ABSTRACT
Subretinal fibrosis is a major untreatable cause of poor outcomes in neovascular age-related macular degeneration. Mouse models of subretinal fibrosis all possess a degree of invasiveness and tissue damage not typical of fibrosis progression. This project characterises JR5558 mice as a model to study subretinal fibrosis. Fundus and optical coherence tomography (OCT) imaging was used to non-invasively track lesions. Lesion number and area were quantified with ImageJ. Retinal sections, wholemounts and Western blots were used to characterise alterations. Subretinal lesions expand between 4 and 8 weeks and become established in size and location around 12 weeks. Subretinal lesions were confirmed to be fibrotic, including various cell populations involved in fibrosis development. Müller cell processes extended from superficial retina into subretinal lesions at 8 weeks. Western blotting revealed increases in fibronectin (4 wk and 8 wk, p < 0.001), CTGF (20 wks, p < 0.001), MMP2 (12 wks and 20 wks p < 0.05), αSMA (12 wks and 20 wks p < 0.05) and GFAP (8 wk and 12 wk, p ≤ 0.01), consistent with our immunofluorescence results. Intravitreal injection of Aflibercept reduced subretinal lesion growth. Our study provides evidence JR5558 mice have subretinal fibrotic lesions that grow between 4 and 8 weeks and confirms this line to be a good model to study subretinal fibrosis development and assess treatment options.
Subject(s)
Disease Models, Animal , Fibrosis , Retina , Tomography, Optical Coherence , Animals , Mice , Tomography, Optical Coherence/methods , Retina/pathology , Retina/metabolism , Receptors, Vascular Endothelial Growth Factor/metabolism , Fibronectins/metabolism , Ependymoglial Cells/metabolism , Ependymoglial Cells/pathology , Connective Tissue Growth Factor/metabolism , Connective Tissue Growth Factor/genetics , Macular Degeneration/pathology , Macular Degeneration/metabolism , Matrix Metalloproteinase 2/metabolism , Intravitreal Injections , Glial Fibrillary Acidic Protein/metabolism , Actins/metabolism , Mice, Inbred C57BL , Recombinant Fusion ProteinsABSTRACT
The ependyma lining the third ventricle (3V) in the mediobasal hypothalamus plays a crucial role in energy balance and glucose homeostasis. It is characterized by a high functional heterogeneity and plasticity, but the underlying molecular mechanisms governing its features are not fully understood. Here, 5481 hypothalamic ependymocytes were cataloged using FACS-assisted scRNAseq from fed, 12h-fasted, and 24h-fasted adult male mice. With standard clustering analysis, typical ependymal cells and ß2-tanycytes appear sharply defined, but other subpopulations, ß1- and α-tanycytes, display fuzzy boundaries with few or no specific markers. Pseudospatial approaches, based on the 3V neuroanatomical distribution, enable the identification of specific versus shared tanycyte markers and subgroup-specific versus general tanycyte functions. We show that fasting dynamically shifts gene expression patterns along the 3V, leading to a spatial redistribution of cell type-specific responses. Altogether, we show that changes in energy status induce metabolic and functional switches in tanycyte subpopulations, providing insights into molecular and functional diversity and plasticity within the tanycyte population.
Subject(s)
Ependymoglial Cells , Fasting , Lipid Metabolism , Neurons , Animals , Ependymoglial Cells/metabolism , Male , Fasting/metabolism , Mice , Neurons/metabolism , Ependyma/metabolism , Ependyma/cytology , Hypothalamus/metabolism , Hypothalamus/cytology , Mice, Inbred C57BL , Energy Metabolism , Third Ventricle/metabolism , Glucose/metabolismABSTRACT
Retinitis pigmentosa (RP), an inherited retinal disease, affects 1,5 million people worldwide. The initial mutation-driven photoreceptor degeneration leads to chronic inflammation, characterized by Müller cell activation and upregulation of CD44. CD44 is a cell surface transmembrane glycoprotein and the primary receptor for hyaluronic acid. It is involved in many pathological processes, but little is known about CD44's retinal functions. CD44 expression is also increased in Müller cells from our Pde6bSTOP/STOP RP mouse model. To gain a more detailed understanding of CD44's role in healthy and diseased retinas, we analyzed Cd44-/- and Cd44-/-Pde6bSTOP/STOP mice, respectively. The loss of CD44 led to enhanced photoreceptor degeneration, reduced retinal function, and increased inflammatory response. To understand the underlying mechanism, we performed proteomic analysis on isolated Müller cells from Cd44-/- and Cd44-/-Pde6bSTOP/STOP retinas and identified a significant downregulation of glutamate transporter 1 (SLC1A2). This downregulation was accompanied by higher glutamate levels, suggesting impaired glutamate homeostasis. These novel findings indicate that CD44 stimulates glutamate uptake via SLC1A2 in Müller cells, which in turn, supports photoreceptor survival and function.
