Expression of beta-amyloid precursor and Bcl-2 proto-oncogene proteins in rat retinas after intravitreal injection of aminoadipic acid.

In order to investigate the role of glia in relation to factors that affect the expression of beta-amyloid precursor protein (betaAPP) and B cell lymphoma oncogene protein (Bcl-2) in the central nervous tissue, the patterns of expression of betaAPP and Bcl-2 in developing and mature rat retinas were studied immunocytochemically after intravitreal injection of alpha-aminoadipic acid (alpha-AAA), a glutamate analogue and gliotoxin that is known to cause injury of retinal Müller glial cells. In normal developing retinas, betaAPP and Bcl-2 were expressed primarily but transiently in a small number of neurons in the ganglion cell layer during the first postnatal week. Immunoreactivity of betaAPP and Bcl-2 appeared in the endfeet and proximal part of the radial processes of Müller glial cells from the second postnatal week onwards. In rats that received intravitreal injection of alpha-AAA at birth, there was a loss of immunoreactivity to vimentin, and a delayed expressed on betaAPP or Bcl-2 in Muller glial cells until 3-5 weeks post-injection. Immunoreactive neurons were also observed in the inner retina especially in the ganglion cell layer from 5 to 35 days after injection. A significant reduction in numerical density of cells with large somata in the ganglion cell layer was observed in the neonatally injected retinas at P56, which was accompanied by an increased immunostaining in radial processes of Müller glial cells. In contrast, no detectable changes in the expression of betaAPP and Bcl-2 were observed in retina that received alpha-AAA as adults. These results indicate that the gliotoxin alpha-AAA has long lasting effects on the expression of betaAPP and Bcl-2 in Müller glial cells as well as neurons in the developing but not mature retinas. The loss of vimentin and delayed expression of betaAPP and Bcl-2 in developing Müller glial cells suggests that the metabolic integrity of Müller cells was temporarily compromised, which may have adverse effects on developing neurons that are vulnerable or dependent on trophic support from the Müller glial cells.     

Neural stem cell properties of Müller glia in the mammalian retina: regulation by Notch and Wnt signaling

The retina in adult mammals, unlike those in lower vertebrates such as fish and amphibians, is not known to support neurogenesis. However, when injured, the adult mammalian retina displays neurogenic changes, raising the possibility that neurogenic potential may be evolutionarily conserved and could be exploited for regenerative therapy. Here, we show that Müller cells, when retrospectively enriched from the normal retina, like their radial glial counterparts in the central nervous system (CNS), display cardinal features of neural stem cells (NSCs), i.e., they self-renew and generate all three basic cell types of the CNS. In addition, they possess the potential to generate retinal neurons, both in vitro and in vivo. We also provide direct evidence, by transplanting prospectively enriched injury-activated Müller cells into normal eye, that Müller cells have neurogenic potential and can generate retinal neurons, confirming a hypothesis, first proposed in lower vertebrates. This potential is likely due to the NSC nature of Müller cells that remains dormant under the constraint of non-neurogenic environment of the adult normal retina. Additionally, we demonstrate that the mechanism of activating the dormant stem cell properties in Müller cells involves Wnt and Notch pathways. Together, these results identify Müller cells as latent NSCs in the mammalian retina and hence, may serve as a potential target for cellular manipulation for treating retinal degeneration.     

Sonic hedgehog promotes stem-cell potential of Müller glia in the mammalian retina

Müller glia have been demonstrated to display stem-cell properties after retinal damage. Here, we report this potential can be regulated by Sonic hedgehog (Shh) signaling. Shh can stimulate proliferation of Müller glia through its receptor and target gene expressed on them, furthermore, Shh-treated Müller glia are induced to dedifferentiate by expressing progenitor-specific markers, and then adopt cell fate of rod photoreceptor. Inhibition of signaling by cyclopamine inhibits proliferation and dedifferentiation. Intraocular injection of Shh promotes Müller glia activation in the photoreceptor-damaged retina, Shh also enhances neurogenic potential by producing more rhodopsin-positive photoreceptors from Müller glia-derived cells. Together, these results provide evidences that Müller glia act as potential stem cells in mammalian retina, Shh may have therapeutic effects on these cells for promoting the regeneration of retinal neurons.     

Morphological differentiation of the Müller cell: Golgi and electron microscopy study in the chick retina

The sequence of morphological differentiation of Müller cells in the chick retina was investigated in relation to the differentiation of the retinal neurons using the Golgi method. From the beginning of differentiation, the Müller cell develops spurs and lateral processes. Some of these glial processes become transformed into accessory prolongations of the Müller cell. From the 17th or 18th day of incubation, the morphology of the Müller cells is similar to that of the adult retina. On the basis of their inner prolongation, two types of Müller cells were identified. The first type, with diffuse and abundant descending processes, is identical to that described classically. The second type is a cell characterized by sparse and scanty inner ramifications. This report also describes electron microscopic observations of Müller cells and their enwrapping relationship with the axons of the optic nerve fiber layer.     

