Retinal and olfactory bulb precursor cells show distinct responses to FGF-2 and laminin

We analyzed whether the embryonic (E12.5-E14.5) mouse retina possesses genuine neural stem cells and how they respond to defined growth factors and extracellular matrix molecules. Whereas most combinations produced no or limited cell survival and proliferation in culture, FGF-2 plus heparin and laminin stimulated proliferation and the formation of aggregates composed, after two days, of 95.2% nestin-positive cells. However, cells in these aggregates could only be passaged poorly, lost nestin expression and proliferative capacity, and differentiated into neurons. Under the same conditions, olfactory bulb precursor cells divided efficiently and could be expanded. These data suggest that, in addition to FGF-2 and laminin, embryonic retinal neuroepithelial cells need additional extrinsic and/or intrinsic regulators to maintain cell proliferation and self-renewal.     

Autophagy is not universally required for phosphatidyl-serine exposure and apoptotic cell engulfment during neural development

Apoptosis and autophagy are physiological processes implicated in the maintenance of cell and tissue homeostasis. We took advantage of the existence of multiple phases of

developmental cell death in the embryonic chick retina and of the availability of shortterm organotypic retinal cultures to approach the possible relationship between

apoptosis and autophagy during neural development. We examined retinas at embryonic day 5, an early stage at which cell death is related to eye morphogenesis and to retinal

ganglion cell generation, as well as at embryonic day 9, when cell death is associated with neurotrophic support of the retinal ganglion cells. Exposure to 3-methyl-adenine, a

classical inhibitor of autophagy, elicited a selective accumulation of apoptotic bodies in the dorsotemporal area of embryonic day 5 retinas where neurogenesis is taking place.

This accumulation was correlated with a blockage of phosphatidyl-serine presentation and, consequently, with a lack of engulfment of the dying cells by their neighbors. In

striking contrast, none of these phenomena were observed in association with cell death in the optic nerve and optic fissure at embryonic day 5, or in embryonic day 9 retinas.

Our data suggest that autophagy is essential for phosphatidyl-serine presentation by apoptotic cells during the phase of cell death associated to neurogenesis, but this is not a

universal requirement for all phases of cell death occurring during retinal development.     

Rod photoreceptor development in vitro: intrinsic properties of proliferating neuroepithelial cells change as development proceeds in the rat retina

We describe a reaggregated cell culture system in which retinal neuroepithelial cells from embryonic rats proliferate extensively and give rise to rod photoreceptors on the same schedule in vitro as they do in vivo. Both the proliferative potential of the embryonic neuroepithelial cells and the timing of their differentiation into rods are not changed by the presence of a 50-fold excess of neonatal neural retinal cells, although many more of the embryonic cells develop into rods in these circumstances. In such mixed-age cultures, dividing neonatal cells proliferate much less and give rise to rods much sooner than do dividing embryonic cells, suggesting that the dividing cells at the two ages are intrinsically different. These and other findings suggest that both cell-cell interactions and an intrinsic program in neuroepithelial cells determine cell fate in the developing rat retina.     

Diffusible rod-promoting signals in the developing rat retina.

We previously developed a reaggregate cell culture system in which embryonic rat retinal neuroepithelial cells proliferate and give rise to opsin-expressing rod photoreceptor cells (rods) on the same schedule in vitro as they do in vivo. We showed that the proportion of neuroepithelial cells in the embryonic day 15 (E15) retina that differentiated into opsin+ rods after 5-6 days in such cultures increased by approximately 40-fold when the E15 cells were cultured in the presence of an excess of postnatal day 1 (P1) neural retinal cells. In the present study, we have further analyzed this rod-promoting activity of neonatal neural retinal cells. We show that the activity is mediated by a diffusible signal(s) that seems to act over a relatively short distance. Whereas neonatal (P1-P3) neural retina has rod-promoting activity, E15 and adult neural retina, neonatal thymus, cerebrum and cerebellum do not. Finally, we show that neonatal neural retina promotes rod but not amacrine cell development.     

