Postmitotic Retinal Cells Research Articles

Unlocking the role of lactate: metabolic pathways, signaling, and gene regulation in postmitotic retinal cells.

The Warburg effect, which was first described a century ago, asserts that mitotic tumor cells generate higher quantities of lactate. Intriguingly, even in typical physiological circumstances, postmitotic retinal photoreceptor cells also produce elevated levels of lactate. Initially classified as metabolic waste, lactate has since gained recognition as a significant intracellular signaling mediator and extracellular ligand. This current review endeavors to provide a concise overview and discourse on the following topics: the localization of lactate-producing enzymes, the functional significance of these enzymes, the signaling functions of lactate, and its impact on the gene expression of photoreceptors in retinal cells.

Chromatin Remodeling Enzyme Snf2h Is Essential for Retinal Cell Proliferation and Photoreceptor Maintenance.

Chromatin remodeling complexes are required for many distinct nuclear processes such as transcription, DNA replication, and DNA repair. However, the contribution of these complexes to the development of complex tissues within an organism is poorly characterized. Imitation switch (ISWI) proteins are among the most evolutionarily conserved ATP-dependent chromatin remodeling factors and are represented by yeast Isw1/Isw2, and their vertebrate counterparts Snf2h (Smarca5) and Snf2l (Smarca1). In this study, we focused on the role of the Snf2h gene during the development of the mammalian retina. We show that Snf2h is expressed in both retinal progenitors and post-mitotic retinal cells. Using Snf2h conditional knockout mice (Snf2h cKO), we found that when Snf2h is deleted, the laminar structure of the adult retina is not retained, the overall thickness of the retina is significantly reduced compared with controls, and the outer nuclear layer (ONL) is completely missing. The depletion of Snf2h did not influence the ability of retinal progenitors to generate all the differentiated retinal cell types. Instead, the Snf2h function is critical for the proliferation of retinal progenitor cells. Cells lacking Snf2h have a defective S-phase, leading to the entire cell division process impairments. Although all retinal cell types appear to be specified in the absence of the Snf2h function, cell-cycle defects and concomitantly increased apoptosis in Snf2h cKO result in abnormal retina lamination, complete destruction of the photoreceptor layer, and consequently, a physiologically non-functional retina.

Chromatin remodeler Chd7 regulates photoreceptor development and outer segment length

Mutations in the chromatin remodeling factor CHD7 are the predominant cause of CHARGE syndrome, a congenital disorder that frequently includes ocular coloboma. Although CHD7 is known to be required for proper ocular morphogenesis, its role in retinal development has not been thoroughly investigated. Given that individuals with CHARGE syndrome can experience visual impairment even in the absence of coloboma, a better understanding of CHD7 function in the retina is needed. In this study, we characterized the expression pattern of Chd7 in the developing zebrafish and mouse retina and documented ocular and retinal phenotypes in Chd7 loss-of-function mutants. Zebrafish Chd7 was expressed throughout the retinal neuroepithelium when retinal progenitor cells were actively proliferating, and later in subsets of newly post-mitotic retinal cells. At stages of retinal development when most retinal cell types had terminally differentiated, Chd7 expression remained strong in the ganglion cell layer and in some cells in the inner nuclear layer. Intriguingly, strong expression of Chd7 was also observed in the outer nuclear layer where it was co-expressed with markers of post-mitotic cone and rod photoreceptors. Expression of mouse CHD7 displayed a similar pattern, including expression in the ganglion cells, subsets of inner nuclear layer cells, and in the distal outer nuclear layer as late as P15. Two different mutant chd7 zebrafish lines were characterized for ocular and retinal defects. These mutants displayed microphthalmia, reduced numbers of cone photoreceptors, and truncated rod and cone photoreceptor outer segments. Reduced cone photoreceptor number and abnormal outer segments were also observed in heterozygous Chd7 mutant mice. Taken together, our results in zebrafish and mouse reveal a conserved, previously undescribed role for Chd7 in retinal development and photoreceptor outer segment morphogenesis. Moreover, our work suggests an avenue of future investigation into the pathogenesis of visual system defects in CHARGE syndrome.

