Expression of neurturin, glial cell line-derived neurotrophic factor, and their receptor components

PURPOSE. Dysregulation of neurturin (NTN) expression has been linked to photoreceptor apoptosis in a mouse model of inherited retinal degeneration. To investigate the extent to which any such dysregulation depends on the nature of the apoptotic trigger, the expression of NTN, glial cell line-derived neurotrophic factor (GDNF), and their corresponding receptor components were compared in a rat model of light-induced retinal degeneration. METHODS. Retinal expression of NTN, GDNF, their corresponding receptors GFR␣-2 and -1, the transmembrane receptor tyrosine kinase (Ret), and cSrc-p60, a member of the cytoplasmic protein-tyrosine kinases family, were analyzed by Western blot analysis and immunocytochemistry in cyclic light-and dark-reared rats in the presence and absence of intense light exposure. RESULTS. All components for NTN-mediated signaling activation are present in rat photoreceptors and retinal pigment epithelium, the cells primarily affected by light-induced damage. The expression levels of GDNF, its receptor components, and NTN, were not affected by light-induced stress. However, GFR␣-2 expression strikingly increased with the extent of retinal damage, especially at the photoreceptors, in contrast to decreased levels that were observed previously in an inherited degeneration model. CONCLUSIONS. The present study indicates that the expression of receptors of the GDNF family is independently regulated in normal and light-damaged rat retina, and in conjunction with previous work, suggests that the pattern of modulation of these genes during photoreceptor degeneration is determined by the nature of the apoptotic trigger. Such differential responses to different modes of retinal degeneration may reflect influences of the neurotrophic system on photoreceptor survival or in the regulation of neuronal plasticity. (Invest Ophthalmol Vis Sci. 2004;45:1240 -1246) DOI:10.1167/iovs.03-1122 G DNF and neurturin (NTN) are members of the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFL) of neurotrophic factors. GFLs have been shown to influence the development of enteric, sympathetic, parasympathetic, and sensory neurons (for review see Ref. 1). They generally signal through a multicomponent receptor system consisting of the receptor tyrosine kinase Ret and a highaffinity ligand binding glycosyl-phosphatidylinositol (GPI)-linked coreceptor (GFR␣). GDNF-mediated bioactivity involves signaling molecules of the src-family of protein-tyrosine kinases; and, in particular, p60 Src has been shown to interact with activated Ret. 2 GDNF and NTN are expressed in a wide variety of tissues including the retina, suggesting an implication in diverse biological processes. 6 Upregulation of NTN mRNA expression was associated with progressive retinal neurodegeneration, but GFR␣-2 mRNA levels remained lower than in age-matched nondegenerative control retinas. On the assumption that increased NTN expression is a survivalpromoting response of the retina to the onset of degeneration, its potential neurotrophic effect on photoreceptors might be constrained by the persistently low GFR␣-2 levels in rd retinas. Alternatively, because NTN also signals through the GDNF receptor (GFR␣-1) but through a low-affinity interaction, 1 it is possible that increased NTN is limited in its efficacy by failure to activate sufficient survival-promoting pathways through the GFR␣-1 receptors. To assess the extent to which such modulations of expression of GFL members and their receptors are dependent on the nature of the apoptotic trigger, we have compared expression patterns of NTN, GDNF, and their receptor components in a model of photoreceptor cell death induced by exposure to intense light. In rats, light-induced retinal damage is rhodopsinmediated and dependent on light intensity, wave length and duration of the exposure, period of dark adaptation before exposure, and the exposure schedule. 8 -12 The effects were studied of both the type I (damaging both the photoreceptors and the retinal pigment epithelium) and type II (characterized by the loss of visual cells only) light-induced damage regimens on the expression of two members of the GDNF family. The retinal distributions of NTN, GDNF, and their receptor components were assessed by immunoblot and immunocytochemistry in control and light-stressed rat retinas

XIAP Protection of Photoreceptors in Animal Models of Retinitis Pigmentosa

BACKGROUND: Retinitis pigmentosa (RP) is a blinding genetic disorder that is caused by the death of photoreceptors in the outer nuclear layer of the retina. To date, 39 different genetic loci have been associated with the disease, and 28 mutated genes have been identified. Despite the complexity of the underlying genetic basis for RP, the final common pathway is photoreceptor cell death via apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, P23H and S334ter rhodopsin transgenic rat models of RP were used to test the neuroprotective effects of anti-apoptotic gene therapy. Adeno-associated viruses (AAV) carrying the X-linked inhibitor of apoptosis (XIAP) or green fluorescent protein (GFP) were delivered subretinally into the eye of transgenic rat pups. Histological and functional measures were used to assess neuroprotection. XIAP is known to block apoptosis by inhibiting the action of caspases-3, -7 and -9. The results show that XIAP gene therapy provides long-term neuroprotection of photoreceptors at both structural and functional levels. CONCLUSIONS/SIGNIFICANCE: Our gene therapy strategy targets the apoptotic cascade, which is the final common pathway in all forms of retinitis pigmentosa. This strategy holds great promise for the treatment of RP, as it allows for the broad protection of photoreceptors, regardless of the initial disease causing mutation

