stone
- Department of Cell and Developmental Biology, Vanderbilt University, Nashville TN 37232, USA
| | - Hannah H. Lee
- Department of Ophthalmology and Visual Sciences, Vanderbilt University Medical Center, Nashville TN 37232, USA
| | - Edward M. Levine
- Department of Cell and Developmental Biology, Vanderbilt University, Nashville TN 37232, USA
- Department of Ophthalmology and Visual Sciences, Vanderbilt University Medical Center, Nashville TN 37232, USA
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Yin Z, Kang J, Xu H, Huo S, Xu H. Recent progress of principal techniques used in the study of Müller glia reprogramming in mice. CELL REGENERATION (LONDON, ENGLAND) 2024; 13:30. [PMID: 39663301 PMCID: PMC11635068 DOI: 10.1186/s13619-024-00211-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/24/2024] [Revised: 11/21/2024] [Accepted: 11/26/2024] [Indexed: 12/13/2024]
Abstract
In zebrafish, Müller glia (MG) cells retain the ability to proliferate and de-differentiate into retinal progenitor-like cells, subsequently differentiating into retinal neurons that can replace those damaged or lost due to retinal injury. In contrast, the reprogramming potential of MG in mammals has been lost, with these cells typically responding to retinal damage through gliosis. Considerable efforts have been dedicated to achieving the reprogramming of MG cells in mammals. Notably, significant advancements have been achieved in reprogramming MG cells in mice employing various methodologies. At the same time, some inevitable challenges have hindered identifying accurate MG cell reprogramming rather than the illusion, let alone improving the reprogramming efficiency and maturity of daughter cells. Recently, several strategies, including lineage tracking, multi-omics techniques, and functional analysis, have been developed to investigate the MG reprogramming process in mice. This review summarizes both the advantages and limitations of these novel strategies for analyzing MG reprogramming in mice, offering insights into enhancing the reliability and efficiency of MG reprogramming.
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Affiliation(s)
- Zhiyuan Yin
- Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Southwest Eye Hospital, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, P.R. China
| | - Jiahui Kang
- Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Southwest Eye Hospital, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, P.R. China
| | - Haoan Xu
- School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China
| | - Shujia Huo
- Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Southwest Eye Hospital, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, P.R. China.
| | - Haiwei Xu
- Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Southwest Eye Hospital, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, P.R. China.
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Shang, F, Schallek J. Characterization of the Retinal Circulation of the Mouse. Invest Ophthalmol Vis Sci 2024; 65:3. [PMID: 39620830 PMCID: PMC11613998 DOI: 10.1167/iovs.65.14.3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2024] [Accepted: 09/30/2024] [Indexed: 12/06/2024] Open
Abstract
Purpose Mice are highly used in retinal research because, like humans, mice have vascularized retinas and choroidal circulation. Although the retinal circulation has been well-characterized in development, its stability during adulthood is less understood. To examine this network, we quantified several key metrics of the trilaminar vasculature. Methods We used mice (n = 15) with transgenic fluorescent NG2-DsRed (JX: #00824), a vascular-associated label in the retina. One eye per mouse was imaged using confocal microscopy (Nikon A1 Ti2 Eclipse) and traced with ImageJ SNT tools. Using an adaptive optics scanning light ophthalmoscope, additional mice (n = 3) were imaged at single-cell resolution within the living eye to measure the same vasculature. Results Across mice, we found a stable retinal circulation that formed and maintained a trilaminar stratification throughout early adulthood at all eccentricities. Bridging these layers, microvessels had five distinct anatomical branching patterns. The superficial, intermediate, and deep plexuses increased in density with depth: 16.14 ± 3.61 mm/mm2, 22.14 ± 6.86 mm/mm2, and 31.01 ± 6.24 mm/mm2, respectively. This patterning was not impacted by eccentricity or age (13-61 weeks). Similar metrics were achieved using adaptive optics scanning light ophthalmoscope in vivo with the same analysis pipeline. Conclusions The mouse retinal vasculature was stable up to 50 weeks of age, providing a robust and extensive baseline dataset with which models of retinal vascular and neural disease may be compared. Vessels connecting the laminae were more complex than previously reported and represented a uniquely vulnerable population due to their relatively low density.
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Affiliation(s)
- Fei Shang,
- Department of Neuroscience, University of Rochester, Rochester, NY, United States
- Center for Visual Science, University of Rochester, Rochester, NY, United States
| | - Jesse Schallek
- Department of Neuroscience, University of Rochester, Rochester, NY, United States
- Center for Visual Science, University of Rochester, Rochester, NY, United States
- Flaum Eye Institute, University of Rochester, Rochester, NY, United States
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Kothurkar AA, Patient GS, Noel NCL, Krzywańska AM, Carr BJ, Chu CJ, MacDonald RB. 'Iterative Bleaching Extends Multiplexity' facilitates simultaneous identification of all major retinal cell types. J Cell Sci 2024; 137:jcs263407. [PMID: 39540305 PMCID: PMC11827602 DOI: 10.1242/jcs.263407] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2024] [Accepted: 11/03/2024] [Indexed: 11/16/2024] Open
Abstract
To understand the multicellular composition of tissues, and how it is altered during development, ageing and/or disease, we must visualise the complete cellular landscape. Currently, this is hindered by our limited ability to combine multiple cellular markers. To overcome this, we adapted a highly multiplexed immunofluorescence (IF) technique called 'Iterative Bleaching Extends Multiplexity' (IBEX) to the zebrafish retina. We optimised fluorescent antibody micro-conjugation to perform sequential rounds of labelling on a single tissue to simultaneously visualise all major retinal cell types with 11 cell-specific antibodies. We further adapted IBEX to be compatible with fluorescent transgenic reporter lines, in situ hybridisation chain reaction (HCR), and whole-mount immunofluorescence (WMIF). We applied IBEX at multiple stages to study the spatial and temporal relationships between glia and neurons during retinal development. Finally, we demonstrate the utility of IBEX across species by testing it on the turquoise killifish (Nothobranchius furzeri) and African clawed frog (Xenopus laevis) to glean large amounts of information from precious tissues. These techniques will revolutionise our ability to visualise multiple cell types in any organism where antibodies are readily available.
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Affiliation(s)
| | - Gregory S. Patient
- Institute of Ophthalmology, University College London, London EC1V 9EL, UK
| | - Nicole C. L. Noel
- Institute of Ophthalmology, University College London, London EC1V 9EL, UK
| | | | - Brittany J. Carr
- Department of Ophthalmology & Visual Sciences, University of Alberta, Edmonton, AB T5H 3V9, Canada
| | - Colin J. Chu
- Institute of Ophthalmology, University College London, London EC1V 9EL, UK
| | - Ryan B. MacDonald
- Institute of Ophthalmology, University College London, London EC1V 9EL, UK
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11
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Bills JD, Seifert AW, Morris AC. Retinal neuroanatomy of two emerging model organisms, the spiny mouse (Acomys dimidiatus) and the Mongolian gerbil (Meriones unguiculatus). Exp Eye Res 2024; 247:110055. [PMID: 39159803 DOI: 10.1016/j.exer.2024.110055] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2024] [Revised: 08/12/2024] [Accepted: 08/16/2024] [Indexed: 08/21/2024]
Abstract
Current research using animal models to investigate retinal cell biology and model retinal degenerative diseases largely utilize small mammals that are nocturnal and lack the ability to restore lost vision. In contrast, the Mongolian gerbil (Meriones) is a diurnal rodent with good photopic vision, and the spiny mouse (Acomys) is a small desert-dwelling rodent with remarkable regenerative capabilities. The goal of this study was to identify antibodies that detect retinal cell classes in Meriones and Acomys, and to describe the retinal anatomy of these two species in comparison to outbred laboratory mice (Mus musculus). Immunohistochemistry was performed on retinal sections with antibodies for various retinal cell types. Sections were imaged by light, fluorescence, and confocal microscopy. Cell density, morphology, and placement were compared between species qualitatively and quantitatively. Our analyses revealed a classic assembly of retinal cells in Meriones and Acomys, with a few deviations compared to Mus. Meriones displayed the highest density of cones and Acomys the lowest. A higher density of bipolar cell bodies in the proximal portion of the inner nuclear layer was observed in both Acomys and Meriones compared to Mus, and both species exhibited an increase in amacrine cell density compared to Mus. Our results provide a foundation for future research into the visual system adaptations of these interesting species.
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Affiliation(s)
- Jessica D Bills
- Department of Biology, University of Kentucky, Lexington, KY 40506-0225, USA
| | - Ashley W Seifert
- Department of Biology, University of Kentucky, Lexington, KY 40506-0225, USA
| | - Ann C Morris
- Department of Biology, University of Kentucky, Lexington, KY 40506-0225, USA.
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Beachum AN, Salazar G, Nachbar A, Krause K, Klose H, Meyer K, Maserejian A, Ross G, Boyd H, Weigel T, Ambaye L, Miller H, Coutinho-Budd J. Glia multitask to compensate for neighboring glial cell dysfunction. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.09.06.611719. [PMID: 39314422 PMCID: PMC11418964 DOI: 10.1101/2024.09.06.611719] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 09/25/2024]
Abstract
As glia mature, they undergo glial tiling to abut one another without invading each other's boundaries. Upon the loss of the secreted neurotrophin Spätzle3 (Spz3), Drosophila cortex glia transform morphologically and lose their intricate interactions with neurons and surrounding glial subtypes. Here, we reveal that all neighboring glial cell types (astrocytes, ensheathing glia, and subperineurial glia) react by extending processes into the previous cortex glial territory to compensate for lost cortex glial function and reduce the buildup of neuronal debris. However, the loss of Spz3 alone is not sufficient for glia to cross their natural borders, as blocking CNS growth via nutrient-restriction blocks the aberrant infiltration induced by the loss of Spz3. Surprisingly, even when these neighboring glia divert their cellular resources beyond their typical borders to take on new compensatory roles, they are able to multitask to continue to preserve their own normal functions to maintain CNS homeostasis.
