Synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) by isolated normal and rd mouse retinal photoreceptor neurons in culture

Cultures of dissociated retinal neurons and photoreceptors from homozygous wild-type, heterozygous rd/+ and homozygous rd/rd retinas have been used to investigate the capacity of isolated photoreceptor cells to synthesize and secrete the interphotoreceptor retinoid-binding protein (IRBP). Retinal cells were dissociated on postnatal day 2 and grown in chemically defined medium in the absence of glial and pigmented epithelial cells. Expression of IRBP immunoreactive materials in these cultures was cell type-specific and developmentally regulated. Thus increasing numbers of rod photoreceptor cells showed immunoreactivity during the first week in culture, whereas nonphotoreceptor cell types remained consistently negative. Photoreceptor immunoreactivity could be detected in permeated (detergent-treated) as well as in unpermeated preparations, the latter suggesting that some IRBP is associated with the photoreceptor cell surface. These materials appeared to be loosely bound to the photoreceptors, since they disappeared when the cultures were exposed for 6 hr to IRBP-free medium but not when they were exposed to IRBP-containing medium. IRBP synthesis and secretion could be demonstrated by analyzing either cell extracts or conditioned medium by "slot blot" and Western blot techniques using affinity purified antibodies against bovine IRBP as well as by fluorographic analysis after metabolic labeling of the cultures with 35S-methionine. Comparisons of cultures from the different genotypes showed many similarities, including the abundance of IRBP-immunoreactive photoreceptors in 7 day cultures. However, immunochemical analysis showed lower conditioned medium/cell extract IRBP ratios in rd/rd cultures, an observation consistent with previous reports suggesting that IRBP secretion may be deficient in rd/rd photoreceptor cells.     

Rhodopsin, 11-cis vitamin A, and interstitial retinol-binding protein (IRBP) during retinal development in normal and rd mutant mice

Biochemical and immunological techniques were used to determine the emergence of interstitial retinol binding protein (IRBP), rhodopsin, and stored retinyl esters (all-trans and 11-cis) during retinal development in normal and rd mice. IRBP could be demonstrated at embryonic Day 17 (E17), corresponding to an early stage of inner segment development. Although all-trans retinyl esters were present earlier, 11-cis retinyl esters did not appear until postnatal Days 6-7 (P6-P7), corresponding to rod outer segment (ROS) disc formation. Rhodopsin was detected at the same developmental stage. The proportion of 11-cis retinyl esters reached a maximum of 40-50% at P15-P20. Thereafter, the proportion dropped, due to more rapid accumulation of the all-trans isomer. Rhodopsin and IRBP increased in parallel with ROS elongation up to P25, when the ROS had reached their mature lengths. The increases then continued up to P40-P50. In rd (retinal degeneration) mice, IRBP and rhodopsin were identical with the controls until P12, but then dropped as the photoreceptors degenerated. Synthesis and secretion of IRBP in vitro was less than 10% of the controls in rd retinas at P26, when only 4-5% of the photoreceptors survived. The quantities of retinyl esters (mainly stearate and palmitate in the ratio of 6:1, respectively) stored in dark-adapted mouse eyes progressively increased as the animals aged, representing 0.5 mole eq. of the rhodopsin at 8 months. Although retinyl esters (11-cis and all-trans) also accumulated in rd mouse eyes up to P12, little further increase occurred. At P93, the retinyl esters (0.01 nmole X eye-1) were only 4% of the controls at P91. A peak in the proportion of 11-cis isomer occurred at P10-P20, but it averaged only 15% of the total ester and declined to 5% at P93. These findings support the hypothesis that IRBP is synthesized by the rods and cones, and suggest that its synthesis and secretion are initiated when the photoreceptor inner segments start to differentiate. 11-cis Retinoids and rhodopsin do not appear until the outer segments start to form. It is suggested that in the rd mouse the absence of photoreceptors, perhaps coupled with lack of normal interphotoreceptor matrix, leads to a loss in the ability of the pigment epithelium to store retinyl esters.     