Subject(s)
Ependymoglial Cells , Hyaluronan Receptors , Retinitis Pigmentosa , Signal Transduction , Animals , Hyaluronan Receptors/metabolism , Hyaluronan Receptors/genetics , Mice , Ependymoglial Cells/metabolism , Signal Transduction/physiology , Retinitis Pigmentosa/metabolism , Retinitis Pigmentosa/pathology , Retinitis Pigmentosa/genetics , Mice, Knockout , Mice, Inbred C57BL , Photoreceptor Cells, Vertebrate/metabolism , Cell Survival/physiology , Mice, Transgenic , Retina/metabolism , Retina/pathologyABSTRACT
Disruptions in energy homeostasis can lead to diseases like obesity and diabetes, affecting millions of people each year. Tanycytes, the adult stem cells in the hypothalamus, play crucial roles in assisting hypothalamic neurons in maintaining energy balance. Although tanycytes have been extensively studied in rodents, our understanding of human tanycytes remains limited. In this study, we utilized single-cell transcriptomics data to explore the heterogeneity of human embryonic tanycytes, investigate their gene regulatory networks, analyze their intercellular communication, and examine their developmental trajectory. Our analysis revealed the presence of two clusters of ß tanycytes and three clusters of α tanycytes in our dataset. Surprisingly, human embryonic tanycytes displayed significant similarities to mouse tanycytes in terms of marker gene expression and transcription factor activities. Trajectory analysis indicated that α tanycytes were the first to be generated, giving rise to ß tanycytes in a dorsal-ventral direction along the third ventricle. Furthermore, our CellChat analyses demonstrated that tanycytes generated earlier along the developmental lineages exhibited increased intercellular communication compared to those generated later. In summary, we have thoroughly characterized the heterogeneity of human embryonic tanycytes from various angles. We are confident that our findings will serve as a foundation for future research on human tanycytes.
Subject(s)
Ependymoglial Cells , Single-Cell Analysis , Transcriptome , Humans , Ependymoglial Cells/metabolism , Ependymoglial Cells/cytology , Gene Regulatory Networks , Mice , Animals , Gene Expression Profiling , Cell Communication/genetics , Hypothalamus/metabolism , Hypothalamus/cytologyABSTRACT
Purpose: The purpose of this study was to investigate the ocular morphological characteristics of Col4a3-/- mice as a model of Alport syndrome (AS) and the potential pathogenesis. Methods: The expression of collagen IV at 8, 12, and 21 weeks of age was evaluated by immunohistochemistry in wild-type (WT) and Col4a3-/- mice. Hematoxylin and eosin (H&E) staining and thickness measurements were performed to assess the thickness of anterior lens capsule and retina. Ultrastructure analysis of corneal epithelial basement membrane, anterior lens capsule, internal limiting membrane (ILM), and retinal pigment epithelium (RPE) basement membrane was performed using transmission electron microscopy. Finally, Müller cell activation was evaluated by glial fibrillary acidic protein (GFAP) expression. Results: Collagen IV was downregulated in the corneal epithelial basement membrane and ILM of Col4a3-/- mice. The hemidesmosomes of Col4a3-/- mice corneal epithelium became flat and less electron-dense than those of the WT group. Compared with those of the WT mice, the anterior lens capsules of Col4a3-/- mice were thinner. Abnormal structure was detected at the ILM Col4a3-/- mice, and the basal folds of the RPE basement membrane in Col4a3-/- mice were thicker and shorter. The retinas of Col4a3-/- mice were thinner than those of WT mice, especially within 1000 µm away from the optic nerve. GFAP expression enhanced in each age group of Col4a3-/- mice. Conclusions: Our results suggested that Col4a3-/- mice exhibit ocular anomalies similar to patients with AS. Additionally, Müller cells may be involved in AS retinal anomalies. Translational Relevance: This animal model could provide an opportunity to understand the underlying mechanisms of AS ocular disorders and to investigate potential new treatments.