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Affiliation(s)
- Allison N. Beachum
- Department of Neuroscience, University of Virginia, Charlottesville, VA 22903
| | - Gabriela Salazar
- Department of Neuroscience, University of Virginia, Charlottesville, VA 22903
| | - Amelia Nachbar
- Department of Neuroscience, University of Virginia, Charlottesville, VA 22903
| | - Kevin Krause
- Department of Neuroscience, University of Virginia, Charlottesville, VA 22903
| | - Hannah Klose
- Department of Neuroscience, University of Virginia, Charlottesville, VA 22903
| | - Kate Meyer
- Department of Neuroscience, University of Virginia, Charlottesville, VA 22903
| | | | - Grace Ross
- Department of Biology, University of Vermont, Burlington, VT 05405
| | - Hannah Boyd
- Department of Biology, University of Vermont, Burlington, VT 05405
| | - Thaddeus Weigel
- Department of Neuroscience, University of Virginia, Charlottesville, VA 22903
| | - Lydia Ambaye
- Department of Biology, University of Vermont, Burlington, VT 05405
| | - Hayes Miller
- Department of Neuroscience, University of Virginia, Charlottesville, VA 22903
| | - Jaeda Coutinho-Budd
- Department of Neuroscience, University of Virginia, Charlottesville, VA 22903
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13
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Ruffini A, Dvoriashyna M, Govetto A, Romano MR, Repetto R. A Mathematical Model of Interstitial Fluid Flow and Retinal Tissue Deformation in Macular Edema. Invest Ophthalmol Vis Sci 2024; 65:19. [PMID: 39254963 PMCID: PMC11401122 DOI: 10.1167/iovs.65.11.19] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/11/2024] Open
Abstract
Purpose This study aims to develop a mathematical model to elucidate fluid circulation in the retina, focusing on the movement of interstitial fluid (comprising water and albumin) to understand the mechanisms underlying exudative macular edema (EME). Methods The model integrates physiological factors such as retinal pigment epithelium (RPE) pumping, osmotic pressure gradients, and tissue deformation. It accounts for spatial variability in hydraulic conductivity (HC) across the retina and incorporates the structural role of Müller cells (MCs) in maintaining retinal stability. Results The model predicts that tissue deformation is maximal at the center of the fovea despite fluid exudation from blood capillaries occurring elsewhere, aligning with clinical observations. Additionally, the model suggests that spatial variability in HC across the thickness of the retina plays a protective role against fluid accumulation in the fovea. Conclusions Despite inherent simplifications and uncertainties in parameter values, this study represents a step toward understanding the pathophysiology of EME. The findings provide insights into the mechanisms underlying fluid dynamics in the retina and fluid accumulation in the foveal region, showing that the specific conformation of Müller cells is likely to play a key role.
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Affiliation(s)
- Alessia Ruffini
- Department of Civil, Chemical and Environmental Engineering, University of Genoa, Genoa, Italy
| | - Mariia Dvoriashyna
- School of Mathematics, University of Edinburgh, Edinburgh, United Kingdom
| | - Andrea Govetto
- Department of Biomedical Sciences, Humanitas University, Milan, Italy
| | - Mario R Romano
- Department of Biomedical Sciences, Humanitas University, Milan, Italy
| | - Rodolfo Repetto
- Department of Civil, Chemical and Environmental Engineering, University of Genoa, Genoa, Italy
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14
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Kozlowski C, Hadyniak SE, Kay JN. Retinal neurons establish mosaic patterning by excluding homotypic somata from their dendritic territories. Cell Rep 2024; 43:114615. [PMID: 39133615 PMCID: PMC11440617 DOI: 10.1016/j.celrep.2024.114615] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2023] [Revised: 06/01/2024] [Accepted: 07/24/2024] [Indexed: 08/21/2024] Open
Abstract
In vertebrate retina, individual neurons of the same type are distributed regularly across the tissue in a pattern known as a mosaic. Establishment of mosaics during development requires cell-cell repulsion among homotypic neurons, but the mechanisms underlying this repulsion remain unknown. Here, we show that two mouse retinal cell types, OFF and ON starburst amacrine cells, establish mosaic spacing by using their dendritic arbors to repel neighboring homotypic somata. Using transgenic tools and single-cell labeling, we identify a developmental period when starburst somata are contacted by neighboring starburst dendrites; these serve to exclude somata from settling within the neighbor's dendritic territory. Dendrite-soma exclusion is mediated by MEGF10, a cell-surface molecule required for starburst mosaic patterning. Our results implicate dendrite-soma exclusion as a key mechanism underlying starburst mosaic spacing and raise the possibility that this could be a general mechanism for mosaic patterning across many cell types and species.
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Affiliation(s)
- Christopher Kozlowski
- Departments of Neurobiology, Ophthalmology, and Cell Biology, Duke University School of Medicine, Durham, NC 27710, USA
| | - Sarah E Hadyniak
- Departments of Neurobiology, Ophthalmology, and Cell Biology, Duke University School of Medicine, Durham, NC 27710, USA
| | - Jeremy N Kay
- Departments of Neurobiology, Ophthalmology, and Cell Biology, Duke University School of Medicine, Durham, NC 27710, USA.
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15
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Holden JM, Wareham LK, Calkins DJ. Morphological and electrophysiological characterization of a novel displaced astrocyte in the mouse retina. Glia 2024; 72:1356-1370. [PMID: 38591270 PMCID: PMC11081821 DOI: 10.1002/glia.24536] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2023] [Revised: 03/08/2024] [Accepted: 03/30/2024] [Indexed: 04/10/2024]
Abstract
Astrocytes throughout the central nervous system are heterogeneous in both structure and function. This diversity leads to tissue-specific specialization where morphology is adapted to the surrounding neuronal circuitry, as seen in Bergman glia of the cerebellum and Müller glia of the retina. Because morphology can be a differentiating factor for cellular classification, we recently developed a mouse where glial-fibrillary acidic protein (GFAP)-expressing cells stochastically label for full membranous morphology. Here we utilize this tool to investigate whether morphological and electrophysiological features separate types of mouse retinal astrocytes. In this work, we report on a novel glial population found in the inner plexiform layer and ganglion cell layer which expresses the canonical astrocyte markers GFAP, S100β, connexin-43, Sox2 and Sox9. Apart from their retinal layer localization, these cells are unique in their radial distribution. They are notably absent from the mid-retina but are heavily concentrated near the optic nerve head, and to a lesser degree the peripheral retina. Additionally, their morphology is distinct from both nerve fiber layer astrocytes and Müller glia, appearing more similar to amacrine cells. Despite this structural similarity, these cells lack protein expression of common neuronal markers. Additionally, they do not exhibit action potentials, but rather resemble astrocytes and Müller glia in their small amplitude, graded depolarization to both light onset and offset. Their structure, protein expression, physiology, and intercellular connections suggest that these cells are astrocytic, displaced from their counterparts in the nerve fiber layer. As such, we refer to these cells as displaced retinal astrocytes.
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Affiliation(s)
- Joseph Matthew Holden
- Department of Ophthalmology and Visual Sciences, Vanderbilt University Medical Center, Nashville, TN 37212
- Vanderbilt Neuroscience Graduate Program, Vanderbilt University, Nashville, TN 37212
| | - Lauren Katie Wareham
- Department of Ophthalmology and Visual Sciences, Vanderbilt University Medical Center, Nashville, TN 37212
| | - David John Calkins
- Department of Ophthalmology and Visual Sciences, Vanderbilt University Medical Center, Nashville, TN 37212
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16
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Stone ML, Lee HH, Levine E. Agarose disk electroporation method for ex vivo retinal tissue cultured at the air-liquid interface reveals electrical stimulus-induced cell cycle reentry in retinal cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.12.21.572865. [PMID: 38187784 PMCID: PMC10769434 DOI: 10.1101/2023.12.21.572865] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/09/2024]
Abstract
It is advantageous to culture the ex vivo murine retina along with many other tissue types at the air-liquid interface. However, gene delivery to these cultures can be challenging. Electroporation is a fast and robust method of gene delivery, but typically requires submergence in a liquid buffer to allow electric current flow. We have developed a submergence-free electroporation technique using an agarose disk that allows for efficient gene delivery to the ex vivo murine retina. This method advances our ability to use ex vivo retinal tissue for genetic studies and can easily be adapted for any tissue cultured at an air-liquid interface. We found an increased ability to transfected Muller glia at 14 days ex vivo and an increase in BrdU incorporation in Muller glia following electrical stimulation. Use of this method has revealed valuable insights on the state of ex vivo retinal tissues and the effects of electrical stimulation on retinal cells.
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17
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Shinozaki Y, Namekata K, Guo X, Harada T. Glial cells as a promising therapeutic target of glaucoma: beyond the IOP. FRONTIERS IN OPHTHALMOLOGY 2024; 3:1310226. [PMID: 38983026 PMCID: PMC11182302 DOI: 10.3389/fopht.2023.1310226] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/09/2023] [Accepted: 12/18/2023] [Indexed: 07/11/2024]
Abstract
Glial cells, a type of non-neuronal cell found in the central nervous system (CNS), play a critical role in maintaining homeostasis and regulating CNS functions. Recent advancements in technology have paved the way for new therapeutic strategies in the fight against glaucoma. While intraocular pressure (IOP) is the most well-known modifiable risk factor, a significant number of glaucoma patients have normal IOP levels. Because glaucoma is a complex, multifactorial disease influenced by various factors that contribute to its onset and progression, it is imperative that we consider factors beyond IOP to effectively prevent or slow down the disease's advancement. In the realm of CNS neurodegenerative diseases, glial cells have emerged as key players due to their pivotal roles in initiating and hastening disease progression. The inhibition of dysregulated glial function holds the potential to protect neurons and restore brain function. Consequently, glial cells represent an enticing therapeutic candidate for glaucoma, even though the majority of glaucoma research has historically concentrated solely on retinal ganglion cells (RGCs). In addition to the neuroprotection of RGCs, the proper regulation of glial cell function can also facilitate structural and functional recovery in the retina. In this review, we offer an overview of recent advancements in understanding the non-cell-autonomous mechanisms underlying the pathogenesis of glaucoma. Furthermore, state-of-the-art technologies have opened up possibilities for regenerating the optic nerve, which was previously believed to be incapable of regeneration. We will also delve into the potential roles of glial cells in the regeneration of the optic nerve and the restoration of visual function.
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Affiliation(s)
- Youichi Shinozaki
- Visual Research Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Kazuhiko Namekata
- Visual Research Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Xiaoli Guo
- Visual Research Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Takayuki Harada
- Visual Research Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
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18
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Kozlowski C, Hadyniak SE, Kay JN. Retinal neurons establish mosaic patterning by excluding homotypic somata from their dendritic territory. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.11.17.567616. [PMID: 38014021 PMCID: PMC10680827 DOI: 10.1101/2023.11.17.567616] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/29/2023]
Abstract
In vertebrate retina, individual neurons of the same type are distributed regularly across the tissue in a pattern known as a mosaic. Establishment of mosaics during development requires cell-cell repulsion among homotypic neurons, but the mechanisms underlying this repulsion remain unknown. Here we show that two mouse retinal cell types, OFF and ON starburst amacrine cells, establish mosaic spacing by using their dendritic arbors to repel neighboring homotypic somata. Using newly-generated transgenic tools and single cell labeling, we identify a transient developmental period when starburst somata receive extensive contacts from neighboring starburst dendrites; these serve to exclude somata from settling within the neighbor's dendritic territory. Dendrite-soma exclusion is mediated by MEGF10, a cell-surface molecule required for starburst mosaic patterning. Our results implicate dendrite-soma exclusion as a key mechanism underlying starburst mosaic spacing, and suggest that this could be a general mechanism for mosaic patterning across many cell types and species.
