In the absence of Sonic hedgehog, p53 induces apoptosis and inhibits retinal cell proliferation, cell-cycle exit and differentiation in zebrafish

Background

Sonic hedgehog (Shh) signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation.

Methodology/Principal Findings

Analysis of the zebrafish shh ^−/− mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh ^−/− mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh ^−/− mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh ^−/− mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53 ^−/− shh ^−/− mutant retina suggesting the effect of p53 on retinal differentiation.

Conclusions

Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina. Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.     

PKCζ Mediates Breakdown of Outer Blood-Retinal Barriers in Diabetic Retinopathy

Aims/hypothesis

Diabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia.

Methods

Studies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor.

Results

PKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death.

Conclusions

In the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology.     

Tropisms of AAV for Subretinal Delivery to the Neonatal Mouse Retina and Its Application for In Vivo Rescue of Developmental Photoreceptor Disorders

Background

Adeno-associated virus (AAV) is well established as a vehicle for in vivo gene transfer into the mammalian retina. This virus is promising not only for gene therapy of retinal diseases, but also for in vivo functional analysis of retinal genes. Previous reports have shown that AAV can infect various cell types in the developing mouse retina. However, AAV tropism in the developing retina has not yet been examined in detail.

Methodology/Principal Findings

We subretinally delivered seven AAV serotypes (AAV2/1, 2/2, 2/5, 2/8, 2/9, 2/10, and 2/11) of AAV-CAG-mCherry into P0 mouse retinas, and quantitatively evaluated the tropisms of each serotype by its infecting degree in retinal cells. After subretinal injection of AAV into postnatal day 0 (P0) mouse retinas, various retinal cell types were efficiently transduced with different AAVs. Photoreceptor cells were efficiently transduced with AAV2/5. Retinal cells, except for bipolar and Müller glial cells, were efficiently transduced with AAV2/9. Horizontal and/or ganglion cells were efficiently transduced with AAV2/1, AAV2/2, AAV2/8, AAV2/9 and AAV2/10. To confirm the usefulness of AAV-mediated gene transfer into the P0 mouse retina, we performed AAV-mediated rescue of the Cone-rod homeobox gene knockout (Crx KO) mouse, which exhibits an outer segment formation defect, flat electroretinogram (ERG) responses, and photoreceptor degeneration. We injected an AAV expressing Crx under the control of the Crx 2kb promoter into the neonatal Crx KO retina. We showed that AAV mediated-Crx expression significantly decreased the abnormalities of the Crx KO retina.

Conclusion/Significance

In the current study, we report suitable AAV tropisms for delivery into the developing mouse retina. Using AAV2/5 in photoreceptor cells, we demonstrated the possibility of gene replacement for the developmental disorder and subsequent degeneration of retinal photoreceptors caused by the absence of Crx.     

Altered miRNA expression in canine retinas during normal development and in models of retinal degeneration

Background

Although more than 246 loci/genes are associated with inherited retinal diseases, the mechanistic events that link genetic mutations to photoreceptor cell death are poorly understood. miRNAs play a relevant role during retinal development and disease. Thus, as a first step in characterizing miRNA involvement during disease expression and progression, we examined miRNAs expression changes in normal retinal development and in four canine models of retinal degenerative disease.

Results

The initial microarray analysis showed that 50 miRNAs were differentially expressed (DE) early (3 vs. 7 wks) in normal retina development, while only 2 were DE between 7 and 16 wks, when the dog retina is fully mature. miRNA expression profiles were similar between dogs affected with xlpra2, an early-onset retinal disease caused by a microdeletion in RPGRORF15, and normal dogs early in development (3 wks) and at the peak of photoreceptor death (7 wks), when only 2 miRNAs were DE. However, the expression varied much more markedly during the chronic cell death stage at 16 wks (118 up-/55 down-regulated miRNAs). Functional analyses indicated that these DE miRNAs are associated with an increased inflammatory response, as well as cell death/survival. qRT-PCR of selected apoptosis-related miRNAs (“apoptomirs”) confirmed the microarray results in xlpra2, and extended the analysis to the early-onset retinal diseases rcd1 (PDE6B-mutation) and erd (STK38L-mutation), as well as the slowly progressing prcd (PRCD-mutation). The results showed up-regulation of anti-apoptotic (miR-9, -19a, -20, -21, -29b, -146a, -155, -221) and down-regulation of pro-apoptotic (miR-122, -129) apoptomirs in the early-onset diseases and, with few exceptions, also in the prcd-mutants.

