Stimulation of functional neuronal regeneration from Müller glia in adult mice.

Many retinal diseases lead to the loss of retinal neurons and cause visual impairment. The adult mammalian retina has little capacity for regeneration. By contrast, teleost fish functionally regenerate their retina following injury, and Müller glia (MG) are the source of regenerated neurons. The proneural transcription factor Ascl1 is upregulated in MG after retinal damage in zebrafish and is necessary for regeneration. Although Ascl1 is not expressed in mammalian MG after injury, forced expression of Ascl1 in mouse MG induces a neurogenic state in vitro and in vivo after NMDA (N-methyl-d-aspartate) damage in young mice. However, by postnatal day 16, mouse MG lose neurogenic capacity, despite Ascl1 overexpression. Loss of neurogenic capacity in mature MG is accompanied by reduced chromatin accessibility, suggesting that epigenetic factors limit regeneration. Here we show that MG-specific overexpression of Ascl1, together with a histone deacetylase inhibitor, enables adult mice to generate neurons from MG after retinal injury. The MG-derived neurons express markers of inner retinal neurons, synapse with host retinal neurons, and respond to light. Using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we show that the histone deacetylase inhibitor promotes accessibility at key gene loci in the MG, and allows more effective reprogramming. Our results thus provide a new approach for the treatment of blinding retinal diseases.

MicroRNAs miR-25, let-7 and miR-124 regulate the neurogenic potential of Müller glia in mice.

Müller glial cells (MG) generate retinal progenitor (RPC)-like cells after injury in non-mammalian species, although this does not occur in the mammalian retina. Studies have profiled gene expression in these cells to define genes that may be relevant to their differences in neurogenic potential. However, less is known about differences in micro-RNA (miRNA) expression. In this study, we compared miRNAs from RPCs and MG to identify miRNAs more highly expressed in RPCs, and others more highly expressed in MG. To determine whether these miRNAs are relevant to the difference in neurogenic potential between these two cell types, we tested them in dissociated cultures of MG using either mimics or antagomiRs to increase or reduce expression, respectively. Among the miRNAs tested, miR-25 and miR-124 overexpression, or let-7 antagonism, induced Ascl1 expression and conversion of ∼40% of mature MG into a neuronal/RPC phenotype. Our results suggest that the differences in miRNA expression between MG and RPCs contribute to their difference in neurogenic potential, and that manipulations in miRNAs provide a new tool with which to reprogram MG for retinal regeneration.

Cell-replacement therapy and neural repair in the retina.

Visual impairment severely affects the quality of life of patients and their families and is also associated with a deep economic impact. The most common pathologies responsible for visual impairment and legally defined blindness in developed countries include age-related macular degeneration, glaucoma and diabetic retinopathy. These conditions share common pathophysiological features: dysfunction and loss of retinal neurons. To date, two main approaches are being taken to develop putative therapeutic strategies: neuroprotection and cell replacement. Cell replacement is a novel therapeutic approach to restore visual capabilities to the degenerated adult neural retina and represents an emerging field of regenerative neurotherapy. The discovery of a population of proliferative cells in the mammalian retina has raised the possibility of harnessing endogenous retinal stem cells to elicit retinal repair. Furthermore, the development of suitable protocols for the reprogramming of differentiated somatic cells to a pluripotent state further increases the therapeutic potential of stem-cell-based technologies for the treatment of major retinal diseases. Stem-cell transplantation in animal models has been most effectively used for the replacement of photoreceptors, although this therapeutic approach is also being used for inner retinal pathologies. In this review, we discuss recent advances in the development of cell-replacement approaches for the treatment of currently incurable degenerative retinal diseases.

Primary Cell Cultures to Study the Regeneration Potential of Murine Müller Glia after MicroRNA Treatment.

Müller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates such as fish, MG-driven regeneration occurs naturally; in mammals, however, stimulation with certain factors or genetic/epigenetic manipulation is required. Since MG comprise only 5% of the retinal cell population, there is a need for model systems that allow the study of this cell population exclusively. One of these model systems is primary MG cultures that are reproducible and can be used for a variety of applications, including molecule/factor screening and identification, testing of compounds or factors, cell monitoring, and/or functional tests. This model is used to study the potential of murine MG to convert into retinal neurons after supplementation or inhibition of microRNAs (miRNAs) via transfection of artificial miRNAs or their inhibitors. The use of MG-specific reporter mice in combination with immunofluorescent labeling and single-cell RNA sequencing (scRNA-seq) confirmed that 80%-90% of the cells found in these cultures are MG. Using this model, it was discovered that miRNAs can reprogram MG into retinal progenitor cells (RPCs), which subsequently differentiate into neuronal-like cells. The advantages of this technique are that miRNA candidates can be tested for their efficiency and outcome before their usage in in vivo applications.