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Affiliation(s)
- Christopher Kozlowski
- Departments of Neurobiology, Ophthalmology, and Cell Biology, Duke University School of Medicine, Durham, NC 27710 USA
| | - Sarah E Hadyniak
- Departments of Neurobiology, Ophthalmology, and Cell Biology, Duke University School of Medicine, Durham, NC 27710 USA
| | - Jeremy N Kay
- Departments of Neurobiology, Ophthalmology, and Cell Biology, Duke University School of Medicine, Durham, NC 27710 USA
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19
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Rupareliya VP, Singh AA, Butt AM, A H, Kumar H. The "molecular soldiers" of the CNS: Astrocytes, a comprehensive review on their roles and molecular signatures. Eur J Pharmacol 2023; 959:176048. [PMID: 37758010 DOI: 10.1016/j.ejphar.2023.176048] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2023] [Revised: 08/24/2023] [Accepted: 09/13/2023] [Indexed: 09/29/2023]
Abstract
For a long time, neurons held the position of central players in the nervous system. Since there are far more astrocytes than neurons in the brain, it makes us wonder if these cells just take up space and support the neurons or if they are actively participating in central nervous system (CNS) homeostasis. Now, astrocytes' contribution to CNS physiology is appreciated as they are known to regulate ion and neurotransmitter levels, synapse formation and elimination, blood-brain barrier integrity, immune function, cerebral blood flow, and many more. In many neurological and psychiatric disorders, astrocyte functions are altered. Advancements in microscopic and transcriptomic tools revealed populations of astrocytes with varied morphology, electrophysiological properties, and transcriptomic profiles. Neuron-circuit-specific functions and neuron-specific interactions of astroglial subpopulations are found, which suggests that diversity is essential in carrying out diverse region-specific CNS functions. Investigations on heterogeneous astrocyte populations are revealing new astrocyte functions and their role in pathological conditions, opening a new therapeutic avenue for targeting neurological conditions. The true extent of astrocytic heterogeneity and its functional implications are yet to be fully explored. This review summarizes essential astrocytic functions and their relevance in pathological conditions and discusses astrocytic diversity in relation to morphology, function, and gene expression throughout the CNS.
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Affiliation(s)
- Vimal P Rupareliya
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER)-Ahmedabad, Gandhinagar, Gujarat 382355, India
| | - Aditya A Singh
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER)-Ahmedabad, Gandhinagar, Gujarat 382355, India
| | - Ayub Mohammed Butt
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER)-Ahmedabad, Gandhinagar, Gujarat 382355, India
| | - Hariharan A
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER)-Ahmedabad, Gandhinagar, Gujarat 382355, India
| | - Hemant Kumar
- Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER)-Ahmedabad, Gandhinagar, Gujarat 382355, India.
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20
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Mani A, Yang X, Zhao TA, Leyrer ML, Schreck D, Berson DM. A circuit suppressing retinal drive to the optokinetic system during fast image motion. Nat Commun 2023; 14:5142. [PMID: 37612305 PMCID: PMC10447436 DOI: 10.1038/s41467-023-40527-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2022] [Accepted: 07/26/2023] [Indexed: 08/25/2023] Open
Abstract
Optokinetic nystagmus (OKN) assists stabilization of the retinal image during head rotation. OKN is driven by ON direction selective retinal ganglion cells (ON DSGCs), which encode both the direction and speed of global retinal slip. The synaptic circuits responsible for the direction selectivity of ON DSGCs are well understood, but those sculpting their slow-speed preference remain enigmatic. Here, we probe this mechanism in mouse retina through patch clamp recordings, functional imaging, genetic manipulation, and electron microscopic reconstructions. We confirm earlier evidence that feedforward glycinergic inhibition is the main suppressor of ON DSGC responses to fast motion, and reveal the source for this inhibition-the VGluT3 amacrine cell, a dual neurotransmitter, excitatory/inhibitory interneuron. Together, our results identify a role for VGluT3 cells in limiting the speed range of OKN. More broadly, they suggest VGluT3 cells shape the response of many retinal cell types to fast motion, suppressing it in some while enhancing it in others.
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Affiliation(s)
- Adam Mani
- Department of Neuroscience, Brown University, Providence, RI, USA
| | - Xinzhu Yang
- Department of Neuroscience, Brown University, Providence, RI, USA
| | - Tiffany A Zhao
- Department of Neuroscience, Brown University, Providence, RI, USA
| | - Megan L Leyrer
- Department of Neuroscience, Brown University, Providence, RI, USA
| | - Daniel Schreck
- Department of Neuroscience, Brown University, Providence, RI, USA
| | - David M Berson
- Department of Neuroscience, Brown University, Providence, RI, USA.
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21
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Cullen PF, Sun D. Astrocytes of the eye and optic nerve: heterogeneous populations with unique functions mediate axonal resilience and vulnerability to glaucoma. FRONTIERS IN OPHTHALMOLOGY 2023; 3:1217137. [PMID: 37829657 PMCID: PMC10569075 DOI: 10.3389/fopht.2023.1217137] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/14/2023]
Abstract
The role of glia, particularly astrocytes, in mediating the central nervous system's response to injury and neurodegenerative disease is an increasingly well studied topic. These cells perform myriad support functions under physiological conditions but undergo behavioral changes - collectively referred to as 'reactivity' - in response to the disruption of neuronal homeostasis from insults, including glaucoma. However, much remains unknown about how reactivity alters disease progression - both beneficially and detrimentally - and whether these changes can be therapeutically modulated to improve outcomes. Historically, the heterogeneity of astrocyte behavior has been insufficiently addressed under both physiological and pathological conditions, resulting in a fragmented and often contradictory understanding of their contributions to health and disease. Thanks to increased focus in recent years, we now know this heterogeneity encompasses both intrinsic variation in physiological function and insult-specific changes that vary between pathologies. Although previous studies demonstrate astrocytic alterations in glaucoma, both in human disease and animal models, generally these findings do not conclusively link astrocytes to causative roles in neuroprotection or degeneration, rather than a subsequent response. Efforts to bolster our understanding by drawing on knowledge of brain astrocytes has been constrained by the primacy in the literature of findings from peri-synaptic 'gray matter' astrocytes, whereas much early degeneration in glaucoma occurs in axonal regions populated by fibrous 'white matter' astrocytes. However, by focusing on findings from astrocytes of the anterior visual pathway - those of the retina, unmyelinated optic nerve head, and myelinated optic nerve regions - we aim to highlight aspects of their behavior that may contribute to axonal vulnerability and glaucoma progression, including roles in mitochondrial turnover and energy provisioning. Furthermore, we posit that astrocytes of the retina, optic nerve head and myelinated optic nerve, although sharing developmental origins and linked by a network of gap junctions, may be best understood as distinct populations residing in markedly different niches with accompanying functional specializations. A closer investigation of their behavioral repertoires may elucidate not only their role in glaucoma, but also mechanisms to induce protective behaviors that can impede the progressive axonal damage and retinal ganglion cell death that drive vision loss in this devastating condition.
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Affiliation(s)
- Paul F. Cullen
- Department of Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical School, Boston, MA, United States
| | - Daniel Sun
- Department of Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical School, Boston, MA, United States
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22
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Kugler E, Bravo I, Durmishi X, Marcotti S, Beqiri S, Carrington A, Stramer B, Mattar P, MacDonald RB. GliaMorph: a modular image analysis toolkit to quantify Müller glial cell morphology. Development 2023; 150:dev201008. [PMID: 36625162 PMCID: PMC10110500 DOI: 10.1242/dev.201008] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2022] [Accepted: 01/03/2023] [Indexed: 01/11/2023]
Abstract
Cell morphology is crucial for all cell functions. This is particularly true for glial cells as they rely on complex shape to contact and support neurons. However, methods to quantify complex glial cell shape accurately and reproducibly are lacking. To address this, we developed the image analysis pipeline 'GliaMorph'. GliaMorph is a modular analysis toolkit developed to perform (1) image pre-processing, (2) semi-automatic region-of-interest selection, (3) apicobasal texture analysis, (4) glia segmentation, and (5) cell feature quantification. Müller glia (MG) have a stereotypic shape linked to their maturation and physiological status. Here, we characterized MG on three levels: (1) global image-level, (2) apicobasal texture, and (3) regional apicobasal vertical-to-horizontal alignment. Using GliaMorph, we quantified MG development on a global and single-cell level, showing increased feature elaboration and subcellular morphological rearrangement in the zebrafish retina. As proof of principle, we analysed expression changes in a mouse glaucoma model, identifying subcellular protein localization changes in MG. Together, these data demonstrate that GliaMorph enables an in-depth understanding of MG morphology in the developing and diseased retina.
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Affiliation(s)
- Elisabeth Kugler
- Institute of Ophthalmology, University College London, 11-43 Bath St, Greater London EC1V 9EL, UK
| | - Isabel Bravo
- Institute of Ophthalmology, University College London, 11-43 Bath St, Greater London EC1V 9EL, UK
| | - Xhuljana Durmishi
- Institute of Ophthalmology, University College London, 11-43 Bath St, Greater London EC1V 9EL, UK
| | - Stefania Marcotti
- Randall Centre for Cell & Molecular Biophysics, King's College London, New Hunt's House, London SE1 1UL, UK
| | - Sara Beqiri
- Institute of Ophthalmology, University College London, 11-43 Bath St, Greater London EC1V 9EL, UK
| | - Alicia Carrington
- Institute of Ophthalmology, University College London, 11-43 Bath St, Greater London EC1V 9EL, UK
| | - Brian Stramer
- Randall Centre for Cell & Molecular Biophysics, King's College London, New Hunt's House, London SE1 1UL, UK
| | - Pierre Mattar
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, K1H 8M5, Canada
- Ottawa Hospital Research Institute (OHRI), Ottawa, ON, K1H 8L6, Canada
| | - Ryan B. MacDonald
- Institute of Ophthalmology, University College London, 11-43 Bath St, Greater London EC1V 9EL, UK
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23
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Marchese NA, Ríos MN, Guido ME. Müller glial cell photosensitivity: a novel function bringing higher complexity to vertebrate retinal physiology. JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY 2023. [DOI: 10.1016/j.jpap.2023.100162] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
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24
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Kugler E, Breitenbach EM, MacDonald R. Glia Cell Morphology Analysis Using the Fiji GliaMorph Toolkit. Curr Protoc 2023; 3:e654. [PMID: 36688682 PMCID: PMC10108223 DOI: 10.1002/cpz1.654] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Glial cells are the support cells of the nervous system. Glial cells typically have elaborate morphologies that facilitate close contacts with neighboring neurons, synapses, and the vasculature. In the retina, Müller glia (MG) are the principal glial cell type that supports neuronal function by providing a myriad of supportive functions via intricate cell morphologies and precise contacts. Thus, complex glial morphology is critical for glial function, but remains challenging to resolve at a sub-cellular level or reproducibly quantify in complex tissues. To address this issue, we developed GliaMorph as a Fiji-based macro toolkit that allows 3D glial cell morphology analysis in the developing and mature retina. As GliaMorph is implemented in a modular fashion, here we present guides to (a) setup of GliaMorph, (b) data understanding in 3D, including z-axis intensity decay and signal-to-noise ratio, (c) pre-processing data to enhance image quality, (d) performing and examining image segmentation, and (e) 3D quantification of MG features, including apicobasal texture analysis. To allow easier application, GliaMorph tools are supported with graphical user interfaces where appropriate, and example data are publicly available to facilitate adoption. Further, GliaMorph can be modified to meet users' morphological analysis needs for other glial or neuronal shapes. Finally, this article provides users with an in-depth understanding of data requirements and the workflow of GliaMorph. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Download and installation of GliaMorph components including example data Basic Protocol 2: Understanding data properties and quality 3D-essential for subsequent analysis and capturing data property issues early Basic Protocol 3: Pre-processing AiryScan microscopy data for analysis Alternate Protocol: Pre-processing confocal microscopy data for analysis Basic Protocol 4: Segmentation of glial cells Basic Protocol 5: 3D quantification of glial cell morphology.
