Thioredoxin rod-derived cone viability factor protects against photooxidative retinal damage.

Rod-derived cone viability factor (RdCVF) is a trophic factor of the thioredoxins family that promotes the survival of cone photoreceptors. It is encoded by the nucleoredoxin-like gene 1 Nxnl1 which also encodes by alternative splicing a long form of RdCVF (RdCVFL), a thioredoxin enzyme that interacts with TAU. The known role of thioredoxins in the defense mechanism against oxidative damage led us to examine the retinal phenotype of the Nxnl1(-/-) mice exposed to photooxidative stress. Here we found that, in contrast to wild-type mice, the rod photoreceptors of Nxnl1(-/-) mice are more sensitive to light after exposure to 1700 or 2500 lx. The delivery of RdCVF by AAV to mice deficient of Nxnl1(-/-) protects rod photoreceptors from light damage. Interestingly, the RdCVF2L protein, encoded by the paralog gene Nxnl2, is able to reduce TAU phosphorylation, as does RdCVFL, but does not protect the rod from light damage. Our result shows that the Nxnl1 gene, through the thioredoxin RdCVFL, is part of an endogenous defense mechanism against photooxidative stress that is likely of great importance for human vision.

The disruption of the rod-derived cone viability gene leads to photoreceptor dysfunction and susceptibility to oxidative stress.

Rod-derived cone viability factor (RdCVF) is a thioredoxin-like protein, which has therapeutic potential for rod-cone dystrophies such as retinitis pigmentosa (RP). Cone loss in rodent models of RP is effectively reduced by RdCVF treatment. In this study, we investigate the physiological role of RdCVF in the retina by analyzing the phenotype of the mouse lacking the RdCVF gene, Nxnl1. Although the mice do not show an obvious developmental defect, an age-related reduction of both cone and rod function and a delay in the dark-adaptation of the retina are recorded by electroretinogram (ERG). This functional change is accompanied by a 17% reduction in cone density and a 20% reduction in thickness of the outer nuclear layer. The transcriptome of the retina reveals early changes in the expression of genes involved in programmed cell death, stress-response and redox-signaling, which is followed by a generalized injury response with increased microglial activation, GFAP, FGF2 and lipid peroxidation levels. Furthermore, cones of the mice lacking Nxnl1 are more sensitive to oxidative stress with a reduction of 65% in the cone flicker ERG amplitude measured under hyperoxic conditions. We show here that the RdCVF gene, in addition to therapeutic properties, has an essential role in photoreceptor maintenance and resistance to retinal oxidative stress.

GOAnno: GO annotation based on multiple alignment.

UNLABELLED: GOAnno is a web tool that automatically annotates proteins according to the Gene Ontology (GO) using evolutionary information available in hierarchized multiple alignments. GO terms present in the aligned functional subfamily can be cross-validated and propagated to obtain highly reliable predicted GO annotation based on the GOAnno algorithm. AVAILABILITY: The web tool and a reduced version for local installation are freely available at http://igbmc.u-strasbg.fr/GOAnno/GOAnno.html SUPPLEMENTARY INFORMATION: The website supplies a detailed explanation and illustration of the algorithm at http://igbmc.u-strasbg.fr/GOAnno/GOAnnoHelp.html.

DNA repair in the degenerating mouse retina.

In light of different recent results suggesting that the adult mammalian central nervous system can produce new neurons, possibly as an endogenous repair mechanism, we investigated whether neurogenesis occurs in response to photoreceptor degeneration in the rd1 mouse, a model of human-inherited retinal dystrophy. Bromodeoxy-Uridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression experiments detected cell proliferation in the extreme peripheral retina, in both wt and rd1 retina, independent of degeneration. BrdU incorporation and PCNA expression also occurred in rd1 photoreceptors. Our results strongly suggest that these photoreceptors undergo DNA repair: p53, PCNA, and DNA ligase IV are expressed before photoreceptor death, consistent with a model where photoreceptors expressing the rd1 mutation activate a process of DNA repair but which is overwhelmed by the disease mutation leading to apoptotic death. The existence of such a balance offers potential new targets for neuroprotective approaches.

Rod-cone interactions: developmental and clinical significance.

During the last decade, numerous research reports have considerably improved our knowledge about the physiopathology of retinal degenerations. Three non-mutually exclusive general areas dealing with therapeutic approaches have been proposed; gene therapy, pharmacology and retinal transplantations. The first approach involving correction of the initial mutation, will need a great deal of time and further development before becoming a therapeutic tool in human clinical practice. The observation that cone photoreceptors, even those seemingly unaffected by any described anomaly, die secondarily to rod disappearance related to mutations expressed specifically in the latter, led us to study the interactions between these two photoreceptor populations to search for possible causal links between rod degeneration and cone death. These in vivo and in vitro studies suggest that paracrine interactions between both cell types exist and that rods are necessary for continued cone survival. Since the role of cones in visual perception is essential, pending the identification of the factors mediating these interactions underway, rod replacement by transplantation and/or neuroprotection by trophic factors or alternative pharmacological means appear as promising approaches for limiting secondary cone loss in currently untreatable blinding conditions.