Common and distinct factors regulate expression of mRNA for ETV5 and GDNF, Sertoli cell proteins essential for spermatogonial stem cell maintenance

Ets variant gene 5 (ETV5) and glial cell-derived neurotrophic factor (GDNF) are produced in Sertoli cells and required for maintenance and self-renewal of spermatogonial stem cells (SSCs) in mice. Fibroblast growth factors (FGFs) have been reported to stimulate Etv5 mRNA expression, and FSH was shown to stimulate Gdnf mRNA in Sertoli cell cultures, but there is no other information on factors that regulate these key Sertoli cell proteins necessary for stem cell maintenance. In this study, we investigated regulation of ETV5 and GDNF using the TM4 murine Sertoli cell line. FGF2 stimulated a time- and dose-dependent increase in Etv5 mRNA expression, with a maximal 8.3-fold increase at 6 h following 25 ng/ml FGF2 treatment. This FGF2 dose also stimulated Gdnf mRNA at 48 h. FGF2 effects on Etv5 and Gdnf mRNA were partially mediated through mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-signaling cascades. Specific inhibitors of MAPK (PD98059) and PI3K (wortmannin) pathways reduced Etv5 and Gdnf mRNA expression in FGF2-treated cells. Epidermal growth factor (EGF) stimulated Etv5 mRNA but not Gdnf mRNA. TNFalpha and IL-1beta stimulated Gdnf mRNA, but had no effect on Etv5 mRNA. Other hormonal regulators of Sertoli cells such as testosterone, triiodothyronine and activin A did not affect Etv5 or Gdnf mRNA expression. Results with primary Sertoli cell cultures confirmed findings obtained with the TM4 cell line, validating the use of the TM4 model to examine regulation of Etv5 and Gdnf mRNA expression. In conclusion, we have identified common and unique pathways that regulate Etv5 and Gdnf mRNA in Sertoli cells, and FGFs are emerging as key regulators of the Sertoli cell proteins that control SSCs.     

Branching morphogenesis of the ureteric epithelium during kidney development is coordinated by the opposing functions of GDNF and Sprouty1

Branching of ureteric bud-derived epithelial tubes is a key morphogenetic process that shapes development of the kidney. Glial cell line-derived neurotrophic factor (GDNF) initiates ureteric bud formation and promotes subsequent branching morphogenesis. Exactly how GDNF coordinates branching morphogenesis is unclear. Here we show that the absence of the receptor tyrosine kinase antagonist Sprouty1 (Spry1) results in irregular branching morphogenesis characterized by both increased number and size of ureteric bud tips. Deletion of Spry1 specifically in the epithelium is associated with increased epithelial Wnt11 expression as well as increased mesenchymal Gdnf expression. We propose that Spry1 regulates a Gdnf/Ret/Wnt11-positive feedback loop that coordinates mesenchymal-epithelial dialogue during branching morphogenesis. Genetic experiments indicate that the positive (GDNF) and inhibitory (Sprouty1) signals have to be finely balanced throughout renal development to prevent hypoplasia or cystic hyperplasia. Epithelial cysts develop in Spry1-deficient kidneys that share several molecular characteristics with those observed in human disease, suggesting that Spry1 null mice may be useful animal models for cystic hyperplasia.     