Role of Müller glia in neuroprotection and regeneration in the retina

Glial cells are thought to protect neurons from various neurological insults. When there is injury to retina, Müller cells, which are the predominant glial element in the retina, undergo significant morphological, cellular and molecular changes. Some of these changes reflect Müller cell involvement in protecting the retina from further damage. Müller cells express growth factors, neurotransmitter transporters and antioxidant agents that could have an important role in preventing excitotoxic damage to retinal neurons. Moreover, Müller cells contact to endothelial cells to facilitate the neovascularization process during hypoxic conditions. Finally, recent studies have pointed to a role of Müller cells in retina regeneration after damage, dedifferentiating to progenitor cells and then giving rise to different neuronal cell types. In this article we will review the role of Müller glia in neuroprotection and regeneration after damage in the retina.     

The nephroblastoma overexpressed gene (NOV/ccn3) protein associates with Notch1 extracellular domain and inhibits myoblast differentiation via Notch signaling pathway

We demonstrate a novel interaction of the nephroblastoma overexpressed gene (NOV), a member of the CCN gene family, with the Notch signaling pathway. NOV associates with the epidermal growth factor-like repeats of Notch1 by the CT (C-terminal cysteine knot) domain. The promoters of HES1 and HES5, which are the downstream transducers of Notch signaling, were activated by NOV. Expressions of NOV and Notch1 were concomitant in the presomitic mesoderm and later in the myocytes and chondrocytes, suggesting their synergistic effects in mesenchymal cell differentiation. In C2/4 myogenic cells, elevated expression of NOV led to down-regulation of MyoD and myogenin, resulting in inhibition of myotube formation. These results indicate that NOV-Notch1 association exerts a positive effect on Notch signaling and consequently suppresses myogenesis.     

Identification of CD44 as a cell surface marker for Müller glia precursor cells

In the retina, both neurons and glia differentiate from a common progenitor population. CD44 cell surface antigen is a hyaluronic acid receptor expressed on mature Müller glial cells. We found that in the developing mouse retina, expression of CD44 was transiently observed at or around birth in a subpopulation of c-kit-positive retinal progenitor cells. During in vitro culture, purified CD44/c-kit-positive retinal progenitor cells exclusively differentiated into Müller glial cells and not into neurons, suggesting that CD44 marks a subpopulation of retinal progenitor cells that are fated to become glia. Over-expression of CD44 inhibited the extension of processes by Müller glial cells and neurons. Notch signaling is known to be involved in the specification of retinal progenitors into a glial fate. Activation of Notch signaling increased the number of CD44-positive cells, and treatment with the Notch signal inhibitor, DAPT, at early, but not later, stages of retinal development abolished both CD44-positive cells and Müller glial cells. Together, CD44 was identified as an early cell surface marker of the Müller glia lineage, and Notch signalling was involved in commitment of retinal progenitor cells to CD44 positive Müller glial precursor cells.     

Atoh7 promotes retinal Müller cell differentiation into retinal ganglion cells

Glaucoma is one of the leading eye diseases due to the death of retinal ganglion cells. Increasing evidence suggests that retinal Müller cells exhibit the characteristics of retinal progenitor cells and can differentiate to neurons in injured retinas under certain conditions. However, the number of ganglion cells differentiated from retinal Müller cells falls far short of therapeutic needs. This study aimed to promote the differentiation of retinal Müller cells into ganglion cells by introducing Atoh7 into the stem cells dedifferentiated from retinal Müller cells. Rat retinal Müller cells were isolated and dedifferentiated into stem cells, which were transfected with PEGFP-N1 or PEGFP-N1-Atoh7 vector, and then further induced to differentiate into ganglion cells. The proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of control transfected or untransfected cells. In summary, Atoh7 promotes the differentiation of retinal Müller cells into retinal ganglion cells. This may open a new avenue for gene therapy of glaucoma by promoting optic nerve regeneration.     

Molecular Signals for Development of Neuronal Circuitry in the Retina

In this review article, we summarize recently accumulated knowledge regarding the molecular mechanisms, which control retinal development. Retinal neurons are born in two waves of cytogenesis. In the first wave, neurons of cone circuitry are generated, whereas in the second wave, rod circuitry is added. Neurons generated in these two waves of cytogenesis differ in many respects, including the molecular cues used for migrational guidance. The neurons generated in the second phase of proliferation are arranged in radial columns associated with Müller cells, whereas those of the first phase are often found outside the radial columns. Certain early born cone photoreceptors may form templates for the arrangement of additional mosaics of other cell types. These mosaic arrangements of cell bodies are subsequently refined by lateral displacement of cells and apoptosis. Müller cells may play an important role in directing migration of second phase neurons within groups of radial columns and also in guiding the projections of these neurons so that specific connections are formed. The Müller cell's ability to exert these influences perhaps resides in a variety of cell adhesion molecules such as L1/NgCAM, F11, and 5A11, which are expressed on the surface of Müller cells and retinal neurons. CAMs also promote neurite outgrowth through second messenger pathways.     

