optimizing-tear-collection-in-mice-for-mrna-and-protein-analysis

Optimizing Tear Collection in Mice for mRNA and Protein Analysis

The tear film is a highly dynamic biofluid capable of reflecting pathology-associated molecular changes, not only in the ocular surface but also in other ...

CINVESTAV Sede Sur

D-serine is an N-methyl-D-aspartate (NMDA) receptor coagonist predominantly produced by glial cells in the brain and the retina. Whereas a role for D-serine as a modulator of NMDA receptors in neurons has been suggested, its function in glial cells has not been analyzed. We here show that D-serine modulates gene expression in Müller glial cells from the retina through the induction of transcription factor CREB phosphorylation and the expression of the immediate-early gene c-fos. Pharmacological analysis indicates that D-serine effect involves NMDA receptor activation. Comparison of the effect of D-serine in Müller cells, hippocampal astrocytes and hippocampal neurons suggests that D-serine could function as a retinal NMDA receptor coagonist activating functionally relevant transcription factor pathways in glial cells.

D-serine regulates CREB phosphorylation induced by NMDA receptor activation in Müller glia from the retina.

In the adult retina, N-methyl-D-aspartate (NMDA) neurotoxicity induces Müller cell reactive gliosis which is characterized by changes in gene expression that lead to proliferation and affect retinal physiology. The amino acid D-serine is synthesized in Müller cells and modulates these processes acting as a coagonist of NMDA receptors. We have found that the transcription factor DREAM (downstream regulatory element antagonist modulator), which acts as a transcriptional repressor by binding as a tetramer to regulatory elements located in the promoter region of target genes, is expressed in these cells and that its DNA-binding activity is modulated by NMDA receptor activation. Consistently, immunocytochemical analysis demonstrates that NMDA receptor activation induces changes in the nuclear localization of this transcription factor. DREAM is a pleiotropic transcription factor capable to repress and activate genes involved in several physiological events in different tissues. These results link, for the first time, this transcription factor with NMDA-receptor activation. Given the relevance of glutamatergic transmission in the retina and the remarkable functional plasticity of Müller cells, these findings support the notion that the NMDA receptor-dependent modulation of DREAM activity could play a role in relevant physiological processes ranging from retinal response to injury to differentiation capacity of retinal progenitor cells.

D-Serine/N-methyl-D-aspartate receptor signaling decreases DNA-binding activity of the transcriptional repressor DREAM in Müller glia from the retina.

Postnatal retinal Müller glia are considered to be retinal progenitors as they retain the ability to dedifferentiate, proliferate, and differentiate to new retinal glia and neurons after injury. The proliferation and differentiation processes are coordinated by several extrinsic factors and neurotransmitters, including glutamate. Thus, the appropriate numbers and proportions of the different cell types are generated to form a functional retina during development and during injury repair. Here we analyze the changes in the proliferation of postnatal Müller glia-derived progenitors after activation of the N-methyl-D-aspartate (NMDA) glutamate receptors.

NMDA receptor mediates proliferation and CREB phosphorylation in postnatal Müller glia-derived retinal progenitors.

Abuse of toluene-containing inhalants is associated to various cognitive impairments that have been partly associated to deviation of the hippocampal neurogenesis processes during adulthood. In the present study we analyzed the effect of chronic toluene exposure (6000ppm) on cell proliferation and migration in the other selected area of the rodent brain where neurogenesis persist throughout adulthood, the subventricular zone of the lateral ventricle (SVZ). We used an anti-Ki67 antibody to evaluate SVZ cell proliferation, BrdU to evaluate cell survival and double-staining with BrdU and the migration marker doublecortin (DCX) to evaluate migration, by immunofluorescence 2h, 1, 5, 10 or 15 days after 20 sessions of toluene exposure. We found that toluene induced an initial burst of cell proliferation in the SVZ but not a significant increase in migration toward the rostral migratory stream (RMS) or the number of cells that migrate to the olfactory bulb. In addition, we detected a small number of new migrating cells in the corpus callosum and striatum of control mice that was similar in toluene-exposed brains. These results may underline the homeostatic capabilities of the populations of dividing cells, previously demonstrated using other drugs of abuse and demonstrate that toluene misuse can alter cellular proliferation in the postnatal brain.

Chronic toluene exposure induces cell proliferation in the mice SVZ but not migration through the RMS.