Glial cell line-derived neurotrophic factor induces histologic and functional protection of rod photoreceptors in the rd/rd mouse.

PURPOSE: To evaluate the neuroprotective potential of glial cell line-derived neurotrophic factor (GDNF) in the retinal degeneration (rd/rd) mouse model of human retinitis pigmentosa. METHODS: Subretinal injections of GDNF were made into rd/rd mice at 13 and 17 days of age and electroretinograms (ERGs) recorded at 22 days. Control mice received saline vehicle injections or underwent no procedure. At 23 days of age, retinas from treated and control mice were fixed and processed for wholemount immunohistochemistry using an anti-rod opsin antibody, and rod numbers were estimated using an unbiased stereological systematic random approach. Subsequent to counting, immunolabeled retinas were re-embedded and sectioned in a transverse plane and the numbers of rods recalculated. RESULTS: Although ERGs could not be recorded from sham-operation or nonsurgical rd/rd mice at 22 days of age, detectable responses (both a- and b-waves) were observed in 4 of 10 GDNF-treated mice. Stereological assessment of immunolabeled rods at 23 days showed that control rd/rd retinas contained 41,880+/-3,890 (mean +/- SEM; n = 6), phosphate-buffered saline (PBS)-injected retinas contained 61,165+/-4,932 (n = 10; P < 0.001 versus control retinas) and GDNF-injected retinas contained 89,232+/-8,033 (n = 10; P < 0.001 versus control retinas, P < 0.002 versus PBS). This increase in rod numbers after GDNF treatment was confirmed by cell counts obtained from frozen sections. CONCLUSIONS: GDNF exerts both histologic and functional neuroprotective effects on rod photoreceptors in the rd/rd mouse. Thus rescue was demonstrated in an animal model of inherited retinal degeneration in which the gene defect was located within the rods themselves, similar to most forms of human retinitis pigmentosa. GDNF represents a candidate neurotrophic factor for palliating some forms of hereditary human blindness.

Fibroblast growth factor receptor 4 (FGFR4) is expressed in adult rat and human retinal photoreceptors and neurons.

The fibroblast growth factor (FGF) family, with its prototype members acidic FGF (FGF-1) and basic FGF (FGF-2), binds to four related receptor tyrosine kinases, termed FGFR1, R2, R3, and R4, expressed on most types of cells in tissue culture. In many respects, the FGFR appear similar to other growth factor receptors; thus, dimerization of receptor monomers on ligand binding is likely to be a requisite for activation of the kinase domains, leading to receptor trans-phosphorylation. Within the central nervous system (CNS), including retina, FGFR1 and R2 have been widely described as the predominant forms. FGFR4 is reported to be strongly expressed only during early stages of development, and apart from one small region (the lateral habenular nucleus) is not detectable in adult CNS. Screening of different neural and nonneural tissues by reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that whereas FGFR1 and R2 were strongly expressed in adult cortex, cerebellum, retina, and kidney, robust FGFR4 expression was only seen in retina and kidney. FGFR4 mRNA was present within fractions of the outer and inner nuclear layers isolated from adult rat retinas, and could also be detected in pure photoreceptor cultures prepared from young rat retinas. On the contrary, FGFR4 mRNA could not be detected in primary cultures of retinal Müller glia or pigment epithelium, indicating specific enrichment in retinal neurons. In situ hybridization studies of adult rat retina showed FGFR4 expression in all retinal cellular layers, especially prominent in the outer nuclear layer. FGFR4 protein was detected by immunoblotting of homogenates of rat retina, with specific antibody binding to bands at 115, 47, and 30 kDa. FGFR4 mRNA and protein were also reliably detected in postmortem adult human retina. The potential roles of these signal transduction molecules in FGF-induced biological responses in the retina are discussed.

MDM2 expression during mouse embryogenesis and the requirement of p53.