Neuroprotection of Insulin against Oxidative Stress-induced Apoptosis in Cultured Retinal Neurons： Involvement of Phosphoinositide 3-kinase/Akt Signal Pathway

In order to investigate the neuroprotection of insulin in retinal neurons,we used retinal neuronalculture as a model system to study the protective effects of insulin against H_2O_2-induced cytotoxicity andapoptotic death.Primary retinal neuronal cultures were grown from retinas of 0-2-day old Sprague-Dawleyrats.Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay.Apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling assay,and byDNA laddering analysis.Phosphoinositide 3-kinase (PI3K) activity was measured using phosphoinositide4,5-bisphophate and [γ-~(32)P]ATP as substrate.Western blot analysis with anti-phospho-Akt (pS473) antibodywas performed to examine the level of phosphorylated Akt.We observed that treatment with 100μM H_2O_2for 24 h significantly decreased cell viability and induced apoptotic death of retinal neurons,and that pretreatmentwith 10 nM insulin significantly inhibited or attenuated H_2O_2-induced cytotoxicity and apoptosis.Pretreatmentwith LY294002,a specific PI3K inhibitor,abolished the cytoprotective effect of insulin.Insulin also stronglyactivated both PI3K and the downstream effector Akt.These results suggest that insulin protects retinalneurons from oxidative stress-induced apoptosis and that the PI3K/Akt signal pathway is involved in insulin-mediated retinal neuroprotection.     

Insulin rescues retinal neurons from apoptosis by a phosphatidylinositol 3-kinase/Akt-mediated mechanism that reduces the activation of caspase-3.

The ability of insulin to protect neurons from apoptosis was examined in differentiated R28 cells, a neural cell line derived from the neonatal rat retina. Apoptosis was induced by serum deprivation, and the number of pyknotic cells was counted. p53 and Akt were examined by immunoblotting after serum deprivation and insulin treatment, and caspase-3 activation was examined by immunocytochemistry. Serum deprivation for 24 h caused approximately 20% of R28 cells to undergo apoptosis, detected by both pyknosis and activation of caspase-3. 10 nm insulin maximally reduced the amount of apoptosis with a similar potency as 1.3 nm (10 ng/ml) insulin-like growth factor 1, which acted as a positive control. Insulin induced serine phosphorylation of Akt, through the phosphatidylinositol (PI) 3-kinase pathway. Inhibition of PI 3-kinase with wortmannin or LY294002 blocked the ability of insulin to rescue the cells from apoptosis. SN50, a peptide inhibitor of NF-kappaB nuclear translocation, blocked the rescue effect of insulin, but neither insulin or serum deprivation induced phosphorylation of IkappaB. These results suggest that insulin is a survival factor for retinal neurons by activating the PI 3-kinase/Akt pathway and by reducing caspase-3 activation. The rescue effect of insulin does not appear to be mediated by NF-kappaB or p53. These data suggest that insulin provides trophic support for retinal neurons through a PI 3-kinase/Akt-dependent pathway.     

ATP induces the death of developing avian retinal neurons in culture via activation of P2X7 and glutamate receptors

Previous data suggest that nucleotides are important mitogens in the developing retina. Here, the effect of ATP on the death of cultured chick embryo retina cells was investigated. In cultures obtained from retinas of 7-day-old chick embryos (E7) that were cultivated for 2 days (E7C2), both ATP and BzATP induced a ～30 % decrease in cell viability that was time- and dose-dependent and that could be blocked by 0.2 mM oxidized ATP or 0.3 μM KN-62. An increase in cleaved caspase-3 levels and in the number of TUNEL-positive cells was observed when cultures were incubated with 3 mM ATP and immunolabeling for cleaved-caspase 3 was observed over neurons but not over glial cells. ATP-dependent cell death was developmentally regulated, the maximal levels being detected by E7C2-3. Nucleotides were able to increase neuronal ethidium bromide and sulforhodamine B uptake in mixed and purified neuronal cultures, an effect that was blocked by the antagonists Brilliant Blue G and oxidized ATP. In contrast, nucleotide-induced cell death was observed only in mixed cultures, but not in purified cultures of neurons or glia. ATP-induced neuronal death was blocked by the glutamatergic antagonists MK801 and DNQX and activation of P2X7 receptors by ATP decreased the uptake of [³H]-d-aspartate by cultured glial cells with a concomitant accumulation of it in the extracellular medium. These results suggest that ATP induces apoptosis of chick embryo retinal neurons in culture through activation of P2X7 and glutamate ionotropic receptors. Involvement of a P2X7 receptor-mediated inhibition of the glial uptake of glutamate is suggested.     