Human adult dental pulp stem cells (DPSCs) are self-renewing stem cells that originate from the neural crest during development and remain within the dental pulp niche through adulthood. Due to their multi-lineage differentiation potential and their relative ease of access they represent an exciting alternative for autologous stem cell-based therapies in neurodegenerative diseases. In animal models, DPSCs transplanted into the brain differentiate into functional neurons or astrocytes in response to local environmental cues that appear to influence the fate of the surviving cells. Here we tested the hypothesis that DPSCs might be able to respond to factors present in the retina enabling the regenerative potential of these cells. We evaluated the response of DPSCs to conditioned media from organotypic explants from control and chemically damaged rat retinas. To evaluate cell differentiation, we analyzed the expression of glial fibrillary acidic protein (GFAP), early neuronal and retinal markers (polysialic acid-neural cell adhesion molecule (PSA-NCAM); Pax6; Ascl1; NeuroD1) and the late photoreceptor marker rhodopsin, by immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). Exposure of DPSC cultures to conditioned media from control retinas induced a 39% reduction on the number of DPSCs that expressed GFAP; the expression of Pax6, Ascl1, PSA-NCAM or NeuroD1 was undetectable or did not change significantly. Expression of rhodopsin was not detectable in control or after exposure of the cultures with retinal conditioned media. By contrast, 44% of DPSCs exposed to conditioned media from damaged retinas were immunopositive to this protein. This response could not be reproduced when conditioned media from Müller-enriched primary cultures was used. Finally, quantitative RT-PCR was performed to compare the relative expression of glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in DPSC co-cultured with retinal organotypic explants, where BDNF mRNA expression was significantly upregulated in retinal-exposed cultures. Our data demonstrate that DPSC cultures respond to cues from the rat retina and differentiate to express retinal neuronal markers.

Human dental pulp stem cells respond to cues from the rat retina and differentiate to express the retinal neuronal marker rhodopsin.

Primary cilia are specialized organelles that extend from the cell surface and concentrate signal transduction components. In the nervous system, primary cilia-associated signals, such as sonic hedgehog (Shh), regulate cell proliferation and neuronal fate. Primary cilia assembly and maintenance require a multi-subunit intraflagellar transport (IFT) protein complex. Defects in primary cilia and IFT proteins are associated to severe pathological phenotypes. In the retina, the study of primary cilia has been mainly restricted to the specialized photoreceptor outer segment. The presence and physiological role of primary cilia in other retinal cells have not been clearly elucidated. Müller cells are the main glia of the retina where they exert distinct functions to maintain homeostasis. In pathological conditions, Müller cells mount a unique regenerative response through the processes of dedifferentiation, proliferation, and differentiation into neuronal lineages. The involvement of IFT proteins or a primary cilium in these processes has not been explored. In this study, we used mature Müller glia primary cultures to reveal the presence of the primary cilia by immunoreactivity to acetylated α-tubulin and γ-tubulin, which localize to the axoneme and ciliar basal body, respectively. We demonstrate that si-RNA-mediated downregulation of IFT20 gene expression, a main component of the IFT machinery, blocks Shh-induced Müller cell proliferation. We present evidence that IFT20 ablation impairs the dedifferentiation capacity of Müller cells induced by Shh and by glutamate. Our demonstration that Müller glia expresses IFT20 and harbors primary cilia, and opens new venues of research on the role of primary cilia in the retina.

Primary cilia in rat mature Müller glia: downregulation of IFT20 expression reduces sonic hedgehog-mediated proliferation and dedifferentiation potential of Müller glia primary cultures.

Cell-type determination is a complex process driven by the combinatorial effect of extrinsic signals and the expression of transcription factors and regulatory genes. MicroRNAs (miRNAs) are non-coding RNAs that, generally, inhibit the expression of target genes and have been involved, among other processes, in cell identity acquisition. To search for candidate miRNAs putatively involved in mice rod photoreceptor and Müller glia (MG) identity, we compared miRNA expression profiles between late-stage retinal progenitor cells (RPCs), CD73-immunopositive (CD73+) rods and postnatal MG. We found a close similarity between RPCs and CD73+ miRNA expression profiles but a divergence between CD73+ and MG miRNA signatures. We validated preferentially expressed miRNAs in the CD73+ subpopulation (miR-182, 183, 124a, 9(∗), 181c and 301b(∗)) or MG (miR-143, 145, 214, 199a-5p, 199b(∗), and 29a). Taking advantage of the unique capacity of MG to dedifferentiate into progenitor-like cells that can be differentiated to a rod phenotype in response to external cues, we evaluated changes of selected miRNAs in MG-derived progenitors (MGDP) during neuronal differentiation. We found decreased levels of miR-143 and 145, but increased levels of miR-29a in MGDP. In MGDPs committed to early neuronal lineages we found increased levels of miR-124a and upregulation of miR-124a, 9(∗) and 181c during MGDP acquisition of rod phenotypes. Furthermore, we demonstrated that ectopic miR-124 expression is sufficient to enhance early neuronal commitment of MGDP. Our data reveal a dynamic regulation of miRNAs in MGDP through early and late neuronal commitment and miRNAs that could be potential targets to exploit the silent neuronal differentiation capacity of MG in mammals.