A Comparative Analysis of Reactive Müller Glia Gene Expression After Light Damage and microRNA-Depleted Müller Glia-Focus on microRNAs.

Müller glia (MG) are the predominant glia in the neural retina and become reactive after injury or in disease. microRNAs (miRNAs) are translational repressors that regulate a variety of processes during development and are required for MG function. However, no data is available about the MG miRNAs in reactive gliosis. Therefore, in this study, we aimed to profile miRNAs and mRNAs in reactive MG 7 days after light damage. Light damage was performed for 8 h at 10,000 lux; this leads to rapid neuronal loss and strong MG reactivity. miRNAs were profiled using the Nanostring platform, gene expression analysis was conducted via microarray. We compared the light damage dataset with the dataset of Dicer deleted MG in order to find similarities and differences. We found: (1) The vast majority of MG miRNAs declined in reactive MG 7 days after light damage. (2) Only four miRNAs increased after light damage, which included miR-124. (3) The top 10 genes found upregulated in reactive MG after light damage include Gfap, Serpina3n, Ednrb and Cxcl10. (4) The miRNA decrease in reactive MG 7 days after injury resembles the profile of Dicer-depleted MG after one month. (5) The comparison of both mRNA expression datasets (light damage and Dicer-cKO) showed 1,502 genes were expressed under both conditions, with Maff , Egr2, Gadd45b, and Atf3 as top upregulated candidates. (6) The DIANA-TarBase v.8 miRNA:RNA interaction tool showed that three miRNAs were found to be present in all networks, i.e., after light damage, and in the combined data set; these were miR-125b-5p, let-7b and let-7c. Taken together, results show there is an overlap of gene regulatory events that occur in reactive MG after light damage (direct damage of neurons) and miRNA-depleted MG (Dicer-cKO), two very different paradigms. This suggests that MG miRNAs play an important role in a ubiquitous MG stress response and manipulating these miRNAs could be a first step to attenuate gliosis.

Developmental changes in the accessible chromatin, transcriptome and Ascl1-binding correlate with the loss in Müller Glial regenerative potential.

Diseases and damage to the retina lead to losses in retinal neurons and eventual visual impairment. Although the mammalian retina has no inherent regenerative capabilities, fish have robust regeneration from Müller glia (MG). Recently, we have shown that driving expression of Ascl1 in adult mouse MG stimulates neural regeneration. The regeneration observed in the mouse is limited in the variety of neurons that can be derived from MG; Ascl1-expressing MG primarily generate bipolar cells. To better understand the limits of MG-based regeneration in mouse retinas, we used ATAC- and RNA-seq to compare newborn progenitors, immature MG (P8-P12), and mature MG. Our analysis demonstrated developmental differences in gene expression and accessible chromatin between progenitors and MG, primarily in neurogenic genes. Overexpression of Ascl1 is more effective in reprogramming immature MG, than mature MG, consistent with a more progenitor-like epigenetic landscape in the former. We also used ASCL1 ChIPseq to compare the differences in ASCL1 binding in progenitors and reprogrammed MG. We find that bipolar-specific accessible regions are more frequently linked to bHLH motifs and ASCL1 binding. Overall, our analysis indicates a loss of neurogenic gene expression and motif accessibility during glial maturation that may prevent efficient reprogramming.

Frataxin overexpression in Müller cells protects retinal ganglion cells in a mouse model of ischemia/reperfusion injury in vivo.

Müller cells are critical for retinal function and neuronal survival but can become detrimental in response to retinal ischemia and increased oxidative stress. Elevated oxidative stress increases expression of the mitochondrial enzyme frataxin in the retina, and its overexpression is neuroprotective after ischemia. Whether frataxin expression in Müller cells might improve their function and protect neurons after ischemia is unknown. The aim of this study was to evaluate the effect of frataxin overexpression in Müller cells on neuronal survival after retinal ischemia/reperfusion in the mouse in vivo. Retinal ischemia/reperfusion was induced in mice overexpressing frataxin in Müller cells by transient elevation of intraocular pressure. Retinal ganglion cells survival was determined 14 days after lesion. Expression of frataxin, antioxidant enzymes, growth factors and inflammation markers was determined with qRT-PCR, Western blotting and immunohistochemistry 24 hours after lesion. Following lesion, there was a 65% increase in the number of surviving RGCs in frataxin overexpressing mice. Improved survival was associated with increased expression of the antioxidant enzymes Gpx1 and Sod1 as well as the growth factors Cntf and Lif. Additionally, microglial activation was decreased in these mice. Therefore, support of Müller cell function constitutes a feasible approach to reduce neuronal degeneration after ischemia.