Conclusions

Our results suggest that apoptomirs might be expressed by diseased retinas in an attempt to counteract the degenerative process. The pattern of expression in diseased retinas mirrored the morphology and cell death kinetics previously described for these diseases. This study suggests that common miRNA regulatory mechanisms may be involved in retinal degeneration processes and provides attractive opportunities for the development of novel miRNA-based therapies to delay the progression of the degenerative process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-172) contains supplementary material, which is available to authorized users.     

Distinct signature of altered homeostasis in aging rod photoreceptors: implications for retinal diseases

Background

Advanced age contributes to clinical manifestations of many retinopathies and represents a major risk factor for age-related macular degeneration, a leading cause of visual impairment and blindness in the elderly. Rod photoreceptors are especially vulnerable to genetic defects and changes in microenvironment, and are among the first neurons to die in normal aging and in many retinal degenerative diseases. The molecular mechanisms underlying rod photoreceptor vulnerability and potential biomarkers of the aging process in this highly specialized cell type are unknown.

Methodology/Principal Findings

To discover aging-associated adaptations that may influence rod function, we have generated gene expression profiles of purified rod photoreceptors from mouse retina at young adult to early stages of aging (1.5, 5, and 12 month old mice). We identified 375 genes that showed differential expression in rods from 5 and 12 month old mouse retina compared to that of 1.5 month old retina. Quantitative RT-PCR experiments validated expression change for a majority of the 25 genes that were examined. Macroanalysis of differentially expressed genes using gene class testing and protein interaction networks revealed overrepresentation of cellular pathways that are potentially photoreceptor-specific (angiogenesis and lipid/retinoid metabolism), in addition to age-related pathways previously described in several tissue types (oxidative phosphorylation, stress and immune response).

Conclusions/Significance

Our study suggests a progressive shift in cellular homeostasis that may underlie aging-associated functional decline in rod photoreceptors and contribute to a more permissive state for pathological processes involved in retinal diseases.     

Generation,Purification and Transplantation of Photoreceptors Derived from Human Induced Pluripotent Stem Cells

Background

Inherited and acquired retinal degenerations are frequent causes of visual impairment and photoreceptor cell replacement therapy may restore visual function to these individuals. To provide a source of new retinal neurons for cell based therapies, we developed methods to derive retinal progenitors from human ES cells.

Methodology/Physical Findings

In this report we have used a similar method to direct induced pluripotent stem cells (iPS) from human fibroblasts to a retinal progenitor fate, competent to generate photoreceptors. We also found we could purify the photoreceptors derived from the iPS cells using fluorescence activated cell sorting (FACS) after labeling photoreceptors with a lentivirus driving GFP from the IRBP cis-regulatory sequences. Moreover, we found that when we transplanted the FACS purified iPSC derived photoreceptors, they were able to integrate into a normal mouse retina and express photoreceptor markers.

Conclusions

This report provides evidence that enriched populations of human photoreceptors can be derived from iPS cells.     

Tauroursodeoxycholic acid (TUDCA) protects photoreceptors from cell death after experimental retinal detachment

Background

Detachment of photoreceptors from the underlying retinal pigment epithelium is seen in various retinal disorders such as retinal detachment and age-related macular degeneration and leads to loss of photoreceptors and vision. Pharmacologic inhibition of photoreceptor cell death may prevent this outcome. This study tests whether systemic administration of tauroursodeoxycholic acid (TUDCA) can protect photoreceptors from cell death after experimental retinal detachment in rodents.