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Affiliation(s)
- Elisabeth Kugler
- Institute of Ophthalmology, University College London, Greater London, UK
| | | | - Ryan MacDonald
- Institute of Ophthalmology, University College London, Greater London, UK
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25
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Križaj D, Cordeiro S, Strauß O. Retinal TRP channels: Cell-type-specific regulators of retinal homeostasis and multimodal integration. Prog Retin Eye Res 2023; 92:101114. [PMID: 36163161 PMCID: PMC9897210 DOI: 10.1016/j.preteyeres.2022.101114] [Citation(s) in RCA: 17] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2022] [Revised: 08/03/2022] [Accepted: 08/08/2022] [Indexed: 02/05/2023]
Abstract
Transient receptor potential (TRP) channels are a widely expressed family of 28 evolutionarily conserved cationic ion channels that operate as primary detectors of chemical and physical stimuli and secondary effectors of metabotropic and ionotropic receptors. In vertebrates, the channels are grouped into six related families: TRPC, TRPV, TRPM, TRPA, TRPML, and TRPP. As sensory transducers, TRP channels are ubiquitously expressed across the body and the CNS, mediating critical functions in mechanosensation, nociception, chemosensing, thermosensing, and phototransduction. This article surveys current knowledge about the expression and function of the TRP family in vertebrate retinas, which, while dedicated to transduction and transmission of visual information, are highly susceptible to non-visual stimuli. Every retinal cell expresses multiple TRP subunits, with recent evidence establishing their critical roles in paradigmatic aspects of vertebrate vision that include TRPM1-dependent transduction of ON bipolar signaling, TRPC6/7-mediated ganglion cell phototransduction, TRP/TRPL phototransduction in Drosophila and TRPV4-dependent osmoregulation, mechanotransduction, and regulation of inner and outer blood-retina barriers. TRP channels tune light-dependent and independent functions of retinal circuits by modulating the intracellular concentration of the 2nd messenger calcium, with emerging evidence implicating specific subunits in the pathogenesis of debilitating diseases such as glaucoma, ocular trauma, diabetic retinopathy, and ischemia. Elucidation of TRP channel involvement in retinal biology will yield rewards in terms of fundamental understanding of vertebrate vision and therapeutic targeting to treat diseases caused by channel dysfunction or over-activation.
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Affiliation(s)
- David Križaj
- Departments of Ophthalmology, Neurobiology, and Bioengineering, University of Utah, Salt Lake City, USA
| | - Soenke Cordeiro
- Institute of Physiology, Faculty of Medicine, Christian-Albrechts-University Kiel, Germany
| | - Olaf Strauß
- Experimental Ophthalmology, Department of Ophthalmology, Charité - Universitätsmedizin Berlin, a Corporate Member of Freie Universität, Humboldt-University, The Berlin Institute of Health, Berlin, Germany.
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26
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Abstract
Visual information processing in the retina requires the rhythmic expression of clock genes. The intrinsic retinal circadian clock is independent of the master clock located in the hypothalamic suprachiasmatic nucleus and emerges from retinal cells, including glia. Less clear is how glial oscillators influence the daily regulation of visual information processing in the mouse retina. Here, we demonstrate that the adult conditional deletion of the gene Bmal1 in GLAST-positive glial cells alters retinal physiology. Specifically, such deletion was sufficient to lower the amplitude of the electroretinogram b-wave recorded under light-adapted conditions. Furthermore, recordings from > 20,000 retinal ganglion cells (RGCs), the retina output, showed a non-uniform effect on RGCs activity in response to light across different cell types and over a 24-h period. Overall, our results suggest a new role of a glial circadian gene in adjusting mammalian retinal output throughout the night-day cycle.
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27
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Yang S, Qi S, Wang C. The role of retinal Müller cells in diabetic retinopathy and related therapeutic advances. Front Cell Dev Biol 2022; 10:1047487. [PMID: 36531955 PMCID: PMC9757137 DOI: 10.3389/fcell.2022.1047487] [Citation(s) in RCA: 24] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2022] [Accepted: 11/24/2022] [Indexed: 11/19/2023] Open
Abstract
Diabetic retinopathy (DR) is a significant complication of diabetes. During the pathogenesis of retinal microangiopathy and neuronopathy, activated retinal Müller cells (RMCs) undergo morphological and structural changes such as increased expression of glial fibrillary acidic protein, disturbance of potassium and water transport regulation, and onset of production of a large number of inflammatory and vascular growth factors as well as chemokines. Evidently, activated RMCs are necessary for the pathogenesis of DR; therefore, exploring the role of RMCs in DR may provide a new target for the treatment thereof. This article reviews the mechanism of RMCs involvement in DR and the progress in related treatments.
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Affiliation(s)
| | - Shounan Qi
- Department of Ophthalmology, The Second Hospital of Jilin University, Changchun, China
| | - Chenguang Wang
- Department of Ophthalmology, The Second Hospital of Jilin University, Changchun, China
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28
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Procyk CA, Rodgers J, Zindy E, Lucas RJ, Milosavljevic N. Quantitative characterisation of ipRGCs in retinal degeneration using a computation platform for extracting and reconstructing single neurons in 3D from a multi-colour labeled population. Front Cell Neurosci 2022; 16:1009321. [PMID: 36385954 PMCID: PMC9664085 DOI: 10.3389/fncel.2022.1009321] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2022] [Accepted: 09/30/2022] [Indexed: 12/24/2022] Open
Abstract
Light has a profound impact on mammalian physiology and behavior. Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin, rendering them sensitive to light, and are involved in both image-forming vision and non-image forming responses to light such as circadian photo-entrainment and the pupillary light reflex. Following outer photoreceptor degeneration, the death of rod and cone photoreceptors results in global re-modeling of the remnant neural retina. Although ipRGCs can continue signaling light information to the brain even in advanced stages of degeneration, it is unknown if all six morphologically distinct subtypes survive, or how their dendritic architecture may be affected. To answer these questions, we generated a computational platform-BRIAN (Brainbow Analysis of individual Neurons) to analyze Brainbow labeled tissues by allowing objective identification of voxels clusters in Principal Component Space, and their subsequent extraction to produce 3D images of single neurons suitable for analysis with existing tracing technology. We show that BRIAN can efficiently recreate single neurons or individual axonal projections from densely labeled tissue with sufficient anatomical resolution for subtype quantitative classification. We apply this tool to generate quantitative morphological information about ipRGCs in the degenerate retina including soma size, dendritic field size, dendritic complexity, and stratification. Using this information, we were able to identify cells whose characteristics match those reported for all six defined subtypes of ipRGC in the wildtype mouse retina (M1-M6), including the rare and complex M3 and M6 subtypes. This indicates that ipRGCs survive outer retinal degeneration with broadly normal morphology. We additionally describe one cell in the degenerate retina which matches the description of the Gigantic M1 cell in Humans which has not been previously identified in rodent.
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Affiliation(s)
- Christopher A. Procyk
- Ocular Cell and Gene Therapy Group, Centre for Gene Therapy and Regenerative Medicine, King’s College London, Guy’s Hospital, London, United Kingdom
| | - Jessica Rodgers
- Faculty of Biology Medicine and Health, Centre for Biological Timing and Division of Neuroscience, University of Manchester, Manchester, United Kingdom
| | - Egor Zindy
- Centre for Microscopy and Molecular Imaging, Université Libre de Bruxelles, Brussels, Belgium
| | - Robert J. Lucas
- Faculty of Biology Medicine and Health, Centre for Biological Timing and Division of Neuroscience, University of Manchester, Manchester, United Kingdom
| | - Nina Milosavljevic
- Faculty of Biology Medicine and Health, Centre for Biological Timing and Division of Neuroscience, University of Manchester, Manchester, United Kingdom
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29
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Fitzpatrick MJ, Kerschensteiner D. Homeostatic plasticity in the retina. Prog Retin Eye Res 2022; 94:101131. [PMID: 36244950 DOI: 10.1016/j.preteyeres.2022.101131] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2022] [Revised: 09/25/2022] [Accepted: 09/28/2022] [Indexed: 02/07/2023]
Abstract
Vision begins in the retina, whose intricate neural circuits extract salient features of the environment from the light entering our eyes. Neurodegenerative diseases of the retina (e.g., inherited retinal degenerations, age-related macular degeneration, and glaucoma) impair vision and cause blindness in a growing number of people worldwide. Increasing evidence indicates that homeostatic plasticity (i.e., the drive of a neural system to stabilize its function) can, in principle, preserve retinal function in the face of major perturbations, including neurodegeneration. Here, we review the circumstances and events that trigger homeostatic plasticity in the retina during development, sensory experience, and disease. We discuss the diverse mechanisms that cooperate to compensate and the set points and outcomes that homeostatic retinal plasticity stabilizes. Finally, we summarize the opportunities and challenges for unlocking the therapeutic potential of homeostatic plasticity. Homeostatic plasticity is fundamental to understanding retinal development and function and could be an important tool in the fight to preserve and restore vision.
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30
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Demais V, Pohl A, Wunderlich KA, Pfaller AM, Kaplan L, Barthélémy A, Dittrich R, Puig B, Giebel B, Hauck SM, Pfrieger FW, Grosche A. Release of VAMP5-positive extracellular vesicles by retinal Müller glia in vivo. J Extracell Vesicles 2022; 11:e12254. [PMID: 36043482 PMCID: PMC9428896 DOI: 10.1002/jev2.12254] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2022] [Revised: 06/25/2022] [Accepted: 07/18/2022] [Indexed: 11/11/2022] Open
Abstract
Cell-cell interactions in the central nervous system are based on the release of molecules mediating signal exchange and providing structural and trophic support through vesicular exocytosis and the formation of extracellular vesicles. The specific mechanisms employed by each cell type in the brain are incompletely understood. Here, we explored the means of communication used by Müller cells, a type of radial glial cells in the retina, which forms part of the central nervous system. Using immunohistochemical, electron microscopic, and molecular analyses, we provide evidence for the release of distinct extracellular vesicles from endfeet and microvilli of retinal Müller cells in adult mice in vivo. We identify VAMP5 as a Müller cell-specific SNARE component that is part of extracellular vesicles and responsive to ischemia, and we reveal differences between the secretomes of immunoaffinity-purified Müller cells and neurons in vitro. Our findings suggest extracellular vesicle-based communication as an important mediator of cellular interactions in the retina.