Regulation of retinal amacrine cell generation by miR-216b and Foxn3.

The mammalian retina contains a complex mixture of different types of neurons. We find that microRNA miR-216b is preferentially expressed in postmitotic retinal amacrine cells in the mouse retina, and expression of miR-216a/b and miR-217 in retina depend in part on Ptf1a, a transcription factor required for amacrine cell differentiation. Surprisingly, ectopic expression of miR-216b directed the formation of additional amacrine cells and reduced bipolar neurons in the developing retina. We identify the Foxn3 mRNA as a retinal target of miR-216b by Argonaute PAR-CLIP and reporter analysis. Inhibition of Foxn3, a transcription factor, in the postnatal developing retina by RNAi increased the formation of amacrine cells and reduced bipolar cell formation. Foxn3 disruption by CRISPR in embryonic retinal explants also increased amacrine cell formation, whereas Foxn3 overexpression inhibited amacrine cell formation prior to Ptf1a expression. Co-expression of Foxn3 partially reversed the effects of ectopic miR-216b on retinal cell formation. Our results identify Foxn3 as a novel regulator of interneuron formation in the developing retina and suggest that miR-216b likely regulates Foxn3 and other genes in amacrine cells.

Aerobic Glycolysis in the Retina: Functional Roles of Pyruvate Kinase Isoforms.

One hundred years ago, Otto Heinrich Warburg observed that postmitotic retinal cells are the highest oxygen-consuming cells in the body. He compared these cells to actively growing mitotic tumor cells since both cells reprogram glucose for anabolic processes, which include lipid, protein, and RNA/DNA synthesis, and for antioxidant metabolism. To achieve this metabolic reprogramming, cancer cells preferentially express a less active dimeric form, the M2 isoform of pyruvate kinase (PKM2), which shuttles glucose toward the accumulation of glycolytic intermediates that redirect cell activities into anabolic processes. Similar to cancer cells, retinal photoreceptors predominantly express the M2 isoform of PKM2. This isoform performs both metabolic and non-metabolic functions in photoreceptor cells. This review focuses on the metabolic and non-metabolic roles of pyruvate kinases in photoreceptor cell functions.

Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in vivo Genome Editing.

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.

Induction of rod versus cone photoreceptor-specific progenitors from retinal precursor cells

During development, multipotent progenitors undergo temporally-restricted differentiation into post-mitotic retinal cells; however, the mechanisms of progenitor division that occurs during retinogenesis remain controversial. Using clonal analyses (lineage tracing and single cell cultures), we identify rod versus cone lineage-specific progenitors derived from both adult retinal stem cells and embryonic neural retinal precursors. Taurine and retinoic acid are shown to act in an instructive and lineage-restricted manner early in the progenitor lineage hierarchy to produce rod-restricted progenitors from stem cell progeny. We also identify an instructive, but lineage-independent, mechanism for the specification of cone-restricted progenitors through the suppression of multiple differentiation signaling pathways. These data indicate that exogenous signals play critical roles in directing lineage decisions and resulting in fate-restricted rod or cone photoreceptor progenitors in culture. Additional factors may be involved in governing photoreceptor fates in vivo.

The Retinal Pigment Epithelium Is a Notch Signaling Niche in the Mouse Retina

Notch signaling in neural progenitor cell is triggered by ligands expressed in adjacent cells. To identify the sources of active Notch ligands in the mouse retina, we negatively regulated Notch ligand activity in various neighbors of retinal progenitor cells (RPCs) by eliminating mindbomb E3 ubiquitin protein ligase 1 (Mib1). Mib1-deficient retinal cells failed to induce Notch activation in intra-lineage RPCs, which prematurely differentiated into neurons; however, Mib1 in post-mitotic retinal ganglion cells was not important. Interestingly, Mib1 in the retinal pigment epithelium (RPE) also contributed to Notch activationin adjacent RPCs by supporting the localizationofactive Notch ligands at RPE-RPC contacts. Combining this RPE-driven Notch signaling and intra-retinal Notch signaling, we propose a model in which one RPC daughter receives extra Notch signals from the RPE to become an RPC, whereas its sister cell receives only a subthreshold level of intra-retinal Notch signal and differentiates into a neuron.