Methodology/Principal Findings

Retinal detachment was created in rats by subretinal injection of hyaluronic acid. The animals were treated daily with vehicle or TUDCA (500 mg/kg). TUNEL staining was used to evaluate cell death. Photoreceptor loss was evaluated by measuring the relative thickness of the outer nuclear layer (ONL). Macrophage recruitment, oxidative stress, cytokine levels, and caspase levels were also quantified. Three days after detachment, TUDCA decreased the number of TUNEL-positive cells compared to vehicle (651±68/mm² vs. 1314±68/mm², P = 0.001) and prevented the reduction of ONL thickness ratio (0.84±0.03 vs. 0.65±0.03, P = 0.002). Similar results were obtained after 5 days of retinal detachment. Macrophage recruitment and expression levels of TNF-a and MCP-1 after retinal detachment were not affected by TUDCA treatment, whereas increases in activity of caspases 3 and 9 as well as carbonyl-protein adducts were almost completely inhibited by TUDCA treatment.

Conclusions/Significance

Systemic administration of TUDCA preserved photoreceptors after retinal detachment, and was associated with decreased oxidative stress and caspase activity. TUDCA may be used as a novel therapeutic agent for preventing vision loss in diseases that are characterized by photoreceptor detachment.     

Transplantation of photoreceptor and total neural retina preserves cone function in P23H rhodopsin transgenic rat

Background

Transplantation as a therapeutic strategy for inherited retinal degeneration has been historically viewed to restore vision as a method by replacing the lost retinal cells and attempting to reconstruct the neural circuitry with stem cells, progenitor cells and mature neural retinal cells.

Methods and Findings

We present evidence for an alternative strategy aimed at preventing the secondary loss of cones, the most crucial photoreceptors for vision, by transplanting normal photoreceptors cells into the eye of the P23H rat, a model of dominant retinitis pigmentosa. We carried out transplantation of photoreceptors or total neural retina in 3-month-old P23H rats and evaluated the function and cell counts 6 months after surgery. In both groups, cone loss was significantly reduced (10%) in the transplanted eyes where the cone outer segments were found to be considerably longer. This morphological effect correlated with maintenance of the visual function of cones as scored by photopic ERG recording, but more precisely with an increase in the photopic b-wave amplitudes by 100% and 78% for photoreceptor transplantation and whole retinal transplantation respectively.

Conclusions

We demonstrate here that the transplanted tissue prevents the loss of cone function, which is further translated into cone survival.     

Altering TET dioxygenase levels within physiological range affects DNA methylation dynamics of HEK293 cells

The TET family of dioxygenases (TET1/2/3) can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and has been shown to be involved in active and passive DNA demethylation. Here, we demonstrate that altering TET dioxygenase levels within physiological range can affect DNA methylation dynamics of HEK293 cells. Overexpression of TET1 increased global 5hmC levels and was accompanied by mild DNA demethylation of promoters, gene bodies and CpG islands. Conversely, the simultaneous knockdown of TET1, TET2, and TET3 led to decreased global 5hmC levels and mild DNA hypermethylation of above-mentioned regions. The methylation changes observed in the overexpression and knockdown studies were mostly non-reciprocal and occurred with different preference depending on endogenous methylation and gene expression levels. Single-nucleotide 5hmC profiling performed on a genome-wide scale revealed that TET1 overexpression induced 5mC oxidation without a distribution bias among genetic elements and structures. Detailed analysis showed that this oxidation was related to endogenous 5hmC levels. In addition, our results support the notion that the effects of TET1 overexpression on gene expression are generally unrelated to its catalytic activity.     

Inactivation of MARCH5 Prevents Mitochondrial Fragmentation and Interferes with Cell Death in a Neuronal Cell Model

Purpose

To study the impact of the mitochondrial ubiquitin ligase MARCH5 on mitochondrial morphology and induction of apoptosis using an in vitro model of neuronal precursor cells exposed to glaucoma-relevant stress conditions.

Methods

RGC5 cells transfected with expression constructs for MARCH5, MARCH5^H43W, Dpr1^K38A or vector control were exposed to either elevated pressure of 30 mmHg, oxidative stress caused by mitochondrial electron transport chain (ETC) inhibition, or hypoxia-reoxygenation conditions. Mitochondrial morphology of RGC5 cells was analyzed following staining of the mitochondrial marker cytochrome c and photoactivatable GFP (PAGFP) diffusion assay. Induction of apoptotic cell death in these cells was determined by analyzing the release of cytochrome c from mitochondria into the cytosol and flow cytometry.