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Affiliation(s)
- Valerie Demais
- Plateforme Imagerie In Vitro, CNRS UAR 3156, NeuropôleUniversity of StrasbourgStrasbourgFrance
| | - Anne Pohl
- Department of Physiological GenomicsBioMedical Center BMCLudwig‐Maximilian UniversityPlanegg‐MartinsriedGermany
- Institute of Human GeneticsUniversity of RegensburgRegensburgGermany
| | - Kirsten A. Wunderlich
- Department of Physiological GenomicsBioMedical Center BMCLudwig‐Maximilian UniversityPlanegg‐MartinsriedGermany
| | - Anna M. Pfaller
- Department of Physiological GenomicsBioMedical Center BMCLudwig‐Maximilian UniversityPlanegg‐MartinsriedGermany
| | - Lew Kaplan
- Department of Physiological GenomicsBioMedical Center BMCLudwig‐Maximilian UniversityPlanegg‐MartinsriedGermany
| | - Amelie Barthélémy
- Centre National de la Recherche ScientifiqueUniversité de StrasbourgInstitut des Neurosciences Cellulaires et IntégrativesStrasbourgFrance
| | - Robin Dittrich
- Institute for Transfusion MedicineUniversity Hospital EssenUniversity of Duisburg‐EssenEssenGermany
| | - Berta Puig
- Neurology DepartmentExperimental Research in Stroke and Inflammation (ERSI)University Medical Center Hamburg‐EppendorfHamburgGermany
| | - Bernd Giebel
- Institute for Transfusion MedicineUniversity Hospital EssenUniversity of Duisburg‐EssenEssenGermany
| | - Stefanie M. Hauck
- Metabolomics and Proteomics Core and Research Unit Protein ScienceHelmholtz‐Zentrum MünchenMünchenGermany
| | - Frank W. Pfrieger
- Plateforme Imagerie In Vitro, CNRS UAR 3156, NeuropôleUniversity of StrasbourgStrasbourgFrance
- Centre National de la Recherche ScientifiqueUniversité de StrasbourgInstitut des Neurosciences Cellulaires et IntégrativesStrasbourgFrance
| | - Antje Grosche
- Department of Physiological GenomicsBioMedical Center BMCLudwig‐Maximilian UniversityPlanegg‐MartinsriedGermany
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31
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Salazar G, Ross G, Maserejian AE, Coutinho-Budd J. Quantifying Glial-Glial Tiling Using Automated Image Analysis in Drosophila. Front Cell Neurosci 2022; 16:826483. [PMID: 35401121 PMCID: PMC8987577 DOI: 10.3389/fncel.2022.826483] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Accepted: 01/31/2022] [Indexed: 11/20/2022] Open
Abstract
Not only do glia form close associations with neurons throughout the central nervous system (CNS), but glial cells also interact closely with other glial cells. As these cells mature, they undergo a phenomenon known as glial tiling, where they grow to abut one another, often without invading each other’s boundaries. Glial tiling occurs throughout the animal kingdom, from fruit flies to humans; however, not much is known about the glial-glial interactions that lead to and maintain this tiling. Drosophila provide a strong model to investigate glial-glial tiling, where tiling occurs both among individual glial cells of the same subtype, as well as between those of different subtypes. Furthermore, the spatial segregation of the CNS allows for the unique ability to visualize and manipulate inter-subtype interactions. Previous work in Drosophila has suggested an interaction between cortex glia and astrocytes, where astrocytes cross the normal neuropil-cortex boundary in response to dysfunctional cortex glia. Here, we further explore this interaction by implementing an automated pipeline to more fully characterize this astrocyte-cortex glial relationship. By quantifying and correlating the extent of cortex glial dysfunction and aberrant astrocyte infiltration using automated analysis, we maximize the size of the quantified dataset to reveal subtle patterns in astrocyte-cortex glial interactions. We provide a guide for creating and validating a fully-automated image analysis pipeline for exploring these interactions, and implement this pipeline to describe a significant correlation between cortex glial dysfunction and aberrant astrocyte infiltration, as well as demonstrate variations in their relationship across different regions of the CNS.
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Affiliation(s)
- Gabriela Salazar
- Department of Biology, The University of Vermont, Burlington, VT, United States.,Vermont Complex Systems Center, The University of Vermont, Burlington, VT, United States
| | - Grace Ross
- Department of Biology, The University of Vermont, Burlington, VT, United States
| | - Ariana E Maserejian
- Department of Biology, The University of Vermont, Burlington, VT, United States
| | - Jaeda Coutinho-Budd
- Department of Biology, The University of Vermont, Burlington, VT, United States
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32
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Tworig JM, Feller MB. Müller Glia in Retinal Development: From Specification to Circuit Integration. Front Neural Circuits 2022; 15:815923. [PMID: 35185477 PMCID: PMC8856507 DOI: 10.3389/fncir.2021.815923] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Accepted: 12/23/2021] [Indexed: 01/21/2023] Open
Abstract
Müller glia of the retina share many features with astroglia located throughout the brain including maintenance of homeostasis, modulation of neurotransmitter spillover, and robust response to injury. Here we present the molecular factors and signaling events that govern Müller glial specification, patterning, and differentiation. Next, we discuss the various roles of Müller glia in retinal development, which include maintaining retinal organization and integrity as well as promoting neuronal survival, synaptogenesis, and phagocytosis of debris. Finally, we review the mechanisms by which Müller glia integrate into retinal circuits and actively participate in neuronal signaling during development.
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Affiliation(s)
- Joshua M. Tworig
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, United States
- *Correspondence: Joshua M. Tworig,
| | - Marla B. Feller
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, United States
- Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, United States
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33
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Benfey N, Foubert D, Ruthazer ES. Glia Regulate the Development, Function, and Plasticity of the Visual System From Retina to Cortex. Front Neural Circuits 2022; 16:826664. [PMID: 35177968 PMCID: PMC8843846 DOI: 10.3389/fncir.2022.826664] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Accepted: 01/07/2022] [Indexed: 11/13/2022] Open
Abstract
Visual experience is mediated through a relay of finely-tuned neural circuits extending from the retina, to retinorecipient nuclei in the midbrain and thalamus, to the cortex which work together to translate light information entering our eyes into a complex and dynamic spatio-temporal representation of the world. While the experience-dependent developmental refinement and mature function of neurons in each major stage of the vertebrate visual system have been extensively characterized, the contributions of the glial cells populating each region are comparatively understudied despite important findings demonstrating that they mediate crucial processes related to the development, function, and plasticity of the system. In this article we review the mechanisms for neuron-glia communication throughout the vertebrate visual system, as well as functional roles attributed to astrocytes and microglia in visual system development and processing. We will also discuss important aspects of glial function that remain unclear, integrating the knowns and unknowns about glia in the visual system to advance new hypotheses to guide future experimental work.
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Xin Y, He Q, Liang H, Zhang K, Guo J, Zhong Q, Chen D, Li J, Liu Y, Chen S. m 6A epitranscriptomic modification regulates neural progenitor-to-glial cell transition in the retina. eLife 2022; 11:79994. [PMID: 36459087 PMCID: PMC9718531 DOI: 10.7554/elife.79994] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2022] [Accepted: 11/13/2022] [Indexed: 12/03/2022] Open
Abstract
N 6-methyladenosine (m6A) is the most prevalent mRNA internal modification and has been shown to regulate the development, physiology, and pathology of various tissues. However, the functions of the m6A epitranscriptome in the visual system remain unclear. In this study, using a retina-specific conditional knockout mouse model, we show that retinas deficient in Mettl3, the core component of the m6A methyltransferase complex, exhibit structural and functional abnormalities beginning at the end of retinogenesis. Immunohistological and single-cell RNA sequencing (scRNA-seq) analyses of retinogenesis processes reveal that retinal progenitor cells (RPCs) and Müller glial cells are the two cell types primarily affected by Mettl3 deficiency. Integrative analyses of scRNA-seq and MeRIP-seq data suggest that m6A fine-tunes the transcriptomic transition from RPCs to Müller cells by promoting the degradation of RPC transcripts, the disruption of which leads to abnormalities in late retinogenesis and likely compromises the glial functions of Müller cells. Overexpression of m6A-regulated RPC transcripts in late RPCs partially recapitulates the Mettl3-deficient retinal phenotype. Collectively, our study reveals an epitranscriptomic mechanism governing progenitor-to-glial cell transition during late retinogenesis, which is essential for the homeostasis of the mature retina. The mechanism revealed in this study might also apply to other nervous systems.
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Affiliation(s)
- Yanling Xin
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceGuangzhouChina
| | - Qinghai He
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceGuangzhouChina
| | - Huilin Liang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceGuangzhouChina
| | - Ke Zhang
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceGuangzhouChina
| | - Jingyi Guo
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceGuangzhouChina
| | - Qi Zhong
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceGuangzhouChina
| | - Dan Chen
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceGuangzhouChina
| | - Jinyan Li
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceGuangzhouChina
| | - Yizhi Liu
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceGuangzhouChina
| | - Shuyi Chen
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual ScienceGuangzhouChina
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35
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Kicková E, Sadeghi A, Puranen J, Tavakoli S, Sen M, Ranta VP, Arango-Gonzalez B, Bolz S, Ueffing M, Salmaso S, Caliceti P, Toropainen E, Ruponen M, Urtti A. Pharmacokinetics of Pullulan-Dexamethasone Conjugates in Retinal Drug Delivery. Pharmaceutics 2021; 14:pharmaceutics14010012. [PMID: 35056906 PMCID: PMC8779473 DOI: 10.3390/pharmaceutics14010012] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2021] [Revised: 12/11/2021] [Accepted: 12/17/2021] [Indexed: 12/11/2022] Open
Abstract
The treatment of retinal diseases by intravitreal injections requires frequent administration unless drug delivery systems with long retention and controlled release are used. In this work, we focused on pullulan (≈67 kDa) conjugates of dexamethasone as therapeutic systems for intravitreal administration. The pullulan-dexamethasone conjugates self-assemble into negatively charged nanoparticles (average size 326 ± 29 nm). Intravitreal injections of pullulan and pullulan-dexamethasone were safe in mouse, rat and rabbit eyes. Fluorescently labeled pullulan particles showed prolonged retention in the vitreous and they were almost completely eliminated via aqueous humor outflow. Pullulan conjugates also distributed to the retina via Müller glial cells when tested in ex vivo retina explants and in vivo. Pharmacokinetic simulations showed that pullulan-dexamethasone conjugates may release free and active dexamethasone in the vitreous humor for over 16 days, even though a large fraction of dexamethasone may be eliminated from the eye as bound pullulan-dexamethasone. We conclude that pullulan based drug conjugates are promising intravitreal drug delivery systems as they may reduce injection frequency and deliver drugs into the retinal cells.