We compared mouse embryonic expression of the MDM2 proto-oncogene, p21WAF1/CIP1 and their transcriptional regulator, p53. MDM2 expression is ubiquitous from 7.5 to 11.5 days post coitum (dpc) and more restricted from 12.5 dpc, with the highest levels in the testes and neural tube. From 14.5 to 18.5 dpc, the nasal respiratory epithelium expresses high levels of MDM2 RNA and protein and p21WAF1/CIP1 RNA, in both wild type and p53 null embryos. MDM2 expression during development is tissue-specific and, like p21WAF1/CIP1, is independent of p53. MDM2 may have a developmental role after 6.5 dpc, when MDM2 null mice die (Jones, S.N., Roe, A.E., Donehower, L.A., Bradley, A., 1995. Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206-208; Montes de Oca Luna, R., Wagner, D.S., Lozano, G., 1995. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203-206).

Normal retina releases a diffusible factor stimulating cone survival in the retinal degeneration mouse.

The role of cellular interactions in the mechanism of secondary cone photoreceptor degeneration in inherited retinal degenerations in which the mutation specifically affects rod photoreceptors was studied. We developed an organ culture model of whole retinas from 5-week-old mice carrying the retinal degeneration mutation, which at this age contain few remaining rods and numerous surviving cones cocultured with primary cultures of mixed cells from postnatal day 8 normal-sighted mice (C57BL/6) retinas or retinal explants from normal (C57BL/6) or dystrophic (C3H/He) 5-week-old mice. After 7 days, the numbers of residual cone photoreceptors were quantified after specific peanut lectin or anti-arrestin antibody labeling by using an unbiased stereological approach. Examination of organ cultured retinas revealed significantly greater numbers of surviving cones (15-20%) if cultured in the presence of retinas containing normal rods as compared with controls or cocultures with rod-deprived retinas. These data indicate the existence of a diffusible trophic factor released from retinas containing rod cells and acting on retinas in which only cones are present. Because cones are responsible for high acuity and color vision, such data could have important implications not only for eventual therapeutic approaches to human retinal degenerations but also to define interactions between retinal photoreceptor types.

Expression of p21(WAF1/CIP1) during mouse odontogenesis.

p21(WAF1/CIP1) is a cyclin-dependent kinase (Cdk) inhibitor. This protein may function during development as an inducible growth inhibitor that contributes to cell cycle exit and differentiation. The expression pattern of p21 during mouse embryogenesis was correlated with terminal differentiation of multiple cell lineages including skeletal muscles, cartilage, skin and nasal epithelium. p21 expression was analyzed by in situ hybridization during odontogenesis as well as during in vitro tooth development in chemically defined medium with or without retinoic acid. p21 transcripts were detected in the restricted area of the inner dental epithelium during late cap and initial bell stages and then confined to the post-mitotic odontoblasts and ameloblasts. The replicating cells were devoid of any signal. The distribution of p21 mRNA in vitro, whatever the culture conditions, was similar to the in vivo pattern. p21 protein immunolocalization was superimposed on the transcripts distribution but more restricted in ameloblasts. TGFbeta1 is known to induce p21 expression. During dental cytodifferentiations, TGFbeta1 and p21 expressions overlap. Growth inhibition by TGFbeta1 may be associated with p21 induction.

The MDM2 C-terminal region binds to TAFII250 and is required for MDM2 regulation of the cyclin A promoter.

MDM2 proto-oncogene expression is aberrant in many human tumors. Its normal role is to modulate the functions of p53. The N terminus of MDM2 interacts with p53, whereas the properties of the rest of the molecule are poorly understood. We show that MDM2 binds to the general transcription factor TFIID in vivo. The C-terminal Ring finger interacts with TAFII250/CCG1, and the central acidic domain interacts with TBP. Expression of MDM2 activates the cyclin A gene promoter but not c-fos, showing that the effects of MDM2 are specific. Deletion of the C-terminal region of MDM2 abolishes activation, showing that the C-terminal domain of MDM2 is functionally important. We found that increasing MDM2 expression to higher levels inhibits the cyclin A promoter. Inhibition appears to result from titration of general transcription factors because MDM2 overexpression inhibits c-fos as well as other promoters in vivo and basal transcription in vitro. The mechanisms of repression of the cyclin A and fos promoters appear to be different. Cyclin A repression is lost by deleting the C terminus, whereas that of c-fos is lost by removal of the acidic domain. These results reinforce the conclusion that the C terminus of MDM2 mediates effects on the cyclin A promoter. MDM2 transformed cells contain elevated levels of cyclin A mRNA, showing that activation occurs under physiological conditions. There is a positive correlation between MDM2 binding to TAFII250 and MDM2 activation of the cyclin A promoter. The C-terminal region of MDM2, which contains the Ring finger, interacts with TAFII250 and is required for regulation of the cyclin A promoter by MDM2. Our results link the activity of MDM2, a transforming protein implicated in many human tumors, with cyclin A, a regulator of the cell cycle.

Functional interactions between p53 and the TFIIH complex are affected by tumour-associated mutations.