Results

Exposure of RGC5 cells to oxidative stress conditions as well as to elevated pressure resulted in the fragmentation of the mitochondrial network in control cells as well as in cells expressing MARCH5. In cells expressing inactive MARCH5^H43W or inactive Drp^K38A, mitochondrial fragmentation was significantly blocked and mitochondrial morphology was comparable to that of control cells under normal conditions. Exposure of RGC5 cells to elevated pressure or oxidative stress conditions induced apoptotic cell death as assessed by cytochrome c release and DNA staining, while expression of dominant-negative MARCH5^H43W or Drp1^K38A did significantly delay cell death.

Conclusion

Preventing mitochondrial fragmentation through interference with the mitochondrial fission machinery protects neuronal cells from programmed cell death following exposure to stressors physiologically relevant to the pathogenesis of glaucoma.     

Deletion of Aldose Reductase from Mice Inhibits Diabetes-Induced Retinal Capillary Degeneration and Superoxide Generation

Purpose

Pharmacologic inhibition of aldose reductase (AR) previously has been studied with respect to diabetic retinopathy with mixed results. Since drugs can have off-target effects, we studied the effects of AR deletion on the development and molecular abnormalities that contribute to diabetic retinopathy. Since recent data suggests an important role for leukocytes in the development of the retinopathy, we determined also if AR in leukocytes contributes to leukocyte-mediated death of retinal endothelial cells in diabetes.

Methods

Wild-type (WT; C57BL/6J) and AR deficient (AR^−/−) mice were made diabetic with streptozotocin. Mice were sacrificed at 2 and 10 months of diabetes to evaluate retinal vascular histopathology, to quantify retinal superoxide production and biochemical and physiological abnormalities in the retina, and to assess the number of retinal endothelial cells killed by blood leukocytes in a co-culture system.

Results

Diabetes in WT mice developed the expected degeneration of retinal capillaries, and increased generation of superoxide by the retina. Leukocytes from diabetic WT mice also killed more retinal endothelial cells than did leukocytes from nondiabetic animals (p<0.0001). Deletion of AR largely (P<0.05) inhibited the diabetes-induced degeneration of retinal capillaries, as well as the increase in superoxide production by retina. AR-deficiency significantly inhibited the diabetes-induced increase in expression of inducible nitric oxide synthase (iNOS) in retina, but had no significant effect on expression of intercellular adhesion molecule-1 (ICAM-1), phosphorylated p38 MAPK, or killing of retinal endothelial cells by leukocytes.

Conclusions

AR contributes to the degeneration of retinal capillaries in diabetic mice. Deletion of the enzyme inhibits the diabetes-induced increase in expression of iNOS and of superoxide production, but does not correct a variety of other pro-inflammatory abnormalities associated with the development of diabetic retinopathy.     

Novel rodent models for macular research

Background

Many disabling human retinal disorders involve the central retina, particularly the macula. However, the commonly used rodent models in research, mouse and rat, do not possess a macula. The purpose of this study was to identify small laboratory rodents with a significant central region as potential new models for macular research.

Methodology/Principal Findings

Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli, laboratory rodents less commonly used in retinal research, were subjected to confocal scanning laser ophthalmoscopy (cSLO), fluorescein and indocyanine green angiography, and spectral-domain optical coherence tomography (SD-OCT) using standard equipment (Heidelberg Engineering HRA1 and Spectralis™) adapted to small rodent eyes. The existence of a visual streak-like pattern was assessed on the basis of vascular topography, retinal thickness, and the topography of retinal ganglion cells and cone photoreceptors. All three species examined showed evidence of a significant horizontal streak-like specialization. cSLO angiography and retinal wholemounts revealed that superficial retinal blood vessels typically ramify and narrow into a sparse capillary net at the border of the respective area located dorsal to the optic nerve. Similar to the macular region, there was an absence of larger blood vessels in the streak region. Furthermore, the thickness of the photoreceptor layer and the population density of neurons in the ganglion cell layer were markedly increased in the visual streak region.

Conclusions/Significance

The retinal specializations of Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli resemble features of the primate macula. Hence, the rodents reported here may serve to study aspects of macular development and diseases like age-related macular degeneration and diabetic macular edema, and the preclinical assessment of therapeutic strategies.     