MicroRNA changes through Müller glia dedifferentiation and early/late rod photoreceptor differentiation.

Fyn kinase is widely expressed in neuronal and glial cells of the brain, where it exerts multiple functional roles that affect fundamental physiological processes. The aim of our study was to investigate the, so far unknown, functional role of Fyn in the retina. We report that Fyn is expressed, in vivo, in a subpopulation of Müller glia. We used a mouse model of Fyn genetic ablation and Müller-enriched primary cultures to demonstrate that Fyn deficiency induces morphological alterations in the mature retina, a reduction in the thickness of the outer and inner nuclear layers and alterations in postnatal Müller cell physiology. These include shortening of Müller cell processes, a decrease in cell proliferation, inactivation of the Akt signal transduction pathway, a reduced number of focal adhesions points and decreased adhesion of these cells to the ECM. As abnormalities in Müller cell physiology have been previously associated to a compromised retinal function we evaluated behavioral responses to visual stimulation. Our results associate Fyn deficiency with impaired visual optokinetic responses under scotopic and photopic light conditions. Our study reveals novel roles for Fyn kinase in retinal morphology and Müller cell physiology and suggests that Fyn is required for optimal visual processing.

Fyn kinase genetic ablation causes structural abnormalities in mature retina and defective Müller cell function.

Nasal olfactory stem and neural progenitor cells (NOS/PCs) are considered possible tools for regenerative stem cell therapies in neurodegenerative diseases. Neurogenesis is a complex process regulated by extrinsic and intrinsic signals that include DNA-methylation and other chromatin modifications that could be experimentally manipulated in order to increase neuronal differentiation. The aim of the present study was the characterization of primary cultures and consecutive passages (P2-P10) of NOS/PCs isolated from male Swiss-Webster (mNOS/PCs) or healthy humans (hNOS/PCs). We evaluated and compared cellular morphology, proliferation rates and the expression pattern of pluripotency-associated markers and DNA methylation-associated gene expression in these cultures. Neuronal differentiation was induced by exposure to all-trans retinoic acid and forskolin for 7 days and evaluated by morphological analysis and immunofluorescence against neuronal markers MAP2, NSE and MAP1B. In response to the inductive cues mNOS/PCs expressed NSE (75.67%) and MAP2 (35.34%); whereas the majority of the hNOS/PCs were immunopositive to MAP1B. Treatment with procainamide, a specific inhibitor of DNA methyltransferase 1 (DNMT1), increases in the number of forskolin'/retinoic acid-induced mature neuronal marker-expressing mNOS/PCs cells and enhances neurite development in hNOS/PCs. Our results indicate that mice and human nasal olfactory stem/progenitors cells share pluripotency-related gene expression suggesting that their application for stem cell therapy is worth pursuing and that DNA methylation inhibitors could be efficient tools to enhance neuronal differentiation from these cells.

Pharmacological inhibition of DNA methyltransferase 1 promotes neuronal differentiation from rodent and human nasal olfactory stem/progenitor cell cultures.

The neural retina hosts a unique specialized type of macroglial cell that not only preserves retinal homeostasis, function, and integrity but also may serve as a source of new neurons during regenerative processes: the Müller cell. Precise microRNA-driven mechanisms of gene regulation impel and direct the processes of Müller glia lineage acquisition from retinal progenitors during development, the triggering of their response to retinal degeneration and, in some cases, Müller cell reprogramming and regenerative events. In this review we survey the recent reports describing, through functional assays, the regulatory role of microRNAs in Müller cell physiology, differentiation potential, and retinal pathology. We discuss also the evidence based on expression analysis that points out the relevance of a Müller glia-specific microRNA signature that would orchestrate these processes.

microRNA expression in the neural retina: Focus on Müller glia.

microRNA profiling has identified cell-specific expression patterns that could represent molecular signatures triggering the acquisition of a specific phenotype; in other words, of cellular identity and its associated function. Several groups have hypothesized that retinal cell phenotyping could be achieved through the determination of the global pattern of miRNA expression across specific cell types in the adult retina. This is especially relevant for Müller glia in the context of retinal damage, as these cells undergo dramatic changes of gene expression in response to injury, that render them susceptible to acquire a progenitor-like phenotype and be a source of new neurons.We describe a method that combines an experimental protocol for excitotoxic-induced retinal damage through N-methyl-D-aspartate subretinal injection with magnetic-activated cell sorting (MACS) of Müller cells and RNA isolation for microRNA profiling. Comparison of microRNA patterns of expression should allow Müller cell phenotyping under different experimental conditions.