Subject(s)
Basement Membrane , Collagen Type IV , Disease Models, Animal , Mice, Knockout , Nephritis, Hereditary , Animals , Nephritis, Hereditary/pathology , Nephritis, Hereditary/genetics , Nephritis, Hereditary/metabolism , Collagen Type IV/genetics , Collagen Type IV/metabolism , Collagen Type IV/deficiency , Mice , Basement Membrane/metabolism , Basement Membrane/pathology , Basement Membrane/ultrastructure , Retinal Pigment Epithelium/pathology , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/ultrastructure , Microscopy, Electron, Transmission , Mice, Inbred C57BL , Lens Capsule, Crystalline/metabolism , Lens Capsule, Crystalline/pathology , Lens Capsule, Crystalline/ultrastructure , Epithelium, Corneal/pathology , Epithelium, Corneal/ultrastructure , Epithelium, Corneal/metabolism , Glial Fibrillary Acidic Protein/metabolism , Glial Fibrillary Acidic Protein/genetics , Retina/pathology , Retina/metabolism , Retina/ultrastructure , Autoantigens/genetics , Autoantigens/metabolism , Ependymoglial Cells/pathology , Ependymoglial Cells/metabolism , Ependymoglial Cells/ultrastructure , Immunohistochemistry , MaleABSTRACT
Purpose: Lipid nanoparticles (LNPs) show promise in their ability to introduce mRNA to drive protein expression in specific cell types of the mammalian eye. Here, we examined the ability of mRNA encapsulated in LNPs with two distinct formulations to drive gene expression in mouse and human retina and other ocular tissues. Methods: We introduced mRNA-carrying LNPs into two biological systems. Intravitreal injections were tested to deliver LNPs into the mouse eye. Human retinal pigment epithelium (RPE) and retinal explants were used to assess mRNA expression in human tissue. We analyzed specificity of expression using histology, immunofluorescence, and imaging. Results: In mice, mRNAs encoding GFP and ciliary neurotrophic factor (CNTF) were specifically expressed by Müller glia and RPE. Acute inflammatory changes measured by microglia distribution (Iba-1) or interleukin-6 (IL-6) expression were not observed 6 hours post-injection. Human RPE also expressed high levels of GFP. Human retinal explants expressed GFP in cells with apical and basal processes consistent with Müller glia and in perivascular cells consistent with macrophages. Conclusions: We demonstrated the ability to reliably transfect subpopulations of retinal cells in mouse eye tissues in vivo and in human ocular tissues. Of significance, intravitreal injections were sufficient to transfect the RPE in mice. To our knowledge, we demonstrate delivery of mRNA using LNPs in human ocular tissues for the first time. Translational Relevance: Ocular gene-replacement therapies using non-viral vector methods are a promising alternative to adeno-associated virus (AAV) vectors. Our studies show that mRNA LNP delivery can be used to transfect retinal cells in both mouse and human tissues without inducing significant inflammation. This methodology could be used to transfect retinal cell lines, tissue explants, mice, or potentially as gene-replacement therapy in a clinical setting in the future.
Subject(s)
Intravitreal Injections , Nanoparticles , RNA, Messenger , Retinal Pigment Epithelium , Animals , Humans , RNA, Messenger/administration & dosage , RNA, Messenger/metabolism , Mice , Retinal Pigment Epithelium/metabolism , Nanoparticles/chemistry , Mice, Inbred C57BL , Ciliary Neurotrophic Factor/genetics , Ciliary Neurotrophic Factor/metabolism , Ciliary Neurotrophic Factor/administration & dosage , Retina/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Ependymoglial Cells/metabolism , Gene Transfer Techniques , LiposomesABSTRACT
Generation of neurons through direct reprogramming has emerged as a promising therapeutic approach for treating neurodegenerative diseases. In this study, we present an efficient method for reprogramming retinal glial cells into neurons. By suppressing Notch signaling by disrupting either Rbpj or Notch1/2, we induced mature Müller glial cells to reprogram into bipolar- and amacrine-like neurons. We demonstrate that Rbpj directly activates both Notch effector genes and genes specific to mature Müller glia while indirectly repressing expression of neurogenic basic helix-loop-helix (bHLH) factors. Combined loss of function of Rbpj and Nfia/b/x resulted in conversion of nearly all Müller glia to neurons. Last, inducing Müller glial proliferation by overexpression of dominant-active Yap promotes neurogenesis in both Rbpj- and Nfia/b/x/Rbpj-deficient Müller glia. These findings demonstrate that Notch signaling and NFI factors act in parallel to inhibit neurogenic competence in mammalian Müller glia and help clarify potential strategies for regenerative therapies aimed at treating retinal dystrophies.