MicroRNA-restricted transgene expression in the retina

Background

Gene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions.

Methodology/Principal Findings

To this end, we generated AAV2/5 vectors expressing EGFP and containing four tandem copies of miR-124 or miR-204 complementary sequences in the 3′UTR of the transgene expression cassette. These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrate that miR-124 and miR-204 target sequences can efficiently restrict AAV2/5-mediated transgene expression to retinal pigment epithelium and photoreceptors, respectively, in mice and pigs. Interestingly, transgene restriction was observed at low vector doses relevant to therapy.

Conclusions

We conclude that microRNA-mediated regulation of transgene expression can be applied in the retina to either restrict to a specific cell type the robust expression obtained using ubiquitous promoters or to provide an additional layer of gene expression regulation when using cell-specific promoters.     

Epigenetic,Genetic and Environmental Interactions in Esophageal Squamous Cell Carcinoma from Northeast India

Background

Esophageal squamous cell carcinoma (ESCC) develops as a result of complex epigenetic, genetic and environmental interactions. Epigenetic changes like, promoter hypermethylation of multiple tumour suppressor genes are frequent events in cancer, and certain habit-related carcinogens are thought to be capable of inducing aberrant methylation. However, the effects of environmental carcinogens depend upon the level of metabolism by carcinogen metabolizing enzymes. As such key interactions between habits related factors and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of genes are likely. However, this remains largely unexplored in ESCC. Here, we studied the interaction of various habits related factors and polymorphism of GSTM1/GSTT1 genes towards inducing promoter hypermethylation of multiple tumour suppressor genes.

Methodology/Principal Findings

The study included 112 ESCC cases and 130 age and gender matched controls. Conditional logistic regression was used to calculate odds ratios (OR) and multifactor dimensionality reduction (MDR) was used to explore high order interactions. Tobacco chewing and smoking were the major individual risk factors of ESCC after adjusting for all potential confounding factors. With regards to methylation status, significantly higher methylation frequencies were observed in tobacco chewers than non chewers for all the four genes under study (p<0.01). In logistic regression analysis, betel quid chewing, alcohol consumption and null GSTT1 genotypes imparted maximum risk for ESCC without promoter hypermethylation. Whereas, tobacco chewing, smoking and GSTT1 null variants were the most important risk factors for ESCC with promoter hypermethylation. MDR analysis revealed two predictor models for ESCC with promoter hypermethylation (Tobacco chewing/Smoking/Betel quid chewing/GSTT1 null) and ESCC without promoter hypermethylation (Betel quid chewing/Alcohol/GSTT1) with TBA of 0.69 and 0.75 respectively and CVC of 10/10 in both models.

Conclusion

Our study identified a possible interaction between tobacco consumption and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of tumour suppressor genes in ESCC.     

The Balance of Apoptotic and Necrotic Cell Death in Mycobacterium tuberculosis Infected Macrophages Is Not Dependent on Bacterial Virulence

Background

An important mechanism of Mycobacterium tuberculosis pathogenesis is the ability to control cell death pathways in infected macrophages: apoptotic cell death is bactericidal, whereas necrotic cell death may facilitate bacterial dissemination and transmission.

Methods

We examine M.tuberculosis control of spontaneous and chemically induced macrophage cell death using automated confocal fluorescence microscopy, image analysis, flow cytometry, plate-reader based vitality assays, and M.tuberculosis strains including H37Rv, and isogenic virulent and avirulent strains of the Beijing lineage isolate GC1237.

Results

We show that bacterial virulence influences the dynamics of caspase activation and the total level of cytotoxicity. We show that the powerful ability of M.tuberculosis to inhibit exogenously stimulated apoptosis is abrogated by loss of virulence. However, loss of virulence did not influence the balance of macrophage apoptosis and necrosis – both virulent and avirulent isogenic strains of GC1237 induced predominantly necrotic cell death compared to H37Rv which induced a higher relative level of apoptosis.

Conclusions

This reveals that macrophage necrosis and apoptosis are independently regulated during M. tuberculosis infection of macrophages. Virulence affects the level of host cell death and ability to inhibit apoptosis but other strain-specific characteristics influence the ultimate mode of host cell death and alter the balance of apoptosis and necrosis.