A Spontaneous Nonhuman Primate Model of Myopic Foveoschisis.

Purpose: Foveoschisis involves the pathologic splitting of retinal layers at the fovea, which may occur congenitally in X-linked retinoschisis (XLRS) or as an acquired complication of myopia. XLRS is attributed to functional loss of the retinal adhesion protein retinoschisin 1 (RS1), but the pathophysiology of myopic foveoschisis is unclear due to the lack of animal models. Here, we characterized a novel nonhuman primate model of myopic foveoschisis through clinical examination and multimodal imaging followed by morphologic, cellular, and transcriptional profiling of retinal tissues and genetic analysis. Methods: We identified a rhesus macaque with behavioral and anatomic features of myopic foveoschisis, and monitored disease progression over 14 months by fundus photography, fluorescein angiography, and optical coherence tomography (OCT). After necropsy, we evaluated anatomic and cellular changes by immunohistochemistry and transcriptomic changes using single-nuclei RNA-sequencing (snRNA-seq). Finally, we performed Sanger and whole exome sequencing with focus on the RS1 gene. Results: Affected eyes demonstrated posterior hyaloid traction and progressive splitting of the outer plexiform layer on OCT. Immunohistochemistry showed increased GFAP expression in Müller glia and loss of ramified Iba-1+ microglia, suggesting macro- and microglial activation with minimal photoreceptor alterations. SnRNA-seq revealed gene expression changes predominantly in cones and retinal ganglion cells involving chromatin modification, suggestive of cellular stress at the fovea. No defects in the RS1 gene or its expression were detected. Conclusions: This nonhuman primate model of foveoschisis reveals insights into how acquired myopic traction leads to phenotypically similar morphologic and cellular changes as congenital XLRS without alterations in RS1.

XLRS Rat with Rs1-/Y Exon-1-Del Shows Failure of Early Postnatal Outer Retina Development.

We generated a Long Evans transgenic rat with targeted deletion of the whole Rs1 exon-1 and evaluated the pathological retinal phenotype of this Rs1-/Y rat model of X-linked retinoschisis (XLRS). The Rs1-/Y rat exhibited very early onset and rapidly progressive photoreceptor degeneration. The outer limiting membrane (OLM) was disrupted and discontinuous by post-natal day (P15) and allowed photoreceptor nuclei to dislocate from the outer nuclear layers (ONL) into the sub-retinal side of the OLM. Dark-adapted electroretinogram (ERG) a-wave and b-wave amplitudes were considerably reduced to only 20-25% of WT by P17. Microglia and Müller glial showed cell marker activation by P7. Intravitreal application of AAV8-RS1 at P5-6 induced RS1 expression by P15 and rescued the inner nuclear layer (INL) and outer plexiform layer (OPL) cavity formation otherwise present at P15, and the outer-retinal structure was less disrupted. This Rs1-/Y exon-1-del rat model displays substantially faster rod cell loss compared to the exon-1-del Rs1-KO mouse. Most unexpected was the rapid appearance of schisis cavities between P7 and P15, and then cavities rapidly disappeared by P21/P30. The rat model provides clues on the molecular and cellular mechanisms underlying XLRS pathology in this model and points to a substantial and early changes to normal retinal development.

MicroRNA and diabetic retinopathy-biomarkers and novel therapeutics.

Diabetic retinopathy (DR) accounts for ~80% of legal blindness in persons aged 20-74 years and is associated with enormous social and health burdens. Current therapies are invasive, non-curative, and in-effective in 15-25% of DR patients. This review outlines the potential utility of microRNAs (miRNAs) as biomarkers and potential therapy for diabetic retinopathy. miRNAs are small noncoding forms of RNA that may play a role in the pathogenesis of DR by altering the level of expression of genes via single nucleotide polymorphism and regulatory loops. A majority of miRNAs are intracellular and specific intracellular microRNAs have been associated with cellular changes associated with DR. Some microRNAs are extracellular and called circulatory microRNAs. Circulatory miRNAs have been found to be differentially expressed in serum and bodily fluid in patients with diabetes mellitus (DM) with and without retinopathy. Some miRNAs have been associated with the severity of DR, and future studies may reveal whether circulatory miRNAs could serve as novel reliable biomarkers to detect or predict retinopathy progression. Therapeutic strategies can be developed utilizing the natural miRNA/long noncoding RNA (lncRNA) regulatory loops. miRNAs and lncRNAs are two major families of the non-protein-coding transcripts. They are regulatory molecules for fundamental cellular processes via a variety of mechanisms, and their expression and function are tightly regulated. The recent evidence indicates a cross-talk between miRNAs and lncRNAs. Therefore, dysregulation of miRNAs and lncRNAs is critical to human disease pathogenesis, such as diabetic retinopathy. miRNAs are long-distance communicators and reprogramming agents, and they embody an entirely novel paradigm in cellular and tissue signaling and interaction. By targeting specific miRNAs, whole pathways implicated in the pathogenesis of DR may potentially be altered. Understanding the endogenous roles of miRNAs in the pathogenesis of diabetic retinopathy could lead to novel diagnostic and therapeutic approaches to managing this frequently blinding retinal condition.