Subject(s)
Cellular Reprogramming , Ependymoglial Cells , NFI Transcription Factors , Neuroglia , Neurons , Receptors, Notch , Retina , Signal Transduction , Animals , NFI Transcription Factors/metabolism , NFI Transcription Factors/genetics , Mice , Retina/metabolism , Retina/cytology , Ependymoglial Cells/metabolism , Ependymoglial Cells/cytology , Neuroglia/metabolism , Receptors, Notch/metabolism , Neurons/metabolism , Neurons/cytology , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Neurogenesis , YAP-Signaling Proteins/metabolism , Cell ProliferationABSTRACT
This study aims to explore the effect of Lycii Fructus and Salviae Miltiorrhizae Radix et Rhizoma(LFSMR), a drug pair possesses the function of nourishing Yin, promoting blood circulation, and brightening the eyes, in treating retinitis pigmentosa(RP)by inhibiting the gliosis of Müller cells(MCs) and inducing their reprogramming and differentiation into various types of retinal nerve cells. Twelve C57 mice were used as the normal control group, and 48 transgenic RP(rd10) mice were randomly divided into the model group, positive control group, and low and high dose LFSMR groups, with 12 mice in each group. HE staining was used to detect pathological changes in the retina, and an electroretinogram was used to detect retinal function. Retinal optical coherence tomography was used to detect retinal thickness and perform fundus photography, and laser speckle perfusion imaging was used to detect local retinal blood flow. Digital PCR was used to detect gene expression related to retinal nerve cells, and immunofluorescence was used to detect protein expression related to retinal nerve cells. LFSMR could significantly improve the pathological changes, increase the amplitude of a and b waves, increase the retinal thickness, restore retinal damage, and increase retinal blood flow in mice with RP lesions. LFSMR could also significantly inhibit the m RNA expression of the glial fibrillary acidic protein( GFAP) during the pathogenesis of RP and upregulate m RNA expression of sex determining region Y box protein 2(SOX2), paired box protein 6(Pax6),rhodopsin, protein kinase C-α(PKCα), syntaxin, and thymic cell antigen 1. 1(Thy1. 1). LFSMR could significantly inhibit GFAP protein expression and enhance protein expression of SOX2, Pax6, rhodopsin, PKCα, syntaxin, and Thy1. 1. It could also reverse the pathological changes in the retina of rd10 mice, improve retinal function and fundus performance, increase retinal thickness, enhance local retinal blood flow, and exert therapeutic effects on RP. The mechanism of action of LFSMR may be related to inhibiting the gliosis of MCs and promoting their reprogramming and differentiation into various types of retinal nerve cells.
Subject(s)
Drugs, Chinese Herbal , Ependymoglial Cells , Lycium , Mice, Inbred C57BL , Retinitis Pigmentosa , Salvia miltiorrhiza , Animals , Mice , Ependymoglial Cells/drug effects , Ependymoglial Cells/metabolism , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/administration & dosage , Lycium/chemistry , Retinitis Pigmentosa/drug therapy , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/metabolism , Retinitis Pigmentosa/physiopathology , Salvia miltiorrhiza/chemistry , Male , Retina/drug effects , Rhizome/chemistry , HumansABSTRACT
OBJECTIVES: In Western society, high-caloric diets rich in fats and sugars have fueled the obesity epidemic and its related disorders. Disruption of the body-brain communication, crucial for maintaining glucose and energy homeostasis, arises from both obesogenic and genetic factors, leading to metabolic disorders. Here, we investigate the role of hypothalamic tanycyte shuttles between the pituitary portal blood and the third ventricle cerebrospinal fluid in regulating energy balance. METHODS: We inhibited vesicle-associated membrane proteins (VAMP1-3)-mediated release in tanycytes by expressing the botulinum neurotoxin type B light chain (BoNT/B) in a Cre-dependent manner in tanycytes. This was achieved by injecting either TAT-Cre in the third ventricle or an AAV1/2 expressing Cre under the control of the tanycyte-specific promoter iodothyronine deiodinase 2 into the lateral ventricle of adult male mice. RESULTS: In male mice fed a standard diet, targeted expression of BoNT/B in adult tanycytes blocks leptin transport into the mediobasal hypothalamus and results in normal-weight central obesity, including increased food intake, abdominal fat deposition, and elevated leptin levels but no marked change in body weight. Furthermore, BoNT/B expression in adult tanycytes promotes fatty acid storage, leading to glucose intolerance and insulin resistance. Notably, these metabolic disturbances occur despite a compensatory increase in insulin secretion, observed both in response to exogenous glucose boluses in vivo and in isolated pancreatic islets. Intriguingly, these metabolic alterations are associated with impaired spatial memory in BoNT/B-expressing mice. CONCLUSIONS: These findings underscore the central role of tanycytes in brain-periphery communication and highlight their potential implication in the age-related development of type 2 diabetes and cognitive decline. Our tanycytic BoNT/B mouse model provides a robust platform for studying how these conditions progress over time, from prediabetic states to full-blown metabolic and cognitive disorders, and the mechanistic contribution of tanycytes to their development. The recognition of the impact of tanycytic transcytosis on hormone transport opens new avenues for developing targeted therapies that could address both metabolic disorders and their associated cognitive comorbidities, which often emerge or worsen with advancing age.