The p53 tumour suppressor is mutated in the majority of human tumours. p53's proposed role as the guardian of the genome is reflected in its multiple effects on transcription genome stability, cell growth and survival. We show that p53 interacts both physically and functionally with the TFIIH complex. There are multiple protein-protein contacts, involving two regions of p53 and three subunits of TFIIH, ERCC2 (XPD), ERCC3 (XPB) and p62. p53 and its C-terminus (amino acids 320-393) inhibit both of the TFIIH helicases and in vitro transcription in the absence of TFIIH. Transcription inhibition is overcome by TFIIH. The N-terminal region of p53 (1-320), lacking the C-terminus, is inactive on its own, yet apparently affects the activity of the C-terminus in the native protein. Interestingly, mutant p53s that are frequently found in tumours are less efficient inhibitors of the helicases and transcription. We hypothesize that the interactions provide an immediate and direct link for p53 to the multiple functions of TFIIH in transcription, DNA repair and possibly the cell cycle.

Repression and redirection of Saccharomyces cerevisiae tRNA synthesis from upstream of the transcriptional start site.

Derivatives of the Saccharomyces cerevisiae SUP4 tRNATyr gene with binding sites for the transcription regulatory protein GCN4 located upstream of the transcriptional start site have been constructed. The effect of GCN4 on transcription of these genes by purified RNA polymerase III and transcription factors (TF) IIIB and IIIC has been analyzed. GCN4 effectively blocks initiation of transcription only when prebound to sites that overlap with the binding site of TFIIIB. Residual GCN4-repressed transcription is significantly redirected to nearby downstream sites, the selection of which depends on the location of bound GCN4. That prebound repressing GCN4 redirects, instead of merely blocking, the TFIIIC-dependent interaction of TFIIIB with DNA has been directly demonstrated by footprinting. The effect of GCN4 on transcription persists after it has been stripped off its DNA-binding site: once it has been redirected, DNA-bound TFIIIB remains in place, a consequence of the fact that it binds extraordinarily tightly to DNA without recognizing specific DNA sequence.

Diverse molecular mechanisms of inhibition of NF-kappa B/DNA binding complexes by I kappa B proteins.

The activity of the rel/NF-kappa B/dorsal family of kappa B site binding proteins is regulated by I kappa B proteins. The ankyrin repeat motif identified I kappa B family members, which include I kappa B alpha (pp40/MAD-3), I kappa B gamma, and bcl3, directly associated with kappa B site binding proteins, resulting in specific DNA-binding inhibition of rel, p50, or p65 dimers. We report that I kappa B gamma, containing eight ankyrin repeats, mediates a reversible inhibition of (p50)2-DNA complex but cannot displace preformed DNA-protein complexes. I kappa B alpha and bcl3, on the other hand, can displace preformed DNA-protein complexes. I kappa B alpha specifically displaces (p65)2 or p50/p65-DNA complexes but requires the C-terminal 37 amino acids in addition to the ankyrin repeat domain. Human bcl3 protein specifically displaces (p50)2-DNA complexes. Because I kappa B alpha and bcl3 can displace preformed (p65)2 or (p50)2-DNA complexes, respectively, we propose that they can act as repressors or antirepressors of NF-kappa B-induced gene expression.

Saccharomyces cerevisiae transcription factors IIIB and IIIC bend the DNA of a tRNA(Gln) gene.

The ability of Saccharomyces cerevisiae transcription factors IIIC and IIIB to bend DNA at their binding sites in a tRNA(Gln) gene has been analyzed by two methods that are based on the anomalous electrophoretic mobility of bent DNA in a tight gel network. Both transcription factors bend DNA, TFIIIB more than TFIIIC. The effective centers of the transcription factor IIIB- and IIIC-induced bends are not in helical phase.

Structural analysis of the human inter-alpha-trypsin inhibitor light-chain gene.

The human inter-alpha-trypsin inhibitor (ITI) light-chain gene, which codes for the two proteins alpha 1-microglobulin (protein HC) and ITI-derived human inhibitor of 30 kDa (HI-30), was isolated from a human genomic library. This gene, present as a single copy in the human genome, is composed of 10 exons and 9 introns distributed over 20 kbp. A single transcriptional initiation site was identified in the 5'-flanking region which contained promoter elements, but no typical TATA box. However a sequence equivalent to the TATA box is present on both sense and anti-sense strands in the 5'-flanking region of the first exon coding for HI-30. The exon-intron organization suggests that the regions coding for protein HC and other members of the lipocalin superfamily evolved from a common ancestral gene that is probably different from that coding for HI-30. These data suggest that two distinct ancestral genes could have existed and fused during evolution. Several direct and one inverted repeats are also found within this gene, as well as potential glucocorticoid-receptor binding sites.