Insulin activates caspase-3 by a phosphatidylinositol 3'-kinase-dependent pathway

Activation of the caspase proteases by c-Jun N-terminal kinase 1 (JNK1) has been proposed as a mechanism of apoptotic cell death. Here we report that insulin activates caspase-3 by a pathway requiring phosphatidylinositol 3'-kinase (PI3-kinase). JNK1 assays demonstrated that insulin treatment of myeloma cells induced 3-fold activation of JNK1. Inhibition of PI3-kinase with wortmannin and LY294002 blocked insulin-dependent activation of JNK1. Caspase assays demonstrated that insulin increased caspase-3 activity 3-fold and that inhibition of PI3-kinase blocked this effect. Cell death was doubled by insulin and was due to a 3-fold increase in apoptosis of cells in the G1/G0 phase of the cell cycle. Inhibition of PI3-kinase completely blocked this effect. Finally, inhibition of caspase-3 with benzyloxycarbonyl-Asp-2,6-dichlorobenzoyloxymethylketone blocked cell death due to insulin. Taken together, these findings indicate that insulin activates caspase-3 by a PI3-kinase-dependent pathway resulting in increased apoptosis and cell death.     

Cell cycle inhibition and retinoblastoma protein overexpression prevent Purkinje cell death in organotypic slice cultures

Purkinje cells are vulnerable to a number of physical, chemical, and genetic insults during development and maturity. Normal development of these cells depends on the cell-cell interactions between granule and astroglial cell populations. Apoptotic death in Purkinje neurons had been shown to be associated with cell cycle activation, and new DNA synthesis is associated with Purkinje cell death in staggerer and lurcher mutant mice. Here using an in vitro organotypic slice culture model from 9 (P9) and 4 days (P4) old postnatal rats we show that the cyclin dependent kinase (cdk) inhibitors (roscovitine, olomoucine, and flavopiridol) protect the Purkinje cells from cell death. The results are more pronounced in the cerebellar sections from P4 rats. Analysis of Purkinje neurons in sections from P4 rats after 1 week of culturing showed that while there were very limited calbindin positive neurons in the untreated sections the cdk inhibitor treated sections had a notably higher number. Although treatment with cdk inhibitors inhibited Purkinje cell loss significantly, the morphology of these neurons was abnormal, with stunted dendrites and axons. Since the retinoblastoma protein (Rb) is the major pocket protein involved in determining the differentiated state of neurons we examined the effect of over-expressing Rb in the organotypic cultures. Rb overexpression significantly inhibited the Purkinje cell death and these neurons maintained their normal morphology. Thus our studies show that the cell death in Purkinje neurons observed in organotypic cultures is cell cycle dependent and the optimal survival requires Rb.     

Neuronal influence on B and H human blood-group antigen expression in rat cochlear cultures

Summary The presence of B and H human blood-group antigens was analyzed by immunocytochemistry in rat cochleas developing either in vivo or in vitro. Developing animals, on embryonic day (E) 18 and postnatal day (P) 3, were used for in vivo studies. For in vitro studies, cochleas were removed at E18 and placed for 3 or 8 days in organotypic culture either directly or after partial spiral ganglion removal. Results from epithelial regions from cochleas developing in vivo were similar to those observed in corresponding areas of direct organotypic cultures where the innervation from spiral ganglion neurons was present. Antibodies to human blood group antigens, anti B and anti AB, selectively labeled hair cells. The intensity of labeling was weak at E18, but increased at P3 in vivo or after 3–8 days in organotypic culture. Anti H antibodies showed weak labeling of the apical surface of hair cells and other epithelial cells at E18; this labeling also increased at P3 or after 3–8 days in culture. In contrast, the non-innervated regions from organotypic cultures, where ganglia were partially removed, exhibited an epithelial disorganization and no hair cell labeling with any of the antibodies studied. The present findings suggest that human blood-group antigen expression on developing cochlear hair cells of rats may be related to afferent nerve fiber influence.     