Müller glial microRNAs are required for the maintenance of glial homeostasis and retinal architecture.

To better understand the roles of microRNAs in glial function, we used a conditional deletion of Dicer1 (Dicer-CKOMG) in retinal Müller glia (MG). Dicer1 deletion from the MG leads to an abnormal migration of the cells as early as 1 month after the deletion. By 6 months after Dicer1 deletion, the MG form large aggregations and severely disrupt normal retinal architecture and function. The most highly upregulated gene in the Dicer-CKOMG MG is the proteoglycan Brevican (Bcan) and overexpression of Bcan results in similar aggregations of the MG in wild-type retina. One potential microRNA that regulates Bcan is miR-9, and overexpression of miR-9 can partly rescue the effects of Dicer1 deletion on the MG phenotype. We also find that MG from retinitis pigmentosa patients display an increase in Brevican immunoreactivity at sites of MG aggregation, linking the retinal remodeling that occurs in chronic disease with microRNAs.

The microRNA expression profile of mouse Müller glia in vivo and in vitro.

The profile of miRNAs in mature glia is not well characterized, and most studies have been done in cultured glia. In order to identify the miRNAs in adult and young (postnatal day 11/12) Müller glia of the neural retina, we isolated the Müller glia from Rlbp-CreER: Stopf/f-tdTomato mice by means of fluorescent activated cell sorting and analyzed their miRNAs using NanoStrings Technologies®. In freshly isolated adult Müller glia, we identified 7 miRNAs with high expression levels in the glia, but very low levels in the retinal neurons. These include miR-204, miR-9, and miR-125-5p. We also found 15 miRNAs with high levels of expression in both neurons and glia, and many miRNAs that were enriched in neurons and expressed at lower levels in Müller glia, such as miR-124. We next compared miRNA expression of acutely isolated Müller glia with those that were maintained in dissociated culture for 8 and 14 days. We found that most miRNAs declined in vitro. Interestingly, some miRNAs that were not highly expressed in adult Müller glia increased in cultured cells. Our results thus show the miRNA profile of adult Müller glia and the effects of cell culture on their levels.

Neurogenic potential of stem/progenitor-like cells in the adult mammalian eye.

The neural retina as part of the brain has received a great deal of attention since quiescent neural stem cells/progenitor cells (NSC/PCs) were discovered in this non-neurogenic region. Herein, we particularly feature the adult rodent eye and provide an overview of all putative neuronal progenitor-like cells attributed to the various ocular areas that have been identified during the last decade. These neuronal progenitor-like cells include the pigmented cells of the ciliary body (CB), as well as the pigmented cells of the iris and the retinal pigment epithelium (RPE). Within the retina, the Müller cells, the specialized macroglia of the vertebrate eye, display neurogenic potential, i.e. de-differentiation into retinal neurons following exogenous stimulation. In addition, retinal astrocytes, which are immigrants from the brain and do not arise from a common retinal progenitor show signs of de-differentiation after injury. Interestingly, microglial cells, the immune competent cells of the central nervous system (CNS), feature neurogenic potential in vitro. Moreover, it appears that this potential can also be initially induced by injury in vivo, both in the brain and the retina. This review summarizes characteristics of various endogenous progenitor-like cells reported in in vitro and in vivo studies. A focus is placed on in vivo studies with a special regard to cellular responses after exogenous stimulation, such as growth factor treatment or injury. Finally, we discuss therapeutic potential of these cells with respect to cell replacement strategies and putative clinical application.

In situ dividing and phagocytosing retinal microglia express nestin, vimentin, and NG2 in vivo.