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Affiliation(s)
- Eva Kicková
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Italy; (E.K.); (S.S.); (P.C.)
| | - Amir Sadeghi
- School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Yliopistonranta 1C, 70211 Kuopio, Finland; (A.S.); (J.P.); (V.-P.R.); (E.T.); (M.R.)
| | - Jooseppi Puranen
- School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Yliopistonranta 1C, 70211 Kuopio, Finland; (A.S.); (J.P.); (V.-P.R.); (E.T.); (M.R.)
| | - Shirin Tavakoli
- Drug Research Program, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, 00710 Helsinki, Finland;
| | - Merve Sen
- Centre for Ophthalmology, Institute for Ophthalmic Research, University of Tübingen, Elfriede-Aulhorn-Str. 7, D-72076 Tübingen, Germany; (M.S.); (B.A.-G.); (S.B.); (M.U.)
| | - Veli-Pekka Ranta
- School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Yliopistonranta 1C, 70211 Kuopio, Finland; (A.S.); (J.P.); (V.-P.R.); (E.T.); (M.R.)
| | - Blanca Arango-Gonzalez
- Centre for Ophthalmology, Institute for Ophthalmic Research, University of Tübingen, Elfriede-Aulhorn-Str. 7, D-72076 Tübingen, Germany; (M.S.); (B.A.-G.); (S.B.); (M.U.)
| | - Sylvia Bolz
- Centre for Ophthalmology, Institute for Ophthalmic Research, University of Tübingen, Elfriede-Aulhorn-Str. 7, D-72076 Tübingen, Germany; (M.S.); (B.A.-G.); (S.B.); (M.U.)
| | - Marius Ueffing
- Centre for Ophthalmology, Institute for Ophthalmic Research, University of Tübingen, Elfriede-Aulhorn-Str. 7, D-72076 Tübingen, Germany; (M.S.); (B.A.-G.); (S.B.); (M.U.)
| | - Stefano Salmaso
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Italy; (E.K.); (S.S.); (P.C.)
| | - Paolo Caliceti
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Italy; (E.K.); (S.S.); (P.C.)
| | - Elisa Toropainen
- School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Yliopistonranta 1C, 70211 Kuopio, Finland; (A.S.); (J.P.); (V.-P.R.); (E.T.); (M.R.)
| | - Marika Ruponen
- School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Yliopistonranta 1C, 70211 Kuopio, Finland; (A.S.); (J.P.); (V.-P.R.); (E.T.); (M.R.)
| | - Arto Urtti
- School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Yliopistonranta 1C, 70211 Kuopio, Finland; (A.S.); (J.P.); (V.-P.R.); (E.T.); (M.R.)
- Drug Research Program, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, 00710 Helsinki, Finland;
- Institute of Chemistry, St. Petersburg State University, Petergof, Universitetskii pr. 26, 198504 St. Petersburg, Russia
- Correspondence:
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Tworig JM, Coate C, Feller MB. Excitatory neurotransmission activates compartmentalized calcium transients in Müller glia without affecting lateral process motility. eLife 2021; 10:73202. [PMID: 34913435 PMCID: PMC8806189 DOI: 10.7554/elife.73202] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2021] [Accepted: 12/15/2021] [Indexed: 11/13/2022] Open
Abstract
Neural activity has been implicated in the motility and outgrowth of glial cell processes throughout the central nervous system. Here, we explore this phenomenon in Müller glia, which are specialized radial astroglia that are the predominant glial type of the vertebrate retina. Müller glia extend fine filopodia-like processes into retinal synaptic layers, in similar fashion to brain astrocytes and radial glia that exhibit perisynaptic processes. Using two-photon volumetric imaging, we found that during the second postnatal week, Müller glial processes were highly dynamic, with rapid extensions and retractions that were mediated by cytoskeletal rearrangements. During this same stage of development, retinal waves led to increases in cytosolic calcium within Müller glial lateral processes and stalks. These regions comprised distinct calcium compartments, distinguished by variable participation in waves, timing, and sensitivity to an M1 muscarinic acetylcholine receptor antagonist. However, we found that motility of lateral processes was unaffected by the presence of pharmacological agents that enhanced or blocked wave-associated calcium transients. Finally, we found that mice lacking normal cholinergic waves in the first postnatal week also exhibited normal Müller glial process morphology. Hence, outgrowth of Müller glial lateral processes into synaptic layers is determined by factors that are independent of neuronal activity. When it comes to studying the nervous system, neurons often steal the limelight; yet, they can only work properly thanks to an ensemble cast of cell types whose roles are only just emerging. For example, ‘glial cells’ – their name derives from the Greek word for glue – were once thought to play only a passive, supporting function in nervous tissues. Now, growing evidence shows that they are, in fact, integrated into neural circuits: their activity is influenced by neurons, and, in turn, they help neurons to function properly. The role of glial cells is becoming clear in the retina, the thin, light-sensitive layer that lines the back of the eye and relays visual information to the brain. There, beautifully intricate Müller glial cells display fine protrusions (or ‘processes') that intermingle with synapses, the busy space between neurons where chemical messengers are exchanged. These messengers can act on Müller cells, triggering cascades of molecular events that may influence the structure and function of glia. This is of particular interest during development: as Müller cells mature, they are exposed to chemicals released by more fully formed retinal neurons. Tworig et al. explored how neuronal messengers can influence the way Müller cells grow their processes. To do so, they tracked mouse retinal glial cells ‘live’ during development, showing that they were growing fine, highly dynamic processes in a region rich in synapses just as neurons and glia increased their communication. However, using drugs to disrupt this messaging for a short period did not seem to impact how the processes grew. Extending the blockade over a longer timeframe also did not change the way Müller cells developed, with the cells still acquiring their characteristic elaborate process networks. Taken together, these results suggest that the structural maturation of Müller glial cells is not impacted by neuronal signaling, giving a more refined understanding of how glia form in the retina and potentially in the brain.
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Affiliation(s)
- Joshua M Tworig
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
| | - Chandler Coate
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
| | - Marla B Feller
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
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Thébault S. Minireview: Insights into the role of TRP channels in the retinal circulation and function. Neurosci Lett 2021; 765:136285. [PMID: 34634394 DOI: 10.1016/j.neulet.2021.136285] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Revised: 08/25/2021] [Accepted: 08/28/2021] [Indexed: 12/17/2022]
Abstract
Consistent with their wide distribution throughout the CNS, transcripts of all transient receptor potential (TRP) cation channel superfamily members have been detected in both neuronal and non-neuronal cells of the mammalian retina. Evidence shows that members of the TRPC (canonical, TRPC1/4/5/6), TRPV (vanilloid, TRPV1/2/4), TRPM (melastatin, TRPM1/2/3/5), TRPA (ankyrin, TRPA1), and TRPP (polycystin, TRPP2) subfamilies contribute to retinal function and circulation in health and disease, but the relevance of most TRPs has yet to be determined. Their principal role in light detection is far better understood than their participation in the control of intraocular pressure, retinal blood flow, oxidative stress, ion homeostasis, and transmitter signaling for retinal information processing. Moreover, if the therapeutic potential of targeting some TRPs to treat various retinal diseases remains speculative, recent studies highlight that vision restoration strategies are very likely to benefit from the thermo- and mechanosensitive properties of TRPs. This minireview focuses on the evidence of the past 5 years about the role of TRPs in the retina and retinal circulation, raises some possibilities about the function of TRPs in the retina, and discusses the potential sources of endogenous stimuli for TRPs in this tissue, as a reflection for future studies.
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Affiliation(s)
- Stéphanie Thébault
- Instituto de Neurobiología, Universidad Nacional Autónoma de México (UNAM), Campus UNAM-Juriquilla, 76230 Querétaro, Mexico.
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Kugler EC, Greenwood J, MacDonald RB. The "Neuro-Glial-Vascular" Unit: The Role of Glia in Neurovascular Unit Formation and Dysfunction. Front Cell Dev Biol 2021; 9:732820. [PMID: 34646826 PMCID: PMC8502923 DOI: 10.3389/fcell.2021.732820] [Citation(s) in RCA: 104] [Impact Index Per Article: 26.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2021] [Accepted: 09/01/2021] [Indexed: 12/15/2022] Open
Abstract
The neurovascular unit (NVU) is a complex multi-cellular structure consisting of endothelial cells (ECs), neurons, glia, smooth muscle cells (SMCs), and pericytes. Each component is closely linked to each other, establishing a structural and functional unit, regulating central nervous system (CNS) blood flow and energy metabolism as well as forming the blood-brain barrier (BBB) and inner blood-retina barrier (BRB). As the name suggests, the “neuro” and “vascular” components of the NVU are well recognized and neurovascular coupling is the key function of the NVU. However, the NVU consists of multiple cell types and its functionality goes beyond the resulting neurovascular coupling, with cross-component links of signaling, metabolism, and homeostasis. Within the NVU, glia cells have gained increased attention and it is increasingly clear that they fulfill various multi-level functions in the NVU. Glial dysfunctions were shown to precede neuronal and vascular pathologies suggesting central roles for glia in NVU functionality and pathogenesis of disease. In this review, we take a “glio-centric” view on NVU development and function in the retina and brain, how these change in disease, and how advancing experimental techniques will help us address unanswered questions.
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Affiliation(s)
- Elisabeth C Kugler
- Institute of Ophthalmology, Faculty of Brain Sciences, University College London, London, United Kingdom
| | - John Greenwood
- Institute of Ophthalmology, Faculty of Brain Sciences, University College London, London, United Kingdom
| | - Ryan B MacDonald
- Institute of Ophthalmology, Faculty of Brain Sciences, University College London, London, United Kingdom
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DeSantis DF, Smith CJ. Tetris in the Nervous System: What Principles of Neuronal Tiling Can Tell Us About How Glia Play the Game. Front Cell Neurosci 2021; 15:734938. [PMID: 34512272 PMCID: PMC8430210 DOI: 10.3389/fncel.2021.734938] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2021] [Accepted: 08/09/2021] [Indexed: 11/14/2022] Open
Abstract
The precise organization and arrangement of neural cells is essential for nervous system functionality. Cellular tiling is an evolutionarily conserved phenomenon that organizes neural cells, ensuring non-redundant coverage of receptive fields in the nervous system. First recorded in the drawings of Ramon y Cajal more than a century ago, we now have extensive knowledge of the biochemical and molecular mechanisms that mediate tiling of neurons. The advent of live imaging techniques in both invertebrate and vertebrate model organisms has enhanced our understanding of these processes. Despite advancements in our understanding of neuronal tiling, we know relatively little about how glia, an essential non-neuronal component of the nervous system, tile and contribute to the overall spatial arrangement of the nervous system. Here, we discuss lessons learned from neurons and apply them to potential mechanisms that glial cells may use to tile, including cell diversity, contact-dependent repulsion, and chemical signaling. We also discuss open questions in the field of tiling and what new technologies need to be developed in order to better understand glial tiling.