Inhibition of PI3K/Akt Pathway Impairs G2/M Transition of Cell Cycle in Late Developing Progenitors of the Avian Embryo Retina

PI3K/Akt is an important pathway implicated in the proliferation and survival of cells in the CNS. Here we investigated the participation of the PI3K/Akt signal pathway in cell cycle of developing retinal progenitors. Immunofluorescence assays performed in cultures of chick embryo retinal cells and intact tissues revealed the presence of phosphorylated Akt and 4E-BP1 in cells with typical mitotic profiles. Blockade of PI3K activity with the chemical inhibitor LY 294002 (LY) in retinal explants blocked the progression of proliferating cells through G2/M transition, indicated by an expressive increase in the number of cells labeled for phosphorylated histone H3 in the ventricular margin of the retina. No significant level of cell death could be detected at this region. Retinal explants treated with LY for 24 h also showed a significant decrease in the expression of phospho-Akt, phospho-GSK-3 and the hyperphosphorylated form of 4E-BP1. Although no change in the expression of cyclin B1 was detected, a significant decrease in CDK1 expression was noticed after 24 h of LY treatment both in retinal explants and monolayer cultures. Our results suggest that PI3K/Akt is an active pathway during proliferation of retinal progenitors and its activity appears to be required for proper CDK1 expression levels and mitosis progression of these cells.     

Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF, lack NGF receptors but are supported by a liver-derived neurotrophic factor

Explant and dissociated neuron-enriched cultures of nodose ganglia (inferior or distal sensory ganglion of the Xth cranial nerve) were established from chick embryos taken between embryonic Day 4 (E4) and Day 16 (E16). The response of each type of culture to nerve growth factor (NGF) was examined over this developmental range. At the earliest ages taken (E4-E6), NGF elicited modest neurite outgrowth from ganglion explants cultured in collagen gel for 24 hr, although the effect of NGF on ganglia taken from E4 chicks was only marginally greater than spontaneous neurite extension from control ganglia of the same developmental age. The response of nodose explants to NGF was maximal at E6-E7, but declined to a negligible level in ganglia taken from E9-E10 or older chick embryos. In dissociated neuron-enriched cultures, nodose ganglion neurons were unresponsive to NGF throughtout the entire developmental age range between E5 and E12. In contrast to the lack of effect of NGF, up to 50% of nodose ganglion neurons survived and produced extensive neurites in dissociated cultures, on either collagen- or polylysine-coated substrates, in the presence of extracts of late embryonic or early posthatched chick liver (E18-P7). Antiserum to mouse NGF did not block the neurotrophic activity of chick (or rat or bovine) liver extracts. Whether cultured with chick liver extract alone or with chick liver extract plus NGF, nodose ganglion neurons taken from E6-E12 chick embryos and maintained in culture for 2 days were devoid of NGF receptors, as assessed by autoradiography of cultures incubated with 125I-NGF. Under similar conditions 70-95% of spinal sensory neurons (dorsal root ganglion--DRG) were heavily labeled. 2+     

Semaphorin 3A and neurotrophins: a balance between apoptosis and survival signaling in embryonic DRG neurons

Large numbers of neurons are eliminated by apoptosis during nervous system development. For instance, in the mouse dorsal root ganglion (DRG), the highest incidence of cell death occurs between embryonic days 12 and 14 (E12-E14). While the cause of cell death and its biological significance in the nervous system is not entirely understood, it is generally believed that limiting quantities of neurotrophins are responsible for neuronal death. Between E12 and E14, developing DRG neurons pass through tissues expressing high levels of axonal guidance molecules such as Semaphorin 3A (Sema3A) while navigating to their targets. Here, we demonstrate that Sema3A acts as a death-inducing molecule in neurotrophin-3 (NT-3)-, brain-derived neurotrophic factor (BDNF)- and nerve growth factor (NGF)-dependent E12 and E13 cultured DRG neurons. We show that Sema3A most probably induces cell death through activation of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway, and that this cell death is blocked by a moderate increase in NGF concentration. Interestingly, increasing concentrations of other neurotrophic factors, such as NT-3 or BDNF, do not elicit similar effects. Our data suggest that the number of DRG neurons is determined by a fine balance between neurotrophins and Semaphorin 3A, and not only by neurotrophin levels.     