Disturbances of colonic motility in mouse models of Hirschsprung's disease

Mutations in genes encoding members of the GDNF and endothelin-3 (Et-3) signaling pathways can cause Hirschsprung's disease, a congenital condition associated with an absence of enteric neurons in the distal gut. GDNF signals through Ret, a receptor tyrosine kinase, and Et-3 signals through endothelin receptor B (Ednrb). The effects of Gdnf, Ret, and ET-3 haploinsufficiency and a null mutation in ET-3 on spontaneous motility patterns in adult and developing mice were investigated. Video recordings were used to construct spatiotemporal maps of spontaneous contractile patterns in colon from postnatal and adult mice in vitro. In Ret(+/-) and ET-3(+/-) mice, which have normal numbers of enteric neurons, colonic migrating motor complexes (CMMCs) displayed similar properties under control conditions and following inhibition of nitric oxide synthase (NOS) activity to wild-type mice. In the colon of Gdnf(+/-) mice and in the ganglionic region of ET-3(-/-) mice, there was a 50-60% reduction in myenteric neuron number. In Gdnf(+/-) mice, CMMCs were present, but abnormal, and the proportion of myenteric neurons containing NOS was not different from that of wild-type mice. In the ganglionic region of postnatal ET-3(-/-) mice, CMMCs were absent, and the proportion of myenteric neurons containing NOS was over 100% higher than in wild-type mice. Thus impairments in spontaneous motility patterns in the colon of Gdnf(+/-) mice and in the ganglionic region of ET-3(-/-) mice are correlated with a reduction in myenteric neuron density.     

Gdnf Haploinsufficiency Causes Hirschsprung-Like Intestinal Obstruction and Early-Onset Lethality in Mice

Hirschsprung disease (HSCR) is a common congenital disorder that results in intestinal obstruction and lethality, as a result of defective innervation of the gastrointestinal (GI) tract. Despite its congenital origin, the molecular etiology of HSCR remains elusive for >70% of patients. Although mutations in the c-RET receptor gene are frequently detected in patients with HSCR, mutations in the gene encoding its ligand (glial cell line-derived neurotrophic factor [GDNF]), are rarely found. In an effort to establish a possible link between human HSCR and mutations affecting the Gdnf locus, we studied a large population of mice heterozygous for a Gdnf null mutation. This Gdnf(+/-) mutant cohort recapitulates complex features characteristic of HSCR, including dominant inheritance, incomplete penetrance, and variable severity of symptoms. The lack of one functioning Gdnf allele causes a spectrum of defects in gastrointestinal motility and predisposes the mutant mice to HSCR-like phenotypes. As many as one in five Gdnf(+/-) mutant mice die shortly after birth. Using a transgenic marking strategy, we identified hypoganglionosis of the gastrointestinal tract as a developmental defect that renders the mutant mice susceptible to clinical symptoms of HSCR. Our findings offer a plausible way to link an array of seemingly disparate features characteristic of a complex disease to a much more narrowly defined genetic cause. These findings may have general implications for the genetic analysis of cause and effect in complex human diseases.     

Antioxidants slow photoreceptor cell death in mouse models of retinitis pigmentosa

Retinitis pigmentosa (RP) is a heterogeneous group of diseases in which one of a wide variety of mutations selectively causes rod photoreceptor cell death. After rods die, cone photoreceptors gradually die resulting in blindness. Antioxidants reduce cone cell death in rd1/rd1 mice indicating that cones die from oxidative damage in that model of rapidly progressive RP. In this study, we sought to determine if this observation could be generalized to models of other types of RP, rd10/rd10 mice, a model of more slowly progressive recessive RP, and Q344ter mice, a model of rapidly progressive dominant RP. Compared to appropriate vehicle-treated controls, rd10/rd10 and Q344ter mice treated between P18 and P35 with a mixture of antioxidants previously found to be effective in rd1/rd1 mice showed significantly greater cone survival. Antioxidant-treated rd10/rd10 mice showed preservation of cone function as shown by a significant increase in photopic ERG b-wave amplitudes, and surprisingly showed temporary preservation of scotopic a-wave amplitudes, prolonged rod survival, and slowed depletion of rhodopsin mRNA. These data suggest that oxidative damage contributes to cone cell death regardless of the disease causing mutation that leads to the demise of rods, and that in more slowly progressive rod degenerations, oxidative damage may also contribute to rod cell death. Protection from oxidative damage may be a broadly applicable treatment strategy in RP.     