A complement factor H homolog, heparan sulfation, and syndecan maintain inversin compartment boundaries in C. elegans cilia.

Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly. Canonical disease models suggest that defective interactions between complement factor H (CFH) and cell surface heparan sulfate (HS) result in increased alternative complement pathway activity, cytolytic damage, and tissue inflammation in the retina. Although these factors are thought to contribute to increased disease risk, multiple studies indicate that noncanonical mechanisms that result from defective CFH and HS interaction may contribute to the progression of AMD as well. A total of 60 ciliated sensory neurons in the nematode Caenorhabditis elegans detect chemical, olfactory, mechanical, and thermal cues in the environment. Here, we find that a C. elegans CFH homolog localizes on CEP mechanosensory neuron cilia where it has noncanonical roles in maintaining inversin/NPHP-2 within its namesake proximal compartment and preventing inversin/NPHP-2 accumulation in distal cilia compartments in aging adults. CFH localization and maintenance of inversin/NPHP-2 compartment integrity depend on the HS 3-O sulfotransferase HST-3.1 and the transmembrane proteoglycan syndecan/SDN-1. Defective inversin/NPHP-2 localization in mouse and human photoreceptors with CFH mutations indicates that these functions and interactions may be conserved in vertebrate sensory neurons, suggesting that previously unappreciated defects in cilia structure may contribute to the progressive photoreceptor dysfunction associated with CFH loss-of-function mutations in some AMD patients.

Unique molecular signatures of microRNAs in ocular fluids and plasma in diabetic retinopathy.

The main objective of this pilot study was to identify circulatory microRNAs in aqueous or plasma that were reflecting changes in vitreous of diabetic retinopathy patients. Aqueous, vitreous and plasma samples were collected from a total of 27 patients undergoing vitreoretinal surgery: 11 controls (macular pucker or macular hole patients) and 16 with diabetes mellitus(DM): DM-Type I with proliferative diabetic retinopathy(PDR) (DMI-PDR), DM Type II with PDR(DMII-PDR) and DM Type II with nonproliferative DR(DMII-NPDR). MicroRNAs were isolated using Qiagen microRNeasy kit, quantified on BioAnalyzer, and profiled on Affymetrix GeneChip miRNA 3.0 microarrays. Data were analyzed using Expression Console, Transcriptome Analysis Console, and Ingenuity Pathway Analysis. The comparison analysis of circulatory microRNAs showed that out of a total of 847 human microRNA probes on the microarrays, common microRNAs present both in aqueous and vitreous were identified, and a large number of unique microRNA, dependent on the DM type and severity of retinopathy. Most of the dysregulated microRNAs in aqueous and vitreous of DM patients were upregulated, while in plasma, they were downregulated. Dysregulation of miRNAs in aqueous did not appear to be a good representative of the miRNA abundance in vitreous, or plasma, although a few potential candidates for common biomarkers stood out: let-7b, miR-320b, miR-762 and miR-4488. Additionally, each of the DR subtypes showed miRNAs that were uniquely dysregulated in each fluid (i.e. aqueous: for DMII-NPDR was miR-455-3p; for DMII-PDR was miR-296, and for DMI-PDR it was miR-3202). Pathway analysis identified TGF-beta and VEGF pathways affected. The comparative profiling of circulatory miRNAs showed that a small number of them displayed differential presence in diabetic retinopathy vs. controls. A pattern is emerging of unique molecular microRNA signatures in bodily fluids of DR subtypes, offering promise for the use of ocular fluids and plasma for diagnostic and therapeutic purposes.