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Affiliation(s)
- Dana F DeSantis
- Center for Stem Cells and Regenerative Medicine, University of Notre Dame, Notre Dame, IN, United States.,Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, United States
| | - Cody J Smith
- Center for Stem Cells and Regenerative Medicine, University of Notre Dame, Notre Dame, IN, United States.,Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, United States
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40
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Li Z, Ungerer M, Faßbender J, Wenhart C, Holthoff HP, Muench G. Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs. PLoS One 2021; 16:e0255363. [PMID: 34347814 PMCID: PMC8336840 DOI: 10.1371/journal.pone.0255363] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2021] [Accepted: 07/14/2021] [Indexed: 11/27/2022] Open
Abstract
The standard histological processing procedure, which produces excellent staining of sections for most tissues, fails to yield satisfactory results in adult mouse orbits or eyeballs. Here, we show that a protocol using tissue block staining and domestic adhesive tapes resulted in qualified integral serial cryo-sections of whole orbits or eyeballs, and the fine structures were well preserved. The histological processing protocol comprises paraformaldehyde fixation, ethylenediaminetetraacetic acid decalcification, tissue block staining with hematoxylin and eosin, embedding, adhesive tape aided sectioning, and water-soluble mounting. This protocol was proved to be the best in comparison with seven other related existing histological traditional or non-traditional processing methods, according to the staining slice quality. We observed a hundred percent success rate in sectioning, collection, and mounting with this method. The reproducibility tested on qualified section success rates and slice quality scores confirmed that the technique is reliable. The feasibility of the method to detect target molecules in orbits was verified by successful trial tests on block immunostaining and adhesive tape-aided sectioning. Application of this protocol in joints, brains, and so on,—the challenging integral sectioning tissues, also generated high-quality histological staining sections.
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Affiliation(s)
- Zhongmin Li
- Advancecor GmbH, Martinsried, Germany
- * E-mail:
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41
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Paisley CE, Kay JN. Seeing stars: Development and function of retinal astrocytes. Dev Biol 2021; 478:144-154. [PMID: 34260962 DOI: 10.1016/j.ydbio.2021.07.007] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2021] [Revised: 07/06/2021] [Accepted: 07/08/2021] [Indexed: 02/06/2023]
Abstract
Throughout the central nervous system, astrocytes adopt precisely ordered spatial arrangements of their somata and arbors, which facilitate their many important functions. Astrocyte pattern formation is particularly important in the retina, where astrocytes serve as a template that dictates the pattern of developing retinal vasculature. Thus, if astrocyte patterning is disturbed, there are severe consequences for retinal angiogenesis and ultimately for vision - as seen in diseases such as retinopathy of prematurity. Here we discuss key steps in development of the retinal astrocyte population. We describe how fundamental developmental forces - their birth, migration, proliferation, and death - sculpt astrocytes into a template that guides angiogenesis. We further address the radical changes in the cellular and molecular composition of the astrocyte network that occur upon completion of angiogenesis, paving the way for their adult functions in support of retinal ganglion cell axons. Understanding development of retinal astrocytes may elucidate pattern formation mechanisms that are deployed broadly by other axon-associated astrocyte populations.
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Affiliation(s)
- Caitlin E Paisley
- Departments of Neurobiology, Ophthalmology, and Cell Biology, Duke University School of Medicine, Durham, NC, 27710, USA
| | - Jeremy N Kay
- Departments of Neurobiology, Ophthalmology, and Cell Biology, Duke University School of Medicine, Durham, NC, 27710, USA.
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42
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Neurovascular regulation in diabetic retinopathy and emerging therapies. Cell Mol Life Sci 2021; 78:5977-5985. [PMID: 34230991 DOI: 10.1007/s00018-021-03893-9] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2021] [Revised: 06/15/2021] [Accepted: 06/25/2021] [Indexed: 12/11/2022]
Abstract
Diabetic retinopathy (DR) is the leading cause of vision loss in working adults in developed countries. The disease traditionally classified as a microvascular complication of diabetes is now widely recognized as a neurovascular disorder resulting from disruption of the retinal neurovascular unit (NVU). The NVU comprising retinal neurons, glia and vascular cells coordinately regulates blood flow, vascular density and permeability to maintain homeostasis. Disturbance of the NVU during DR can lead to vision-threatening clinical manifestations. A limited number of signaling pathways have been identified for intercellular communication within the NVU, including vascular endothelial growth factor (VEGF), the master switch for angiogenesis. VEGF inhibitors are now widely used to treat DR, but their limited efficacy implies that other signaling molecules are involved in the pathogenesis of DR. By applying a novel screening technology called comparative ligandomics, we recently discovered secretogranin III (Scg3) as a unique DR-selective angiogenic and vascular leakage factor with therapeutic potential for DR. This review proposes neuron-derived Scg3 as the first diabetes-selective neurovascular regulator and discusses important features of Scg3 inhibition for next-generation disease-targeted anti-angiogenic therapies of DR.
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43
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Choi JIV, Tchernookova BK, Kumar W, Kiedrowski L, Goeke C, Guizzetti M, Larson J, Kreitzer MA, Malchow RP. Extracellular ATP-Induced Alterations in Extracellular H + Fluxes From Cultured Cortical and Hippocampal Astrocytes. Front Cell Neurosci 2021; 15:640217. [PMID: 33994945 PMCID: PMC8120152 DOI: 10.3389/fncel.2021.640217] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2020] [Accepted: 03/19/2021] [Indexed: 12/18/2022] Open
Abstract
Small alterations in the level of extracellular H+ can profoundly alter neuronal activity throughout the nervous system. In this study, self-referencing H+-selective microelectrodes were used to examine extracellular H+ fluxes from individual astrocytes. Activation of astrocytes cultured from mouse hippocampus and rat cortex with extracellular ATP produced a pronounced increase in extracellular H+ flux. The ATP-elicited increase in H+ flux appeared to be independent of bicarbonate transport, as ATP increased H+ flux regardless of whether the primary extracellular pH buffer was 26 mM bicarbonate or 1 mM HEPES, and persisted when atmospheric levels of CO2 were replaced by oxygen. Adenosine failed to elicit any change in extracellular H+ fluxes, and ATP-mediated increases in H+ flux were inhibited by the P2 inhibitors suramin and PPADS suggesting direct activation of ATP receptors. Extracellular ATP also induced an intracellular rise in calcium in cultured astrocytes, and ATP-induced rises in both calcium and H+ efflux were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin. Replacement of extracellular sodium with choline did not significantly reduce the size of the ATP-induced increases in H+ flux, and the increases in H+ flux were not significantly affected by addition of EIPA, suggesting little involvement of Na+/H+ exchangers in ATP-elicited increases in H+ flux. Given the high sensitivity of voltage-sensitive calcium channels on neurons to small changes in levels of free H+, we hypothesize that the ATP-mediated extrusion of H+ from astrocytes may play a key role in regulating signaling at synapses within the nervous system.
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Affiliation(s)
- Ji-In Vivien Choi
- Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, United States.,Stritch School of Medicine, Loyola University, Maywood, IL, United States
| | - Boriana K Tchernookova
- Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, United States
| | - Wasan Kumar
- Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, United States
| | - Lech Kiedrowski
- Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, United States.,Spot Cells LLC, Chicago, IL, United States
| | - Calla Goeke
- VA Portland Health Care System, Portland, OR, United States.,Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR, United States
| | - Marina Guizzetti
- VA Portland Health Care System, Portland, OR, United States.,Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR, United States
| | - John Larson
- Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, United States
| | - Matthew A Kreitzer
- Department of Biology, Indiana Wesleyan University, Marion, IN, United States
| | - Robert Paul Malchow
- Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, United States.,Department Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, United States
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44
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Yamagata M, Yan W, Sanes JR. A cell atlas of the chick retina based on single-cell transcriptomics. eLife 2021; 10:e63907. [PMID: 33393903 PMCID: PMC7837701 DOI: 10.7554/elife.63907] [Citation(s) in RCA: 90] [Impact Index Per Article: 22.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Accepted: 01/01/2021] [Indexed: 12/14/2022] Open
Abstract
Retinal structure and function have been studied in many vertebrate orders, but molecular characterization has been largely confined to mammals. We used single-cell RNA sequencing (scRNA-seq) to generate a cell atlas of the chick retina. We identified 136 cell types plus 14 positional or developmental intermediates distributed among the six classes conserved across vertebrates - photoreceptor, horizontal, bipolar, amacrine, retinal ganglion, and glial cells. To assess morphology of molecularly defined types, we adapted a method for CRISPR-based integration of reporters into selectively expressed genes. For Müller glia, we found that transcriptionally distinct cells were regionally localized along the anterior-posterior, dorsal-ventral, and central-peripheral retinal axes. We also identified immature photoreceptor, horizontal cell, and oligodendrocyte types that persist into late embryonic stages. Finally, we analyzed relationships among chick, mouse, and primate retinal cell classes and types. Our results provide a foundation for anatomical, physiological, evolutionary, and developmental studies of the avian visual system.
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Affiliation(s)
- Masahito Yamagata
- Center for Brain Science and Department of Molecular and Cellular Biology, Harvard UniversityCambridgeUnited States
| | - Wenjun Yan
- Center for Brain Science and Department of Molecular and Cellular Biology, Harvard UniversityCambridgeUnited States
| | - Joshua R Sanes
- Center for Brain Science and Department of Molecular and Cellular Biology, Harvard UniversityCambridgeUnited States
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45
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Lankford CK, Laird JG, Inamdar SM, Baker SA. A Comparison of the Primary Sensory Neurons Used in Olfaction and Vision. Front Cell Neurosci 2020; 14:595523. [PMID: 33250719 PMCID: PMC7676898 DOI: 10.3389/fncel.2020.595523] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2020] [Accepted: 10/06/2020] [Indexed: 12/18/2022] Open
Abstract
Vision, hearing, smell, taste, and touch are the tools used to perceive and navigate the world. They enable us to obtain essential resources such as food and highly desired resources such as mates. Thanks to the investments in biomedical research the molecular unpinning’s of human sensation are rivaled only by our knowledge of sensation in the laboratory mouse. Humans rely heavily on vision whereas mice use smell as their dominant sense. Both modalities have many features in common, starting with signal detection by highly specialized primary sensory neurons—rod and cone photoreceptors (PR) for vision, and olfactory sensory neurons (OSN) for the smell. In this chapter, we provide an overview of how these two types of primary sensory neurons operate while highlighting the similarities and distinctions.
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Affiliation(s)
- Colten K Lankford
- Department of Biochemistry, University of Iowa, Iowa City, IA, United States
| | - Joseph G Laird
- Department of Biochemistry, University of Iowa, Iowa City, IA, United States
| | - Shivangi M Inamdar
- Department of Biochemistry, University of Iowa, Iowa City, IA, United States
| | - Sheila A Baker
- Department of Biochemistry, University of Iowa, Iowa City, IA, United States.,Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, United States
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46
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An efficient inducible RPE-Selective cre transgenic mouse line. Exp Eye Res 2020; 202:108370. [PMID: 33264655 DOI: 10.1016/j.exer.2020.108370] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2020] [Revised: 10/30/2020] [Accepted: 11/20/2020] [Indexed: 12/22/2022]
Abstract
Cre-mediated modulation of gene function in the murine retinal pigment epithelium (RPE) has been widely used, but current postnatal RPE-selective Cre driver lines suffer from limited recombination efficiency and/or ectopic or mosaic expression. We sought to generate a transgenic mouse line with consistently efficient RPE-selective Cre activity that could be temporally regulated. We used ϕC31 integrase to insert a DNA construct encoding a human BEST1 promoter fragment driving a Cre recombinase estrogen receptor fusion (BEST1-CreERT2) at the Rosa26 locus of C57BL/6J mice. Rosa26BEST1-CreERT2 mice were bred with a tdTomato reporter line and to mice with a Cre-conditional allele of Tfam. 4-hydroxytamoxifen or vehicle was delivered by four consecutive daily intraperitoneal injections. TdTomato was robustly expressed in the RPE of mice of both sexes for inductions beginning at P14 (males 90.7 ± 4.5%, females 84.7 ± 3.2%) and at 7 weeks (males 84.3 ± 7.0%, females 82 ± 3.6%). <0.6% of Muller glia also expressed tdTomato, but no tdTomato fluorescence was observed in other ocular cells or in multiple non-ocular tissues, with the exception of sparse foci in the testis. No evidence of retinal toxicity was observed in mice homozygous for the transgene induced beginning at P14 and assessed at 7-10 months. RPE-selective ablation of Tfam beginning at P14 led to reduced retinal thickness at 8 months of age and diminished retinal electrical responses at 12 months, as expected. These findings demonstrate that we have generated a mouse line with consistently efficient, tamoxifen-mediated postnatal induction of Cre recombination in the RPE and a small fraction of Muller glia. This line should be useful for temporally regulated modulation of gene function in the murine RPE.