Crucial role of TrkB ligands in the survival and phenotypic differentiation of developing locus coeruleus noradrenergic neurons

The role of glial cell-line derived neurotrophic factor (GDNF) and neurotrophins in the development of locus coeruleus noradrenergic neurons was evaluated. We found that two neurotrophic factors previously reported to prevent the degeneration of lesioned adult central noradrenergic neurons, GDNF and neurotrophin 3 (NT3), do not play significant roles in the prenatal development of locus coeruleus noradrenergic neurons, as demonstrated by: (1) the lack of alterations in double Gdnf/Nt3 null mutant mice; and (2) the lack of survival-promoting effects of GDNF and/or NT3 in rat E13.5 primary cultures. In contrast, null mutant mice for TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor and neurotrophin 4, displayed a clear loss of locus coeruleus noradrenergic neurons. In accordance with this, treatment of rat E13.5 primary cultures with TrkB ligands prevented the early loss of noradrenergic neurons and maintained their survival for up to 6 days in vitro. Moreover, an additional 5-10-fold increase in the number of tyrosine hydroxylase positive noradrenergic neurons was detected after 12 hours in culture. This second effect of TrkB ligands involved neither proliferation nor survival, because the number of BrdU- or TUNEL-positive noradrenergic neurons did not change and the effect was elicited by delayed administration of either factor. Because TrkB ligands increased the number of tyrosine hydroxylase-positive cells expressing Phox2a, a paired homeodomain protein required for the development of locus coeruleus noradrenergic neurons, but did not affect the number of Phox2a-positive tyrosine hydroxylase-negative cells, our results suggest that the second effect of TrkB ligands may involve promoting or inducing a noradrenergic phenotype. In summary, our findings suggest that, unlike NT3 and GDNF, TrkB ligands are required and sufficient to promote the development of central noradrenergic neurons.     

An extracellular retinol-binding glycoprotein in the eyes of mutant rats with retinal dystrophy: development, localization, and biosynthesis

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein in the interphotoreceptor matrix of bovine, human, monkey, and rat eyes. It may transport retinol between the retinal pigment epithelium and the neural retina. In light-reared Royal College of Surgeons (RCS) and RCS retinal dystrophy gene (rdy)+ rats, the amount of IRBP in the interphotoreceptor matrix increased in corresponding proportion to the amount of total rhodopsin through postnatal day 22 (P22). In the RCS-rdy+ rats, the amount increased slightly after P23. However, in the RCS rats there was a rapid fall in the quantity of IRBP as the photoreceptors degenerated between P23 and P29. No IRBP was detected by immunocytochemistry in rats at P28. The amount of rhodopsin fell more slowly. Although retinas from young RCS and RCS-rdy+ rats were able to synthesize and secrete IRBP, this ability was lost in retinas from older RCS rats (P51, P88) but not their congenic controls. The photoreceptor cells have degenerated at these ages in the RCS animals, and may therefore be the retinal cells responsible for IRBP synthesis. The putative function of IRBP in the extracellular transport of retinoids during the visual cycle is consistent with a defect in retinol transport in the RCS rat reported by others.     

Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development

The Tet family of enzymes (Tet1/2/3) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Mouse embryonic stem cells (mESCs) highly express Tet1 and have an elevated level of 5hmC. Tet1 has been implicated in ESC maintenance and lineage specification in?vitro but its precise function in development is not well defined. To establish the role of Tet1 in pluripotency and development, we have generated Tet1 mutant mESCs and mice. Tet1(-/-) ESCs have reduced levels of 5hmC and subtle changes in global gene expression, and are pluripotent and support development of live-born mice in tetraploid complementation assay, but display skewed differentiation toward trophectoderm in?vitro. Tet1 mutant mice are viable, fertile, and grossly normal, though some mutant mice have a slightly smaller body size at birth. Our data suggest that Tet1 loss leading to a partial reduction in 5hmC levels does not affect pluripotency in ESCs and is compatible with embryonic and postnatal development.     