Effects of intravitreal injection of human CD34+ bone marrow stem cells in a murine model of diabetic retinopathy.

Human CD34 + stem cells are mobilized from bone marrow to sites of tissue ischemia and play an important role in tissue revascularization. This study used a murine model to test the hypothesis that intravitreal injection of human CD34 + stem cells harvested from bone marrow (BMSCs) can have protective effects in eyes with diabetic retinopathy. Streptozotocin-induced diabetic mice (C57BL/6J) were used as a model for diabetic retinopathy. Subcutaneous implantation of Alzet pump, loaded with Tacrolimus and Rapamycin, 5 days prior to intravitreal injection provided continuous systemic immunosuppression for the study duration to avoid rejection of human cells. Human CD34 + BMSCs were harvested from the mononuclear cell fraction of bone marrow from a healthy donor using magnetic beads. The CD34 + cells were labeled with enhanced green fluorescent protein (EGFP) using a lentiviral vector. The right eye of each mouse received an intravitreal injection of 50,000 EGFP-labeled CD34 + BMSCs or phosphate buffered saline (PBS). Simultaneous multimodal in vivo retinal imaging system consisting of fluorescent scanning laser ophthalmoscopy (enabling fluorescein angiography), optical coherence tomography (OCT) and OCT angiography was used to confirm the development of diabetic retinopathy and study the in vivo migration of the EGFP-labeled CD34 + BMSCs in the vitreous and retina following intravitreal injection. After imaging, the mice were euthanized, and the eyes were removed for immunohistochemistry. In addition, microarray analysis of the retina and retinal flat mount analysis of retinal vasculature were performed. The development of retinal microvascular changes consistent with diabetic retinopathy was visualized using fluorescein angiography and OCT angiography between 5 and 6 months after induction of diabetes in all diabetic mice. These retinal microvascular changes include areas of capillary nonperfusion and late leakage of fluorescein dye. Multimodal in vivo imaging and immunohistochemistry identified EGFP-labeled cells in the superficial retina and along retinal vasculature at 1 and 4 weeks following intravitreal cell injection. Microarray analysis showed changes in expression of 162 murine retinal genes following intravitreal CD34 + BMSC injection when compared to PBS-injected control. The major molecular pathways affected by intravitreal CD34 + BMSC injection in the murine retina included pathways implicated in the pathogenesis of diabetic retinopathy including Toll-like receptor, MAP kinase, oxidative stress, cellular development, assembly and organization pathways. At 4 weeks following intravitreal injection, retinal flat mount analysis showed preservation of the retinal vasculature in eyes injected with CD34 + BMSCs when compared to PBS-injected control. The study findings support the hypothesis that intravitreal injection of human CD34 + BMSCs results in retinal homing and integration of these human cells with preservation of the retinal vasculature in murine eyes with diabetic retinopathy.

Effects of aging and environmental tobacco smoke exposure on ocular and plasma circulatory microRNAs in the Rhesus macaque.

Purpose: To identify changes induced by environmental tobacco smoke (ETS) in circulatory microRNA (miRNA) in plasma and ocular fluids of the Rhesus macaque and compare these changes to normal age-related changes. Tobacco smoke has been identified as the leading environmental risk factor for age-related macular degeneration (AMD). Methods: All Rhesus macaques were housed at the California National Primate Research Center (CNPRC), University of California, Davis. Four groups of animals were used: Group 1 (1-3 years old), Group 2 (19-28 years old), Group 3 (10-16 years old), and Group 4 (middle aged, 9-14 years old). Group 4 was exposed to smoke for 1 month. Ocular fluids and plasma samples were collected, miRNAs isolated, and expression data obtained using Affymetrix miRNA GeneTitan Array Plates 4.0. Bioinformatics analysis was done on the Affymetrix Expression Console (EC), Transcriptome Analysis Software (TAS) using ANOVA for candidate miRNA selection, followed by Ingenuity Pathway Analysis (IPA). Results: The expression of circulatory miRNAs showed statistically significant changes with age and ETS. In the plasma samples, 45 miRNAs were strongly upregulated (fold change >±1.5, p<0.05) upon ETS exposure. In the vitreous, three miRNAs were statistically significantly downregulated with ETS, and two of them (miR-6794 and miR-6790) were also statistically significantly downregulated with age. Some retinal layers exhibited a thinning trend measured with optical coherence tomography (OCT) imaging. The pathways activated were IL-17A, VEGF, and recruitment of eosinophils, Th2 lymphocytes, and macrophages. Conclusions: ETS exposure of Rhesus macaques resulted in statistically significant changes in the expression of the circulatory miRNAs, distinct from those affected by aging. The pathways activated appear to be common for ETS and AMD pathogenesis. These data will be used to develop an animal model of early dry AMD.

Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease.

PURPOSE: To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells. METHODS: CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay. RESULTS: Five gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% ± 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected. CONCLUSIONS: The CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.

Intravitreal Administration of Human Bone Marrow CD34+ Stem Cells in a Murine Model of Retinal Degeneration.

PURPOSE: Intravitreal murine lineage-negative bone marrow (BM) hematopoietic cells slow down retinal degeneration. Because human BM CD34+ hematopoietic cells are not precisely comparable to murine cells, this study examined the effect of intravitreal human BM CD34+ cells on the degenerating retina using a murine model. METHODS: C3H/HeJrd1/rd1 mice, immunosuppressed systemically with tacrolimus and rapamycin, were injected intravitreally with PBS (n = 16) or CD34+ cells (n = 16) isolated from human BM using a magnetic cell sorter and labeled with enhanced green fluorescent protein (EGFP). After 1 and 4 weeks, the injected eyes were imaged with scanning laser ophthalmoscopy (SLO)/optical coherence tomography (OCT) and tested with electroretinography (ERG). Eyes were harvested after euthanasia for immunohistochemical and microarray analysis of the retina. RESULTS: In vivo SLO fundus imaging visualized EGFP-labeled cells within the eyes following intravitreal injection. Simultaneous OCT analysis localized the EGFP-labeled cells on the retinal surface resulting in a saw-toothed appearance. Immunohistochemical analysis of the retina identified EGFP-labeled cells on the retinal surface and adjacent to ganglion cells. Electroretinography testing showed a flat signal both at 1 and 4 weeks following injection in all eyes. Microarray analysis of the retina following cell injection showed altered expression of more than 300 mouse genes, predominantly those regulating photoreceptor function and maintenance and apoptosis. CONCLUSIONS: Intravitreal human BM CD34+ cells rapidly home to the degenerating retinal surface. Although a functional benefit of this cell therapy was not seen on ERG in this rapidly progressive retinal degeneration model, molecular changes in the retina associated with CD34+ cell therapy suggest potential trophic regenerative effects that warrant further exploration.

Comparison of In Vivo Gene Expression Profiling of RPE/Choroid following Intravitreal Injection of Dexamethasone and Triamcinolone Acetonide.

Purpose. To identify retinal pigment epithelium (RPE)/choroid genes and their relevant expression pathways affected by intravitreal injections of dexamethasone and triamcinolone acetonide in mice at clinically relevant time points for patient care. Methods. Differential gene expression of over 34,000 well-characterized mouse genes in the RPE/choroid of 6-week-old C57BL/6J mice was analyzed after intravitreal steroid injections at 1 week and 1 month postinjection, using Affymetrix Mouse Genome 430 2.0 microarrays. The data were analyzed using GeneSpring GX 12.5 and Ingenuity Pathway Analysis (IPA) microarray analysis software for biologically relevant changes. Results. Both triamcinolone and dexamethasone caused differential activation of genes involved in "Circadian Rhythm Signaling" pathway at both time points tested. Triamcinolone (TAA) uniquely induced significant changes in gene expression in "Calcium Signaling" (1 week) and "Glutamate Receptor Signaling" pathways (1 month). In contrast, dexamethasone (Dex) affected the "GABA Receptor Signaling" (1 week) and "Serotonin Receptor Signaling" (1 month) pathways. Understanding how intraocular steroids affect the gene expression of RPE/choroid is clinically relevant. Conclusions. This in vivo study has elucidated several genes and pathways that are potentially altering the circadian rhythms and several other neurotransmitter pathways in RPE/choroid during intravitreal steroid injections, which likely has consequences in the dysregulation of RPE function and neurodegeneration of the retina.