Subject(s)
Energy Metabolism , Ependymoglial Cells , Glucose , Homeostasis , Animals , Male , Mice , Glucose/metabolism , Ependymoglial Cells/metabolism , Cognition/drug effects , Leptin/metabolism , Mice, Inbred C57BL , Hypothalamus/metabolism , Obesity/metabolismABSTRACT
BACKGROUND: Estrogen secretion by the ovaries regulates the hypothalamic-pituitary-gonadal axis during the reproductive cycle, influencing gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion, and also plays a role in regulating metabolism. Here, we establish that hypothalamic tanycytes-specialized glia lining the floor and walls of the third ventricle-integrate estrogenic feedback signals from the gonads and couple reproduction with metabolism by relaying this information to orexigenic neuropeptide Y (NPY) neurons. METHODS: Using mouse models, including mice floxed for Esr1 (encoding estrogen receptor alpha, ERα) and those with Cre-dependent expression of designer receptors exclusively activated by designer drugs (DREADDs), along with viral-mediated, pharmacological and indirect calorimetric approaches, we evaluated the role of tanycytes and tanycytic estrogen signaling in pulsatile LH secretion, cFos expression in NPY neurons, estrous cyclicity, body-weight changes and metabolic parameters in adult females. RESULTS: In ovariectomized mice, chemogenetic activation of tanycytes significantly reduced LH pulsatile release, mimicking the effects of direct NPY neuron activation. In intact mice, tanycytes were crucial for the estrogen-mediated control of GnRH/LH release, with tanycytic ERα activation suppressing fasting-induced NPY neuron activation. Selective knockout of Esr1 in tanycytes altered estrous cyclicity and fertility in female mice and affected estrogen's ability to inhibit refeeding in fasting mice. The absence of ERα signaling in tanycytes increased Npy transcripts and body weight in intact mice and prevented the estrogen-mediated decrease in food intake as well as increase in energy expenditure and fatty acid oxidation in ovariectomized mice. CONCLUSIONS: Our findings underscore the pivotal role of tanycytes in the neuroendocrine coupling of reproduction and metabolism, with potential implications for its age-related deregulation after menopause. SIGNIFICANCE STATEMENT: Our investigation reveals that tanycytes, specialized glial cells in the brain, are key interpreters of estrogen signals for orexigenic NPY neurons in the hypothalamus. Disrupting tanycytic estrogen receptors not only alters fertility in female mice but also impairs the ability of estrogens to suppress appetite. This work thus sheds light on the critical role played by tanycytes in bridging the hormonal regulation of cyclic reproductive function and appetite/feeding behavior. This understanding may have potential implications for age-related metabolic deregulation after menopause.
Subject(s)
Ependymoglial Cells , Estrogen Receptor alpha , Fertility , Luteinizing Hormone , Signal Transduction , Animals , Estrogen Receptor alpha/metabolism , Estrogen Receptor alpha/genetics , Female , Mice , Fertility/physiology , Ependymoglial Cells/metabolism , Signal Transduction/physiology , Luteinizing Hormone/metabolism , Estrous Cycle/physiology , Estrous Cycle/metabolism , Neuropeptide Y/metabolism , Ovariectomy , Neurons/metabolism , Hypothalamus/metabolism , Mice, Inbred C57BL , Gonadotropin-Releasing Hormone/metabolismABSTRACT
Semaphorin6A (Sema6A) is a repulsive guidance molecule that plays many roles in central nervous system, heart and bone development, as well as immune system responses and cell signaling in cancer. Loss of Sema6A or its receptor PlexinA2 in zebrafish leads to smaller eyes and improper retinal patterning. Here, we investigate a potential role for the Sema6A intracellular domain in zebrafish eye development and dissect which phenotypes rely on forward signaling and which rely on reverse signaling. We performed rescue experiments on zebrafish Sema6A morphants with either full-length Sema6A (Sema6A-FL) or Sema6A lacking its intracellular domain (Sema6A-ΔC). We identified that the intracellular domain is not required for eye size and retinal patterning, however it is required for retinal integrity, the number and end feet strength of Müller glia and protecting against retinal cell death. This novel function for the intracellular domain suggests a role for Sema6A reverse signaling in zebrafish eye development.