Cooperation between GDNF/Ret and ephrinA/EphA4 signals for motor-axon pathway selection in the limb

Establishment of limb innervation by motor neurons involves a series of hierarchical axon guidance decisions by which motor-neuron subtypes evaluate peripheral guidance cues and choose their axonal trajectory. Earlier work indicated that the pathway into the dorsal limb by lateral motor column (LMC[l]) axons requires the EphA4 receptor, which mediates repulsion elicited by ephrinAs expressed in ventral limb mesoderm. Here, we implicate glial-cell-line-derived neurotrophic factor (GDNF) and its receptor, Ret, in the same guidance decision. In Gdnf or Ret mutant mice, LMC(l) axons follow an aberrant ventral trajectory away from dorsal territory enriched in GDNF, showing that the GDNF/Ret system functions as an instructive guidance signal for motor axons. This phenotype is enhanced in mutant mice lacking Ret and EphA4. Thus, Ret and EphA4 signals cooperate to enforce the precision of the same binary choice in motor-axon guidance.     

Progesterone Attenuates Microglial-Driven Retinal Degeneration and Stimulates Protective Fractalkine-CX3CR1 Signaling

Retinitis pigmentosa (RP) is a degenerative disease leading to photoreceptor cell loss. Mouse models of RP, such as the rd10 mouse (B6.CXBl-Pde6brd10/J), have enhanced our understanding of the disease, allowing for development of potential therapeutics. In 2011, our group first demonstrated that the synthetic progesterone analogue ‘Norgestrel’ is neuroprotective in two mouse models of retinal degeneration, including the rd10 mouse. We have since elucidated several mechanisms by which Norgestrel protects stressed photoreceptors, such as upregulating growth factors. This study consequently aimed to further characterize Norgestrel’s neuroprotective effects. Specifically, we sought to investigate the role that microglia might play; for microglial-derived inflammation has been shown to potentiate neurodegeneration. Dams of post-natal day (P) 10 rd10 pups were given a Norgestrel-supplemented diet (80mg/kg). Upon weaning, pups remained on Norgestrel. Tissue was harvested from P15-P50 rd10 mice on control or Norgestrel-supplemented diet. Norgestrel-diet administration provided significant retinal protection out to P40 in rd10 mice. Alterations in microglial activity coincided with significant protection, implicating microglial changes in Norgestrel-induced neuroprotection. Utilizing primary cultures of retinal microglia and 661W photoreceptor-like cells, we show that rd10 microglia drive neuronal cell death. We reveal a novel role of Norgestrel, acting directly on microglia to reduce pro-inflammatory activation and prevent neuronal cell death. Norgestrel effectively suppresses cytokine, chemokine and danger-associated molecular pattern molecule (DAMP) expression in the rd10 retina. Remarkably, Norgestrel upregulates fractalkine-CX3CR1 signaling 1 000-fold at the RNA level, in the rd10 mouse. Fractalkine-CX3CR1 signaling has been shown to protect neurons by regulating retinal microglial activation and migration. Ultimately, these results present Norgestrel as a promising treatment for RP, with dual actions as a neuroprotective and anti-inflammatory agent in the retina.     

Differential effects of nutritional folic acid deficiency and moderate hyperhomocysteinemia on aortic plaque formation and genome-wide DNA methylation in vascular tissue from ApoE-/- mice

Low folate intake is associated with vascular disease. Causality has been attributed to hyperhomocysteinemia. However, human intervention trials have failed to show the benefit of homocysteine-lowering therapies. Alternatively, low folate may promote vascular disease by deregulating DNA methylation. We investigated whether folate could alter DNA methylation and atherosclerosis in ApoE null mice. Mice were fed one of six diets (n?=?20 per group) for 16?weeks. Basal diets were either control (C; 4% lard) or high fat (HF; 21% lard and cholesterol, 0.15%) with different B-vitamin compositions: (1) folic acid and B-vitamin replete, (2) folic acid deficient (-F), (3) folic acid, B6 and B12 deficient (-F-B). -F diets decreased plasma (up to 85%; P?相似文献   

Increased expression of glial cell line-derived neurotrophic factor protects against oxidative damage-induced retinal degeneration