GSTM1 and GSTM5 Genetic Polymorphisms and Expression in Age-Related Macular Degeneration.

PURPOSE: Previously, two cytosolic antioxidant enzymes, Glutathione S-transferase Mu 1 (GSTM1) and Mu 5 (GSTM5), were reduced in retinas with age-related macular degeneration (AMD). This study compared genomic copy number variations (gCNV) of these two antioxidant enzymes in AMD versus controls. METHODS: Genomic copy number (gCN) assays were performed using Taqman Gene Copy Number Assays (Applied Biosystems, Darmstadt, Germany) in technical quadruplicate for both GSTM1 and GSTM5. Peripheral leukocyte RNA levels were compared with controls in technical triplicates. Statistical comparisons were performed in SAS v9.2 (SAS Institute Inc., Cary, NC). RESULTS: A large percentage of patients in both AMD and age-matched control groups had no copies of GSTM1 (0/0). The mean gCN of GSTM1 was 1.40 (range 0-4) and 1.61 (range 0-5) for AMD and control, respectively (p = 0.29). A greater percentage of control patients had > 3 gCNs of GSTM1 compared with AMD, respectively (15.3% versus 3.0%, p = 0.004). The gCN of GSTM5 was 2 in all samples except one control sample. The relative quantification of GSTM1 and GSTM5 mRNA from peripheral blood leukocytes in patients showed significant differences in relative expression in AMD versus control (p < 0.05). Peripheral blood leukocyte mRNA and gCN were not significantly correlated (p = 0.27). CONCLUSION: Since high copy numbers of GSTM1 are found more frequently in controls than in AMD, it is possible that high copy number leads to increased retinal antioxidant defense. Genomic polymorphisms of GSTM1 and GSTM5 do not significantly affect the peripheral blood leukocyte mRNA levels.

Localization of complement factor H gene expression and protein distribution in the mouse outer retina.

PURPOSE: To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina. METHODS: Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC. RESULTS: Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh(-/-) eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh(-/-) mice. Greatly reduced Cfh protein immunohistological signals in the Cfh(-/-) eyes also supported the specificity of the Cfh protein distribution results. CONCLUSIONS: Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC.

Age-dependent increase in miRNA-34a expression in the posterior pole of the mouse eye.

PURPOSE: MicroRNA-34a (miR-34a) has been implicated in neurodegeneration. MiR-34a belongs to a signaling network involving p53 and Sirt-1. This network responds to DNA damage with further downstream signals that induce senescence or apoptosis. Our goal was to measure the expression level of miR-34a in the mouse retina and RPE as a function of age. METHODS: The age-dependent change in miR-34a expression was quantified using a real-time PCR (RT-PCR) assay on microRNA isolates from eye tissue: the retina and RPE/choroid (4, 18, 24, and 32 months of age). Tissue localization of miR-34a was determined by in situ hybridization (ISH) for a series of time points. Expression of the miR-34a target gene Sirt1 was analyzed using RT-PCR and immunohistochemistry. RESULTS: MiR-34a examined with real-time PCR showed a linear increase in expression with age when compared to that of 4-month-old mice. However, the level of expression between the 24 and 32-month-old animals showed mild downregulation. An age-related increase in miR-34a expression was confirmed in the mouse eye using in situ hybridization. An inverse relationship between the levels of expression of miR-34a and its target Sirt1 mRNA was found at 18 and 24 months of age. CONCLUSIONS: Our data showed that miR-34a expression increased in the retina and RPE with age. The level of DNA damage in mitochondria in the retina and RPE followed a similar time course. This suggests that miR-34a may play a role in the senescence and apoptosis of the retina and RPE cells in the aging eye.

Enhanced innate antiviral gene expression, IFN-α, and cytolytic responses are predictive of mucosal immune recovery during simian immunodeficiency virus infection.