Inactivation of the Akt survival pathway during photoreceptor apoptosis in the retinal degeneration mouse. Invest Ophthalmol Vis Sci 2006

PURPOSE. Previous work has indicated that the serine-threonine protein kinase Akt is a general mediator of cellular survival signals and that loss of Akt-mediated signaling can lead to the activation of apoptosis. This study was conducted to establish whether regulation of the Akt survival pathway mechanisms is implicated in the induction of apoptosis during photoreceptor cell death in the rd mouse model of retinal degeneration. METHODS. Quantitative Western blot analysis and immunocytochemistry were used to examine the activation status and localization of key components of the Akt signaling cascade (Akt, BAD, Forkhead [FKHR], HSP27, mitogen-activated protein (MAP) kinase kinase-3 and -6 (MKK3/6), the tumor-suppressor phosphatase PTEN, and the cytoplasmic protein-tyrosine kinase cSrc-p60), in the retina of the rd mouse in comparison with the control. The time points examined spanned the period of photoreceptor degeneration. RESULTS. In the period up to the peak of photoreceptor apoptosis at postnatal day 15, dysregulation of the survival pathway was identified at several levels, including deactivation of both Akt itself and its downstream transcription factor target Forkhead (FKHR) and activation of the upstream negative regulator PTEN. CONCLUSIONS. Taken in conjunction with previous studies, the data support a model in which photoreceptor cell death in the rd mouse is the result of combined inactivation of the Akt survival pathway and the activation of the two major apoptotic pathways. (Invest Ophthalmol Vis Sci

Generation of light-sensitive photoreceptor phenotypes by genetic modification of human adult ocular stem cells with Crx

Purpose: this study compared the effect of the transcription factor Crx (cone, rod homeobox) on the differentiation of human adult corneal (hCSC) and retinal (hRSC) stem cells into functional photoreceptors. Methods: stem cells isolated from postmortem human corneas and retinal ciliary bodies were maintained in serum-free culture and genetically modified by electroporation to express exogenous epitope-tagged murine Crx. Expression of stem cell markers (Pax6, Oct3/4, and proliferating cell nuclear antigen [PCNA]), neuronal markers (nestin, neuron-specific class III beta-tubulin, Map2 a/b, and neurofilament), and photoreceptor-specific markers (rhodopsin, cyclic nucleotide-gated cation channel-3, blue-cone opsin, and beta-6-PDE) was evaluated by immunocytochemistry. A cGMP enzyme-linked immunoassay was used to assess phototransduction cascade activity by measurement of light-induced hydrolysis of cGMP. Results: expression of the stem cell markers of proliferation and pluripotency Pax6, PCNA, and Oct3/4 was decreased by exogenous Crx expression in both hCSCs and hRSCs. Correspondingly, the expression of the mature neuronal markers Map2 a/b and neurofilament was increased. Both hCSCs and hRSCs displayed photoreceptor-specific immunolabeling. However, light-activated GMP hydrolysis was observed only in hRSCs after exogenous expression of Crx. Conclusions: the present study extends previous findings that exogenous Crx expression can promote differentiation of human retina-derived stem cells into light-sensitive photoreceptor phenotypes. Although Crx can induce human cornea-derived stem cells to express photoreceptor-specific proteins, it does not seem to be sufficient to direct their differentiation into functional photoreceptors. Nevertheless, this study demonstrates that genetic modification of adult human retinal stem cells can cause differentiation into light-sensitive photoreceptor phenotypes

Modulated expression of secreted frizzled-related proteins in human retinal degeneration