Subject(s)
Protein Domains , Retina , Semaphorins , Zebrafish Proteins , Zebrafish , Animals , Zebrafish/metabolism , Zebrafish/embryology , Semaphorins/metabolism , Semaphorins/genetics , Retina/metabolism , Zebrafish Proteins/metabolism , Zebrafish Proteins/genetics , Signal Transduction , Ependymoglial Cells/metabolism , Ependymoglial Cells/cytologyABSTRACT
The adult hippocampus generates new granule cells (aGCs) with functional capabilities that convey unique forms of plasticity to the preexisting circuits. While early differentiation of adult radial glia-like cells (RGLs) has been studied extensively, the molecular mechanisms guiding the maturation of postmitotic neurons remain unknown. Here, we used a precise birthdating strategy to study aGC differentiation using single-nuclei RNA sequencing. Transcriptional profiling revealed a continuous trajectory from RGLs to mature aGCs, with multiple immature stages bearing increasing levels of effector genes supporting growth, excitability, and synaptogenesis. Analysis of differential gene expression, pseudo-time trajectory, and transcription factors (TFs) revealed critical transitions defining four cellular states: quiescent RGLs, proliferative progenitors, immature aGCs, and mature aGCs. Becoming mature aGCs involved a transcriptional switch that shuts down pathways promoting cell growth, such SoxC TFs, to activate programs that likely control neuronal homeostasis. aGCs overexpressing Sox4 or Sox11 remained immature. Our results unveil precise molecular mechanisms driving adult RGLs through the pathway of neuronal differentiation.
Subject(s)
Cell Differentiation , Hippocampus , Neurogenesis , Neurons , SOXC Transcription Factors , Animals , Hippocampus/metabolism , Hippocampus/cytology , Neurons/metabolism , Neurons/cytology , SOXC Transcription Factors/metabolism , SOXC Transcription Factors/genetics , Cell Differentiation/genetics , Neurogenesis/genetics , Mice , Transcription, Genetic , Gene Expression Profiling , Transcription Factors/metabolism , Transcription Factors/genetics , Ependymoglial Cells/metabolism , Ependymoglial Cells/cytologyABSTRACT
In the injured zebrafish retina, Müller glial cells (MG) reprogram to adopt retinal stem cell properties and regenerate damaged neurons. The strongest zebrafish reprogramming factors might be good candidates for stimulating a similar regenerative response by mammalian MG. Myc proteins are potent reprogramming factors that can stimulate cellular plasticity in differentiated cells; however, their role in MG reprogramming and retina regeneration remains poorly explored. Here, we report that retinal injury stimulates mycb and mych expression and that, although both Mycb and Mych stimulate MG reprogramming and proliferation, only Mych enhances retinal neuron apoptosis. RNA-sequencing analysis of wild-type, mychmut and mycbmut fish revealed that Mycb and Mych regulate â¼40% and â¼16%, respectively, of the genes contributing to the regeneration-associated transcriptome of MG. Of these genes, those that are induced are biased towards regulation of ribosome biogenesis, protein synthesis, DNA synthesis, and cell division, which are the top cellular processes affected by retinal injury, suggesting that Mycb and Mych are potent MG reprogramming factors. Consistent with this, forced expression of either of these proteins is sufficient to stimulate MG proliferation in the uninjured retina.
Subject(s)
Cell Proliferation , Cellular Reprogramming , Ependymoglial Cells , Retina , Zebrafish Proteins , Zebrafish , Animals , Apoptosis/genetics , Cellular Reprogramming/genetics , Ependymoglial Cells/metabolism , Ependymoglial Cells/cytology , Retina/metabolism , Retina/cytology , Retinal Neurons/metabolism , Transcriptome/genetics , Zebrafish Proteins/metabolism , Zebrafish Proteins/geneticsABSTRACT
Mitochondria play critical roles in neural stem/progenitor cell proliferation and fate decisions. The subcellular localization of mitochondria in neural stem/progenitor cells during mitosis potentially influences the distribution of mitochondria to the daughter cells and thus their fates. Therefore, understanding the spatial dynamics of mitochondria provides important knowledge about brain development. In this study, we analyzed the subcellular localization of mitochondria in the fetal human neocortex with a particular focus on the basal radial glial cells (bRGCs), a neural stem/progenitor cell subtype attributed to the evolutionary expansion of the human neocortex. During interphase, bRGCs exhibit a polarized localization of mitochondria that is localized at the base of the process or the proximal part of the process. Thereafter, mitochondria in bRGCs at metaphase show unpolarized distribution in which the mitochondria are randomly localized in the cytoplasm. During anaphase and telophase, mitochondria are still localized evenly, but mainly in the periphery of the cytoplasm. Mitochondria start to accumulate at the cleavage furrow during cytokinesis. These results suggest that the mitochondrial localization in bRGCs is tightly regulated during the cell cycle, which may ensure the proper distribution of mitochondria to the daughter cells and, thus in turn, influence their fates.