Toward therapeutic genome editing in post-mitotic retinal cells

Toward therapeutic genome editing in post-mitotic retinal cells

Loss of MAPK Pathway Activation in Post-Mitotic Retinal Cells as Mechanism in MEK Inhibition-Related Retinopathy in Cancer Patients

Recently, treatment with MEK inhibitors has been shown to be an effective treatment option for metastatic melanoma. Treatment efficacy is dependent on inhibition of MAPK-related melanoma proliferation. However, targeting of MEK can be accompanied by a time-dependent and reversible serous retinopathy of unknown origin.We analyzed the molecular mechanism by which the MEK inhibitor binimetinib may lead to retinopathy, using neuroretina and cell models of retinal pigment epithelium (RPE).Binimetinib inhibited the MAPK pathway while discontinuation of treatment resulted in reactivation. However, cell proliferation was not inhibited correspondingly during binimetinib treatment of ARPE19 cells. Remarkably, post-mitotic neuroretinal tissue displayed a strong MAPK activation that was lost after binimetinib treatment.We propose that binimetinib-associated retinopathy is correlated with inhibition of the MAPK pathway in multiple retinal components. Retinal cells are able to regain the activation after binimetinib treatment, mimicking the reversibility of the retinopathy. As most retinal cells are nonregenerating, other mechanisms than stimulation of proliferation must be involved.

LSD1-Mediated Demethylation of H3K4me2 Is Required for the Transition from Late Progenitor to Differentiated Mouse Rod Photoreceptor.

Epigenetic modifiers can work in concert with transcription factors to control the transition of cells from proliferating progenitors into quiescent terminally differentiated cells. This transition involves changes in histone methylation and one of the key regulators of this is the H3K4me2/1 histone demethylase LSD1. Here, we show that the highest expression of LSD1 occurs in postmitotic retinal cells during the peak period of rod photoreceptor differentiation. Pharmacological inhibition of LSD1 in retinal explants cultured from PN1 to PN8 had three major effects. It prevented the normal decrease in expression of genes associated with progenitor function, it blocked rod photoreceptor development, and it increased expression of genes associated with other retinal cell types. The maintained expression of progenitor genes was associated with a maintained level of H3K4me2 over the gene and its promoter. Among the genes whose expression was maintained was Hes1, a repressor known to block rod photoreceptor development. The inhibition of rod photoreceptor gene expression occurred in spite of the normal expression of transcription factors CRX and NRL, and the normal accumulation of H3K4me2 marks over the promoter and gene body. We suggest that LSD1 acts in concert with a series of nuclear receptors to modify chromatin structure and repress progenitor genes as well as to inhibit ectopic patterns of gene expression in the differentiating postmitotic retinal cells.

Developmental regulation of linkers of the nucleoskeleton to the cytoskeleton during mouse postnatal retinogenesis

Sun proteins and nesprins are two families of proteins whose direct interactions across the nuclear envelope provide for the core of linkers of the nucleoskeleton to the cytoskeleton (LINC complexes) that physically connect the nucleus interior to cytoskeletal networks. Whereas LINC complexes play essential roles in nuclear migration anchorage and underlie normal CNS development, the developmental regulation of their composition remains largely unknown. In this study, we examined the spatiotemporal expression of lamins, Sun proteins and nesprins during postnatal mouse retinal development. Whereas retinal precursor cells mostly express B-type lamins, Sun1, and high molecular weight isoforms of nesprins, post-mitotic retinal cells are characterized by a drastic downregulation of the latter, the expression of A-type lamins, and the strong induction of a specific isoform of nesprin1 late in retinal development. Importantly, our results emphasize different spatiotemporal expression for nesprin1 and nesprin2 and further suggest an important role for KASH-less isoforms of nesprin1 in the CNS. In conclusion, the transition from retinal precursor cells undergoing interkinetic nuclear migration to post-mitotic retinal cells undergoing nuclear translocation and/or anchorage is accompanied by a profound remodeling of LINC complexes composition. This remodeling may reflect different requirements of nuclear dynamics at different stages of CNS development.