BACKGROUND: Following injury, microglia become activated with subsets expressing nestin as well as other neural markers. Moreover, cerebral microglia can give rise to neurons in vitro. In a previous study, we analysed the proliferation potential and nestin re-expression of retinal macroglial cells such as astrocytes and Müller cells after optic nerve (ON) lesion. However, we were unable to identify the majority of proliferative nestin(+) cells. Thus, the present study evaluates expression of nestin and other neural markers in quiescent and proliferating microglia in naïve retina and following ON transection in adult rats in vivo. METHODOLOGY/PRINCIPAL FINDINGS: For analysis of cell proliferation and cells fates, rats received BrdU injections. Microglia in retinal sections or isolated cells were characterized using immunofluorescence labeling with markers for microglia (e.g., Iba1, CD11b), cell proliferation, and neural cells (e.g., nestin, vimentin, NG2, GFAP, Doublecortin etc.). Cellular analyses were performed using confocal laser scanning microscopy. In the naïve adult rat retina, about 60% of resting ramified microglia expressed nestin. After ON transection, numbers of nestin(+) microglia peaked to a maximum at 7 days, primarily due to in situ cell proliferation of exclusively nestin(+) microglia. After 8 weeks, microglia numbers re-attained control levels, but 20% were still BrdU(+) and nestin(+), although no further local cell proliferation occurred. In addition, nestin(+) microglia co-expressed vimentin and NG2, but not GFAP or neuronal markers. Fourteen days after injury and following retrograde labeling of retinal ganglion cells (RGCs) with Fluorogold (FG), nestin(+)NG2(+) microglia were positive for the dye indicating an active involvement of a proliferating cell population in phagocytosing apoptotic retinal neurons. CONCLUSIONS/SIGNIFICANCE: The current study provides evidence that in adult rat retina, a specific resident population of microglia expresses proteins of immature neural cells that are involved in injury-induced cell proliferation and phagocytosis while transdifferentiation was not observed.

Proliferative response of microglia and macrophages in the adult mouse eye after optic nerve lesion.

PURPOSE: The purpose of this in vivo study was to evaluate the proliferative response of immunologic cells during the acute phase after optic nerve (ON) lesion in the neural retina and the ciliary body (CB) in the adult mouse. METHODS: The number of cells obtained 5 to 10 days after ON crush was compared with that counted after intraorbital ON transection. In addition, after ON crush, the time course of in situ proliferating Ki67(+) microglia and macrophages was analyzed from 6 hours up to 10 days. RESULTS: The number of BrdU(+)F4/80(+) retinal microglia and ciliary macrophages increased over time, reaching the peak number 10 days after ON lesion. In the retina, both ON lesion types resulted in a similar number of BrdU(+)F4/80(+) microglia. Approximately 85% of all BrdU(+) cells were identified as F4/80(+) microglia. However, this cell population represented only 30% of all F4/80(+) microglia. The peak of microglial in situ proliferation was found 2 days after ON crush. In the CB, both ON lesion types induced a significant increase in the number of BrdU(+)F4/80(+) macrophages. Of interest, the number of cells after ON transection further increased over time, whereas those after ON crush did not. CONCLUSIONS: ON lesion significantly increased proliferation of F4/80(+) immunologic cells in both the retina and CB. Although no significant differences in cellular response were observed in the retina between both lesion types, ON transection had a more pronounced effect on ciliary macrophages than did ON crush. Therefore, both regions seem not to act in concert during the acute phase after ON lesion.

Optic nerve lesion increases cell proliferation and nestin expression in the adult mouse eye in vivo.

In the naïve adult rodent eye cell proliferation does not occur. The aim of this in vivo study was to evaluate if quiescent putative progenitor-like cells within the adult mouse eye can be activated by optic nerve (ON) injury. For a comprehensive analysis, three areas were assessed: the ON, the neural retina, and the ciliary body (CB). Two lesion types were performed, i.e. intraorbital ON transection, or ON crush lesion, in order to analyse possible differences in cellular response after injury. This mouse study shows, for the first time that ON lesion up-regulates cell proliferation and nestin expression in the mouse eye as compared to naïve controls. Numbers and distribution patterns of BrdU+ cells obtained were similar after both lesion types, suggesting analogous mechanisms of activation. Interestingly, a differential cell proliferative response was observed in the CB. After ON lesion, the absence of BrdU/TUNEL co-labelled cells confirmed that BrdU+ cells were indeed proliferating. Following ON lesion, in the retina approximately 18% of all BrdU+ cells were positive for the neural stem cell/progenitor cell (NSC/PC) marker nestin. The fraction of BrdU+/nestin+ cells in the CB was approximately 26%. Most of the BrdU+/nestin+ cells found in the neural retina were identified as reactive astrocytes and Müller cells. Since reactive glia cells can participate in adult neuro- and gliogenesis this may indicate a potential for regeneration after ON lesion in vivo.