Insulin concentration is critical in culturing human neural stem cells and neurons

Cell culture of human-derived neural stem cells (NSCs) is a useful tool that contributes to our understanding of human brain development and allows for the development of therapies for intractable human brain disorders. Human NSC (hNSC) cultures, however, are not commonly used, mainly because of difficulty with consistently maintaining the cells in a healthy state. In this study, we show that hNSC cultures, unlike NSCs of rodent origins, are extremely sensitive to insulin, an indispensable culture supplement, and that the previously reported difficulty in culturing hNSCs is likely because of a lack of understanding of this relationship. Like other neural cell cultures, insulin is required for hNSC growth, as withdrawal of insulin supplementation results in massive cell death and delayed cell growth. However, severe apoptotic cell death was also detected in insulin concentrations optimized to rodent NSC cultures. Thus, healthy hNSC cultures were only produced in a narrow range of relatively low insulin concentrations. Insulin-mediated cell death manifested not only in all human NSCs tested, regardless of origin, but also in differentiated human neurons. The underlying cell death mechanism at high insulin concentrations was similar to insulin resistance, where cells became less responsive to insulin, resulting in a reduction in the activation of the PI3K/Akt pathway critical to cell survival signaling.     

RAF kinase activity regulates neuroepithelial cell proliferation and neuronal progenitor cell differentiation during early inner ear development

Background

Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood.

Methodology/Principal Findings

By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation.

Conclusions/Significance

We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG.     

Insulin-like growth factor-1 regulation of retinal progenitor cell proliferation and differentiation

Strategies to improve retinal progenitor cell (RPC) capacity to yield proliferative and multipotent pools of cells that can efficiently differentiate into retinal neurons, including photoreceptors, could be vital for cell therapy in retinal degenerative diseases. In this study, we found that insulin-like growth factor-1 (IGF-1) plays a role in the regulation of proliferation and differentiation of RPCs. Our results show that IGF-1 promotes RPC proliferation via IGF-1 receptors (IGF-1Rs), stimulating increased phosphorylation in the PI3K/Akt and MAPK/Erk pathways. An inhibitor experiment revealed that IGF-1-induced RPC proliferation was inhibited when the PI3K/Akt and MAPK/Erk pathways were blocked. Furthermore, under the condition of differentiation, IGF-1-pretreated RPCs prefer to differentiate into retinal neurons, including photoreceptors, in vitro, which is crucial for visual formation and visual restoration. These results demonstrate that IGF-1 accelerates the proliferation of RPCs and IGF-1 pretreated RPCs may have shown an increased potential for retinal neuron differentiation, providing a novel strategy for regulating the proliferation and differentiation of retinal progenitors in vitro and shedding light upon the application of RPCs in retinal cell therapy.     

The perplexed and confused mutations affect distinct stages during the transition from proliferating to post-mitotic cells within the zebrafish retina

To identify and study genes essential for vertebrate retinal development, we are screening zebrafish embryos for mutations that disrupt retinal histogenesis. Key steps in retinogenesis include withdrawal from mitosis by multipotent neuroepithelial cells, specification to particular cell types, migration to the appropriate laminar positions, and molecular and morphological differentiation. In this study, we have identified two recessive mutations that affect the transition of proliferating neuroepithelial cells to postmitotic retinal cells. Both the perplexed and confused mutant phenotypes were initially detectable when the first retinal neuroepithelial cells began to leave the cell cycle. At this time, each mutant retina showed increased cell death and a lack of morphological differentiation. Cell death was found to be apoptotic in both perplexed and confused retinas based on TUNEL analysis and activation of caspase-3. TUNEL-phosphoRb-BrdU colocalization studies indicated that the perplexed mutation caused death in cells transitioning from a proliferative to postmitotic state. For the confused mutation, TUNEL-phosphoRb-BrdU analysis revealed that only a subset of postmitotic cells were induced to activate apoptosis. Mosaic analysis demonstrated that within the retina the perplexed mutation functions noncell-autonomously. Furthermore, whole lens or eye cup transplantations indicated that the retinal defect was intrinsic to the retina. Mosaic analysis with confused embryos showed this mutation acts cell-autonomously. From these studies, we conclude that the perplexed and confused genes are essential at distinct stages during the transition from proliferating to postmitotic cells within the zebrafish retina.