Oxidative damage contributes to retinal cell death in patients with age-related macular degeneration or retinitis pigmentosa. One approach to treatment is to identify and eliminate the sources of oxidative damage. Another approach is to identify treatments that protect cells from multiple sources of oxidative damage. In this study, we investigated the effect of increased expression of glial cell line-derived neurotrophic factor (GDNF) in three models of oxidative damage-induced retinal degeneration. Double transgenic mice with doxycycline-inducible expression of GDNF in the retina were exposed to paraquat, FeSO(4), or hyperoxia, all sources of oxidative damage and retinal cell death. Compared to controls, mice with increased expression of GDNF in the retina showed significant preservation of retinal function measured by electroretinograms, reduced thinning of retinal cell layers, and fewer TUNEL-positive cells indicating less retinal cell death. Mice over-expressing GDNF also showed less staining for acrolein, nitrotyrosine, and 8-hydroxydeoxyguanosine, indicating less oxidative damage to lipids, proteins, and DNA. This suggests that GDNF did not act solely to allow cells to tolerate higher levels of oxidative damage before initiation of apoptosis, but also reduced damage from oxidative stress to critical macromolecules. These data suggest that gene transfer of Gdnf should be considered as a component of therapy for retinal degenerations in which oxidative damage plays a role.     

Sprouty1 is a critical regulator of GDNF/RET-mediated kidney induction

Intercellular signaling molecules and their receptors, whose expression must be tightly regulated in time and space, coordinate organogenesis. Regulators of intracellular signaling pathways provide an additional level of control. Here we report that loss of the receptor tyrosine kinase (RTK) antagonist, Sprouty1 (Spry1), causes defects in kidney development in mice. Spry1(-/-) embryos have supernumerary ureteric buds, resulting in the development of multiple ureters and multiplex kidneys. These defects are due to increased sensitivity of the Wolffian duct to GDNF/RET signaling, and reducing Gdnf gene dosage correspondingly rescues the Spry1 null phenotype. We conclude that the function of Spry1 is to modulate GDNF/RET signaling in the Wolffian duct, ensuring that kidney induction is restricted to a single site. These results demonstrate the importance of negative feedback regulation of RTK signaling during kidney induction and suggest that failures in feedback control may underlie some human congenital kidney malformations.     

Delayed rhodopsin regeneration and altered distribution of interphotoreceptor retinoid binding protein (IRBP) in the mivit/mivit (vitiligo) mouse

Rhodopsin regeneration requires attachment between the retinal pigment epithelium (RPE) and rod outer segments; however, in experimentally induced retinal detachment, rhodopsin regeneration can be restored partially upon addition of IRBP (interphotoreceptor retinoid binding protein). The mivit/mivit (vitiligo) mutant mouse, a model of slowly progressing photoreceptor cell degeneration, has a marked elevation of IRBP at 4 weeks as well as progressive detachment of the retina. The purpose of this study was to determine whether this mutant is capable of regenerating rhodopsin within a few hours following an intense light bleach. Rhodopsin regeneration was determined spectrophotometrically in mice after an intense one hour light bleach followed by 0, 1, 2, 4 or 24 h of dark recovery. IRBP was localized immunohistochemically in fixed frozen tissue at the light microscopic level and in LR Gold embedded tissue at the ultrastructural level. Rhodopsin regeneration experiments indicated that rhodopsin levels following 0, 1, 2 and 4 h dark-recovery were significantly less in mivit/mivit mutants compared with controls. Immunohistochemical detection of IRBP indicated an altered distribution of the protein in the mutant mice compared with controls. There was accumulation in the region of the inner segments in mutant retinas rather than distribution only to the RPE/OS apical regions as in controls. The data suggest that regeneration of rhodopsin is reduced by 4 weeks postnatally in the mivit/mivit mouse. There is partial detachment of the retina at this age; and IRBP, thought to be essential for proper functioning of the visual cycle, is aberrantly distributed in this mutant.