In Vitro Assays for Mouse Müller Cell Phenotyping Through microRNA Profiling in the Damaged Retina.

Müller glial cells exert multiple essential functions in retinal physiology and retinopathies reflecting perhaps the existence of distinct Müller cellular subpopulations. Harnessing Müller cell heterogeneity may serve to enhance new therapeutic approaches for retinal disease.

Müller Cell Molecular Heterogeneity: Facts and Predictions.

Retinal Müller cells secrete extracellular vesicles that can be captured by other Müller cells. In response to a signal that may be deleterious for the retina, Müller glia-derived extracellular vesicles spread instructions to induce gene expression changes in other cells.

Müller Glia to Müller Glia Extracellular Vesicle-Dependent Signaling Induces Multipotency Genes  and l Expression in Response to N-methyl-D-aspartate (NMDA) Exposure.

The eye is a complex organ composed of various cell types, each serving a unique purpose. However, aging brings about structural and functional changes in these cells, leading to discomfort and potential pathology. Alterations in gene expression, influenced by aging and environmental factors, significantly affect cell structure and function. Epigenetics, a field focused on understanding the correlation between changes in gene expression, cell function, and environmental factors, plays a crucial role in unraveling the molecular events responsible for age-related eye changes. This prompts the possibility of developing epigenetic strategies to intervene in these changes or reinstate proper molecular activities. Indeed, research has demonstrated that epigenetic modifications, including DNA methylation, histone modification, and non-coding RNAs, are closely associated with age-related alterations in gene expression and cell function. This review aims to compile and synthesize the most recent body of evidence supporting the role of epigenetics in age-related alterations observed in various components of the eye. Specifically, it focuses on the impact of epigenetic changes in the ocular surface, tear film, aqueous humor, vitreous humor, and lens. Furthermore, it highlights the significant advancements that have been made in the field of epigenetic-based experimental therapies, specifically focusing on their potential for treating pathological conditions in the aging eye.

Article	Year
D-serine regulates CREB phosphorylation induced by NMDA receptor activation in Müller glia from the retina. Neuroscience letters\| PubMed ID: 17920195	2007
D-Serine/N-methyl-D-aspartate receptor signaling decreases DNA-binding activity of the transcriptional repressor DREAM in Müller glia from the retina. Neuroscience letters\| PubMed ID: 18191896	2008
NMDA receptor mediates proliferation and CREB phosphorylation in postnatal Müller glia-derived retinal progenitors. Molecular vision\| PubMed ID: 19365572	2009
Chronic toluene exposure induces cell proliferation in the mice SVZ but not migration through the RMS. Neuroscience letters\| PubMed ID: 24882722	2014
Human dental pulp stem cells respond to cues from the rat retina and differentiate to express the retinal neuronal marker rhodopsin. Neuroscience\| PubMed ID: 25242642	2014
Primary cilia in rat mature Müller glia: downregulation of IFT20 expression reduces sonic hedgehog-mediated proliferation and dedifferentiation potential of Müller glia primary cultures. Cellular and molecular neurobiology\| PubMed ID: 25504432	2015
MicroRNA changes through Müller glia dedifferentiation and early/late rod photoreceptor differentiation. Neuroscience\| PubMed ID: 26708746	2016
Fyn kinase genetic ablation causes structural abnormalities in mature retina and defective Müller cell function. Molecular and cellular neurosciences\| PubMed ID: 26808221	2016
Pharmacological inhibition of DNA methyltransferase 1 promotes neuronal differentiation from rodent and human nasal olfactory stem/progenitor cell cultures. International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience\| PubMed ID: 28161254	2017
microRNA expression in the neural retina: Focus on Müller glia. Journal of neuroscience research\| PubMed ID: 29030949	2018
In Vitro Assays for Mouse Müller Cell Phenotyping Through microRNA Profiling in the Damaged Retina. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29564798	2018
Müller Cell Molecular Heterogeneity: Facts and Predictions. ASN neuro\| PubMed ID: 35673270	2022
Müller Glia to Müller Glia Extracellular Vesicle-Dependent Signaling Induces Multipotency Genes and l Expression in Response to N-methyl-D-aspartate (NMDA) Exposure. ASN neuro\| PubMed ID: 37345290	2023
Epigenetic mechanisms of non-retinal components of the aging eye and novel therapeutic strategies. Experimental eye research\| PubMed ID: 37802281	2023

Monica Lamas

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