Subject(s)
Cell Cycle , Ependymoglial Cells , Mitochondria , Neocortex , Humans , Neocortex/cytology , Neocortex/metabolism , Mitochondria/metabolism , Cell Cycle/physiology , Ependymoglial Cells/metabolism , Ependymoglial Cells/cytology , Neural Stem Cells/metabolism , Neural Stem Cells/cytologyABSTRACT
Antiretroviral treatments have notably extended the lives of individuals with HIV and reduced the occurrence of comorbidities, including ocular manifestations. The involvement of endoplasmic reticulum (ER) stress in HIV-1 pathogenesis raises questions about its correlation with cellular senescence or its role in initiating senescent traits. This study investigated how ER stress and dysregulated autophagy impact cellular senescence triggered by HIV-1 Tat in the MIO-M1 cell line (human Müller glial cells). Cells exposed to HIV-1 Tat exhibited increased vimentin expression combined with markers of ER stress (BiP, p-eIF2α), autophagy (LC3, Beclin-1, p62), and the senescence marker p21 compared to control cells. Western blotting and staining techniques like SA-ß-gal were employed to examine these markers. Additionally, treatments with ER stress inhibitor 4-PBA before HIV-1 Tat exposure led to a decreased expression of ER stress, senescence, and autophagy markers. Conversely, pre-treatment with the autophagy inhibitor 3-MA resulted in reduced autophagy and senescence markers but did not alter ER stress markers compared to control cells. The findings suggest a link between ER stress, dysregulated autophagy, and the initiation of a senescence phenotype in MIO-M1 cells induced by HIV-1 Tat exposure.
Subject(s)
Autophagy , Cellular Senescence , Endoplasmic Reticulum Stress , HIV-1 , tat Gene Products, Human Immunodeficiency Virus , Humans , tat Gene Products, Human Immunodeficiency Virus/metabolism , tat Gene Products, Human Immunodeficiency Virus/genetics , HIV-1/physiology , Cell Line , Ependymoglial Cells/metabolism , Ependymoglial Cells/virology , HIV Infections/virologyABSTRACT
Polyamine (PA) spermidine (SPD) plays a crucial role in aging. Since SPD accumulates in glial cells, particularly in Müller retinal cells (MCs), the expression of the SPD-synthesizing enzyme spermidine synthase (SpdS) in Müller glia and age-dependent SpdS activity are not known. We used immunocytochemistry, Western blot (WB), and image analysis on rat retinae at postnatal days 3, 21, and 120. The anti-glutamine synthetase (GS) antibody was used to identify glial cells. In the neonatal retina (postnatal day 3 (P3)), SpdS was expressed in almost all progenitor cells in the neuroblast. However, by day 21 (P21), the SpdS label was pronouncedly expressed in multiple neurons, while GS labels were observed only in radial Müller glial cells. During early cell adulthood, at postnatal day 120 (P120), SpdS was observed solely in ganglion cells and a few other neurons. Western blot and semi-quantitative analyses of SpdS labeling showed a dramatic decrease in SpdS at P21 and P120 compared to P3. In conclusion, the redistribution of SpdS with aging indicates that SPD is first synthesized in all progenitor cells and then later in neurons, but not in glia. However, MCs take up and accumulate SPD, regardless of the age-associated decrease in SPD synthesis in neurons.
Subject(s)
Ependymoglial Cells , Retina , Spermidine Synthase , Animals , Rats , Spermidine Synthase/metabolism , Spermidine Synthase/genetics , Retina/metabolism , Ependymoglial Cells/metabolism , Aging/metabolism , Spermidine/metabolism , Neuroglia/metabolism , Animals, NewbornABSTRACT
To prevent ocular pathologies, new generation of dietary supplements have been commercially available. They consist of nutritional supplement mixing components known to provide antioxidative properties, such as unsaturated fatty acid, resveratrol or flavonoids. However, to date, few data evaluating the impact of a mixture mainly composed of those components (Nutrof Total®) on the retina are available. Only one in-vivo preclinical study demonstrated that dietary supplementation (DS) prevents the retina from light-induced retinal degeneration; and only one in-vitro study on Müller cells culture showed that glutamate metabolism cycle was key in oxidative stress response. Therefore, we raised the question about the in-vivo effect of DS on glutamate metabolism in the retina. Herein, we showed that the dietary supplementation promotes in-vivo increase of retinal glutamine amount through a higher glutamine synthesis as observed in-vitro on Muller cells. Therefore, we can suggest that the promotion of glutamine synthesis is part of the protective effect of DS against retinal degeneration, acting as a preconditioning mechanism against retinal degeneration.