Notch1 is required in newly postmitotic cells to inhibit the rod photoreceptor fate

Several models of cell fate determination can be invoked to explain how single retinal progenitor cells (RPCs) produce different cell types in a terminal division. To gain insight into this process, the effects of the removal of a cell fate regulator, Notch1, were studied in newly postmitotic cells using a conditional allele of Notch1 (N1-CKO) in mice. Almost all newly postmitotic N1-CKO cells became rod photoreceptors, whereas wild-type (WT) cells achieved a variety of fates. Single cell profiling of wild-type and N1-CKO retinal cells transitioning from progenitor to differentiated states revealed differential expression of inhibitor of DNA binding factors Id1 and Id3, as well as Notch-regulated ankyrin repeat protein (Nrarp). Misexpression of Id1 and Id3 was found to be sufficient to drive production of Müller glial cells and/or RPCs. Moreover, Id1 and Id3 were shown to partially rescue the production of bipolar and Müller glial cells in the absence of Notch1 in mitotic and newly postmitotic cells. Misexpression of Nrarp, a downstream target gene and inhibitor of the Notch signaling pathway, resulted in the overproduction of rod photoreceptors at the expense of Müller glial cells. These data demonstrate that cell fate decisions can be made in newly postmitotic retinal cells, and reveal some of the regulators downstream of Notch1 that influence the choice of rod and non-rod fates. Taken together, our results begin to address how different signals downstream from a common pathway lead to different fate outcomes.

Pαx6 Expression in Postmitotic Neurons Mediates the Growth of Axons in Response to SFRP1

During development, the mechanisms that specify neuronal subclasses are coupled to those that determine their axonal response to guidance cues. Pax6 is a homedomain transcription factor required for the specification of a variety of neural precursors. After cell cycle exit, Pax6 expression is often shut down in the precursor progeny and most postmitotic neurons no longer express detectable levels of the protein. There are however exceptions and high Pax6 protein levels are found, for example, in postmitotic retinal ganglion cells (RGCs), dopaminergic neurons of the olfactory bulb and the limbic system in the telencephalon. The function of Pax6 in these differentiating neurons remains mostly elusive. Here, we demonstrate that Pax6 mediates the response of growing axons to SFRP1, a secreted molecule expressed in several Pax6-positive forebrain territories. Forced expression of Pax6 in cultured postmitotic cortical neurons, which do not normally express Pax6, was sufficient to increment axonal length. Growth was blocked by the addition of anti-SFRP1 antibodies, whereas exogenously added SFRP1 increased axonal growth of Pax6-transfected neurons but not that of control or untransfected cortical neurons. In the reverse scenario, shRNA-mediated knock-down of Pax6 in mouse retinal explants specifically abolished RGCs axonal growth induced by SFRP1, but had no effect on RGCs differentiation and it did not modify the effect of Shh or Netrin on axon growth. Taken together these results demonstrate that expression of Pax6 is necessary and sufficient to render postmitotic neurons competent to respond to SFRP1. These results reveal a novel and unexpected function of Pax6 in postmitotic neurons and situate Pax6 and SFRP1 as pair regulators of axonal connectivity.