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47
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Fadl BR, Brodie SA, Malasky M, Boland JF, Kelly MC, Kelley MW, Boger E, Fariss R, Swaroop A, Campello L. An optimized protocol for retina single-cell RNA sequencing. Mol Vis 2020; 26:705-717. [PMID: 33088174 PMCID: PMC7553720] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2020] [Accepted: 10/08/2020] [Indexed: 10/25/2022] Open
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.
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Affiliation(s)
- Benjamin R. Fadl
- Neurobiology, Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD
| | - Seth A. Brodie
- Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health & NCI-Frederick, Leidos Biomedical Research Inc., Frederick, MD
| | - Michael Malasky
- Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health & NCI-Frederick, Leidos Biomedical Research Inc., Frederick, MD
| | - Joseph F. Boland
- Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health & NCI-Frederick, Leidos Biomedical Research Inc., Frederick, MD
| | - Michael C. Kelly
- Single Cell Analysis Facility, Cancer Research Technology Program, Frederick National Laboratory, Bethesda, MD
| | - Matthew W. Kelley
- Laboratory of Cochlear Development, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD
| | - Erich Boger
- Genomics and Computational Biology Core, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD
| | - Robert Fariss
- Biological Imaging Core, National Eye Institute, National Institutes of Health, Bethesda, MD
| | - Anand Swaroop
- Neurobiology, Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD
| | - Laura Campello
- Neurobiology, Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD
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Choi SH, Kim KY, Perkins GA, Phan S, Edwards G, Xia Y, Kim J, Skowronska-Krawczyk D, Weinreb RN, Ellisman MH, Miller YI, Ju WK. AIBP protects retinal ganglion cells against neuroinflammation and mitochondrial dysfunction in glaucomatous neurodegeneration. Redox Biol 2020; 37:101703. [PMID: 32896719 PMCID: PMC7484594 DOI: 10.1016/j.redox.2020.101703] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2020] [Revised: 08/12/2020] [Accepted: 08/22/2020] [Indexed: 01/10/2023] Open
Abstract
Glaucoma is a leading cause of blindness worldwide in individuals 60 years of age and older. Despite its high prevalence, the factors contributing to glaucoma progression are currently not well characterized. Glia-driven neuroinflammation and mitochondrial dysfunction play critical roles in glaucomatous neurodegeneration. Here, we demonstrated that elevated intraocular pressure (IOP) significantly decreased apolipoprotein A-I binding protein (AIBP; gene name Apoa1bp) in retinal ganglion cells (RGCs), but resulted in upregulation of TLR4 and IL-1β expression in Müller glia endfeet. Apoa1bp-/- mice had impaired visual function and Müller glia characterized by upregulated TLR4 activity, impaired mitochondrial network and function, increased oxidative stress and induced inflammatory responses. We also found that AIBP deficiency compromised mitochondrial network and function in RGCs and exacerbated RGC vulnerability to elevated IOP. Administration of recombinant AIBP prevented RGC death and inhibited inflammatory responses and cytokine production in Müller glia in vivo. These findings indicate that AIBP protects RGCs against glia-driven neuroinflammation and mitochondrial dysfunction in glaucomatous neurodegeneration and suggest that recombinant AIBP may be a potential therapeutic agent for glaucoma.
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Affiliation(s)
- Soo-Ho Choi
- Department of Medicine, University of California San Diego, La Jolla, CA, 92093, USA.
| | - Keun-Young Kim
- National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California San Diego, La Jolla, CA, 92093, USA
| | - Guy A Perkins
- National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California San Diego, La Jolla, CA, 92093, USA
| | - Sébastien Phan
- National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California San Diego, La Jolla, CA, 92093, USA
| | - Genea Edwards
- Hamilton Glaucoma Center and Shiley Eye Institute, Viterbi Family Department of Ophthalmology, University of California San Diego, La Jolla, CA, 92093, USA
| | - Yining Xia
- Department of Medicine, University of California San Diego, La Jolla, CA, 92093, USA
| | - Jungsu Kim
- Department of Medicine, University of California San Diego, La Jolla, CA, 92093, USA
| | - Dorota Skowronska-Krawczyk
- Department of Physiology, Biophysics & Ophthalmology, University of California Irvine, Irvine, CA, 92697, USA
| | - Robert N Weinreb
- Hamilton Glaucoma Center and Shiley Eye Institute, Viterbi Family Department of Ophthalmology, University of California San Diego, La Jolla, CA, 92093, USA
| | - Mark H Ellisman
- National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California San Diego, La Jolla, CA, 92093, USA
| | - Yury I Miller
- Department of Medicine, University of California San Diego, La Jolla, CA, 92093, USA
| | - Won-Kyu Ju
- Hamilton Glaucoma Center and Shiley Eye Institute, Viterbi Family Department of Ophthalmology, University of California San Diego, La Jolla, CA, 92093, USA.
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Abstract
Diabetic retinopathy (DR) is a frequent complication of diabetes mellitus and an increasingly common cause of visual impairment. Blood vessel damage occurs as the disease progresses, leading to ischemia, neovascularization, blood-retina barrier (BRB) failure and eventual blindness. Although detection and treatment strategies have improved considerably over the past years, there is room for a better understanding of the pathophysiology of the diabetic retina. Indeed, it has been increasingly realized that DR is in fact a disease of the retina's neurovascular unit (NVU), the multi-cellular framework underlying functional hyperemia, coupling neuronal computations to blood flow. The accumulating evidence reveals that both neurochemical (synapses) and electrical (gap junctions) means of communications between retinal cells are affected at the onset of hyperglycemia, warranting a global assessment of cellular interactions and their role in DR. This is further supported by the recent data showing down-regulation of connexin 43 gap junctions along the vascular relay from capillary to feeding arteriole as one of the earliest indicators of experimental DR, with rippling consequences to the anatomical and physiological integrity of the retina. Here, recent advancements in our knowledge of mechanisms controlling the retinal neurovascular unit will be assessed, along with their implications for future treatment and diagnosis of DR.
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50
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Collin GB, Gogna N, Chang B, Damkham N, Pinkney J, Hyde LF, Stone L, Naggert JK, Nishina PM, Krebs MP. Mouse Models of Inherited Retinal Degeneration with Photoreceptor Cell Loss. Cells 2020; 9:E931. [PMID: 32290105 PMCID: PMC7227028 DOI: 10.3390/cells9040931] [Citation(s) in RCA: 57] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2020] [Revised: 04/05/2020] [Accepted: 04/07/2020] [Indexed: 12/12/2022] Open
Abstract
Inherited retinal degeneration (RD) leads to the impairment or loss of vision in millions of individuals worldwide, most frequently due to the loss of photoreceptor (PR) cells. Animal models, particularly the laboratory mouse, have been used to understand the pathogenic mechanisms that underlie PR cell loss and to explore therapies that may prevent, delay, or reverse RD. Here, we reviewed entries in the Mouse Genome Informatics and PubMed databases to compile a comprehensive list of monogenic mouse models in which PR cell loss is demonstrated. The progression of PR cell loss with postnatal age was documented in mutant alleles of genes grouped by biological function. As anticipated, a wide range in the onset and rate of cell loss was observed among the reported models. The analysis underscored relationships between RD genes and ciliary function, transcription-coupled DNA damage repair, and cellular chloride homeostasis. Comparing the mouse gene list to human RD genes identified in the RetNet database revealed that mouse models are available for 40% of the known human diseases, suggesting opportunities for future research. This work may provide insight into the molecular players and pathways through which PR degenerative disease occurs and may be useful for planning translational studies.
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Affiliation(s)
- Gayle B. Collin
- The Jackson Laboratory, Bar Harbor, Maine, ME 04609, USA; (G.B.C.); (N.G.); (B.C.); (N.D.); (J.P.); (L.F.H.); (L.S.); (J.K.N.)
| | - Navdeep Gogna
- The Jackson Laboratory, Bar Harbor, Maine, ME 04609, USA; (G.B.C.); (N.G.); (B.C.); (N.D.); (J.P.); (L.F.H.); (L.S.); (J.K.N.)
| | - Bo Chang
- The Jackson Laboratory, Bar Harbor, Maine, ME 04609, USA; (G.B.C.); (N.G.); (B.C.); (N.D.); (J.P.); (L.F.H.); (L.S.); (J.K.N.)
| | - Nattaya Damkham
- The Jackson Laboratory, Bar Harbor, Maine, ME 04609, USA; (G.B.C.); (N.G.); (B.C.); (N.D.); (J.P.); (L.F.H.); (L.S.); (J.K.N.)
- Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
- Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
| | - Jai Pinkney
- The Jackson Laboratory, Bar Harbor, Maine, ME 04609, USA; (G.B.C.); (N.G.); (B.C.); (N.D.); (J.P.); (L.F.H.); (L.S.); (J.K.N.)
| | - Lillian F. Hyde
- The Jackson Laboratory, Bar Harbor, Maine, ME 04609, USA; (G.B.C.); (N.G.); (B.C.); (N.D.); (J.P.); (L.F.H.); (L.S.); (J.K.N.)
| | - Lisa Stone
- The Jackson Laboratory, Bar Harbor, Maine, ME 04609, USA; (G.B.C.); (N.G.); (B.C.); (N.D.); (J.P.); (L.F.H.); (L.S.); (J.K.N.)
| | - Jürgen K. Naggert
- The Jackson Laboratory, Bar Harbor, Maine, ME 04609, USA; (G.B.C.); (N.G.); (B.C.); (N.D.); (J.P.); (L.F.H.); (L.S.); (J.K.N.)
| | - Patsy M. Nishina
- The Jackson Laboratory, Bar Harbor, Maine, ME 04609, USA; (G.B.C.); (N.G.); (B.C.); (N.D.); (J.P.); (L.F.H.); (L.S.); (J.K.N.)
| | - Mark P. Krebs
- The Jackson Laboratory, Bar Harbor, Maine, ME 04609, USA; (G.B.C.); (N.G.); (B.C.); (N.D.); (J.P.); (L.F.H.); (L.S.); (J.K.N.)
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