Subject(s)
Antioxidants , Dietary Supplements , Fatty Acids, Omega-3 , Glutamine , Retina , Retinal Degeneration , Glutamine/metabolism , Animals , Antioxidants/pharmacology , Fatty Acids, Omega-3/administration & dosage , Retinal Degeneration/metabolism , Retinal Degeneration/prevention & control , Retina/metabolism , Retina/drug effects , Oxidative Stress/drug effects , Cells, Cultured , Ependymoglial Cells/metabolism , Ependymoglial Cells/drug effects , Male , Rats , Disease Models, AnimalABSTRACT
Animals inhabiting temperate and high latitudes undergo drastic seasonal changes in energy storage, facilitated by changes in food intake and body mass. Those seasonal changes in the animal's biology are not mere consequences of environmental energy availability but are anticipatory responses to the energetic requirements of the upcoming season and are actively timed by tracking the annual progression in photoperiod. In this review, we discuss how photoperiod is used to control energy balance seasonally and how this is distinct from energy homeostasis. Most notably, we suggest that photoperiodic control of food intake and body mass does not originate from the arcuate nucleus, as for homeostatic appetite control, but is rather to be found in hypothalamic tanycytes. Tanycytes are specialized ependymal cells lining the third ventricle, which can sense metabolites from the cerebrospinal fluid (e.g. glucose) and can control access of circulating signals to the brain. They are also essential in conveying time-of-year information by integrating photoperiod and altering hypothalamic thyroid metabolism, a feature that is conserved in seasonal vertebrates and connects to seasonal breeding and metabolism. We also discuss how homeostatic feedback signals are handled during times of rapid energetic transitions. Studies on leptin in seasonal mammals suggest a seasonal shift in central sensitivity and blood-brain transport, which might be facilitated by tanycytes. This article is part of the Special Issue on "Food intake and feeding states".
Subject(s)
Eating , Energy Metabolism , Photoperiod , Seasons , Animals , Energy Metabolism/physiology , Eating/physiology , Humans , Ependymoglial Cells/metabolism , Ependymoglial Cells/physiology , Homeostasis/physiology , Hypothalamus/metabolism , Hypothalamus/physiology , Neurons/metabolism , Neurons/physiology , Leptin/metabolismABSTRACT
Aging is a major risk factor for the development or the worsening of retinal degenerative conditions. The intricate network of the neural retina determined that the retinal aging is a complicated process. The aim of this study is to delineate the transcriptomic changes of major retinal neurons during aging in C57BL/6 mice at single-cell level. We analyzed the transcriptional profiles of the photoreceptor, bipolar, amacrine, and Müller glial cells of 1.5-2 and 24-30 months old mice using single-cell RNA sequencing technique. We selectively confirmed the differences in gene expression using immunofluorescence staining and RNA in situ hybridization analysis. We found that each retinal cell type had unique changes upon aging. However, they all showed signs of dysregulated glucose and energy metabolism, and perturbed proteostasis. In particular, old Müller glia exhibited the most profound changes, including the upregulation of cell metabolism, stress-responses, antigen-presentation and immune responses and metal ion homeostasis. The dysregulated gliogenesis and differentiation was confirmed by the presence of Müller glia expressing rod-specific genes in the inner nuclear layer and the outer plexiform layer of the old retina. We further pinpointed the specific loss of GABAergic amacrine cells in old retina. Our study emphasized changes of amacrine and Müller glia during retinal aging, provided resources for further research on the molecular and cellular regulatory mechanisms underlying aging-associated retinal deterioration.
Subject(s)
Aging , Amacrine Cells , Energy Metabolism , Mice, Inbred C57BL , Proteostasis , Animals , Amacrine Cells/metabolism , Mice , Aging/physiology , Energy Metabolism/physiology , Ependymoglial Cells/metabolism , Retina/metabolism , GABAergic Neurons/metabolism , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Retinal Degeneration/genetics , In Situ Hybridization , Homeostasis/physiologyABSTRACT
Diabetic retinopathy (DR), a leading cause of visual impairment, demands a profound comprehension of its cellular mechanisms to formulate effective therapeutic strategies. Our study presentes a comprehensive single-cell analysis elucidating the intricate landscape of Müller cells within DR, emphasizing their nuanced involvement. Utilizing scRNA-seq data from both Sprague-Dawley rat models and human patients, we delineated distinct Müller cell clusters and their corresponding gene expression profiles. These findings were further validated through differential gene expression analysis utilizing human transcriptomic data. Notably, certain Müller cell clusters displayed upregulation of the Rho gene, implying a phagocytic response to damaged photoreceptors within the DR microenvironment. This phenomenon was consistently observed across species. Additionally, the co-expression patterns of RHO and PDE6G within Müller cell clusters provided compelling evidence supporting their potential role in maintaining retinal integrity during DR. Our results offer novel insights into the cellular dynamics of DR and underscore Müller cells as promising therapeutic targets for preserving vision in retinal disorders induced by diabetes.