Immunohistochemical Localization of Glycogen Synthase and GSK3β: Control of Glycogen Content in Retina

Glycogen has an important role in energy handling in several brain regions. In the brain, glycogen is localized in astrocytes and its role in several normal and pathological processes has been described, whereas in the retina, glycogen metabolism has been scarcely investigated. The enzyme glycogen phosphorylase has been located in retinal Müller cells; however the cellular location of glycogen synthase (GS) and its regulatory partner, glycogen synthase kinase 3β (GSK3β), has not been investigated. Our aim was to localize these enzymes in the rat retina by immunofluorescence techniques. We found both GS and GSK3β in Müller cells in the synaptic layers, and within the inner segments of photoreceptor cells. The presence of these enzymes in Müller cells suggests that glycogen could be regulated within the retina as in other tissues. Indeed, we showed that glycogen content in the whole retina in vitro was increased by high glucose concentrations, glutamate, and insulin. In contrast, retina glycogen levels were not modified by norepinephrine nor by depolarization with high KCl concentrations. Insulin also induced an increase in glycogen content in cultured Müller cells. The effect of insulin in both, whole retina and cultured Müller cells was blocked by inhibitors of phosphatidyl-inositol 3-kinase, strongly suggesting that glycogen content in retina is modulated by the insulin signaling pathway. The expression of GS and GSK3β in the synaptic layers and photoreceptor cells suggests an important role of GSK3β regulating glycogen synthase in neurons, which opens multiple feasible roles of insulin within the retina.     

CCN3/NOV inhibits BMP-2-induced osteoblast differentiation by interacting with BMP and Notch signaling pathways

We elucidate the role of CCN3/NOV, a member of the CCN family proteins, in osteoblast differentiation using MC3T3-E1 osteoblastic cells. Transduction with CCN3 adenovirus (AdCCN3) alone induced no apparent changes in the expression of osteoblast-related markers, whereas cotransduction with BMP-2 adenovirus (AdBMP-2) and AdCCN3 significantly inhibited the AdBMP-2-induced mRNA expression of Runx2, osterix, ALP, and osteocalcin. Immunoprecipitation-western analysis revealed that CCN3 associated with BMP-2. Compared to transduction with AdBMP-2 alone, cotransduction with AdBMP-2 and AdCCN3 attenuated the expression of phosphorylated Smad1/5/8 and the mRNA for Id1, Id2, and Id3. Transduction with AdCCN3 stimulated the expression of cleaved Notch1, the mRNA expression of Hes1 and Hey1/Hesr1, and the promoter activities of Hes1 and Hey1. The inhibitory effects of CCN3 on the expression of BMP-2-induced osteoblast-related markers were nullified in Hey1-deficient osteoblastic cells. These results indicate that CCN3 exerts inhibitory effects on BMP-2-induced osteoblast differentiation by its involvement of the BMP and Notch signaling pathways.     

Inhibitory effect of CT domain of CCN3/NOV on proliferation and differentiation of osteogenic mesenchymal stem cells, Kusa-A1

CCN3/NOV activates the Notch signal through the carboxyl terminal cysteine-rich (CT) domain. CCN3 transfection to Kusa-A1 inhibited osteogenic differentiation and cell proliferation, which is accompanied by upregulation of Hes/Hey, Notch downstream targets, and p21, a CDK inhibitor. Upregulation of Hes/Hey and p21 was abrogated by the deletion of CT domain. Anti-proliferative activity of CCN3 was also abrogated by CT domain deletion whereas anti-osteogenic activity was not completely abrogated. We found that CT domain-deleted CCN3 still possesses antagonistic effect on BMP-2. These results suggest that CCN3 employs Notch and BMP pathways in anti-osteogenic activity while it inhibits cell proliferation uniquely by Notch/p21 pathway.     

The expression of NOTCH2, HES1 and SOX9 during mouse retinal development

Notch signaling is an important regulator of both developmental and post-developmental processes. In the developing retina, Notch1 is required for the maintenance of retinal progenitor cells and for inhibiting photoreceptor cell fate, while Notch3 is required for inhibiting ganglion cell fate. Here we used immunolabeling coupled with a knock-in reporter approach to obtain a detailed spatiotemporal expression pattern of Notch2 during mouse retinal development. Although previous in situ hybridization studies did not reveal appreciable levels of Notch2 in the developing retina, we detected NOTCH2 protein and reporter expression in early embryonic retinal progenitors that also expressed the Notch downstream gene, HES1. In the postnatal retina, NOTCH2, as well as the Notch downstream genes, HES1 and SOX9, were detected in VSX2/Cyclin D1/SOX2-expressing cells in the postnatal retina, and in the mature retina NOTCH2 was most abundant in Müller glia. Our findings indicate a potential role for Notch2 in the developing and mature retina.     

Mueiller细胞与视网膜功能

Ｍｕｅｌｌｅｒ细胞是视网膜中的主要胶质细胞。除了一般的支持和营养作用外,近年的许多研究表明,在Ｍｕｅｌｌｅｒ细胞和视网膜视风膜神经元之间在着双向的通讯,它们可以直接通过改变细胞外空间神经活性物质的浓度或间接（通过控制神经元的微环境）调制制神经元活动,因此在视网膜功能中起着重要的作用。