The long noncoding RNA RNCR2 directs mouse retinal cell specification

BackgroundRecent work has identified that many long mRNA-like noncoding RNAs (lncRNAs) are expressed in the developing nervous system. Despite their abundance, the function of these ncRNAs has remained largely unexplored. We have investigated the highly abundant lncRNA RNCR2 in regulation of mouse retinal cell differentiation.ResultsWe find that the RNCR2 is selectively expressed in a subset of both mitotic progenitors and postmitotic retinal precursor cells. ShRNA-mediated knockdown of RNCR2 results in an increase of both amacrine cells and Müller glia, indicating a role for this lncRNA in regulating retinal cell fate specification. We further report that RNCR2 RNA, which is normally nuclear-retained, can be exported from the nucleus when fused to an IRES-GFP sequence. Overexpression of RNCR2-IRES-GFP phenocopies the effects of shRNA-mediated knockdown of RNCR2, implying that forced mislocalization of RNCR2 induces a dominant-negative phenotype. Finally, we use the IRES-GFP fusion approach to identify specific domains of RNCR2 that are required for repressing both amacrine and Müller glial differentiation.ConclusionThese data demonstrate that the lncRNA RNCR2 plays a critical role in regulating mammalian retinal cell fate specification. Furthermore, we present a novel approach for generating dominant-negative constructs of lncRNAs, which may be generally useful in the functional analysis of this class of molecules.

Non-SMC condensin I complex proteins control chromosome segregation and survival of proliferating cells in the zebrafish neural retina

BackgroundThe condensation of chromosomes and correct sister chromatid segregation during cell division is an essential feature of all proliferative cells. Structural maintenance of chromosomes (SMC) and non-SMC proteins form the condensin I complex and regulate chromosome condensation and segregation during mitosis. However, due to the lack of appropriate mutants, the function of the condensin I complex during vertebrate development has not been described.ResultsHere, we report the positional cloning and detailed characterization of retinal phenotypes of a zebrafish mutation at the cap-g locus. High resolution live imaging reveals that the progression of mitosis between prometa- to telophase is delayed and that sister chromatid segregation is impaired upon loss of CAP-G. CAP-G associates with chromosomes between prometa- and telophase of the cell cycle. Loss of the interaction partners CAP-H and CAP-D2 causes cytoplasmic mislocalization of CAP-G throughout mitosis. DNA content analysis reveals increased genomic imbalances upon loss of non-SMC condensin I subunits. Within the retina, loss of condensin I function causes increased rates of apoptosis among cells within the proliferative ciliary marginal zone (CMZ) whereas postmitotic retinal cells are viable. Inhibition of p53-mediated apoptosis partially rescues cell numbers in cap-g mutant retinae and allows normal layering of retinal cell types without alleviating their aberrant nuclear sizes.ConclusionOur findings indicate that the condensin I complex is particularly important within rapidly amplifying progenitor cell populations to ensure faithful chromosome segregation. In contrast, differentiation of postmitotic retinal cells is not impaired upon polyploidization.

Influence of microglia on retinal progenitor cell turnover and cell replacement

Microglia within the retina are continually replaced from the bone marrow and are the resident myeloid-derived cells within the retina. Throughout life, microglial function is conditioned by the microenvironment affording immunomodulation to control inflammation as well as functioning to enable normal development and, during adulthood, maintain normal retinal function. In adulthood, recent evidence supports the concept that the retina continues to replace cells to maintain optimal function. Although in some cases after injury, degeneration, or inflammation there remains an inextricable decline in visual function inferring a deficit in cell replacement, the deficit could be explained by microglial cell activation influencing the ability of either retinal progenitor cells or recruited progenitor cells to integrate and differentiate appropriately. Myeloid cell response differs depending on insult: it is evident that during inflammation microglia and the infiltrating myeloid cell function are conditioned by the cytokine environment. Indeed, modulating myeloid cell function therapeutically suppresses disease in experimental models of autoimmunity, whereas in non-inflammatory models microglia have little or no effect on the course of degeneration. The extent of myeloid activation can help determine retinal progenitor cell turnover. Retinal progenitor cells may be isolated from adult human retina, which, albeit limited, display mitotic activity and can differentiate. Microglial activation secreting IL-6 limits progenitor cell turnover and the extent to which differentiation to post-mitotic retinal cells occurs. Such experimental data illustrate the need to develop methods to replenish normal retinal myeloid cell function facilitating integration, either by cell transplantation or by encouraging retinal progenitor cells to recover retinal function.