The mucosa that lines the respiratory and gastrointestinal (GI) tracts is an important portal of entry for pathogens and provides the first line of innate immune defense against infections. Although an abundance of memory CD4(+) T cells at mucosal sites render them highly susceptible to HIV infection, the gut and not the lung experiences severe and sustained CD4(+) T cell depletion and tissue disruption. We hypothesized that distinct immune responses in the lung and gut during the primary and chronic stages of viral infection contribute to these differences. Using the SIV model of AIDS, we performed a comparative analysis of the molecular and cellular characteristics of host responses in the gut and lung. Our findings showed that both mucosal compartments harbor similar percentages of memory CD4(+) T cells and displayed comparable cytokine (IL-2, IFN-γ, and TNF-α) responses to mitogenic stimulations prior to infection. However, despite similar viral replication and CD4(+) T cell depletion during primary SIV infection, CD4(+) T cell restoration kinetics in the lung and gut diverged during acute viral infection. The CD4(+) T cells rebounded or were preserved in the lung mucosa during chronic viral infection, which correlated with heightened induction of type I IFN signaling molecules and innate viral restriction factors. In contrast, the lack of CD4(+) T cell restoration in the gut was associated with dampened immune responses and diminished expression of viral restriction factors. Thus, unique immune mechanisms contribute to the differential response and protection of pulmonary versus GI mucosa and can be leveraged to enhance mucosal recovery.

Genetics and epigenetics of aging and longevity.

Evolutionary theories of aging predict the existence of certain genes that provide selective advantage early in life with adverse effect on lifespan later in life (antagonistic pleiotropy theory) or longevity insurance genes (disposable soma theory). Indeed, the study of human and animal genetics is gradually identifying new genes that increase lifespan when overexpressed or mutated: gerontogenes. Furthermore, genetic and epigenetic mechanisms are being identified that have a positive effect on longevity. The gerontogenes are classified as lifespan regulators, mediators, effectors, housekeeping genes, genes involved in mitochondrial function, and genes regulating cellular senescence and apoptosis. In this review we demonstrate that the majority of the genes as well as genetic and epigenetic mechanisms that are involved in regulation of longevity are highly interconnected and related to stress response.

Potential therapeutic approaches for modulating expression and accumulation of defective lamin A in laminopathies and age-related diseases.

Scientific understanding of the genetic components of aging has increased in recent years, with several genes being identified as playing roles in the aging process and, potentially, longevity. In particular, genes encoding components of the nuclear lamina in eukaryotes have been increasingly well characterized, owing in part to their clinical significance in age-related diseases. This review focuses on one such gene, which encodes lamin A, a key component of the nuclear lamina. Genetic variation in this gene can give rise to lethal, early-onset diseases known as laminopathies. Here, we analyze the literature and conduct computational analyses of lamin A signaling and intracellular interactions in order to examine potential mechanisms for altering or slowing down aberrant Lamin A expression and/or for restoring the ratio of normal to aberrant lamin A. The ultimate goal of such studies is to ameliorate or combat laminopathies and related diseases of aging, and we provide a discussion of current approaches in this review.

Gadd45 proteins: relevance to aging, longevity and age-related pathologies.

The Gadd45 proteins have been intensively studied, in view of their important role in key cellular processes. Indeed, the Gadd45 proteins stand at the crossroad of the cell fates by controlling the balance between cell (DNA) repair, eliminating (apoptosis) or preventing the expansion of potentially dangerous cells (cell cycle arrest, cellular senescence), and maintaining the stem cell pool. However, the biogerontological aspects have not thus far received sufficient attention. Here we analyzed the pathways and modes of action by which Gadd45 members are involved in aging, longevity and age-related diseases. Because of their pleiotropic action, a decreased inducibility of Gadd45 members may have far-reaching consequences including genome instability, accumulation of DNA damage, and disorders in cellular homeostasis - all of which may eventually contribute to the aging process and age-related disorders (promotion of tumorigenesis, immune disorders, insulin resistance and reduced responsiveness to stress). Most recently, the dGadd45 gene has been identified as a longevity regulator in Drosophila. Although further wide-scale research is warranted, it is becoming increasingly clear that Gadd45s are highly relevant to aging, age-related diseases (ARDs) and to the control of life span, suggesting them as potential therapeutic targets in ARDs and pro-longevity interventions.

In vivo gene expression profiling of retina postintravitreal injections of dexamethasone and triamcinolone at clinically relevant time points for patient care.