PURPOSE. Inherited retinal degenerations such as retinitis pigmentosa (RP) are characterized by progressive death of the photoreceptors due to apoptosis. To identify changes in gene expression associated with the degenerative state in RP retinas, expression profiling of apoptosis-related genes was performed using a gridded array technique. METHODS. Total RNAs from RP and control retinas were used to generate radiolabeled cDNA probes to screen gridded membrane arrays of 205 apoptosis-related genes. Reverse transcription–polymerase chain reaction was used to generate probes corresponding to differentially expressed genes for Northern blot analysis and for mRNA in situ hybridization studies of retinal cryosections. Fluorescence immunocytochemistry was performed on retinal sections using available antibodies. RESULTS. By expression profiling, we identified upregulated expression of the mRNA for secreted Frizzled-related protein-2 (SFRP2) in RP retina in comparison with control. By Northern blot analysis, SFRP2 mRNA levels were 2- to 20-fold higher in RP samples than in controls. The localization of SFRP2 mRNA by in situ hybridization varied according to the degree of degeneration, from stratified in relatively well-preserved retinas to diffuse in the highly degenerative state. By immunofluorescence, SFRP2 protein in RP retinas was found mainly to colocalize with the cell adhesion and signal transducing protein �-catenin. CONCLUSIONS. SFRPs can regulate apoptosis in vitro and appear to interact with the Wnt/Frizzled signaling pathway, which includes routes to apoptotic activation. Increased SFRP2 expression in RP retinas suggests that an altered pattern of Wnt signal transduction may be a step in the degenerative process linking causal mutations with eventual photoreceptor demise. (Invest Ophthalmol Vis Sci

Localization of complement 1 inhibitor (C1INH/SERPING1) in human eyes with age-related macular degeneration

Age-related macular degeneration (AMD) is a common degenerative disease resulting in injury to the retina, retinal pigment epithelium and choriocapillaris. Recent data from histopathology, animal models and genetic studies have implicated altered regulation of the complement system as a major factor in the incidence and progression of this disease. A variant in the gene SERPING1, which encodes C1INH, an inhibitor of the classical and lectin pathways of complement activation, was recently shown to be associated with AMD. In this study we sought to determine the localization of C1INH in human donor eyes. Immunofluorescence studies using a monoclonal antibody directed against C1INH revealed localization to photoreceptor cells, inner nuclear layer neurons, choriocapillaris, and choroidal extracellular matrix. Drusen did not exhibit labeling. Genotype at rs2511989 did not appear to affect C1INH abundance or localization, nor was it associated with significant molecular weight differences when evaluated by Western blot. In a small number of eyes (n = 7 AMD and n = 7 control) AMD affection status was correlated with increased abundance of choroidal C1INH. These results indicate that C1INH protein is present in the retina and choroid, where it may regulate complement activation

Association between the SERPING1 gene and age-related macular degeneration: a two-stage case-control study

Background: age-related macular degeneration is the most prevalent form of visual impairment and blindness in developed countries. Genetic studies have made advancements in establishing the molecular cause of this disease, identifying mutations in the complement factor H (CFH) gene and a locus on chromosome 10 encompassing the HTRA1/LOC387715/ARMS2 genes. Variants in complement 3 (C3) and an HLA locus containing both factor B and C2 genes have also been implicated. We aimed to identify further genetic risk factors for this disease.Methods: we used a case—control study design in a UK sample of patients with age-related macular degeneration (n=479) and controls (n=479) and undertook a low-density screen of 32 genes using 93 single nucleotide polymorphisms (SNPs). Genes were selected as candidates on the basis of potential functional relevance to age-related macular degeneration. Significant initial findings were confirmed by replication in an independent US cohort of 248 unrelated patients with disease and 252 controls, and by high-density genotyping around association signals.Findings: the SNP variant rs2511989, located within intron six of the SERPING1 gene, showed highly significant genotypic association with age-related macular degeneration (uncorrected p=4·0×10?5, corrected p=0·00372). We detected no evidence for association between disease and the other 31 candidate genes. The odds ratio for age-related macular degeneration in rs2511989 G/A heterozygotes compared with wild type G/G homozygotes was 0·63 (95% CI 0·47—0·84). A similar comparison of the A/A homozygotes with the wild type yielded an odds ratio of 0·44 (0·31—0·64). We replicated the observed genotypic association in a US cohort (p=0·008). Furthermore, a secondary high-density genotyping study across the SERPING1 gene region identified five additional SNP variants similarly associated with age-related macular degeneration (rs2244169, rs2511990, rs2509897, rs1005510, and rs2511988).Interpretation: genetic variation in SERPING1 significantly alters susceptibility to age-related macular degeneration. SERPING1 encodes the C1 inhibitor, which has a crucial role in inhibition of complement component 1 (C1) and might implicate the classic pathway of complement activation in this diseas