ISL1 and BRN3B co-regulate the differentiation of murine retinal ganglion cells

LIM-homeodomain (HD) and POU-HD transcription factors play crucial roles in neurogenesis. However, it remains largely unknown how they cooperate in this process and what downstream target genes they regulate. Here, we show that ISL1, a LIM-HD protein, is co-expressed with BRN3B, a POU-HD factor, in nascent post-mitotic retinal ganglion cells (RGCs). Similar to the Brn3b-null retinas, retina-specific deletion of Isl1 results in the apoptosis of a majority of RGCs and in RGC axon guidance defects. The Isl1 and Brn3b double null mice display more severe retinal abnormalities with a near complete loss of RGCs, indicating the synergistic functions of these two factors. Furthermore, we show that both Isl1 and Brn3b function downstream of Math5 to regulate the expression of a common set of RGC-specific genes. Whole-retina chromatin immunoprecipitation and in vitro transactivation assays reveal that ISL1 and BRN3B concurrently bind to and synergistically regulate the expression of a common set of RGC-specific genes. Thus, our results uncover a novel regulatory mechanism of BRN3B and ISL1 in RGC differentiation.

Notch activity permits retinal cells to progress through multiple progenitor states and acquire a stem cell property

Signaling through the Notch pathway regulates multiple aspects of development. The vertebrate retina allows an investigation of the basis for these various effects, because the major cell types of the retina arise from a common progenitor that expresses Notch1. The Notch pathway was constitutively activated in distinct populations of retinal cells during development. Prolonged Notch activity in progenitor cells maintained cells in the progenitor state without perturbing temporal identity, promoting early progenitor characteristics early in development and late progenitor characteristics later in development. Eventually, constitutive Notch activation led these cells to acquire characteristics of glial and stem cells. In contrast, reactivating the Notch pathway in newly postmitotic retinal cells promoted mature glial cell formation in a subset of cells. These data suggest that prolonged Notch activity does not disrupt the normal progression of progenitor temporal states, and that down-regulating or overcoming Notch activity is required for proper formation of both neuronal and glial cell fates.

Effect of p75 NTR on the regulation of naturally occurring cell death and retinal ganglion cell number in the mouse eye

Neurotrophins induce neural cell survival and differentiation during retinal development and regeneration through the high-affinity tyrosine kinase (Trk) receptors. On the other hand, nerve growth factor (NGF) binding to the low-affinity neurotrophin receptor p75 (p75 NTR) might induce programmed cell death (PCD) in the early phase of retinal development. In the present study, we examined the retinal cell types that experience p75 NTR-induced PCD and identify them to be postmitotic retinal ganglion cells (RGCs). However, retinal morphology, RGC number, and BrdU-positive cell number in p75 NTR knockout (KO) mouse were normal after embryonic day 15 (E15). In chick retina, migratory RGCs express p75 NTR, whereas layered RGCs express the high-affinity NGF receptor TrkA, which may switch the pro-apoptotic signaling of p75 NTR into a neurotrophic one. In contrast to the chick model, migratory RGCs express TrkA, while stratified RGCs express p75 NTR in mouse retina. However, RGC number in TrkA KO mouse was also normal at birth. We next examined the expression of transforming growth factor β (TGFβ) receptor, which modulates chick RGC number in combination with p75 NTR, but was absent in mouse RGCs. p75 NTR and TrkA seem to be involved in the regulation of mouse RGC number in the early phase of retinal development, but the number may be later adjusted by other molecules. These results suggest the different mechanism of RGC number control between mouse and chick retina.