PURPOSE: To identify retinal genes and their relevant expression pathways affected by intravitreal injections of dexamethasone (Dex) and triamcinolone acetonide (TAA) in mice at clinically relevant time points for patient care. METHODS: Differential gene expressions of over 34,000 well-characterized mouse genes, in the retinas of 6-week-old C57BL/6J mice, were analyzed after intravitreal steroid injections at 1 week and 1 month time points, using mouse genome microarrays. The data were analyzed using commercial microarray analysis software for biologically relevant changes in gene expression pathways. RESULTS: A common gene pathway, with differentially activated genes for both steroids and time points, was "Semaphorin Signaling in Neurons," a member of the "Axonal Guidance Signaling System." At 1 week postinjection a common theme was activation of genes expressed in retinal glial cells, tumor necrosis factor-α, and transforming growth factor-ß signaling pathways and upregulation of stress response proteins (Serpina3n, Cebpd), as well as neuropeptide signaling somatostatin receptor (Sstr2). Unique for Dex was the upregulation of acute phase proteins (Gfap, Cp, Edn2) as well as Plexna2, a semaphorin signaling receptor, whereas EphrinB receptor ephexin 1 (Argef15) was downregulated. Folate signaling appears to be unique for TAA at 1 week (Folh1, Cubn), whereas aryl-hydrocarbon receptor signaling might be important for both steroids at 1 month postinjection. CONCLUSIONS: Understanding the molecular and genetic effects of intraocular steroid treatments is of clinical relevance. This in vivo study has elucidated several genes and pathways that are potentially altering the neuroprotective/neurodegenerative balance between glial and retinal ganglion cells during intravitreal steroid treatment.

An oncogenic role for the phosphorylated h-subunit of human translation initiation factor eIF3.

Dysregulation of protein synthesis has been implicated in oncogenesis through a mechanism whereby "weak" mRNAs encoding proteins involved in cell proliferation are strongly translated when the protein synthesis apparatus is activated. Previous work has determined that many cancer cells contain high levels of eIF3h, a protein subunit of translation initiation factor eIF3, and overexpression of eIF3h malignantly transforms immortal NIH-3T3 cells. This is a general feature of eIF3h, as high levels also affect translation, proliferation, and a number of malignant phenotypes of CHO-K1 and HeLa cells and, most significantly, of a primary prostate cell line. Furthermore, overexpressed eIF3h inhibits Myc-dependent induction of apoptosis of primary prostate cells. eIF3h appears to function through translation, as the initial appearance of overexpressed eIF3h in rapidly induced NIH-3T3 cells correlates tightly with the stimulation of protein synthesis and the generation of malignant phenotypes. This oncogenic potential of eIF3h is enhanced by phosphorylation at Ser(183). Finally, reduction of eIF3h levels in breast and prostate cancer cell lines by short interfering RNA methods reduces their rates of proliferation and anchorage-independent growth in soft agar. The results provide compelling evidence that high eIF3h levels directly stimulate protein synthesis, resulting in the establishment and maintenance of the malignant state in cells.

A strong genetic determinant of hyperoxia-related retinal degeneration on mouse chromosome 6.

PURPOSE: Hyperoxia-related retinal degeneration (HRRD) is a model system in the mouse in which elevated oxygen levels are used to induce retinal degeneration. The hypothesis for the present study was that strain differences in HRRD susceptibility are due to allelic variants of one or more genes in the mouse genome whose human orthologues should be important targets for research and drug development. METHODS: C57BL/6J, A/J, or B.A-Chr6 mice were exposed to 75% oxygen (hyperoxia) or room air for 14 days. After death, one eye was fixed and processed for outer nuclear layer (ONL) thickness measurements. The retina and RPE/choroid were separately dissected from the fellow eye and processed for microarray analysis. Single nucleotide polymorphism (SNP) analysis for transcribed sequences from the C57BL/6J and A/J genomes was conducted using the NIH genome site. RESULTS: C57BL/6J mice developed a significant retinal degeneration in the inferior hemisphere after 14 days of hyperoxia. Under identical conditions, A/J mice exhibited only minor changes. A significant genetic effect was located on chromosome 6. SNP analysis of known transcribed sequences on chromosome 6 combined with microarray expression analysis yielded 33 candidate genes. CONCLUSIONS: A significant genetic effect of susceptibility to HRRD is located on chromosome 6. In silico analysis of transcribed sequences results in a fairly small number of candidate genes.